XXVIIIth GTRV SCIENTIFIC MEETING

MOT DE LA PRESIDENTE
Groupe Thématique
De Recherche sur la Vectorisation
(GTRV)
Association loi de 1901
XXVIIIth GTRV SCIENTIFIC MEETING
Le "Groupe Thématique de Recherche sur les Vecteurs" (G.T.R.V.) a été créé en 1986 à
l'initiative d'éminents chercheurs français, Patrick COUVREUR (Professeur à l'Université de
Paris-Sud), Francis SCHUBER (Directeur de Recherche CNRS, Strasbourg) et Jean
PHILLIPOT (Directeur de Recherche CNRS, Montpellier). Cette initiative n’a eu pour but
depuis lors que de développer et promouvoir les activités de recherche pluridisciplinaires
tournant autour des Nanotechnologies pour la Santé. Aujourd’hui, les nanoparticules pour la
santé sont présentes dans de nombreux domaines allant de l’imagerie, en passant par la
photothérapie, de la régénérescence tissulaire, ou encore la radiothérapie. Le GTRV dont
les membres sont au cœur de ces problématiques a souhaité mieux vous représenter et
rendre les équipes plus visibles en créant une nouvelle société : la Société Française de
Nanomédecine.
Ces journées seront les dernières journées du GTRV qui seront d’un très haut niveau
scientifique avec une demi-journée sur la délivrance intra-dermique hautement à propos
dans un lieu proche de la Cosmetic Valley, une journée dédiée à la thérapie génique non
virale en coordination avec la Société française de Thérapie Cellulaire et Génique, et une
journée dédiée aux systèmes particulaires dans le domaine de l’imagerie.
La Société Française de Nanomédecine aura à cœur de vous proposer des congrès et des
journées de réflexion sur des sujets d’intérêts pour vous.
En 2014, SFnano vous proposera :
December 2-4, 2013
- une journée sur « l’intérêt de la chimie pour les nanomédecines » le 8 avril 2014, journée
coorganisée avec la Société de Chimie Thérapeutique,
- un workshop à Porto en octobre sur les modèles relevants in silico, in vitro et in vivo pour
l’étude de la délivrance de médicaments en fonction du mode d’administration et
University of Orléans – Campus La Source
In collaboration with
- enfin, le 1er congrès annuel qui aura lieu à Nancy sur les thèmes suivants : nanoparticules
et rayonnements, vieillissement et stress oxydant, nanomédecine régénérative.
Notre activité est aujourd’hui à la frontière entre la délivrance de médicaments et nouveaux
outils d’imagerie. Le GTRV qui regroupe les acteurs clefs de ces domaines doit demeurer à
la pointe et à la hauteur de l’enjeu dans ce domaine en forte accélération.
N’hésitez pas à nous aider afin d’œuvrer ensemble pour que nos équipes soient fortes et
acteurs de la mutation des nanomédecines.
Nathalie Mignet
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TABLE OF CONTENTS
GTRV Presidents
GTRV PRESIDENTS .............................................................................................................................................4
1986-1991
:
P. Couvreur
GTRV BOARD 2012-2014 ....................................................................................................................................5
1992-1993
:
F. Schuber
1994-1995
:
Ph. Maincent
1996-1997
:
F. Lescure
PROGRAM MONDAY DECEMBER 2 .......................................................................................................................11
1998-1999
:
J.P. Benoît
PROGRAM TUESDAY DECEMBER 3.......................................................................................................................17
2000-2001
:
G. Barratt
PROGRAM WEDNESDAY DECEMBER 4 .................................................................................................................31
2002-2003
:
D. Scherman
2004-2007
:
D. Betbeder
2008-2011
:
B. Lebleu
2012-2014
:
N. Mignet
SPONSORS ...........................................................................................................................................................7
GTRV SANOFI PRIZE ............................................................................................................................................8
GTRV DEBIOPHARM PRIZE .................................................................................................................................10
POSTERS – SESSION A .......................................................................................................................................40
POSTERS – SESSION B .......................................................................................................................................76
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Dr. Jean-Luc COLL
GTRV BOARD
2012-2014
Pr. Patrick COUVREUR
Founding member
Inserm UJF U823
Institut Albert Bonniot
38042 Grenoble Cedex 8
France
Founding member
CEA LETI
Campus Minatec
38042 Grenoble
France
UMR CNRS 8612, Faculté de Pharmacie
5, Rue Jean-Baptiste Clément
92296 CHATENAY-MALABRY Cedex
France
Website
Dr. Anne-Claude COUFFIN
Dr. Francis SCHUBER
UMR 7199 CNRS/UdS, Faculté de Pharmacie
74, Route du Rhin
67401 ILLKIRCH Cedex
France
Communication
Dr. Benoît FRISCH
Dr. Gillian BARRATT
Honorary member
UMR CNRS 8612, Faculté de Pharmacie
5, Rue Jean-Baptiste Clément
92296 CHATENAY-MALABRY Cedex
France
Vice Treasurer
Laboratoire de Conception et application
de molécules bioactives
Université Strasbourg
UMR CNRS 7199 – Faculté de Pharmacie
74 route du Rhin, BP 60024
67401 Illkirch cedex
France
Dr. Harivardhan LAKKIREDDY
Dr. Nathalie MIGNET
Head of Drug Delivery Technologies and Innovation
Nanotechnologies Pharmaceutical Sciences Department
Sanofi Research & Development
13, Quai Jules Guesde
94403 Vitry-sur-Seine
France
Vice President
Dr. Bernard LEBLEU
Inserm U1022/ UMR 8151
Université Paris Descartes
4, Avenue de l’Observatoire
75006 Paris
France
Dr. Sergio CAPANCIONI
Sponsoring
President
Communication
UMR CNRS 5235 - DIMNP
Université de Montpellier II, cc107
Place Eugène Bataillon,
34293 Montpellier Cedex 5
France
Debio R.P. SA
146, Route du Levant
1920 Martigny
Suisse
Pr. Catherine PASSIRANI
Secretary - Communication
Inserm U1066 - Université d’Angers
10, Rue André Boquel
49100 Angers
France
Dr. Marie-Pierre ROLS
Vice secretary - Communication
Institut de Pharmacologie et de Biologie Structurale
UMR-CNRS 5089
Route de Narbonne, 205
31077 Toulouse
France
Pr. Véronique PRÉAT
Abstract book
Université catholique de Louvain
Louvain Drug Research Institute
Pharmaceutics & Drug Delivery
Avenue E. Mounier, 73 bte B1.73.12
1200 Bruxelles
Organizing committee
Dr. Sylvie BEGU
Treasurer
Institut Gerhardt - UMR5253
UFR des Sciences pharmaceutiques
15, Avenue Charles Flahault BP14491
34093 Montepellier Cedex
France
Pr. Didier BETBEDER
Sponsoring
Patrick Midoux, CBM Orléans
Chantal Pichon, CBM Orléans
Eva J Toth, CBM Orléans
Jean-Luc Coll, Institut Albert Bonniot UJF823, Grenoble
Sylvie Bégu, Université Montpellier
Anne-Claude Couffin, CEA Leti, Grenoble
Catherine Passirani, Université Angers
Nathalie Mignet, Université Paris Descartes
EA 2689 IFR 114 – Laboratoire de Physiologie
Fac. Med. pôle recherche, Lille 2
1, Place de Verdun
59045 Lille Cedex
France
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The GTRV board thanks all its partners
DebioPharm
Sanofi
Malvern Instruments
Cordouan
Nanosight
Horiba
Schaeffer
In cell Art
GTRV SANOFI PRIZE
Xèmes Journées (Angers, 7-8 décembre 1995)
F. Boury (Université de Angers) : « Propriétés rhéologiques de monocouches polymères à l’interface
eau/dichloromethane »
Président du jury : P. Couvreur
XIèmes Journées (Paris, 12-13 décembre 1996)
C. Meunier-Durmort (Ceremod, CNRS, Meudon) : « Lipoadenofection : quel rôle attribuer à
l’adenovirus ? »
Président du jury : M. Vert
XIIèmes Journées (Bruxelles, 11-12 décembre 1997)
H. Soyez (Polymer Materials Research Group, Université de Gand, Belgique) « Poly(2hydroxyethyl)glutamine as carrier for cytotoxic agents »
Président du jury : F. Schuber
XIIIèmes Journées (Paris, 7-8 décembre 1998)
P. Schelté (UMR CNRS 7514, Strasbourg-Illkirch) « Construction liposomique diépitopique et
études immunologiques : vers la mise au point de vaccins entièrement synthétiques »
Président du jury : J.-P. Benoît
XIVèmes Journées (Marseille, 11-12 décembre 1999)
C. Pean (CEA, Saclay) : « Synthèse, analyse conformationnelle et évaluation des propriétés
biologiques de vecteurs à base de cyclodextrines portant une antenne peptidique »
Président du jury : G. Barratt
XVèmes Journées (Paris, 7-8 décembre 2000)
O. Raguin (INSERM-Hôpital Saint Antoine, Paris) : « L’immunociblage simultané de deux
antigènes de différenciation améliore la sélectivité pour les cellules tumorales »
Président du jury : J. Barbet
XVIèmes Journées (Lyon, 13-14 décembre 2001)
C. Charnay (Université de Montpellier) : « Vers de nouveaux systèmes de délivrance : les silices
mésoporeuses à tensioactifs structurants »
Président du jury : J.M. Devoisselle
XVIIèmes Journées (Paris, 12-13 décembre 2002) ex aequo
C. Raffournier (UMR CNRS 8612, Université de Paris-Sud, Châtenay-Malabry): « Solubilisation
d’une émulsion par un détergent : évolutions structurales »
A. Roth (UMR CNRS 7514, Strasbourg-Illkirch) : « Activité anti-tumorale de constructions
liposomales à base de peptides synthétiques »
Président du jury : E. Fattal
XVIIIèmes Journées (Lille, 11-12 décembre 2003)
G. Phan (Service de Pharmacologie et d'Immunologie, CEA, Saclay) : « Amélioration de la
décorporation du plutonium-239 in vivo par vectorisation liposomale optimisée de l’acide
diéthylènediaminopentaacétique (DTPA) »
Président du jury : B. Lebleu
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XIXèmes Journées (Paris, 16-17 décembre 2004)
S. Daoud-Mahammed (UMR CNRS 8612, Université Paris-Sud, Châtenay-Malabry) : « Gels
associatifs originaux à base de cyclodextrines pour la libération prolongée de principes actifs
hydrophobes »
Président du jury : G. Barratt
XXèmes Journées (Montpellier, 1-2 décembre 2005)
C. Bloquel (INSERM U 640 - CNRS UMR 8151, Université René Descartes, Ecole Nationale
Supérieure de Chimie, Paris) : « Plasmid electrotransfer of eye ciliary muscle : principle and uveitis
therapeutic efficacy using hTNF- soluble receptor »
Président du jury : C. Damgé
GTRV DEBIOPHARM PRIZE
XXèmes Journées (Montpellier, 1-2 décembre 2005)
A. Paillard (EA 2465, Faculté des Sciences J. Perrin, Université d’Artois, Lens) : « Evaluation of neutral or
cationic 60 nm nanoparticle as delivery systems of proteins on an in vitro model of blood brain barrier »
Président du jury : D. Betbeder
XXIèmes Journées (Paris, 13-15 décembre 2006)
B. Aussedat (UMR 7613 CNRS, Université Pierre et Marie Curie, Paris) : « Synthèse et évaluation
biologique de nouveaux vecteurs pseudo-peptidiques »
Président du jury : J. Barbet
XXIèmes Journées (Paris, 13-15 décembre 2006)
K. Van Butsele (Centre d’Education et de Recherche sur les Macromolécules, Université de
Liège, Belgique) : « New polymeric architectures for stealth and pH-responsive micelles »
Président du jury : V. Préat
XXIIèmes Journées (Strasbourg, 17-19 décembre 2007)
H. de Martimprey (UMR CNRS 8121, Institut Gustave Roussy, Villejuif) : « Nanoparticules
copolymériques pour la vectorisation in vivo de siRNA dirrigés contre une tumeur »
Président du jury : B. Ducrey
XXIIèmes Journées (Strasbourg, 17-19 décembre 2007)
J. Barbeau (ENSCR, UMR 6226 CNRS, Equipe « Chimie Organique et Supramoléculaire »,
Rennes) : « Archaeosomes : nouvelles formulations liposomiales pour la vectorisation de
principes actifs et de gènes thérapeutiques »
Président du jury : F. Artzner
XXIIIèmes Journées (Angers, 8-10 décembre 2008)
A. Boudier (UMR 5253 CNRS-ENSCM-UM2-UM1, Institut Charles Gerhardt Montpellier,
Montpellier) : « Micelles pour des thérapies par l’interférence appliquées à des cellules
dendritiques primaires »
Président du jury : F. Boury
XXIIIèmes Journées (Angers, 8-10 décembre 2008)
C. Ancla (Institut des Sciences Moléculaires, site ENSCPB, Université de Bordeaux 1, Pessac)
«Insulin delivery from glucose-responsive microgels»
Président du jury : J.P. Benoît
XXIVèmes Journées (Paris, 7-9 décembre 2009)
L. Plapied (Université Catholique de Louvain, Unité de Pharmacie Galénique, Bruxelles,
Belgique) :
« Optimisation de la stratégie de vaccination par voie orale par nanoparticules polymériques :
ciblage des cellules M ou bioadhésion ? »
Président du jury : B. Lebleu
XXVèmes Journées (Toulouse, 6-8 décembre 2010)
C. Rosazza (IPBS Toulouse, France)
« The cytoskeleton is an active partner in gene electortransfer process »
Président du jury : N. Mignet
XXIVèmes Journées (Paris, 7-9 décembre 2009)
P. Castagnos (UMR CNRS 5623, Université Paul Sabatier, Toulouse) :
« Vésicules catanioniques: un système polyvalent pour la délivrance de principes actifs »
Président du jury : E. Fattal
XXVèmes Journées (Toulouse, 6-8 décembre 2010)
H. Marie (UMR CNRS 8612, Chatenay-Malabry, France)
« Magnetic-fluid loaded liposomes for diagnosis and treatment of brain diseases »
Président du jury : C. Passirani
XXVIèmes Journées (Bruxelles, 5-7 décembre 2011)
E. Moysan (University of Angers)
« Nanoparticle-encapsulated gemcitabine for lung cancer treatment »
XXVIIèmes Journées (Paris, 3-5 décembre 2012)
J Lavaud , Institut Albert Bonniot UJF823, Grenoble
« Development and multimodal imaging characterization of relevant models in oncology »
XXVIèmes Journées (Bruxelles, 5-7décembre 2011)
R. Marchione (University Joseph Fournier, Grenoble, France)
« EB virus Zebra protein as a delivery vector for therapeutic proteins »
XXVIIèmes Journées (Paris, 3-5 décembre 2012)
Emilie Secret (ICG Montpellier, France)
« Multi-functionalized porous silicon nanoparticles for cancer therapy applications »
Président du Jury : S. Begu
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DECEMBER 2, 2013
THE SKIN: WHY IS IT SUCH AN EFFECTIVE BARRIER AND HOW CAN IT BE OVERCOME?
13h15
14h00
Dr. Yogeshvar N. Kalia
Registration
Opening session
NATHALIE MIGNET(Présidente GTRV) & CHANTAL PICHON (Directrice ITP)
PATRICE SOULLIÉ (Délégué Régional CNRS) & CHRISTINE ROUSSELLE (VPCS Université Orléans)
SESSION « COSMETICS AND TOPICAL ADMINISTRATION »
Chairperson: Patrick Couvreur
14h30
DR. YOGESHVAR KALIA, University of Geneva, Switzerland
The skin: why is it such an effective barrier and how can it be overcome?
15h10
Topical delivery of ciclosporin A using polymeric micelles and visualization of transport pathways in
the skin
School of Pharmaceutical Sciences, University of Geneva,
30 quai Ernest Ansermet, 1211 Geneva, Switzerland
Yogi.Kalia@unige.ch, www.unige.ch/pharm//sbg/kalia.html
After completing undergraduate and postgraduate studies in Chemistry at the Imperial College of Science,
Technology and Medicine, he held postdoctoral positions in the Dept. of Biochemistry at the University of
Cambridge and the School of Pharmacy at the University of California-San Francisco. He joined the School
of Pharmaceutical Sciences at the University of Geneva in 1996 where he currently holds the position of
Senior Lecturer. His group’s research interests include (i) development of new formulations to increase local
and systemic bioavailability of topical and transdermal therapeutics, (ii) investigation into the influence of
permeant physicochemical properties on electrically-assisted transport across the skin, (iii) development of
new techniques for the non- and minimally invasive delivery of biotechnology-derived therapeutics across
the skin and (iv) synthesis and characterisation of prodrugs optimised for topical and transdermal
administration. Their research is funded by public grants and through industrial collaborations. He has
published ~115 papers, presented ~150 communications at international conferences and he is a coinventor on 4 patents. He serves on the Editorial Boards of the European Journal of Pharmaceutics and
Biopharmaceutics and Expert Opinion on Drug Delivery and he is also a reviewer for several journals in the
pharmaceutical and biomedical fields.
M. Lapteva, V. Santer, K. Mondon, M. Möller, Y. Kalia
15h30
Biodegradable nanoparticles carrying an antioxidant and a fibroblastic growth factor for dermis
reparation in a context of wound healing
M. Berthet, C. Primard, V. Lahaye, B. Fromy, D. Sigaudo-Roussel, B. Verrier
15h50
Coffee break
16h20
New vectors for the immunobiological diagnosis of contact allergy to fragrances
A. Cortial, S. Briançon
16h40
Sponsor presentations
M. Terray, Malvern Instruments
P. Peotta, Nanosight
C. Megier, Horiba, Taille et concentration en Nanoparticules : Outils innovants et perspectives.
S. Aguy, Cordouan
17h35
DR. VALÉRIE ALARD, LVMH Recherche, Orléans, France
Bioavailability of cosmetic active ingredients: what kind of technologies for which goal?
18h15
End of the day
19h15
Welcome cocktail at the City Hall (Courtesy of Mairie d’Orleans)
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The composition and structure of the stratum corneum make it an extremely effective barrier against molecular
transport. As a consequence, therapeutic agents (or, for that matter, cosmeceuticals) that rely on passive
diffusion to reach their target must combine potency and the correct balance of physicochemical properties to
ensure efficacy. They must first enter and cross the stratum corneum before passing into the viable epidermis
(the target for many dermatologic agents). Systemically-acting therapeutics must penetrate further still and
reach the dermis in order to enter the capillary network. The transfer of a molecule from a topically applied
formulation into the body and its subsequent transport involves many different steps but is dominated by two
distinct physical processes: (i) partitioning between the formulation and the stratum corneum and (ii) diffusion
through the biological membrane. The first step is thermodynamic in nature – governed by the relative affinities
of the molecule for the two media – whereas the second is a kinetic process in which the rate of transport is
determined by the molecular diffusivity in the biological matrix. For most molecules, the rate-limiting step is
transit across the stratum corneum. Many potentially useful locally and systemically-acting therapeutic agents,
with good pharmacological activity, lack the requisite physicochemical properties to ensure efficient delivery.
This is also true for certain cosmeceuticals, e.g., polar anti-oxidants with insufficient lipophilicity to favour
partitioning into the stratum corneum. As a result, different methods have been developed in an attempt to
enhance delivery into and transport across the skin. These approaches rely on several strategies including (i)
chemical modification to make molecular physicochemical properties more amenable to entry into the stratum
corneum (e.g., esterification), (ii) use of advanced formulation techniques to encapsulate the active ingredient
(e.g., micelles or liposomes, again to favour partitioning), (iii) incorporation of excipients that alter solubility or act
as chemical penetration enhancers to reversibly perturb the diffusional barrier, (iv) application of an additional
driving force to complement the flux due to the concentration gradient (e.g., the electric potential gradient used
in iontophoresis) and (v) reversible impairment of skin barrier integrity through either mechanical or physical
means (e.g., lasers, microneedles or radio-frequency). Although very different in their approach, in general,
these techniques either enhance partitioning from the formulation or facilitate diffusion in the skin. They have the
ability to significantly expand the range of molecules that can be considered for topical or transdermal
administration and provide new treatment options for the clinician. In this presentation, I will provide an overview
of some of these methods – including the molecules studied and the results achieved.
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TOPICAL DELIVERY OF CICLOSPORIN A USING POLYMERIC MICELLES AND VISUALIZATION OF
TRANSPORT PATHWAYS IN THE SKIN
Maria Lapteva∗ , Verena Santer, Karine Mondon, Michael Möller, and Yogeshvar Kalia†
BIODEGRADABLE NANOPARTICLES CARRYING AN ANTIOXIDANT AND A FIBROBLASTIC GROWTH
FACTOR FOR DERMIS REPARATION IN A CONTEXT OF WOUND HEALING.
†
Morgane Berthet∗ , Charlotte Primard, Vincent Lahaye, Bérengère Fromy,
Dominique Sigaudo-Roussel, and Bernard Verrier
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne – 30 Quai Ansermet - 1211 Geneva - Switzerland
Biologie Tissulaire et Ingénierie Thérapeutique (LBTI) – CNRS : UMR5305 – 7 Passage du Vercors - 69007 Lyon, France
Ciclosporin A (CsA) is a potent immunosuppressant; however, its systemic administration exposes the patient to
severe side effects. Topical delivery offers significant advantages but CsA is a difficult molecule to formulate and
to deliver because of its lipophilicity and poor aqueous solubility. Micelle formulations using a novel amphiphilic
block copolymer (MPEG-dihexPLA) have recently been shown to incorporate several poorly water soluble drugs
with high loading efficiencies and to increase their delivery into the skin.
Aims: (i) To evaluate cutaneous delivery of CsA after topical application of a MPEG- dihexPLA micelle
formulation, (ii) to synthesize a fluorescent analogue of CsA (Fluo-CsA) and (iii) to use confocal laser scanning
microscopy (CLSM) to visualize micelle and drug distribution in the skin and to identify molecular penetration
pathways .
CsA-loaded micelles were prepared by the solvent evaporation method. CsA was quantified by an in-house
HPLC-UV method. Micelle size and morphology were characterized by DLS and TEM. CsA was labelled with
the fluorescent dye, NHS-fluorescein, using 4-vinylbenzylamine as a linker. Fluo-CsA-loaded micelles were
prepared by adding NR- MPEG-dihexPLA to MPEG-dihexPLA copolymer. Skin transport experiments with CsA
and Fluo-CsA loaded micelles were performed using porcine skin. Skin samples exposed to fluorescent micelles
were subsequently observed with a Zeiss LSM 710 confocal microscope.
Micelle formulations with different copolymer content (5-20 mg/ml) were prepared. CsA drug loadings ranged
between 310.93 9.36 and 333.75 5.56 mgCsA/gcopo and drug contents ranged between 1.67 0.03 and 6.23 022
mg/ml. The micelles were found to be spherical with a diameter below 50 nm. The Fluo-CsA loaded micelles
with a 5 mg/ml copolymer content had a similar drug loading to that of CsA micelles. The amount of CsA
deposited in porcine skin after formulation application for 24 h was dependent on the formulation drug content
and the drug loading in the micelles and ranged between 7.4 1.1 and 16.3 3.0 g/cm2. No CsA was detected in
the receiver compartment. The investigation into the skin deposition kinetics showed that the micelle formulation
with the lowest drug content was able to deliver 1.1 0.5 g/cm2 after application for only 1 h, which exceeds by
15-fold the IC90 value in non-CsA sensitive psoriatic patients. The CLSM images suggested that the labelled
copolymer was localized in the uppermost skin layers, whereas Fluo-CsA was released and penetrated deeper
into the skin. Micelles seemed to be preferentially deposited between single corneocytes and larger groups of
corneocyte called clusters. Moreover, the regions situated directly under the inter-cluster junctions showed more
permeability to Fluo- CsA.
CsA was successfully delivered in supra-therapeutic amounts into the skin using nano-sized MPEG-dihexPLA
micelles. CSLM images showed that the copolymer did not cross intact skin whereas the encapsulated drug
seemed to preferentially penetrate through the inter-cluster regions.
Soldiers’ wounds caused by improvised explosive devices proved to be slow to heal. The development of new
tools is therefore essential for a rapid wound healing, limiting the risks of infection. Wound healing is a
sequential process, and our goal is to develop a tool able to manage the critical steps of this process by
promoting (i) the wound reepithelialisation to reduce risks of infection and (ii) to lead to the redevelopment of a
functional dermis in order to limit fibrosis and the weakening of the scar tissue. In this way, reactive oxygen
species (ROS) which are toxic for cells and alter the extracellular matrix composition will be contented and
growth factors will be brought to activate fibroblasts. Those agents will be carried by biodegradable particles
made of poly lactic acid (PLA) to deliver drugs selectively to the specific site(s) of action over a prolonged time
course.
We have developed two models of nanoparticles made of poly lactic acid (PLA) and synthetized by
nanoprecipitation without surfactant, resulting in negatively-charged, monodisperse particles (200-nm). On one
hand, PLA nanoparticles encapsulating the antioxidant lipoïc acid, on the other hand, PLA nanoparticles
carrying a fibroblastic growth factor 2 (FGF2) adsorbed on their surface.
For the first model, the lipoïc acid encapsulation efficiency has been measured by an indirect quantification
method using HPLC. Furthermore, its biological action has been evaluated in vitro on endothelial cells by
quantification of the intracellular ROS, using the H2DCF-DA (2’,7’-dichlorodihydrofluorescein diacetate) reagent.
For the second model, FGF2 has been adsorbed on PLA particles and the adsorption rate has been evaluated
by quantification of the residual protein, using BCA test. Biological action of the adsorbed FGF-2 compared to
non-adsorbed one has been evaluated in vitro by measuring the proliferation of fibroblasts, and in vivo in mice
using a skin lesion model.
Our results suggest a decrease of the intracellular ROS in presence of encapsulated antioxidants, compared to
free-delivered lipoïc acid. Moreover, particles carrying adsorbed FGF2 have shown to improve the cellular
proliferation of fibroblasts in vitro, and in vivo speed up the wound healing in mice.
Those preliminary studies allowed the characterization of the biological activities of therapeutic agents (an
antioxidant and a growth factor) carried by biocompatible particles made of PLA, in vitro and in vivo in the
context of wound healing. The biological activities of those drugs were not damaged by the formulation process,
on the contrary the transport by the particles may increase their effect in vitro.
∗Intervenant
†Auteur correspondant: mberthet@ibcp.fr
‡Auteur correspondant: bernard.verrier@ibcp.fr
∗Intervenant
†Auteur correspondant: Yogi.kalia@unige.ch
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-14-
NEW VECTORS FOR THE IMMUNOBIOLOGICAL DIAGNOSIS OF CONTACT ALLERGY TO FRAGRANCES
Angele Cortial∗1,2, and Stephanie Briancon
1
Laboratoire d’automatique et de génie des procédés (LAGEP) – CNRS : UMR5007, Université Claude Bernard - Lyon I,
Ecole Supérieure Chimie Physique Electronique de Lyon – bat 308G ESCPE-Lyon, 43 bd du 11 Novembre 1918 - 69622 Villeurbanne, France
Centre International de Recherche en Infectiologie (CIRI) – Université Claude Bernard - Lyon I, Inserm : U1111 – 21 avenue Tony Garnier - 69365 Lyon, France
2
The diagnosis of contact allergy to haptens relies on patch-testing. Nevertheless, skin tests display several
caveats, notably they are time consuming and can promote active sensitization. Immunological assays based on
the detection of hapten-specific T cells from the blood of allergic patients proved reliable alternatives to patchtesting. However, such assays are still not in use, mainly due to the highly hydrophobic nature of haptens that
are poorly soluble in conventional culture media.
Here, we have developed new vectors to encapsulate and solubilize a complex mixture of 8 fragrances
(Fragrance Mix (FM)), and we report on their use for the reactivation of FM-specific T cells in a standard ex vivo
immunobiological assay (using peripheral blood mononuclear cells (PBMCs) purified from sensitized patients to
FM).
Two different types of nanoparticles (NP) were generated: i) poly -caprolactone (PCL)NPs by a
nanoprecipitation method, and ii) petrolatum (Pet)-NPs using an emulsification- sonication process. Use of PCLNP-FM, but not control NP, allowed for a robust secondary T cell response in a large number of FM sensitized
individuals (FM+ patch-tests). Importantly, no reactivation was observed from individuals negative to FM.
These results suggest that encapsulation of haptens and complex mixture into NP vectors is promising to
improve the immunobiological diagnosis of contact allergy to fragrances.
BIOAVAILABILITY OF COSMETIC ACTIVE INGREDIENTS: WHAT KIND OF TECHNOLOGIES FOR WHICH GOAL
Valérie Alard
LVMH Recherche, 185 avenue de Verdun - 45800 St Jean de Braye, France
valard@research.lvmh-pc.com
Graduated from Ecole Nationale Supérieure de Chimie de Mulhouse in 1987, she works for the Parfums
Christian Dior Research center and then for LVMH Research for 26 years. During this period she worked
on physico-chemistry, make-up and skincare formulations, suncare Innovative formulations and in vitro
evaluations. She is currently involved in development and formulation of biodelivery systems in
innovative formula.
The goal of cosmetic industry is to find the best compromise between “efficacy” and sensory properties (visual,
touch, smell and sometimes …taste), in a legislative context more and more strict and that generate a very
complex network.
To achieve this goal, a pragmatic approach is necessary, considering the partition coefficient of the active
ingredients, their interactions with all the other cosmetic formulation materials, from oil phases to aqueous
polymers and powders, then the evolution of the structures when the product is applied on the top of skin and,
for sure, the behavior into the skin.
Different analytical measurements are considered to characterize these different steps. Transmission electron
microscopy using freeze fracture to prepare the sample is a relevant and elegant technique to characterize the
different structures at these steps, and their stability during time under different thermal conditions. Franz cells,
using pig ear skin is an efficient tool that allow to evaluate the impact of the modifications of the compositions on
the behavior of the active ingredient in contact with skin.
A practical example of the development of a new delivery system in a range of different formulations, from a
serum to a cream, illustrate this topic and show that using this kind of approach, stabilization of liposomes may
be achieved even in very nice and comfortable creams, and in parallel penetration of a model active ingredient
in the skin could be increased.
∗Intervenant
-15-
-16-
RNAI-BASED NANOMEDICINES FOR TARGETED PERSONALIZED THERAPY
DECEMBER 3, 2013
Prof. Dan Peer
8h30
Registration – Poster installation Session A
SESSION « GENE THERAPY : NON VIRAL VECTORS »
Chairpersons: Patrick Midoux and Pierre Cordelier
9H00
PR. DAN PEER, University of Tel-Aviv, Israel
RNAi-based nanomedicines for targeted personalized therapy
9h40
Development of anti-HIV lipoplexes for the treatment of cervical cancer
A. Lechanteur, T. Furst, B. Evrard, P. Roncarati, P. Delvenne, G. Piel, P. Hubert
10h00
Development of smart siRNA LNCs to improve the cellular uptake into melanoma cells
10h20
Coffee break & Poster session (Session A)
10h50
Efficient delivery of therapeutic small nucleic acids using ketal nucleoside lipid nanoparticles
P. Resnier, J. Bejaud, N. Yilmaz, N. Lautram, P. Legras, T. Benvegnu, J.-P. Benoit, C. Passirani
P. Barthélémy
11h10
Electrotransfer of oligonucleotides into cancer cells
11h30
Utilization of the dynein molecular motor to improve perinuclear accumulation of Dynein
Prof. Dan Peer is an associate professor that leads an NIH-funded lab in the Faculty of Life Science at Tel
Aviv University (TAU). He is also the director of the Leona M. and Harry B. Helmsley Nanotechnology
Research Fund and the director of the Focal Technology Area (FTA) on Nanomedicines for Personalized
Theranostics, a national nanotechnology initiative, which includes 11 academic labs and a grant of $11.5M
that support this effort.
He was recruited to Tel Aviv University from Harvard Medical School in 2008.
Prof. Peer has been a senior affiliate member of the Research Institute of Methodist Hospital in Houston,
Texas since 2011 and a visiting scientist at the Program in Cellular and Molecular Medicine at Children’s
Hospital and Harvard Medical School in Boston, Massachusetts since 2008.
Prof. Dan Peer’s work was among the first to demonstrate systemic delivery of RNA molecules using targeted nanocarriers to the
immune system and he pioneered the use of RNA interference (RNAi) for in vivo validation of new drug targets within the immune
system. He was recruited to TAU in 2008 from Harvard to establish the laboratory of NanoMedicine.
Prof. Peer generated an international recognition and collaboration in inflammatory bowel diseases (IBD) and oncology area. He
received numerous awards; among them he was recognized by the AAAS excellence in Science program for young investigators
and was recently awarded the innovator (2010) and the breakthrough (2011, 2012, 2013) awards from the Kenneth Rainin
Foundation on his pioneering work in inflammatory bowel diseases (IBD).
M. Golzio, S. Pelofy, A. Paganin, S. Chabot, E. Bellard, M.-P. Rols, J. Teissié
Light Chain Associated Sequences functionalized fluospheres
N. Parassol, C. Bienvenu, C. Boglio, S. Fiorucci, D. Cerezo, X.-M. Yu, G. Godeau, J. Greiner,
P. Vierling, S. Noselli, C. Di Giorgio, V. Van De Bor
11h50
He is an editor of several books in the field of nanomedicine, an associate editor of the Journal of Biomedical Nanotechnology,
BMC Biochemistry and Molecular and cellular therapies and on the editorial boards of the Journal of Controlled Release
(Elsevier), Nanotechnology (IOP) , BioMEMs and Biomedical Nanotechnology (Springer) and Cancer Letters (Elsevier). He has
more than 45 pending and granted patents. Some of them have been licensed to several pharmaceutical companies and one is
under a phase II clinical evaluation. In addition, based on his work, 2 spin-off companies were generated LeukoBiosciences in the
US and Quiet Therapeutics in Israel, aiming to bring nanomedicine into clinical practice.
Cationic nanostructured lipid carriers dedicated to siRNA delivery
J. Bruniaux, E. Sulpice, M. Menneteau, I.Texier, X.Gidrol, F. Navarro
12H10-13H40 LUNCH
RNA interference (RNAi)-based approaches have greatly contributed to better understanding of gene
expression and function in vitro. The capability to apply these strategies in vivo in order to validate the
role of specific genes in normal or pathological conditions, and to induce therapeutic gene silencing,
13H50 POSTER SESSION (SESSION A)
CHAIRPERSONS: BERNARD LEBLEU AND TRISTAN MONTIER
opened new avenues for utilizing RNAi as a novel therapeutic modality. However, the translation of RNAi
15h00
DR. EVA VAN ROOIJ, University Medical Center of Utrecht, The Netherlands
from an effective genomic tool into a novel therapeutic modality has been hindered by the difficulty to
15h40
An E3-14.7K peptide that promotes microtubules-mediated transport of plasmid DNA
MicroRNAs as therapeutic targets in cardiovascular disease
increases polyplexes transfection efficiency
L. Pigeon, C. Gonçalves, C. Pichon, P. Midoux
16h00
Effects of a novel archaeal Tetraether-based colipid on the in vivo gene transfer activity of
deliver RNAi molecules into their target tissues by systemic administration, especially to hematopoietic
cells. In this presentation, I will describe some of the challenges and opportunities in modulating
leukocytes response using RNAi and discuss adverse effects such as immuno-toxicity. Special
emphasize will be made on delivery strategies that target subsets of leukocytes such as the integrin-
cationic amphiphiles
targeted and stabilized nanoparticles platform and the gagomers and I will detail examples from
C. Ballet, T. Le Gall, J. Barbeau, S. Barrier, L. Lemiègre, P. Lehn, T. Benvegnu, T. Montier
inflammatory bowel diseases, viral infection and blood cancers.
16h20
Coffee break & Poster session (Session A)
Personalized nanomedicine has the power of combining nanomedicine with clinical and molecular
16h50
New cationic liposomes bubbles for nucleic acids transfer
biomarkers ("OMICS" data) achieving improve prognosis and disease management as well as
A. Delalande, S. Manta, M. Bessodes, P. Midoux, N. Mignet, C. Pichon
17h10
Cyclodextrin-based multicomponent gene vectors: optimizing nanoparticle self-assembling and
site-specific gene delivery
L. Gallego-Yerga, I. Pflueger, C. Tros de Ilarduya, J. M Benito, C. Ortiz Mellet,
individualized drug selection and dosage profiling to ensure maximum efficacy and safety. In this
presentation, I will also detail aspects of personalized nanomedicine both from the drug and the carrier
standpoint.
J. Garcia Fernandez, F. Mendicuti
17h30
DR. STEPHEN C. HYDE, University of Oxford, United Kingdom
18h10
Round table: Perspectives of non-viral vectors
19h15
Bus for Gala DinnerSESSION « Nano-Oncology »
Non-Viral Gene Therapy for Cystic Fibrosis: From Basic Research to Clinical Impact
-17-
-18-
DEVELOPMENT OF ANTI-HPV LIPOPLEXES FOR THE TREATMENT OF CERVICAL CANCER
1
1
1
2
2
DEVELOPMENT OF STEALTH AND PH-SENSITIVE SIRNA LNCS TO IMPROVE THE CELLULAR UPTAKE
INTO MELANOMA CELLS
1
Anna Lechanteur∗ , Tania Furst , Brigitte Evrard , Patrick Roncarati , Philippe Delvenne , Géraldine Piel , and
2
Pascale Hubert
1
1
1
1
1
2
Pauline Resnier∗ , Jérôme Bejaud , Nesrin Yilmaz , Nolwenn Lautram , Pierre Legras ,
Thierry Benvegnu3, Jean-Pierre Benoit1, and Catherine Passirani1
Laboratory of Pharmaceutical Technology and Biopharmacy - CIRM, University of Liège – Belgique
2
Laboratory of Experimental Pathology, GIGA-CANCER, University of Liège – Belgique
1
Human Papillomaviruses (HPV) are responsible for several diseases and some of them (such as HPV16 and
HPV18) can induce cervical cancer. In this case the two HPV E6 and E7 oncoproteins are essential players in
order to immortalize keratinocytes by decreasing tumor suppressor genes (p53 and pRb). Nowadays cervical
cancer is known to be the third most frequent cause of death in women and only prophylactics vaccines exist.
Except classical cancer therapy (surgery and radio/chemotherapy), there is no more treatment if the lesion is
already developed.
Gene therapy is a promising strategy to treat cancer. We focus on RNA interferences (siRNA) to target mRNA
coding for both HPV E6 and E7 oncoproteins. siRNA would be encapsulated in cationic lipidic nanovectors to
form lipoplexes. This association is essential to protect siRNA, to allow the diffusion into the vaginal mucus and
to cross the anionic cellular membrane.
The aim of this study is to develop a local treatment of cervical cancer by siRNA antiE6 and/or antiE7 that would
be encapsulated in cationic nanovectors.
In the first time, we validated the efficacy of both siRNA antiE6 and antiE7 with a commercially transfection
agent (Oligofectamine ®). Some objectives are achieved: high transfection, extinction of E6 and E7 mRNA and
decreased proliferation of cells. However, the induction of apoptosis is quite low. To enhance the rate of cell
death, we will trigger an anti-apoptotic protein with another siRNA.
In the second time, Oligofectamine® had been replaced by liposomes with good physicochemical characteristics
(size, surface charge, stability...). Liposomes composed of DOTAP/DOPE/Cholesterol (1:0,5:0,5; molar ratio)
are preformed and then mixed with siRNA (100 nM; N/P 5). The size of obtained lipoplexes is around 200nm
and the zeta potential is around +45mV. This formulation leads a very efficient transfection (FACS Analysis).
Moreover, a promising efficacy was obtained as the percentage of mRNA is significantly reduced (qPCR
analysis). However, lipoplexes seem to be more toxic (Fixable Viability Stain 450) than Oligofectamine®
complexed with siRNA. In order to decrease this toxicity, a reduced concentration of lipoplexes was tested. We
showed that at a lower concentration, we were able to reduce toxicity while the efficacy remains the same.
In conclusion, even if the results are hopeful, some other tests will have to be done to validate the efficacy of
lipoplexes. Especially, the decrease of proliferation, the induction of apoptosis and the safety on cells will have
to be confirmed. The formulation will then be optimized to obtain the best efficacy/toxicity ratio.
Finally, we have developed a 3D model of cervical lesion and lipoplexes will be inoculated on it. We will analyze
the safety of lipoplexes, its diffusion into the pluristratified epithelium, and the action of the loaded siRNA.
Micro et nanomédecines biomimétiques (MINT) – Inserm : U1066, Université d’Angers – Université d’Angers, 10 rue André Boquel - 49100 Angers, France
2
Service commun d’animalerie hospitalo-universitaire (SCAHU) – Université d’Angers – Rue haute de reculée - 49933 Angers, France
3
Institut des Sciences Chimiques de Rennes – Institut National des Sciences Appliquées (INSA) - Rennes, Université de Rennes 1, CNRS : UMR6226,
Ecole Nationale Supérieure de Chimie de Rennes – Campus de Beaulieu - Bat. 10 Avenue du Général Leclerc - 35042 Rennes Cedex, France
Melanoma remains the most aggressive form of skin cancer that caused worldwide 80% of death by skin
cancer. Indeed, advanced-stage melanoma is often associated with overall median survival of 2-10 monthswith
only 5-10% of overall above 5 years (1-2). This dramatic clinical prognosis can be explained by resistance
phenomena towards the chemotherapeutic agents that are currently in use (1).Nowadays, siRNA toolsfor
alternative treatment were developed to sensitive cancer cells to classic chemotherapy (3). However, the
biodistribution of siRNA is limited and delivery systemsarea necessity to carrynucleic acidsand to target tumor
and metastatic sites.In this way, siRNA lipid nanocapsules (LNCs) were developed recently to encapsulate and
carrysiRNAbysystemic route.
These LNCs were formed with polymers and cationic lipids that interact with anionic siRNA. These siRNA LNCs
had a size of 75 nm, neutral zeta potential and an encapsulation efficiency of 40% with stability over 3 months
(4-5). Moreover, thepegylationof LNCs by postinsertion methodshouldimprove the lifetime in bloodandlead toa
passive tumoral targeting by EPR effect (6). However, the addition of PEG can prevent the internalization of
LNCs into cells.In this work, classical DSPE-PEG with stealth property and new innovative tetraether-pHsensitive-PEG, developed at Rennes, weregraftedon siRNA LNCsby post-insertion method(7).
Stability, siRNA protection against serum nucleases, cellular uptake and lifetime in blood after intravenous
injections were evaluated for siRNA LNCs with these different surface modifications.Encapsulation efficiency of
siRNA was not modified afterpegylationprocess. Moreover, the grafting of PEG on LNC surface improved the
stability of LNCs at 37C and their protection versus blood nucleases. Otherwise, siRNALNCs demonstrated a
good cell uptake with the detection of fluorescent siRNA in all cells after 24h incubation. Finally, CH50 assays
proved the efficacy of PEG to protect LNC against complement recognition compared to non-modified LNCs.
The intravenous injection of LNCs and PEG LNCs into nude mice confirmed a longer lifetime in blood for
pegylated form of siRNA LNCs.All these experimentsconcerning the stealth LNCswill be compared to the ongoing experiments with pH-sensitive LNCs.
1. Matos AM, Francisco AP.2013.ChemMedChem.
2. Rastrelli M, Alaibac M,Stramare R,Chiarion Sileni V,Montesco MC Campana LG, Rossi CR.2013.ISRN Dermatol;616170.
3. Chen Y, Wu JJ, Huang L. 2010.Mol Ther;18:828e34.
4. David S, Resnier P, Guillot A, Pitard B, Benoit JP, Passirani C. 2012.EurJPharmBiopharm;81:448e52.
5. Resnier P, LeQuinio P,Lautram N, André E, Gaillard C, Bastiat G, Benoit J-P, Passirani C. 2013.Submitto Nanotechnology
6. Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. 2000.J Control Release;65:271e84.
7. Barbeau J, Cammas-Marion S, Auvray P, Benvegnu T. 2011. J Drug Deliv:396068.
∗Intervenant
∗Intervenant
-19-
-20-
EFFICIENT DELIVERY OF THERAPEUTIC SMALL NUCLEIC ACIDS USING KETAL NUCLEOSIDE LIPID
NANOPARTICLES
ELECTROTRANSFER OF OLIGONUCLEOTIDES INTO CANCER CELLS
Muriel Golzio∗, Sandrine Pelofy, Aurelie Paganin, Sophie Chabot, Elisabeth Bellard,
Marie-Pierre Rols, and Justin Teissié
Philippe Barthelemy
Institut de pharmacologie et de biologie structurale (IPBS) – CNRS : UMR5089,
Université Paul Sabatier - Toulouse III – 205 Route de Narbonne - 31077 Toulouse, France
Université Bordeaux, INSERM U869 – France
A novel nucleoside lipid derived from dioleyl ketal was synthesized from uridine in three steps starting from
dioleyl ketone. Electronic microscopy studies show that Ketals Nucleoside Lipids (KNL) self-assemble to
formliposome-like structures in aqueous solutions. KNL is able to bind siRNA as demonstrated by
electrophoresis experiment and standard ethidiumbromide fluorescence displacement assay. Transfection
assays of stable hepaticcell lines HupIRF, carrying a luciferase reporter gene demonstrate that KNL is able to
transfect siRNA and exhibits protein knockdown more efficiently than its diester analog (DOTAU) and
lipofectamine.
Interfering RNAs (siRNA, miRNA) have been proposed to revolutionize the medicine by opening new
therapeutic ways. Perhaps the most serious obstacle to siRNA drug development is delivery. Improving delivery
efficiency will likely require greater insight into the mechanisms by which cells take up siRNA.
The biophysical approach described in this work brings a safe and efficient delivery on the targeted organ. The
electrotransfer or the electropermeabilization (EP) consists in the injection of oligonucleotides in a tissue
followed by the application of calibrated electric pulses which permeabilize the membrane of cells and therefore
promote the entry of hydrophilic molecules into the cell.
We demonstrate that electrotransfer of siRNA is highly effective for the inhibition of the expression of the target
gene.EP allows direct transfer of the oligonucleotides in the cytoplasm of cells. EP acts at the same time on the
permeabilization of the plasma membrane of the cells and on the electrophoretic forces that push the negatively
charged oligonucleotides. Our results showed that EP allowed direct entry into the cytoplasm bypassing the
endocytosic pathway.
The results of our observations will allow to give important directives (pulse parameters, chemical modifications)
for RNAi-based clinical protocols implying the transfer of oligonucleotides in target tissues and to have new tools
for targeted therapy.
∗ Intervenant
-21-
-22-
UTILIZATION OF THE DYNEIN MOLECULAR MOTOR TO IMPROVE PERINUCLEAR ACCUMULATION OF
DYNEIN LIGHT CHAIN ASSOCIATED SEQUENCES FUNCTIONALIZED FLUOSPHERES
CATIONIC NANOSTRUCTURED LIPID CARRIERS DEDICATED TO SIRNA DELIVERY
1,2,3,4
Jonathan Bruniaux∗
Nadège Parassol1, Céline Bienvenu2, Cécile Boglio2, Sébastien Fiorucci2,
1
2
2
2
2
Delphine Cerezo , Xiao-Min Yu , Guilhem Godeau , Jacques Greiner , Pierre Vierling ,
1
*2
§1
∗
Stéphane Noselli , Christophe Di Giorgio , and Véronique Van De Bor
1
Institute of Developmental Biology and Cancer (IBDC) – CNRS: UMR6543, Université Nice Sophia Antipolis [UNS] – Parc Valrose - 06108 Nice, France
2Institut de Chimie de Nice (ICN) – CNRS : UMR7272, Université Nice Sophia Antipolis [UNS] – Faculté des Sciences Parc Valrose
28 Avenue Valrose - 06108 Nice, France
A significant challenge in nanotechnology, and more particularly in gene transfer, is to improve drug nuclear
delivery by engineering nanocarriers transported by cytoskeletal motors1. Molecular motors transport various
cargoes including proteins, mRNAs, organelles, vesicles, viruses, to distinct intracellular compartments2.
Cytoplasmic dynein is an anterograde molecular motor which uses the microtubules to actively carry cargoes
from the membrane periphery toward the microtubule organizing center (MTOC) and hence to nucleus. We
developed an assay to visualize the movement of fluospheres (FS) using intracytoplasmic microinjection into
drosophila oocytes coupled with real time videomicroscopy. We designed dynein motor driven FS using different
dynein light chain 8 (LC8) peptide binding motifs as molecular linkers3-6 allowing us to characterize in real time
the efficiency and dynamics of the FS movement according to its linker’s sequence. Results show that sufficient
concentration of highly conserved LC8 binding motif allows unambiguously fast perinuclear nanoparticle’s
accumulation in a microtubule as well as in a dynein dependent mechanism.
1. Cohen, R.N., Rashkin, M.J.Wen, X. Szoka F.C. Molecular motors as drug delivery vehicles. Drug Discovery Today: Technologies 2,
111-118 (2005).
2. Ross, J.L., Ali, M.Y. & Warshaw, D.M. Cargo transport: molecular motors navigate a complex cytoskeleton. Curr Opin Cell Biol
20, 41-47 (2008).
3. Wang, L., Hare, M., Hays, T.S. & Barbar, E. Dynein light chain LC8 promotes assembly of the coiled-coil domain of swallow protein.
Biochemistry 43, 4611-4620 (2004).
4. Fan, J., Zhang, Q., Tochio, H., Li, M. & Zhang, M. Structural basis of diverse sequencedependent target recognition by the 8 kDa
dynein light chain. J Mol Biol 306, 97-108 (2001).
5. Benison, G., Karplus, P.A. & Barbar, E. Structure and dynamics of LC8 complexes with KXTQT-motif peptides: swallow and
dynein intermediate chain compete for a common site. J Mol Biol 371, 457-468 (2007).
6. Rodr´ıguez-Crespo, I., Yelamos, B., Roncal, F., Albar, J.P., Ortiz de Montellano, P.R., Gavilanes, F. Identification of novel cellular
proteins that bind to the LC8 dynein light chain using a pepscan technique. FEBS Lett 503, 135-1417 (2001).
†
1
Université Joseph Fourier (Grenoble 1 UJF) – Université Joseph Fourier - Grenoble I – Université Joseph Fourier - BP 53 – 38041 Grenoble, France
2
INSERM U1038 (INSERM U1038) – Inserm – Grenoble, France
3
CEA DSV/IRTSV/Biomics - Commissariat à l’Énergie Atomique et aux Énergies Alternatives - 17 Rue des Martyrs - 38054 Grenoble, France
4
CEA LETI MINATEC DTBS– Commissariat à l’Énergie Atomique et aux Énergies Alternatives - 17 Rue des Martyrs - 38054 Grenoble, France
Since the discovery of RNA interference mechanism, the use of siRNA in research is a potent tool for studying
the gene functions and/or for identifying new biomarkers of diseased cells. High throughput screening is an
emerging field which requires generic vectors with highly efficient transfection to study thousands different
siRNA. In this way, progresses have been made regarding the design of non-viral vectors (lipids, peptides,
polymers) as delivery agents. The efficiency and specificity of gene-based expression of siRNA have been
improved, while at the same time reducing toxicity. Among these carriers, lipid-based agents, already used as
commercial reagents for in vitro transfection, are emerging as a promising approach for siRNA delivery.
Here, we focus on multifunctional nanostructured lipid carriers with a cationic shell, as a ready-to-use system
allowing simple, fast and stable over time complexation by establishment of electrostatic bonds with anionic
siRNA. These particles demonstrate an outstanding colloidal stability and their cationic charges promote
effective complexation, as e vidence by gel retardation assay, without generating cytotoxicity. Compared to the
commercially available lipoplexes, these highly stable lipid nanoemulsions offer the possibility to combine
several functions into a unique carrier, such as fluorescent particle tracking through the encapsulation of
lipophilic dyes and/or the co-delivery of other drugs. Thereby, the distribution monitoring into cells with
nanocarriers, entrapping lipophilic cyanine derivatives, demonstrate an improved internalization of nucleic acids.
Furthermore, these lipid particles present high in vitro transfection efficiency in several human cell lines, with a
demonstrated down regulation of targeted proteins. Besides, ligand moieties can be grafted onto their surface in
order to selectively target a specific subset of cells. Such cationic lipid nanodroplets open new avenues in the
development of high throughput RNAi screening as well as their translation to clinics.
Intervenant
-23-
-24-
MICRORNAS AS THERAPEUTIC TARGETS IN CARDIOVASCULAR DISEASE
AN E3-14.7K PEPTIDE THAT PROMOTES MICROTUBULES-MEDIATED TRANSPORT OF PLASMID DNA
INCREASES POLYPLEXES TRANSFECTION EFFICIENCY
Eva van Rooij
1
1
1
†1
Lucie Pigeon∗ , Cristine Goncalves , Chantal Pichon , and Patrick Midoux
Hubrecht Institute, the Netherlands
1
Eva van Rooij attended University Hospital Maastricht in the Netherlands where she received a
Ph.D. at the department of Cardiology. She then went on to complete postdoctoral training in
Molecular Biology at UT Southwestern Medical Center in the lab of Dr. Eric Olson where she
served as lead scientist in the studies that linked microRNAs to cardiovascular disease. Her
work subsequently became the foundation of miRagen Therapeutics, Inc., a company focused
on the development of microRNA therapeutics. In the last years Eva van Rooij served as
miRagen’s Senior Director of Biology and co-founder during which she oversaw all the preclinical studies for the company’s microRNA programs. In 2013 she started an academic lab at
the Hubrecht Institute to further unveil the molecular signaling pathways that are relevant for
cardiovascular biology.
Chronic and acute stress to the heart results in a pathological remodeling response accompanied by
hypertrophy, fibrosis, myocyte apoptosis and eventual death from pump failure and arrhythmias. We have
identified signature expression patterns of microRNAs associated with different forms of heart disease. Gainand loss-of-function studies have revealed profound and unexpected functions for these microRNAs in
numerous facets of cardiac biology, providing glimpses of new regulatory mechanisms and potential therapeutic
targets for heart disease.
Disease-inducing cardiac microRNAs can be persistently regulated in vivo through systemic delivery of antimiRs. The therapeutic opportunities for manipulating microRNA biology in the setting of heart disease will be
discussed.
Centre de Biophysique Moléculaire (CBM) – CNRS : UPR4301 – Centre de Biophysique Moléculaire, CNRS UPR4301,
Inserm and University of Orléans - 45071 Orléans, France
Cationic polymers and lipids are promising chemical vectors for gene therapy. However, the limited cytosolic
diffusion of plasmid DNA (pDNA) impairs its delivery to the nucleus. To improve its intracellular trafficking to the
nucleus of the cell, one strategy is to make a pDNA able to interact with cytoskeleton motors, as most viruses
do.
We have identified a 20 amino-acids peptide (P79-98) of the E3-14.7K early adenoviral protein interacting with
the Dynein light chain TCTEL1 via FIP-1. Videomicroscopy and Single Particle Tracking clearly demonstrate that
a P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2h
polyfection whereas pDNA conjugated with a control peptide exhibits short non-directional movements in the
cytosol. Remarkably, the number of transfected cells is enhanced by a factor 2.5 - up to 76% - upon in vitro
polyfection with P79-98/peGFP. No improvement was observed with a peptide that interacts directly to dynein.
In vivo P79-98/pLuc clearly show a 3- to 5-fold transgene expression in skeletal muscles and liver after
intramuscular and tail vein hydrodynamic injection in mice, respectively. Comparatively, P79-98/peGFP
lipofection do not improve transfection suggesting that the peptide is hidden after the multilamellar assembly of
lipoplexes.
Our results demonstrate for the first time that in vitro and in vivo non viral gene transfer can be drastically
increased when pDNA is conjugated with a FIP-1 interacting sequence allowing its migration on microtubules.
This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.
∗Intervenant
†Auteur correspondant: patrick.midoux@cnrs- orleans.fr
-25-
-26-
EFFECTS OF A NOVEL ARCHAEAL TETRAETHER-BASED COLIPID ON THE IN VIVO GENE TRANSFER
ACTIVITY OF CATIONIC AMPHIPHILES
NEW CATIONIC LIPOSOMES BUBBLES FOR NUCLEIC ACIDS TRANSFER
1
1
2,3
1
1
2
1
Chimie Organique et Supramoléculaire (COS, UMR - CNRS 6226) –
Ecole Nationale Supérieure de Chimie de Rennes – 11 allée de Beaulieu, CS 50837 - 35708 Rennes, France
2
Unité INSERM 1078, SFR ScInBioS – Université de Bretagne occidentale – 46 rue Félix Le Dantec, CS51819 - 29218 Brest, France
3Plateforme SynNanoVect, SFR ScInBioS – Université de Bretagne occidentale – Brest, France
Gene therapy is a promising strategy to cure a broad range of acquired and inherited diseases, through the
delivery into target cells of nucleic acids used as pharmaceutical agents. Amongst others, this might be
achieved using multi-modular non-viral lipid-based systems to treat cystic fibrosis.[i] To date, most optimizations
have concerned the structure of cationic lipids[ii] rather than the nature of colipids.
Our work focus on an original archaeal Tetraether derivative used as a colipid in combination with one
monocationic amphiphile. The liposomes obtained, termed archaeosomes, were characterized regarding lipid
self-assembling properties, macroscopic/microscopic structures, DNA condensation/ neutralization/relaxation
abilities, and colloidal stability in presence of serum. In addition, gene transfer experiments were conducted in
mice with lipid/DNA complexes being administered via systemic or local delivery routes. [iii]
The synthesis of the original Tetraether as well as glycine betaine cationic lipid MM18 will be shortly presented.
Several in vivo gene experiments performed in mice using these preparations will be exposed, confirming in
vitro assays. [iv]
For intravenous administration, the presence of Tetraether in a 10/1 ratio dramatically enhanced the transfection
efficiency. Surprisingly, the incorporation of increased quantities of colipid (10/1 up to 10/3) displayed selectivity
changes towards targeted organs.
2
2
1
2
Anthony Delalande∗ , Simona Manta , Michel Bessodes , Patrick Midoux , Nathalie Mignet ,
†1
and Chantal Pichon
1,3
Caroline Ballet∗ , Tony Le Gall , Julie Barbeau , Sylvain Barrier , Loïc Lemiègre ,
2
†1,3
‡2,3
Pierre Lehn , Thierry Benvegnu , and Tristan Montier
1
Centre de biophysique moléculaire (CBM) – CNRS : UPR4301 – Rue Charles Sadron – 45071 Orléans, France
Unité de pharmacologie chimique et génétique et d’imagerie – Inserm : U1022, CNRS : UMR8151, Université Paris V - Paris Descartes,
Ecole Nationale Supérieure de Chimie de Paris – Paris, France
Ultrasound and microbubbles-mediated gene transfer is a non-invasive, targetable and controlled DNA
delivery technique. Under ultrasound microbubbles are known to interact with cells and to permeabilize
the plasma membrane leading to sonoporation [1]. The main limitation of this technique is the low
transfection efficiency of the commercially available microbubbles. Our project concerns the design of
cationic microbubbles with the aim of binding DNA on the microbubble shell and having the capacity to fuse
with cellular membranes. Several microbubble formulations were produced using three kinds of cationic
lipids: Lipid 1 (triple cationic lipid), Lipid 2 (mono charged cationic lipid) and Lipid 3 (fusogenic lipid).
Microbubble stability was assessed by optical observations, counting, sizing and flow cytometry. Their
acoustic activity and interaction in the presence of cells has also been measured by attenuation
measurements and high-speed imaging microscopy. Microbubbles produced by mechanical shaking showed
required properties: a proper stability up to 3 hours, a size distribution centered at 1.9 m and a resonance
frequency around 1.5 MHz. Microbubbles produced presented a Zeta potential of +45 mV when lipid 1
was used and +12 mV with lipid 2. Plasmid DNA molecules were found on the microbubble surface when
observed by confocal microscopy and atomic force microscopy. Flow cytometry analyzes conducted showed
a good DNA binding capacity. In vitro sonoporation of HeLa cells using the produced microbubbles resulted
in almost 30% of GFP transfected cells. In vivo, gene transfer was achieved on a previous animal model
developed on the Achilles tendon [2]. A stable luciferase expression lasting over two weeks was obtained
after local injection of microbubbles bearing DNA and sonoporation. Further experiments on in vivo
ultrasound microbubble imaging and DNA delivery after systemic injection are in progress.
For intranasal administration, MM18-based lipoplexes in highly concentrated formulations, incorporating PEPEG5000 for colloidal stabilization, are the most efficient for gene transfection.
Altogether, the results showed that the Tetraether colipid can provide complexes with different in vivo
transfection abilities depending on the lipid combination, the lipid/colipid molar ratio, and the administration
route. This original colipid appears thus as an innovative modular platform endowed with properties possibly
beneficial for fine-tuning of in vivo lipofection and other biomedical applications.
1.
2.
Delalande, A., et al., Sonoporation at a low mechanical index. Bubble science, Engineering and Technology, 2011. 3(1): p. 3-11.
Delalande, A., et al., Ultrasound and microbubble-assisted gene delivery in Achilles tendons: Long lasting gene expression and
restoration of fibromodulin KO phenotype. J Control Release, 2011. 156(2): p. 223-30.
∗Intervenant
∗Intervenant
†Auteur correspondant: chantal.pichon@cnrs-orleans.fr
-27-
-28-
CYCLODEXTRIN-BASED MULTICOMPONENT GENE VECTORS: OPTIMIZING NANOPARTICLE SELFASSEMBLING AND SITE-SPECIFIC GENE DELIVERY
1
2
3
2
†1
Laura Gallego-Yerga , Iris Pflueger , Conchita Tros De Ilarduya , Juan M Benito∗ , Carmen Ortiz Mellet , Jose
‡2
4
Garcia Fernandez , and Francisco Mendicuti
NON-VIRAL GENE THERAPY FOR CYSTIC FIBROSIS: FROM BASIC RESEARCH TO CLINICAL IMPACT
Stephen C Hyde
UK Cystic Fibrosis Gene Therapy Consortium
Gene Medicine Research Group, John Radcliffe Hospital, University of Oxford, UK
1
2
Dept. Organic Chemistry, Faculty of Chemistry, University of Sevilla – C/ Prof. Garcá Gonzalez 1, 41012 Sevilla, Espagne
Institute for Chemical Research, CSIC - University of Sevilla (IIQ, CSIC - Univ. Sevilla) – Americo Vespucio 49 - 41092 Sevilla, Espagne
3
Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra - Pamplona, Espagne
4
Departamento de Quìmica Fìsica, Universidad de Alcala – Universidad de Alcala - 28871 Alcala de Henares Madrid, Spain, Espagne
Dr Stephen Hyde is the University Lecturer in Gene Therapy at the University of Oxford (UK). His group
was the first to demonstrate that the fundamental defect in CF could be corrected by gene therapy and
has subsequently run four CF non-viral gene therapy clinical trials.
The clinical success of gene therapy critically depends on the development of safe and efficient delivery
systems. Despite their natural ability to infect cells, the use of virus-based carriers is hampered due to
limite their inherent immunogenicity and scaled-up availability. This fact, together with the advent of
nanotechnology, steered research to the design of artificial carriers. Cationic polymers hold a prominent
position within non-viral gene vectors [1], though their intrinsic polydispersity represents a difficulty for
structure-activity relationship (SAR) studies. Inspired in the facial amphiphilicity concept and its potential
to induce predictable self-assembling patterns [2], we have developed a new family of artificial gene
vectors based on molecularly well-defined self-assembling cyclodextrins (CDs) [3]. The strategy relies on
regioselective chemical functionalization of this macrocyclic scaffold to elaborate pre-organized
macromolecules with segregated cationic and lipophilic domains (polycationic amphiphilic cyclodextrins,
paCDs). Co-formulation of paCDs with plasmid DNA furnish small nanoparticles (CDplexes) exhibiting gene
transfer capabilities rivaling those of commercial standards (e.g. PEI) and reduced toxicity [4]. The
synthetic versatility of the concept permitted the elaboration of structurally diverse paCD libraries to
conduct SAR studies [5]. CDplex stability and gene transfer capabilities critically depend on finely tuning
hydrophilic/hydrophobic balance around the paCD scaffold, which can be easily tailored. Herein, we wish to
illustrate the potential of this approach for (i) controlling formation and dissociation of CDplexes, (ii)
engineering their surface topology [6], and (iii) promoting cell-specific gene transfer [7] by manipulating
paCD molecular structure and rationally building multicomponent assemblies.
1.
2.
3.
4.
M.A. Mintzer, E.E. Simanek, Chem. Rev. 2009, 109, 259.
C. Ortiz Mellet, J.M. Benito, J.M. García Fernandez, Chem. Eur. J. 2010, 16, 6728.
C. Ortiz Mellet, J. M. Garc´ıa Fernandez, J.M. Benito, Chem. Soc. Rev. 2011, 40, 1586.
A. Díaz-Moscoso, L. Le Gourrierec, M. Gomez-Garc´ıa, J.M. Benito, P. Balbuena, F. Ortega- Caballero, N. Guilloteau, C. Di Giorgio, P.
Vierling, J. Defaye, C. Ortiz Mellet, J.M. Garc´ıa Fernandez, Chem. Eur. J. 2009, 15, 12871.
5. C. Ortiz Mellet, J.M. Benito, J.M. Garc´ıa Fernandez, Pure Appl. Chem. 2013, 85, 1825-1845, and references therein.
6. C. Aranda, K. Urbiola, A. Méndez Ardoy, J.M. García Fernandez, C. Ortiz Mellet, C. Tros de Ilarduya, Eur. J. Pharm. Biopharm. 2013, in
press
7. A. Díaz-Moscoso, N. Guilloteau, C. Bienvenu, A. Méndez-Ardoy, J.L. Jiménez Blanco, J.M. Benito, L. Le Gourriérec, C. Di Giorgio, P.
Vierling, J. Defaye, C. Ortiz Mellet, J.M. García Fernandez, Biomaterials, 2011, 32, 7263-7273.
Cystic fibrosis (CF) is the most common lethal inherited disease of Caucasian populations, affecting ~80,000
individuals worldwide. Approximately 30% of this population live in the EU. CF individuals, homozygous for
mutations in the CFTR gene, suffer from repeated bacterial infections of the conducting airways leading to lung
failure. Current treatments are focused on symptomatic relief and are associated with a huge treatment burden,
requiring several hours of self-administered therapy daily, and high individual patient costs (€20-60K/year).
Despite this, the current median age at death in France and the UK is ~25 years.
The UK Cystic Fibrosis Gene Therapy Consortium (UKCFGTC) have previously demonstrated proof-of-principle
for CFTR gene replacement therapy – showing correction of the underlying CF chloride channel defect for 1-2
weeks in patients receiving early-generation non-viral gene transfer vectors. Our most advanced non-viral vector
system is a cationic liposome-based vector termed pGM169/GL67A. This system comprises an entirely CG
dinucleotide-free plasmid DNA (pGM169) that directs sustained CFTR lung expression in animal models, and a
cationic liposome mixture (GL67A) the most effective non-viral lung gene transfer agent we identified from a
large in vivo screen. Unlike conventional plasmids, pGM169 does not engage with the CpG-dependent TLR-9
inflammatory pathway and thus has minimal inflammatory effects. GL67A-based formulations are readily and
potently delivered by aerosol generating devices and are thus ideal for lung delivery.
Following successful nonclinical toxicology studies, two clinical studies have been initiated. In an open-label
Phase I/IIa study we have shown that a single dose to the nose and lungs of CF patients administered via a
nebuliser was safe and could correct the CF chloride channel defect for up to 3 months following a single
administration. We have subsequently initiated a Phase IIb study in which pGM169/GL67A is repeatedly (once a
month for 1 year) delivered to the lungs of CF patients. This study, recruiting 130 patients from around the UK, is
the largest CF gene therapy trial ever conducted worldwide. All patients are now recruited, and the study will be
completed in the summer of 2014.
Funding for these studies has been provided by grants to the UKCFGTC from the UK NIHR Efficacy and
Mechanism Evaluation programme and the Cystic Fibrosis Trust.
∗Intervenant
†Auteur correspondant: mellet@us.es
‡Auteur correspondant: jogarcia@iiq.csic.es
-29-
-30-
DECEMBER 4, 2013
RESPONSIVE AND MULTIMODAL IMAGING PROBES BASED ON LANTHANIDE COMPLEXES
Éva Tóth
8h30
Registration – Poster installation - Session B
SESSION « IMAGING »
Chairpersons: Jean-Luc Coll and Eva Jakab-Toth
9h00 DR. EVA JAKAB-TOTH, CBM CNRS Orléans, France
Responsive and multimodal imaging probes based on lanthanide complexes
9h40 Local delivery of large payloads with composite droplets and a clinical ultrasound scanner
C. Errico, M. Bezagu, O. Couture, S. Arseniyadis, J. Cossy, P. Tabeling, M. Tanter
10h00 Adenosine-Squalene nanoparticles and cerebral ischemia: toward a theranostic tool
A. Gaudin, S. Lepêtre, M. Yemisci, M. Boucher, B. Larrat, S. Mériaux, O. Tagit,
N. Hildebrandt, K. Andrieux, T. Dalkara, P. Couvreur
10h20 In vivo characterization of MV-1 magnetosomes as biogenic contrast agents dedicated to high field MRI:
a dose study
M. Boucher, N. Ginet, D. Garcia, D. Pignol, S. Mériaux
10h40 Coffee break & Poster session (Session B)
11h20 PR. KLAAS NICOLAY, Eindhoven University of Technology, The Netherlands
Quantitative MRI for diagnostics and image-guided therapy
12h00 Patrick Boisseau, Presentation of ETP Nanomedecine
12h10 GENERAL ASSEMBLY of GTRV
12h40 LUNCH & Poster session (Session B)
Chairpersons: Chantal Pichon and Nathalie Mignet
14h30 Pr. Eric Allémann, University of Geneva, Switzerland
Microbubbles - From ultrasound contrast agents to new therapeutic modalities.
15h10 Development of multimodal nanocarriers for vectorized radiotherapy in the context glioblastoma
treatment.
D. Sehedic, M. Mougin Degraef, F. Hindré, F.s Davodeau, E. Garcion
15h30 Nanoparticules à luminescence persistante pour la bioimagerie
C. Richard, T. Maldiney, J. Seguin, E. Teston, M. Bessodes, D. Scherman
15h50 GTRV-SANOFI PRIZE
16h00 GTRV-DEBIOPHARM PRIZE
16h15 End of the 2013 GTRV Annual Meeting
-31-
Centre de Biophysique Moléculaire, CNRS, Orléans, France
III
Eva Jakab Toth has been active in the design and physical-chemical characterization of Ln chelates
related to MRI applications with a special focus on understanding the relationships between chemical
III
structure and MRI efficacy of Gd complexes. She has a solid experience in designing highly efficient and
smart MRI imaging probes based on lanthanide complexes. Recently, she reported responsive agents for
the detection of enzymatic activities, extracellular CaII, pyrophosphate, etc.
Currently Eva Jakab Toth is the director of the Centre of Molecular Biophysics, CNRS, Orléans.
In the last two decades, lanthanide coordination chemistry has witnessed a spectacular evolution, largely
promoted by the successful use of lanthanide complexes in biomedical applications. Millions of clinical magnetic
resonance imaging (MRI) examinations are carried out after the injection of Gd3+ chelates. Luminescent
lanthanide complexes are also gaining more and more importance for in vitro optical assays and cellular optical
imaging. Emerging applications in molecular imaging seek a real-time, repeatable, in vivo visualization of
molecules or molecular events at the cellular and tissue level. One important field in molecular imaging involves
the in vivo detection of physico-chemical parameters of tissues, concentration of ions, metabolites, etc. by
applying smart, activatable imaging probes that are responsive to the specific parameter to detect. In contrast to
nuclear imaging modalities, MRI is particularly well adapted to the design of responsive probes, involving Gd3+based or PARACEST (Paramagnetic Chemical Exchange Saturation Transfer) agents. The efficacy (relaxivity or
CEST properties) of the probe has to be selectively influenced, based on coordination chemistry concepts, by
the particular biomarker that we wish to detect. We develop potential smart contrast agents to detect cation or
neurotransmitter concentration changes in the extracellular space or to monitor enzyme activity.
Recently, bimodal imaging has emerged as a novel concept to ascertain observations made in one imaging
modality by a complementary technique. Ideally, bimodal imaging is performed by using bimodal probes that
combine the characteristics required for both imaging modalities within a single molecular entity. Our major
interest is to use lanthanide chelates as bimodal imaging agents for combined MRI and optical detection. Given
the diverse magnetic and optical properties of lanthanide ions, lanthanide complexes are perfectly suited for the
design of MRI and optical bimodal probes. Different approaches to develop lanthanide-based bimodal agents
will be presented.
-32-
LOCAL DELIVERY OF LARGE PAYLOADS WITH COMPOSITE DROPLETS AND A CLINICAL
ULTRASOUND SCANNER
1
2
†1
2
ADENOSINE-SQUALENE NANOPARTICLES AND CEREBRAL ISCHEMIA: TOWARD A THERANOSTIC
TOOL
2
1
Claudia Errico∗ , Marine Bezagu , Olivier Couture , Stellios Arseniyadis , Janine Cossy ,
3
1
Patrick Tabeling , and Mickael Tanter
2
1
Institut Langevin – CNRS : UMR7587, Inserm : E979, ESPCI ParisTech – France
Laboratoire de Chimie Organique (LCO) – ESPCI ParisTech – 10 rue Vauquelin - 75231 Paris, France
3
Laboratoire Microfluidique, MEMS, Nanostructures (MMN) – ESPCI ParisTech, CNRS : UMR7083 –
10 rue Vauquelin - 75231 Paris, France
Delivering agents with a clinical ultrasound scanner would simplify procedures, permit millimetric resolution and
allow perfect registration between imaging and therapy. For example, tattooing tissue with fluorescent markers
under radiological guidance could improve the localization of tumors during surgery. Such a system could also
be used to deliver therapeutic doses of chemotherapy within small, but detectable metastasis. Our objective is
to create ultrasound-inducible carriers that could both be uniformly released with low-energy pulses and deliver
large payloads in-situ.
For this purpose, composite droplets were produced by injecting, in a microfluidic system, a nanoemulsion of an
aqueous solution in perfluorohexane stabilized by a fluorosurfactant. Microfluidic production generates droplets
that are 4 microns in diameter with a polydispersity of 3%. The matrix of perfluorohexane can be vaporized with
low-intensity ultrasound, but also acts as a barrier preventing exchange between the inner and outer media. For
the various studies using this system, composite droplets comprising fluorescein, cyanine, DMSO,
azidocoumarin and gadolinium-DTPA were produced.
The release of fluorescein droplets was performed both in-vitro and in-vivo, to demonstrate the applicability of
the combination of the composition droplets and ultrasound scanner, along with the concept of ultrasound
tattooing. In-vitro, the release of the composite droplets was tested by injecting them in a tissue culture plate
and focusing the ultrasonic scanner at a specific site observed with a fluorescence microscope. The droplets
were released at a pressure of 1.5 MPa PNP within a pulse of two cycles at 5 MHz (less than a microsecond).
The release of the droplets was 3.3mm in width. Along with the delivery, direct ultrafast monitoring with planewave ultrasound imaging was performed at 13 KHz frame rate. Such ultrafast imaging technique can highlight
small and rapid changes induced by the release of perfluorohexane. Up to 70 dB contrast between the target
region and the surrounding tissue was obtained, demonstrating that the release can be monitored within the first
few milliseconds of initiation.
In-vivo, 100 uL of composite droplets(50x106 droplets) were injected in the femoral vein of a rat. The ultrasonic
array was aligned on the liver and pulses of 1.2 to 4.4 MPa at 5 MHz were focused in specific regions. These
millimetric spots were clearly observable on the ultrasound images. The droplets released within the FDA
guidelines for imaging pulses. Ultrafast imaging also provided high contrast between the zone of release and
unaffected regions, up to 27 dB. The release zones were also viewed under an intraoperative camera, currently
used by surgeon. Tattooing tissue with an ultrasound scanner was thus demonstrated.
These studies showed that the composite droplets can deliver large payloads in-vivo and that the process can
be induced, guided and monitored with the same ultrasonic scanner. Such a combination could become a useful
tool for localized tagging and therapy, since ultrasonic scanners are already ubiquitous in hospitals. Additionally,
due to the high isolation between the inner and outer phase of the droplets, these agents could also perform
ultrasound-induced chemical reaction in-situ and generate drugs remotely.
1
2
3
1
3
Institut Galien Paris-Sud UMR CNRS 8612 – Université Paris Sud - Paris XI – 5, rue Jean-Baptiste Clément - 92290 Chatenay-Malabry, France
2
Brain Research Laboratory - Hacetteppe University – Ankara, Turquie
3
Service NEUROSPIN (NEUROSPIN) – CEA – France
4
NanoBioPhotonics – Université Paris Sud - Paris XI – Université d’Orsay 15 Rue Georges Clémenceau Batiment 220 - 91405 Orsay, France
Cerebral ischemia is the third leading cause of death in developed countries [1], and the first cause of
acquired handicap during adulthood. The only clinical treatment currently available, the rt-PA, can only be
used in very restrained conditions, mainly because of a high risk of hemorrhage. It has been proposed
that the use of MRI and MRI contrast agents could be helpful in the earliest decisional steps of stroke
patients handling. On the other hand, adenosine has been for a long time considered as a putative
treatment of the penumbra [2], the zone surrounding the ischemic core. However, most of the strategies
involving adenosine and MRI contrast agents encountered the problems of their inability to cross the BloodBrain Barrier (BBB). Our goals are to covalently link the squalene to the adenosine (“squalenoylation” [3]), in
order to obtain stable nanoparticles according to the amphiphilic structure of the compound, and to
encapsulate USPIO in these nanoparticles to obtain a theranostic tool. This should allow the nanoobjects
to cross the BBB, and finally permit to reach a cerebral concentration in adenosine able to have a
therapeutic effect, and to obtain a MRI signal thanks to the penetration of the USPIO in the brain
parenchyma.
By nanoprecipitating the adenosine-squalene we obtained stable nanoparticles, with a mean diameter
ofc.a. 120 nm, a mean polydipsersity below 0.2 and a mean zeta potential of around -30 mV. Using an in
vitro model of human BBB [4] (hCMEC/D3 cell line) and a transwell collagen-coated system the ability of
the nanoparticles to cross the BBB has been shown and confirmed by the in vivo biodistribution of
radiolabeled nanoparticles in mice. Moreover, using FRET nanoparticles we were able to show that the
nanoparticles were opening during the transcytosis and exocytosed in a molecular form in the brain
parenchyma. The pharmacological efficiency of the adenosine-squalene nanoparticles was studied in a
transient and permanent model of cerebral ischemia in mice, leading to an impressive neuroprotective
effect as shown by a dramatic decrease of the infarct volume. Finally, by co-nanoprecipitating the
adenosine-squalene with USPIO, magnetic nanoparticles are formed and characterized for their properties
of relaxivity.
These results show for the first time that the “squalenoylation” technology, which has already b e e n
applied to the intravenous administration of anticancer compounds [5][6], is competent for the delivery of
hydrophilic drugs to the brain sanctuary, and could became a new theranostic platform for neurological
diseases.
1.
2.
3.
4.
5.
6.
Dirnagl U. et al, Trends Neurosci., 1999, 22: 391-397
Williams-Karnesky R.L. and Stenzel-Poore M.P., Current Neuropharmacology, 2009, 7: 217-227
Couvreur P. et al., Nanoletters, 2006, 6(11): 2544-2548
Weksler B.B. et al, FASEB J., 2005, 19(13): 1872-1874
Reddy L.H. et al., The Journal of Pharmacology and Experimental Therapeutics, 2008, 325(2): 484-490
Arias J.L. et al., ACS Nano, 2011, 5(2): 1513-1521
*Intervenant
†Auteur correspondant: olivier.couture@espci.fr
∗Intervenant
-33-
3
Alice Gaudin∗ , Sinda Lepêtre , Müge Yemisci , Marianne Boucher , Benoît Larrat , Sébastien Meriaux ,
4
4
1
2
1
Oya Tagit , Niko Hildebrandt , Karine Andrieux , Turgay Dalkara , and Patrick Couvreur
-34-
IN VIVO CHARACTERIZATION OF MV-1 MAGNETOSOMES AS BIOGENIC CONTRAST AGENTS
DEDICATED TO HIGH FIELD MRI: A DOSE STUDY
QUANTITATIVE MRI FOR DIAGNOSTICS AND IMAGE-GUIDED THERAPY
Klaas Nicolay
†1
2
2
2
Marianne Boucher∗ , Nicolas Ginet , Daniel Garcia , David Pignol , and Sébastien Mériaux
2
1
1
UNIRS (UNIRS, CEA / DSV / I2BM / NeuroSpin) – CEA – Gif-sur-Yvette, France, France
LBC (LBC, CEA / DSV / IBEB / SBVME) – CEA – Saint-Paul-lez-Durance, France, France
Magnetosomes are superparamagnetic nanoparticles produced by magnetotactic bacteria that exhibit
different characteristics of interest for molecular MRI applications: a perfectly crystalline and regular
nanocrystal of magnetite coated by a natural lipid bilayer, which can be functionalized for biomarkers
targeting [1] (Figure 1). Here, we demonstrate with ultra high field MRI acquisitions the very high in vivo
contrasting efficiency of magnetosomes compared to a commercial SPIO solution (Ferumoxide). Both
magnetosomes and Ferumoxide cannot pass through the blood-brain-barrier because of their relatively
large diameter. Therefore, contrast efficiency of both nanoparticles has been characterized towards their
ability to provide high-resolution 3D angiograms of mouse brain after intravenous injection.
Magnetovibrio blakemorei MV-1 strain is grown at 30 C in defined media following an already published
protocol [1]. Their magnetosomes (MV-1m) were then extracted after bacteria centrifugation using a
permanent magnet. To calibrate the injected doses for in vivo acquisitions, we investigated the MRI
contrast efficiency of both tested nanoparticles by measuring in vitro their transverse relaxivity r2 at 17.2
T (BioSpec scanner, Bruker, Germany): we acquired T2 parametric map (Multi-Slice Multi- Echoes
sequence, TE=7.7 ms, 66 echoes) of phantom containing 12 tubes filled with different concentrations of
contrast agent. In vivo experiments were carried out on Swiss mice at 17.2 T. T2*-weighted images (Fast
Low Angle Shot sequence, TE/TR= 8/680 ms, R = 90x90x180 m3) were acquired pre and post injection
at the tail vein of MV-1m or Ferumoxide (100 L, 20 molFe/kg and 200 molFe/kg), or control solution (100
L of physiological serum). The post–processing included the Frangi filtering method implemented by [2]
to reveal brain vasculature enhancement induced by contrast agent circulating in blood stream.
Department of Biomedical Engineering, Eindhoven University of Technology, and
Center for Imaging Research & Education (CIRE), Eindhoven, the Netherlands
Klaas Nicolay was trained as a physical chemist and has been involved in the use and development of
Magnetic Resonance techniques for most of his scientific career. Presently, he is Professor of Biomedical
NMR at Eindhoven University of Technology and supervising an extensive research and teaching program in
the field of biomedical imaging. Nicolay is member of the councils of the World Molecular Imaging Society
and European Society for Molecular Imaging.
Magnetic Resonance Imaging (MRI) plays in important role in clinical diagnostics and biomedical research. This
presentation will highlight recent developments of quantitative MRI techniques to allow for the objective
evaluation of tissue status in vivo and how this technology can be used to improve disease diagnosis, the
guidance of therapeutic interventions and the assessment of their efficacy. Examples will be drawn from
research in the area of cancer, metabolic diseases and cardiovascular disorders. Advances in MRI contrast
agent design play an important role in the above research and therefore will receive special attention.
Transverse relaxivities r2 (Table 1) show the great contrasting efficiency of MV-1m compared to
Ferumoxide. 3D angiograms visualization presented in Figure 2 reveal that both high doses of MV-1m or
Ferumoxide strongly highlight brain vasculature, whereas for low doses only magnetosomes do. Large
vessels already detected before injection of contrast agent due to endogenous iron in the blood are
enhanced, and smaller ones are only revealed by low dose of MV-1m or by high doses of MV-1m and
Ferumoxide. As expected, the injection of physiological serum does not provide any vasculature
enhancement (control solution).
In conclusion, our relaxometry measurements and in vivo MRI acquisitions performed at ultra high field
confirm that MV-1m exhibit very high contrasting properties compared with a commercially produced SPIO
solution. The observed gain enables to obtain high-resolution angiograms of mouse brain after injection of
significantly lower dose than the one commonly used in rodent studies. These magnetosomes should
therefore provide enough sensitivity for molecular imaging studies.
∗
Intervenant
†Auteur correspondant: marianne.boucher@cea.fr
-35-
-36-
MICROBUBBLES - FROM ULTRASOUND CONTRAST AGENTS TO NEW THERAPEUTIC MODALITIES
DEVELOPMENT OF MULTIMODAL NANOCARRIERS FOR VECTORIZED RADIOTHERAPY IN THE
CONTEXT GLIOBLASTOMA TREATMENT
Eric Allémann
1
2
1
2
Delphine Sehedic∗ , Marie Mougin Degraef , Francois Hindré , Francois Davodeau ,
1
and Emmanuel Garcion
Geneva-Lausanne School of Pharmacy - University of Geneva, 30, quai E.-Ansermet - CH-1211 Geneva 4
e-mail: eric.allemann@unige.ch
http://www.unige.ch/sciences/pharm/fatec/index.html
2
1
UMR 1066 (MINT) – Inserm : UMR1066 – 4 rue Larrey 49933 Angers, France
Centre de Recherche en Cancérologie Nantes - Angers (CRCNA) – Inserm : U892, Université de Nantes – France
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland
Eric Allémann was born in1963. He studied pharmacy at the University of Geneva. From 1990 to 1993, he worked on
his thesis in the field of parenteral polymeric drug delivery systems with extended release properties. He obtained his
Ph.D. in 1993. He went then to the Faculty of Medicine of the University of Sherbrooke (Quebec) for postdoctoral
research in the field of photodynamic therapy of tumours with polymeric drug delivery systems. In 1995, he became
research associate and lecturer at the University of Geneva. In 1997, the Swiss Confederation for the renewal of
academic positions appointed him. He obtained in October 1999 a position of “maître d'enseignement et de recherche”.
Since October 2001, he joined the Geneva research center of the pharmaceutical company Bracco Group, as senior
research scientist. He was involved in therapeutics and targeting projects. In 2004, he was appointed Manager and
was heading the Department of Novel Agents Research at Bracco Research Geneva. In 2009 he became full professor
of Pharmaceutical technology at the Geneva-Lausanne School of Pharmacy, located at the University of Geneva.
He has authored and an co-authored 73 research and review articles and is (co)-inventor for 11 international patents. He is member of the
Editorial Board of the European Journal of Pharmaceutics and Biopharmaceutics, of the International Journal of Pharmaceutics and of the
Journal of Drug Delivery Science and Technology.
Eric Allémann's research interests are focused on the delivery of active substances by means of supramolecular constructs, nanoand
microparticles for the controlled release and drug targeting as well as for contrast agents for various medical imaging techniques.
Microbubbles are gas-filled vesicles or microspheres, originally designed and used as ultrasound contrast
agents for diagnostic purpose. Microbubbles are mostly used in contrast-enhanced ultrasound examinations to
increase the contrast between the blood compartments and the surrounding tissues. Microbubbles are
composed of a gas core (air, sulphurhexafluoride or perfluorocarbon gases) stabilized by a shell, whether made
of proteins (albumin), fatty acids, phospholipids or polymers. Stabilized microbubbles are stored as aqueous
suspensions or reconstituted from a freeze-dried product before use.
The presentation will review the formulation aspect, the interplay between these fragile structures and the
ultrasound waves. It will show the advantages of using this type of contrast agents for clinical diagnosis
procedures as well as for treatment monitoring and treatment follow up. For instance, contrast-enhance
ultrasound examinations can monitor precisely the effect of targeted anti-cancer therapies. Microbubbles can be
designed as blood pool agents freely circulating in the blood stream or they can be tailored with proteins at their
surface in order to provide targeted microbubbles for molecular imaging.
Glioblastoma multiform (GBM) is the most common primary brain tumor in adult with a prevalence of 5
cases/100000 habitants. Current therapy effectiveness is indeed largely limited by resistance mechanisms
combining intrinsic properties of tumour cells to the influence of the microenvironment in which they develop
(e.g. hypoxia). In this work, we deal to target a radioresistance-associated epitope in GBM cells, the CXCR4,
and to develop lipid nanocapsules (LNC) loaded with radionuclides. This investigation was made in the context
of using multimodal emitters that can be used for radiotherapy and imaging: 188rhenium (188Re) displaying
gamma emission that allows its detection by SPECT-CT and 211Astatin (211At) the development of which
alpha-camera are in progress.
As CXCR4 receptor has been associated with radio-resistance and with cancer stem-like cell (CSLC)
occurrence, we focused on the targeting of this protein in a human GBM models (A172, U87MG overexpressing
the CXCR4 and human primary GBM cells). In line with this, by using immunofluorescence flow cytometry, we
found that CXCR4 expression is correlated with radiation doses with 10% CXCR4 positive cells in control versus
41% after a 32Gy treatment. We then developed and characterized 50 nm lipid nanocapsules (LNCs)
functionalized with a function-blocking monoclonal antibody (mAb) directed against CXCR4 (or isotype control).
Those functionalized or function-free LNCs were further combined to 188Re (beta emitter) and 211At (-emitter)
for physicochemical evaluation and capability to stably convey the chosen radiopharmaceutics in vivo.
Encapsulation of 125I (-emitter and halogen) in miniaturization of the process, which will help for the
development of 211At (halogen), demonstrated its feasibility with a stable encapsulation over time of SIB or
more lipophilic SIB-octadécylamine (in C18) in different medium. In vivo efficacy of the different systems is
under investigation after othotopic implantation of human GBM cells in immunodeprived SCID mice and locoregional convective infusion of the radiotherapeutic nano-objects.
Our work combined the interest of targeting CSLC associated epitopes to the one of inhibiting CXCR4 signaling
pathways to overcome radioresistance. It also constitutes a prerequisite for addressing the relevance of
versus -nanocarrier radiotherapy in glioblastoma.
Depending on ultrasound parameters, microbubbles can undergo stable or intertial cavitation. Inertial cavitation
conditions can be used to enhance delivery of drugs or genetic material to cells, a process named sonoporation.
Finally, inertial cavitation with microbubbles can be used to enhance thrombolytic treatments with recombinant
tissue plasminogen activator. Recent studies on sonothrombolysis will be presented during this talk.
∗Intervenant
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-38-
NANOPARTICULES À LUMINESCENCE PERSISTANTE POUR LA BIOIMAGERIE
Cyrille Richard∗, Thomas Maldiney, Johanne Seguin, Eliott Teston, Michel Bessodes,
and Daniel Scherman
POSTERS – SESSION A
A1
FUNCTIONALIZING LIPOSOMES WITH ANTI-CD44 APTAMER FOR ACTIVE DRUG DELIVERY
W ALHAN ALSHAER, HERVÉ HILLAIREAU, SAID ISMAIL, AND ELIAS FATTAL
A2
BEHAVIOR EVALUATION OF A NANOEMULSION-CONTAINIG PDNA IN SERUM ENVIRONMENT
ANDRÉ SILVA, ALÉLIA ROSA , PRISCILA DE PAULA , LOURENA VERÍSSIMO , LUCYMARA AGNEZ-LIMA , AND
ERYVALDO EGITO
A3
RILES, A NOVEL MOLECULAR PROBE TO MONITOR THE KINETIC OF MICRORNA
EXPRESSION OF BREAST CANCER DEVELOPMENT AS WELL AS THE FUNCTIONAL DELIVERY
OF RNAI-BASED DRUGS
SAFIA EZZINE, JEAN MARC MALINGE, PATRICK MIDOUX, CHANTAL PICHON, AND PATRICK BARIL
A4
IDENTIFICATION OF NEW BIO-EFFECTS OF ULTRASOUND AND MICROBUBBLES-ASSISTED
DRUG DELIVERY
ANTHONY DELALANDE, LUCIE PIGEON, CHLOÉ LEDUC, PATRICK TAUC, ERIC DEPREZ, PATRICK MIDOUX, AND
CHANTAL PICHON
A5
DNA MINICIRCLE AS POTENTIAL NEW DECOY ODN FOR TRANSCRIPTION FACTORS
TARGETING.
THOMAS THIBAULT, STÉPHANE BOURG, CHANTAL PICHON, PATRICK MIDOUX, AND JEAN-MARC MALINGE
A6
CONCEPTION OF CATIONIC MICROBUBBLES FOR DNA DELIVERY IN VIVO UNDER
SONOPORATION
SIMONA MANTA, ANTHONY DELALANDE, MATHIAS LEBERTRE, GILLES RENAULT, OLIVIER COUTURE, DANIEL
SCHERMAN, MICHEL FRANCIS BUREAU, PATRICK MIDOUX, CHANTAL PICHON , MICHEL BESSODES, AND
NATHALIE MIGNET
A7
ENHANCED SENSITIVITY OF U87MG GLIOBLASTOMA CELLS TO TEMOZOLOMIDE AFTER
TREATMENT BY SIRNA CARRIED BY LIPID NANOCAPSULES
KHALED MESSAOUDI, ANNE CLAVREUL, PATRICK SAULNIER, JEAN-PIERRE BENOIT, AND FREDERIC LAGARCE
A8
PTG1: NEW ROBUST TRANSFECTION REAGENTS FOR THE PRODUCTION OF
RECOMBINANT PROTEINS AND VIRAL PARTICLES
HERVÉ CHERADAME, PHILIPPE GUÉGAN, FABIAN GROSS, AND PATRICK MIDOUX
A9
CHARACTERIZATION AND STRUCTURE OF SIRNA LIPID NANOCAPSULES FOR GENE
THERAPY
PAULINE RESNIER, PIERRE LEQUINIO, NOLWENN LAUTRAM, EMILIE ANDRÉ, CÉDRIC GAILLARD,
GUILLAUME BASTIAT, JEAN-PIERRE BENOIT, AND CATHERINE PASSIRANI
A10
ADVANTAGES OF USING THE LIPOTHIOUREA DDSTU AS A COLIPID FOR DNA OR SIRNA
DELIVERY
JOHANNE SEGUIN, MICHEL BESSODES, JEAN HERSCOVICI, KARIM FEKIR, ALICE W EYLAND , DANIEL
SCHERMAN, ANDREW MILLER, AND NATHALIE MIGNET
A11
INFLUENCE OF NON-IONIC SURFACTANTS ON THE FORMATION OF MIXED NANO-OB JECTS
WITH AMPHIPHILIC CYCLODEXTRINS
LEILA ZERKOUNE, ANGELINA ANGELOVA, LUC CHOISNARD, ANNABELLE GEZE, DENIS WOUESSIDJEWE, AND
SYLVIANE LESIEUR
Unité de pharmacologie chimique et génétique et d’imagerie (UPCGI) – CNRS : UMR8151 – 4 avenue de l’observatoire - 75006 Paris - France
Les derniers développements dans le domaine de l’imagerie optique du petit animal ont permis de voir
apparaitre plusieurs nouvelles sondes photoniques [1] Les quantum dots [2] et les nanoparticules à
upconversion [3] présentent notamment des alternatives intéressantes aux sondes organiques par leur fort
rendement quantique, ou leur capacité à contourner l’autofluorescence des tissus. [4] Notre équipe a proposé
l’utilisation de nanoparticules à luminescence persistante (PLNP) pour l’imagerie optique en temps réel chez le
petit animal. [5] Comparables à des condensateurs optiques, ces cristaux se chargent sous l’effet d’une
excitation lumineuse et émettent de la lumière sur une période pouvant aller de quelques minutes à plusieurs
heures. Ces propriétés de luminescence persistante permettent une détection optique à travers les tissus, sans
phénomène d’autofluorescence.
Nous rapporterons les premiers exemples d’utilisation de nanoparticules à luminescence persistante pour
l’imagerie du petit animal [5] Nous montrerons comment la fonctionnalisation de leur surface permet le ciblage
de plusieurs lignées cancéreuses [6] et décrirons l’impact du diamètre sur leur biodistribution. [7] Nous
retracerons les étapes qui ont permis de passer de la première génération de nanoparticules, uniquement
visible pendant 1 heure in vivo au matériau intermédiaire détectable 5 fois plus longtemps [8] pour enfin décrire
le matériau de dernière génération, ré-excitable in situ permettant un suivi in vivo pendant plusieurs semaines.
[9]
1 . Weissleder R et al. Nature 2008; 452, 580-589 Medintz IL, et al. Nature Mater. 2005; 4435-446 Zhou J, et al. Chem. Soc. Rev. 2012;
41, 1323-1349
2. Frangioni JV. Curr. Opin. Chem. Biol. 2003; 7, 626-634
3 . le Masne de Chermont Q, et al. Proc. Natl. Acad. Sci. USA., 2007; 104, 9266-9271
4 . Maldiney T, et al. Bioconjugate Chem. 2012; 23, 472-478
5 . Maldiney T, et al. ACS Nano. 2011; 5, 854-862
6. Maldiney T, et al. J. Am. Chem. Soc. 2011; 133, 11810-11815 Maldiney T, et al.
7. French Patent FR1250846, 2012
∗Intervenant
-39-
-40-
A12
STXB TECHNOLOGY FOR TUMOR THERANOSTIC AND IMMUNOTHERAPY
ESTELLE DRANSART, KAMÉLIA KARA ALI, LAURA BRULLÉ, THI TRAN, BIRGIT OBERLEITNER,
OLIVIER COUTURE, VINCENT SEMETEY, ERIC TARTOUR, AND LUDGER JOHANNES
A13
ANTI-PPRV THERAPY BASED ON IN VIVO EFFIENCY OF SIRNA: PROOF OF CONCEPT USING
A SMALL ANIMAL MODEL
CÉCILE MINET, RENATA SERVAN DE ALMEIDA, CARINE HOLZ, RENAUD LANCELOT, GENEVIÈVE LIBEAU,
MATTIAS HALLBRINK, ÜLO LANGEL, AND EMMANUEL ALBINA
A14
A15
A16
A17
A18
A19
A24
EFFECTS OF A NOVEL ARCHAEAL TETRAETHER-BASED COLIPID ON THE IN VIVO GENE
TRANSFER ACTIVITY OF TWO CATIONIC AMPHIPHILES
TONY LE GALL, JULIE BARBEAU, SYLVAIN BARRIER, MATHIEU BERCHEL, LOIC LEMIEGRE, JELENA JEFTIC,
CRISTELLE MERIADEC, FRANCK ARTZNER, DEBORAH GILL, STEPHEN HYDE, CLAUDE FEREC, PIERRE LEHN,
PAUL-ALAIN JAFFRES, THIERRY BENVEGNU, AND TRISTAN MONTIER
A25
OPTIMIZING THE FORMULATION PROTOCOL OF MAGNETIC SIRNA NANOVECTORS USING
EXPERIMENTAL DESIGN METHODOLOGY
STÉPHANIE DAVID, HERVÉ MARCHAIS, DIDIER BEDIN, AND IGOR CHOURPA
A26
STUDY OF NFKB CONTRIBUTION TO TRANSGENE EXPRESSION MEDIATED BY
ELECTROTRANSFER IN MOUSE TIBIAL CRANIAL MUSCLE.
SAYSOUDA MAHINDHORATEP, HAMID AIT BOUDA, NELLY EL SHAFEY, DANIEL SCHERMAN, ANTOINE
KICHLER, CHANTAL PICHON, PATRICK MIDOUX, NATHALIE MIGNET, AND MICHEL FRANCIS BUREAU
A27
BIODISTRIBUTION IN VIVO DE FORMULATIONS LIPIDIQUES FLUORESCENTES APRÈS
INJECTION SYSTÉMIQUE
NAWAL BELMADI
LUNG CANCER TREATMENT BY TARGETING CD44 RECEPTORS: SIRNA DELIVERY BY
HYALURONIC ACIDACID-MODIFIED LIPOPLEXES
THAIS LEITE NASCIMENTO, HERVÉ HILLAIREAU, MAGALI NOIRAY, DELPHINE COURILLEAU, MYRIAM TAVERNA,
SILVIA ARPICCO, AND ELIAS FATTAL
A28
FORMULATION ET BIODISTRIBUTION IN VIVO DE VECTEURS SYNTHETIQUES POUR LE
TRANSFERT DE GENES DANS LE CADRE DE LA THERAPIE GENIQUE DE LA
MUCOVISCIDOSE
NAWAL BELMADI
STUDY OF NFKB CONTRIBUTION TO TRANSGENE EXPRESSION MEDIATED BY
ELECTROTRANSFER IN MOUSE TIBIAL CRANIAL MUSCLE.
SAYSOUDA MAHINDHORATEP, HAMID AIT BOUDA, NELLY EL SHAFEY, DANIEL SCHERMAN, ANTOINE
KICHLER, CHANTAL PICHON, PATRICK MIDOUX, NATHALIE MIGNET, AND MICHEL FRANCIS BUREAU
A29
POLYMERS WITH PH-SENSITIVE DISSOLUTION SWITCH FOR INTRACYTOSOLIC DELIVERY
OF NUCLEIC ACIDS AND PROTEINS: RECENT ADVANCES
GUY ZUBER
A30
DEVELOPMENT AND EVALUATION OF NEW DRUG DELIVERY SYSTEMS OF NITROSOTHIOLS
FOR ORAL ADMINISTRATION
SHEFAAT ULLAH SHAH, MARIE SOCHA, AND STÉPHANE GIBAUD
A31
CHEMICAL GELS OF LIPID NANOPARTICLES FOR THE CONTROLLED DELIVERY OF
BIOLOGICAL AND LIPOPHILIC DRUGS
ISMAIL CHEIBANI, ANTOINE HOANG, MARIE ESCUDÉ, ERIC BAYMA, RACHEL AUZÉLY, AND ISABELLE TEXIERNOGUES
A32
CHITOSAN-BASED NANOPARTICLES: A STRATEGY FOR THE INTRACELLULAR DELIVERY OF
NUCLEOTIDE ANALOGS
GIOVANNA GIACALONE, HERVÉ HILLAIREAU, AND ELIAS FATTAL
A33
THE INTRACELLULAR FATE OF POLYCARBONATE POLYPLEXES MODULATES THE EFFICACY
OF SIRNA
ANTOINE FRÈRE, MICHAL KAWALEC, PAUL PEIXOTO, BRIGITTE EVRARD, PHILIPPE DUBOIS, LAETITIA
MESPOUILLE, DENIS MOTTET, AND GÉRALDINE PIEL
TRIPPARTITE PH-SENSITIVE MICELLES OF POLYETHYLENIMINE AND DOUBLE
HYDROPHILIC BLOCK COPOLYMERS AS GENE DELIVERY SYSTEMS
NAILA MEBAREK, ANNE AUBERT, CORINE GERARDIN, JEAN-MARIE DEVOISSELLE, AND SYLVIE BÉGU
PH-SENSITIVE POLYION COMPLEX MICELLES AS SIRNA VECTORS FOR DC ENGINNEERING
TO INDUCE TOLERANCE
NAILA MEBAREK, RITA VICENTE, ANNE AUBERT, CHRISTIAN JORGENSEN, JEAN-MARIE DEVOISSELLE,
PASCALE LOUIS-PLENCE, AND SYLVIE BÉGU
DELIVERY OF SIRNA IN LUNG METASTASIS BY TARGETED HISTIDINYLATED
LIPOPOLYPLEXES.
MARIE-PIERRE GOSSELIN, CRISTINE GONCALVES, SAFIA EZZINE, VIRGINIE MALARD, CHANTAL PICHON, AND
PATRICK MIDOUX
DEVELOPMENT AND EVALUATION OF NEW DRUG DELIVERY SYSTEMS OF NITROSOTHIOLS
FOR ORAL ADMINISTRATION
SHEFAAT ULLAH SHAH, MARIE SOCHA, AND STÉPHANE GIBAUD
A20
SYNTHESE ET PROPRIETES DE NUCLEOSIDES AMPHIPHILES FLUORES
GUILHEM GODEAU, LAURENT LATXAGUE, CHRISTOPHE DI GIORGIO, AND PHILIPPE BARTHÉLÉMY
A21
EFFECT OF INFLAMMATORY CYTOKINES ON TRANS-ENDOTHELIAL PASSAGE OF NONVIRAL GENE DELIVERY VECTORS IN A SKELETAL MUSCLE ENDOTHELIUM MODEL.
JEAN-PIERRE GOMEZ, MATHIEU BERCHEL, PAUL-ALAIN JAFFRES, HERVÉ CHERADAME, PHILIPPE GUEGAN,
MATHIEU MEVEL, BRUNO PITARD, TRISTAN MONTIER, PIERRE LEHN, CHANTAL PICHON, AND PATRICK
MIDOUX
A22
LIPOPHOSPHORAMIDYL-CYCLODEXTRIN NANOPARTICLES AS NOVEL DRUG DELIVERY
SYSTEMS
MATHIEU LÉTÉVÉ, OLIVIER WATTRAINT, VÉRONIQUE BONNET, FLORENCE DJEDAÏNI-PILARD, AND CATHERINE
SARAZIN
A23
POLYPLEX MIXING: EVIDENCE OF PDNA EXCHANGE BETWEEN POLYPLEXES.
CRISTINE GONÇALVES, LUCIE PIGEON, CHANTAL PICHON, AND PATRICK MIDOUX
-41-
-42-
POSTER A1
FUNCTIONALIZING LIPOSOMES WITH ANTI-CD44 APTAMER FOR ACTIVE DRUG DELIVERY
Walhan Alshaer∗1, Hervé Hillaireau1, Said Ismail2, and Elias Fattal1
POSTER A2
BEHAVIOR EVALUATION OF A NANOEMULSION-CONTAINIG PDNA IN SERUM ENVIRONMENT
1
André Silva∗ , Alélia Rosa, Priscila De Paula, Lourena Veríssimo, Lucymara Agnez-Lima , and Eryvaldo Egito†
1
2
UMR CNRS 8612, Institut Galien Paris-Sud, Faculté Pharmacie – Université Paris XI - Paris – France
Molecular Biology Research Laboratory, Faculty of Medicine, University of Jordan – Amman 11942 - Jordan, Jordanie
Aptamers are nucleic acid ligands which are isolated from combinatorial oligonucleotide libraries by an in vitro
selection method called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers
exhibit highly complex and sophisticate molecular recognition properties and are capable of binding tightly and
specifically to targets ranging from small molecules to complex multimeric structures. Moreover, many studies
showed that aptamers exhibit low toxicity and no immunogenicity in vivo. Consequently, aptamers are
considered promising molecules that can be used in many biomedical applications including molecular
sensors, therapeutics, biomarkers discovery, active targeting agents, high throughput screening, and
regulation of gene expression1,2.
In the last decade Cancer Stem Cells (CSCs) have been receiving a lot of interest due to their cancer initiating
and maintaining capabilities making them the key cells within a malignant mass, by making it more aggressive
and more resistant to anticancer drugs. These cells have been linked to many markers in an attempt to better
characterize them. Such markers include CD44, EpCAM and CD133 among others.
Liposome is one of the most common and successful drug delivery system in nanomedicine. Thereby,
conjugation of aptamers to liposomes to form specific active drug delivery vehicles is believed to be a
promising therapeutic system to target CSCs.
In this study, the SELEX technology was used to isolate RNA aptamers using human recombinant full length
CD44 protein and 2’-F-pyrimidine modified RNA library with a complexity of around 1014 different molecules.
Following eleven iterative rounds of SELEX, the selected aptamers were cloned and sequenced where only
three different sequences were identified. The binding affinity (Kd) of Apt1 was ˜81 nM. Now we are performing
experiments to functionalized liposomes with Apt1 to be used as a model for active drug delivery to CD44
expressing cells.
1 Universidade Federal do Rio Grande do Norte (UFRN) – Laboratório de Sistemas Dispersos. Departamento de Farmacia.
R. Gal Cordeiro de Farias, Petrópolis. Natal/Brasil. 59.012-570, Brésil
Gene therapy is a purpose of treatment that employs viral or non-viral vehicles to deliver a genetic material into
the cell. Problems regarding the safety of viral vectors have guided the research of non-viral ones, like cationic
nanoemulsions (CN). This kind of vehicle interacts electrostatically with the negative charged DNA making
possible the transfection process.
The aim of this work was to evaluate the behavior of lipoplexes built from a cationic nanoemulsion and the
plasmid pIRES2-EGFP in serum environment.
Therefore, the nanoemulsion was prepared by the sonication method. Complexation was done by mixing the
pDNA to the CN at 5.8 Nitrogen:Phosphate ratio in ice bath for 120 min. Efficiency of complexation was
assessed by electrophoresis in 0.7% agarosis gel, being the complete formation of lipoplex represented by no
migration pattern into the gel. The droplet size and the polidispersity index (PI) were measured by Dynamic Light
Scattering (DLS), after 300 fold water dilution. Samples were added of fetal bovine serum (FBS) at 2:1
(FSB:Lipoplex) ratio and incubated at 37 o C for different times from 0 to 30 minutes. At the end of each time 0.5
M EDTA was added to the mix in order to inactivate the serum enzymatic action. All the tests were performed
before and after FBS addition.
The results revealed that homogeneous cationic nanoemulsions (PI 0.19) of 224.2 (2.3) nm size and zeta
potential of 51.64 (3.6) mV were produced. The lipoplexes formation did not change the CN droplet size
(p>0.05), but the zeta potential decreased to 13.25 (7.8) mV. At the used N:P ratio, 100% of lipoplex formation
was achieved. When SFB was added, the size of lipoplexes increased 3.3 fold and the zeta potential was
inverted to negative (-21.53 mV). However, the lipoplex migration pattern was not modified and the profile of
migration was maintained. Besides the EDTA used to inactivate the FBS did not influence the size, zeta
potential or migration pattern of the lipoplexes.
It can be concluded that the CN was able to form lipoplexes and those lipoplexes increase their size in the
presence of serum. The serum also inverted the zeta potential of the lipoplexes formulation. However, no signs
of pDNA degradation were observed. Moreover, the formulation was able to protect its cargo from serum
degradation. Further studies should be performed in order to guarantee that the increase in size of the
lipoplexes will not change the endocytosis process.
∗ Intervenant
∗Intervenant
†Auteur correspondant: socratesegito@gmail.com
-43-
-44-
POSTER A3
POSTER A4
RILES, A NOVEL MOLECULAR PROBE TO MONITOR THE KINETIC OF MICRORNA EXPRESSION OF
BREAST CANCER DEVELOPMENT AS WELL AS THE FUNCTIONAL DELIVERY OF RNAI-BASED
DRUGS
IDENTIFICATION OF NEW BIO-EFFECTS OF ULTRASOUND AND MICROBUBBLES-ASSISTED DRUG
DELIVERY
1
Safia Ezzine∗†, Jean Marc Malinge, Patrick Midoux, Chantal Pichon, and Patrick Baril
1
2
2
1
1
Centre de Biophysique Moléculaire – CNRS : UPR4301 – France
2
MicroRNAs are small nucleic acids can regulate vital biological functions such as embryonic development,
metabolism and cell differentiation. Recently we engineered a plasmid based RNAi-Inducible Luciferase
Expression System (RILES) to monitor the activity of the endogenous RNAi machinery. We customized the
Cumate gene-switch inducible system in such way that it is the miRNA of interest that controls the expression of
a transcriptional repressor. When the targeted microRNA is present in cells, it represses the production of the
repressor and switch-ON the production of the luciferase protein. Using this system, we were the first to
demonstrate that the kinetic of miRNA expression can be monitored noninvasively in mice during development
of a physiopathological process. Current experiments aim now at exploiting this novel in-vivo molecular imaging
system to determine the kinetic of endogenous microRNAs expression during the tumor dissemination of the
4T1 breast cancer. We first established a novel 4T1 stable cell line expressing the luciferase (4T1 -Luc) to better
understand the mode of tumor spread of this cancer model. Primary tumors were found highly heterogeneous
characterized by a biphasic phase of tumor development and apparition of spontaneous pulmonary metastasis
at late time point. The expression profile of three oncomiRs (miR-10b, -23a and -200c) was established in the
4T1 cells using RILES as molecular probes. Functional studies demonstrate that RILES constructs are
functional, respond well to the presence of both agonists (miR mimics) and antagonists (antagomiRs). This
indicate that RILES can be used as molecular probe to evaluate the functional delivery of RNAi-based drugs
both in vitro and in vivo. We are currently screening several homemade non-viral delivery systems capable to
deliver the RILES constructs and RNAi-based oligoribonucleotides to the 4T1 primary tumors. Our objective is to
establish from mice to mice, the expression peak of the microRNAs of interests to administrate pharmacological
inhibitors (antagomiRs) at this specific time point by sonoporation (ultrasound assisted- microbubbles delivery)
or by the use of chemical based vectors. Our project is line with the development of novel era of personalized
medicine aiming at delivering therapeutic drugs according to the expression of pattern of targeted genes.
1
Anthony Delalande∗ , Lucie Pigeon , Chloé Leduc , Patrick Tauc , Eric Deprez , Patrick Midoux , and Chantal
†1
Pichon
Centre de biophysique moléculaire (CBM) – CNRS : UPR4301 – Rue Charles Sadron – 45071 Orléans, France
Laboratoire de Biologie et Pharmacologie Appliquée (LBPA) – CNRS : UMR8113, École normale supérieure de Cachan - ENS Cachan –
61, avenue du Président Wilson - 94235 Cachan, France
Upon ultrasound exposure, gas microbubbles can be expanded, moved and even destroyed. These properties
offer the opportunity of site-specific local drug/gene delivery. Activation of microbubbles under specific
ultrasound beams induces a transient cell membrane permeabilization with a process known as sonoporation.
Transient pores formed at the plasma membrane are supposed to be responsible for inward and outward
transports of molecules [1]. A key to success of this technique lies in understanding mechanisms governing
microbubble-cell interactions. Improving our knowledge will allow us to fully exploit this method for therapy.
Here, we investigate how microbubbles and ultrasound behave towards cells upon sonoporation at 1 MHz of
frequency in presence of MicromarkerTM microbubbles. In the context of gene delivery in HeLa cells, optimal
transgene expression was obtained with the following parameters: 150 kPa, 40% duty cycle and 60 sec of
stimulation time. In addition to cellular massage and shear stress, some microbubbles were translated inside
cells under this condition, as shown previously [2]. We assessed which cellular bio-effects are induced by these
physical stresses. Actin stress fibers formation, an increase of intracellular calcium concentration and ROS
production were observed as already reported by others in the context of ultrasound stimulation [3].
Interestingly, we found that the nucleus was also affected. In line with data obtained by Furosawa et al., DNA
damage sensor -H2AX was transiently detected [4]; the level was dependent on ultrasound dose and the cell
type. The novelty of this work was the determination of ultrasound effect on chromatin compaction by using
HeLa cells stably co-expressing histones H2B-GFP and H2B-Cherry [5]. FLIM-FRET experiments allowed us
concluding that sonoporation was able to reverse the chromatin compaction during mitosis or induced by ATP
depletion and 1 mM MgCl2 treatments. This has been validated by an increase of phosphorylated H2B in
treated cells. Since Adenosine Monophosphate-activated Protein Kinase (AMPK), a mechanical and energy
sensor molecule, is acting upstream of pathways involving H2B and H2AX phosphorylation, we evaluated its
activation [6]. Indeed, AMPK phosphorylation was dependent on ultrasound intensity. The downstream effect of
AMPK activation is currently investigated in our laboratory. Knowing that AMPK can be linked to protein
expression, cell survival and cell cycle regulation, a deep knowledge on the signaling pathway(s) involved here
will be of interest for a safe and fine use of sonoporation for drug/gene delivery.
1. Delalande, A., et al., Ultrasound and microbubble-assisted gene delivery: recent advances and ongoing challenges.
TherapeuticDelivery,2012.3(10):p.1199-215.
2. Delalande, A., et al., Sonoporation at a low mechanical index. Bubble science, Engineering and Technology, 2011. 3(1):p.3-11.
3. Juffermans, L.J., et al., Ultrasound and microbubble-induced intra- and intercellular bioeffects in primary endothelial
cells.UltrasoundMedBiol,2009.35(11):p.1917-27.
4. Furusawa, Y., et al., DNA double-strand breaks induced by cavitational mechanical effects of ultrasound in cancer cell lines.PloSone,
2012.7(1):p.e29012.
5. Lleres, D., et al., Quantitative analysis of chromatin compaction in living cells using FLIM-FRET. J Cell Biol, 2009.187(4):p.48196.
6. Bungard, D., et al., Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation. Science,
2010. 329(5996):p.1201-5.
∗ Intervenant
∗Intervenant
†Auteur correspondant: chantal.pichon@cnrs-orleans.fr
†Auteur correspondant: safia.ezzine@cnrs- orleans.fr
-45-
-46-
POSTER A5
POSTER A6
DNA MINICIRCLE AS POTENTIAL NEW DECOY ODN FOR TRANSCRIPTION FACTORS TARGETING
1
1,2
1
1
Thomas Thibault , Stéphane Bourg , Chantal Pichon , Patrick Midoux , and Jean-Marc
2
Malinge∗†1
1
Centre de biophysique moléculaire (CBM) – CNRS : UPR4301 – Rue Charles Sadron 45071 Orléans, France
Institut de Chimie Organique et Analytique (ICOA) – CNRS : UMR7311, Université d’Orléans – UFR Sciences Rue de Chartres - 45067 Orléans, France
A decoy oligodeoxynucleotide (ODN) serves as a decoy sequence for usually one target transcription factor,
then inhibiting its binding to consensus sequence at the promoter of genes, and in turn hinders the expression of
critical genes implicated in several diseases (cancer, cardiovascular diseases...). In order to increase the
efficiency of such strategy, we thought to design new ODN with the capability of trapping several different
transcription factors by a single decoy molecule and of exhibiting high stability against nucleases. Short doublestranded DNA minicircle could fulfill such requirements, i. e. proteins multitargeting and stability. However, a
production methodology yielding quantitative amounts of short DNA minicircles is lacking.
We report a plasmid free method for production of double-stranded DNA minicircles of less than 250 base pairs
in length from synthetic DNA ODN in a one pot reaction (*). We engineered an enzymatic reaction using
synthetic ODNs which is characterized by : i) the design of blunt-ended linear nicked double-stranded ODN
substrates comprising 5’ phosphorylated overlapping ODNs with the respective corresponding phosphorylated
complementary ODN strands ii) the use of the designed nicked linear DNA substrate in a one pot ligasemediated circularization reaction in the presence of a bending protein followed by step by step addition of
appropriate enzymes for elimination of reaction contaminants iii) recovery of closed DNA minicircles.
Minicircles of any base composition with the freedom to choose the position and nature of sequences of interest
within DNA minicircle are produced. Notably, minicircles can contain transcription factor binding sequences as
exemplified with one to six binding sites for NF-kB or a combination of binding sites for three different
transcription factors (NF-kB, ETS1, STAT3). In addition, the possibility to produce nicked minicircle gives the
opportunity to control the topological constraint to yield negatively supercoiled minicircles. We showed that
minicircle of 95 base pairs containing two binding sequence for NF-kB binds very efficiently two NF-kB (p50)2
dimers and that when two Ets1 binding sites are present together with a NF-kB binding site, these transcription
factors bind efficiently and simultaneously. Another attractive possibility relates to minicircle incorporation of a
variety of base modifications and chemical functionalization (fluorophores, biotin, amino-modifier dT) which is of
interest for application in the field of gene therapy. Work is in progress to make the proof of concept of minicircle
as active nucleic acid in the decoy strategy.
CONCEPTION OF CATIONIC MICROBUBBLES FOR DNA DELIVERY IN VIVO UNDER SONOPORATION
1
2
3
1
Unité de pharmacologie chimique et génétique et d’imagerie – Inserm, CNRS : UMR8151, Université Paris V - Paris Descartes,
Ecole Nationale Supérieure de Chimie de Paris – Paris, France
2
Centre de biophysique moléculaire (CBM) – CNRS : UPR4301 – Rue Charles Sadron - 45071 Orléans, France
3
Transderma – Transderma – France
4
Institut Cochin – CNRS : UMR8104, Inserm : U1016, Université Paris V - Paris Descartes – 22 rue Méchain - 75014 Paris, France
5
Institut Langevin ”ondes et images” – Université Pierre et Marie Curie (UPMC) - Paris VI, ESPCI ParisTech, CNRS : UMR7587,
Université Paris VII - Paris Diderot – 10 Rue Vauquelin - 75231 Paris, France
DNA delivery in vivo via physical means or non-viral vectors still needs improvements in particular to reach deep
tissues. Sonoporation is highly advantageous for this purpose as ultrasounds can be focalized to a particular
tissue leading to targeted gene delivery, without alteration of the environing tissues. Associated with this
technique, protein expression has already been obtained by other groups using a simple co-injection of DNA
and microbubbles.
We wondered if additional interactions of microbubbles with DNA could not provide a higher transfection
efficacy.
Different methods of microbubbles obtention have been investigated: simple mixing, ultrasounds, lyophilisation
and the vialmix system. Association of DNA to these microbubbles has been evaluated by flow cytometry and
optical detection of labeled DNA. Stability was addressed by following the evolution of the microbubbles under
the microscope, taking 5 pictures per condition and using Image J for counting. Three pegylation methods of the
microbubbles were investigated. Addition of pegylated lipid, pegylation of bubbles prior or post DNA interaction
were performed.
We found that the vialmix agitation system led to the less polydisperse microbubbles with a size being in the 1-2
m range and 98% of the microbubbles are under 10m. The cationic microbubbles are stable in vial up to 5h and
1h diluted in water. Labeled DNA was observed at the surface of the microbubbles. Pegylation post
microbubbles formation was found to be the optimal condition to maintain microbubbles integrity and stability.
We addressed here the first steps of microbubble obtention: conception, preparation and characterization of
pegylated cationic microbubbles, which will be dedicated to in vivo gene delivery. We could form a
monodisperse population of microbubbles, able to interact with DNA, while being pegylated.
DNA minicircles comprising less than 250 base pairs.
∗ Intervenant
-47-
5
1
(*) Malinge, J.-M., Thibault, T., Midoux, P and Pichon, C., (2012) Brevet européen CNRS dépot n 12 306 454.5. In vitro production of
∗Intervenant
†Auteur correspondant: malinge@cnrs-orleans.fr
4
Simona Manta∗ , Anthony Delalande , Mathias Lebertre , Gilles Renault , Olivier Couture , Daniel Scherman ,
Michel Francis Bureau1, Patrick Midoux2, Chantal Pichon2, Michel Bessodes1,
1
and Nathalie Mignet
-48-
POSTER A7
ENHANCED SENSITIVITY OF U87MG GLIOBLASTOMA CELLS TO TEMOZOLOMIDE AFTER
TREATMENT BY SIRNA CARRIED BY LIPID NANOCAPSULES
POSTER A8
PTG1: NEW ROBUST TRANSFECTION REAGENTS FOR THE PRODUCTION OF RECOMBINANT
PROTEINS AND VIRAL PARTICLES
†
†1
Khaled Messaoudi∗ , Anne Clavreul, Patrick Saulnier, Jean-Pierre Benoit, and Frederic Lagarce
2
3
4
Hervé Cheradame∗ , Philippe Guégan , Fabian Gross , and Patrick Midoux
Inserm U1066 MINT – Université d’Angers – France
1
POLYTHERAGENE, LAMBE, CNRS UMR8587 Université d’Evry Val d’Essonne, Evry, 94, France
Laboratoire de Chimie des Polymères, CNRS UMR 7610 Université Pierre et Marie Curie, Paris, France
Plateforme de vectorologie, laboratoire de biothérapie UMR 1037/CRCT, CHU Rangueil - 31059 Toulouse, France
4
Centre de Biophysique Moléculaire, CNRS UPR4301, 45071 Orléans, France
2
3
Glioblastoma multiforme (GBM) is the most aggressive brain tumors due to the resistance of tumor cells to
current treatments combining surgery, radiation, and chemotherapy by oral Temozolomide (TMZ). The efficacy
of TMZ for treating GBM is very limited; this is partly due to the high levels of enzyme activity of DNA repair O6methylguanine DNA methyltransferase (MGMT) in tumor cells. On the other hand, overexpression of EGFR and
galectin-1contribute significantly to TMZ resistance. It was shown that reducing the production of these proteins
by small interfering RNA (siRNA) increases tumor sensitivity to TMZ.
Lipid nanocapsules (LNCs) transacylated with chitosan were developed to bind siRNA. The resulted capsules
were characterized (size, zeta potential, quantity of bound siRNA, cell toxicity). Then, an evaluation of EGFR
and Galectin-1 knockdown in U87MG/MGMT- glioma cells and MGMT Knockdown in U87MG/MGMT+ cells
after siRNA treatment were performed by FACS. Finally (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) test was used to determine if the sensitivity of tumor cells to TMZ was improved after siRNA
treatment.
The resulting nanocapsules have a size of 71.60 1.15 nm and zeta potential of 24.70 2.10 mV. The capacity of
LNCs to graft siRNA was evaluated by fluorescence quenching and showed a fixation of 93.1 0.2 % of
suspension of 2.4 M of siRNA with 6 L of LNCs. Cytotoxicity test of the nanocapsules on U87MG/MGMT- and
U87MG/MGMT+ cells showed cell death at concentrations greater than 100 g/mL. Treatment of U87MG/MGMTcells with anti-EGFR and anti-Galectin-1 siRNAs at the concentration of 40 nM resulted in a reduction of EGFR
and Galectin-1 expression by (51.95 6.03) % and (38.48 2.17) % respectively after 4 days of incubation.
Processing U87MG/MGMT+ by anti-MGMT siRNA at the concentration of 5 nM for 4 days reduced the rate of
MGMT by 31.68 2.83 %. The sensitivity of U87MG/MGMT- cells to TMZ was improved by 58.87 1.23 % with
anti-EGFR siRNA and by 56.84 0.41 % with anti-Galectin-1 siRNA. The association of both siRNA resulted in an
increase of 80.61 1.65 % of the sensibility to TMZ (P<0.05). Regarding the U87MG/MGMT+ cells, their
sensitivity to TMZ was increased by 80.77 3.03 % after treatment with anti-MGMT siRNA compared to cells not
treated with siRNA.
This study showed that the reduction in cellular levels of EGFR and galectin-1 and MGMT resulted in an
increase in tumor sensibility to TMZ. The combination of anti-EGFR siRNA and anti-Galectin-1 has shown a
synergistic effect on tumor sensitivity to TMZ. This method could be implemented in future clinical models for
experimental cancer treatment of GBM.
∗Intervenant
†Auteur correspondant: khaled.messaoudi2111@gmail.com
The bioproduction becomes increasingly important in the fields of pharmacy, but also in cosmetics or foods.
Recombinant proteins (r-proteins) such as therapeutic monoclonal antibodies are very promising
biopharmaceutics for cancer treatments and vaccines. Recombinant viruses including AAV and recombinant
lentiviruses (r-lentivirus) are promising vectors for human gene therapy applications. Bioproduction requires
robust transfection, an efficient protocol easy to implement, with good reproducibility for a maximum production
of r-proteins or r-viral particles in a short time with low cytotoxicity. R-proteins are commonly produced after
transfection of mammalians cells (CHO or HEK293-S cells) with a plasmid DNA encoding protein ensuring right
glycosylation. When possible, it can be produced from CHO cells stably expressing the transgene, but such
clones are difficult to establish and their stability varies. R-viral particles are commonly produced upon
transfection of the human HEK293T packaging cells with at least three plasmids DNA. One encodes the gene of
interest, one encodes gag, pol and rev genes and another one encodes the VSV-G envelope gene. After an
incubation period, the virus containing supernatant is collected, concentrated, the virus titre is determined and
the particles are then used for transfections. Bioproduction uses cells which are adapted to grow in suspension.
Polytheragene has developed the PTG1 family which are very powerful transfection reagents. Here we report
production of r-proteins and r-lentiviral vectors by using PTG1 reagents. PTG1.P mixed with plasmid DNA, does
not affect significantly HEK293 cell viability. The transfection efficiency at 2x106 cell density per ml is found to
be globally increased in presence of [PTG1.P + 0.75g/ml DNA] mix and in particular 120 hours post-transfection,
compared to standard assay based on [jet- PEI + 1 g/ml plasmid DNA] mix. Production of r-lentiviral vectors was
carried out upon Tri-transfection with pTRIP-GFP, 8.91 and VSV-G plasmids of HEK293 packaging cells. The
PTG1.L method (adapted to lentivirus production) was compared to either the calcium phosphate or
polyehtyleneimine (PEI25k) methods. The results indicate that the production of r-viral particles is several folds
higher with PTG1.L than with bPEI. Although PTG1.L allowed the production of r-lentiviral vectors with the viral
titre comparable to that obtained by the conventional calcium phosphate method, the great advantage of the
PTG1.L reagent is that due to its low cytotoxicity, two harvests of virus particles are collected within three days.
PTG1 transfection reagents could become the best partners for the production of r-proteins as well as r-viral
particles for research academic or corporate laboratories.
∗Intervenant
†Auteur correspondant: hcheradame@free.fr
-49-
-50-
POSTER A9
POSTER A10
CHARACTERIZATION AND STRUCTURE OF SIRNA LIPID NANOCAPSULES FOR GENE THERAPY
1
Resnier∗
1
1
ADVANTAGES OF USING THE LIPOTHIOUREA DDSTU AS A COLIPID FOR DNA OR SIRNA DELIVERY
1
Pauline
, Pierre Lequinio , Nolwenn Lautram , Emilie André , Cédric
Gaillard2 , Guillaume Bastiat1, Jean-Pierre Benoit1, and Catherine Passirani†1
Johanne Seguin∗, Michel Bessodes, Jean Herscovici, Karim Fekir, Alice Weyland, Daniel Scherman,
Andrew Miller, and Nathalie Mignet†
Unité de pharmacologie chimique et génétique et d’imagerie – Inserm, CNRS : UMR8151, Université Paris V - Paris Descartes,
Ecole Nationale Supérieure de Chimie de Paris – Paris, France
1
2
Micro et nanomédecines biomimétiques (MINT) – Inserm : U1066, Université d’Angers, 10 rue André Boquel - 49100 Angers, France
Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA) – Institut national de la recherche agronomique (INRA) : UR1268 – France
Gene therapy with small interference RNA (siRNA) represents a promising strategy to temporarily inhibit the
expression of proteins implicated incancerogenesisor chemotherapy resistance (1). Even though intra-tumoral
administration could be envisaged, the current studies are performed with the goal of formulating
nanomedicines to be injected intravenously topossivelytarget thetumorsite as well as metastases (2).Synthetic
nanoparticles and liposomes have been widely developed during this last decade. In this work, polymers and
lipids were associated to formulate lipid nanocapsules (LNCs) for encapsulation and systemic delivery of siRNA
(3-4).siRNALNCs were prepared with DOTAP/DOPE lipoplexes, and assays on siRNA dose and lipid/siRNA
charge ratios were performed to improve the stability and encapsulation yield. The observation of siRNA
lipoplexes and LNC morphology were performed bycryo-TEM showing a specific molecular organization of
siRNA DOTAP/DOPE lipoplexes and evidenced specific lipid microstructures. Finally, complement activation
proved the feasibility of apegylationprocess onsiRNALNCs carried out in a passive targeting strategy for futurein
vivoexperiments. No cytotoxicity on B16F10 and SK-Mel28 melanoma cell lines was evidenced up to 100 g/mL
and 500 g/mL, respectively. This work described an adapted siRNA nanocarrier for intravenous administration,
encouraging the pursuit of studies onin vitroandin vivomelanoma models.
DDSTU is a lipothiourea derivative developped for systemic delivery of non-viral gene therapy [1-5].
Formulations based on lipothiourea are weakly charged but still able to interact with DNA, efficiently transfect
cells in vitro and post local delivery in vivo. Recent insertion of thiourea functions in positively charged
cyclodextrin amphiphiles revealed that the assocation of both hydrogen and ionic bonds increased the level of
model protein expression [6].
We wondered if cooperative effects between hydrogen and ionic bond interaction with DNA phosphates could
also be obtained with lipidic auto-assemblies and could incease DNA and siRNA retention in lipoplexes.
To answer this question, we formulated two different cationic lipids DMAPAP and DODAG with DDSTU, used as
a colipid, characterize these formulations by DLS and gel electrophoresis. Finally, the ratio lipid/DNA or required
to achieve transfection in vitro B16 melanoma and CT26 colon carcinoma cell line was determined. The ratio of
lipid/siRNA which provided a best inhibition of luciferase stably transfected B16 and CT26 cell was also
determined.
We found that the more stable formulations in media with serum were obtained for DMAPAP/DDSTU. With this
formulation an enhancement of transfection was observed for the both cell line investigated and better result
were obtained for a charge ratio of 6. Moreover, lipolexes of siRNA showed a higher luciferase inhibition.
1.
2.
3.
4.
Resnier P, et al., 2013. Biomaterials
Morille M, et al., 2009. Biomaterials
David et al., 2012. Eur J Pharm Biopharm
Resnier et al., 2013. Submit to Nanotechnology
Addition of the lipothiourea DDSTU in cationic lipid formulations increases cell transfection while reducing the
amount of cationic charges needed and thus limiting the cytotoxicity. We currently evaluated the efficacy of
these lipoplexes after intratumoral injection in murine models.
1.
2.
3.
4.
5.
6.
Leblond J, Mignet N, Largeau C, Spanedda MV, Seguin J, Scherman D, Herscovici J. Lipopolythioureas: a new non-cationic system for gene
transfer. Bioconjug Chem. 2007, Mar-Apr;18(2):484-93
Kral T, Leblond J, Hof M, Scherman D, Herscovici J, Mignet N. Lipopolythiourea/DNA interaction: a biophysical study. Biophys Chem.
2010;148(1-3):68-73.
Breton M, Leblond J, Seguin J, Midoux P, Scherman D, Herscovici J, Pichon C, Mignet N. Comparative gene transfer between cationic and
thiourea lipoplexes. J Gene Med. 201, Jan;12(1):45-54.
Leblond J, Mignet N, Largeau C, Seguin J, Scherman D, Herscovici J. Lipopolythiourea transfecting agents: lysine thiourea derivatives.
Bioconjug Chem. 2008 Jan;19(1):306-14.
Leblond J, Mignet N, Leseurre L, Largeau C, Bessodes M, Scherman D, Herscovici J.Design, synthesis, and evaluation of enhanced DNA
binding new lipopolythioureas. Bioconjug Chem., 2006 Sep-Oct;17(5):1200-8.
Méndez -Ardoy A, Guilloteau N, Di Giorgio C, Vierling P, Santoyo-Gonz´alez F, Ortiz Mellet C, Garc´ıa Fernandez JM. -Cyclodextrin-based
polycationic amphiphilic ”click” clusters: effect of structural modifications in their DNA complexing and delivery properties. J Org Chem.
2011;76(15):5882-94.
∗ Intervenant
∗Intervenant
†Auteur correspondant: catherine.passirani@univ- angers.fr
†Auteur correspondant: nathalie.mignet@parisdescartes.fr
-51-
-52-
POSTER A11
POSTER A12
INFLUENCE OF NON-IONIC SURFACTANTS ON THE FORMATION OF MIXED NANO-OB JECTS
WITH AMPHIPHILIC CYCLODEXTRINS
STXB TECHNOLOGY FOR TUMOR THERANOSTIC AND IMMUNOTHERAPY
1
Leila
1
Zerkoune∗ ,
1
1
1
2
Angelina Angelova , Luc Choisnard , Annabelle Geze , Denis Wouessidjewe , and
1
Sylviane Lesieur
1
1
2
1
3
Estelle Dransart∗ , Kamélia Kara Ali , Laura Brullé , Thi Tran , Birgit Oberleitner , Olivier Couture , Vincent
Semetey1, Eric Tartour2, and Ludger Johannes4
1
Institut Curie – 26 rue d’Ulm - 75248 Paris, France
Hopital européen Georges Pompidou – Assistance publique - Hôpitaux de Paris (AP-HP), Höpital européen Georges Pompidou,
Université Paris V - Paris Descartes – France
3
Institut Langevin ”ondes et images” – Université Pierre et Marie Curie (UPMC) - Paris VI, ESPCI ParisTech, CNRS : UMR7587,
Université Paris VII - Paris Diderot – 10 Rue Vauquelin - 75231 Paris, France
4
Compartimentation et dynamique cellulaires (UMR144) – CNRS : UMR144,
Université Pierre et Marie Curie (UPMC) - Paris VI – 26 Rue d’Ulm - 75248 Paris, France
2
1 UMR CNRS 8612, Institut Galien Paris-Sud 11 – CNRS : UMR8612 – France
2UMR CNRS 5063 ICMG FR 2607 Université de Grenoble – CNRS : UMR5063 – France
A recent methodology for grafting of aliphatic chains on the secondary face of native cyclodextrins (CD) by
enzymatic transesterification, using thermolysin as a biocatalyzer, has opened new possibilities for the design
of drug nanocarriers with colloidal behaviour [1-3]. Amphiphilic CD derivatives with decanoyl alkyl chains (CDC10) may self-assemble into nanospheres with multilamellar internal organization or in reservoir-type particles,
which can embed lipophilic drugs.
However, the physical stability of these carriers requires the addition of surfactants in the formulation. The
purpose of this work is to study the interaction between -CD-C10 nanospheres and non-ionic surfactants, such
as n-dodecyl -D maltoside (DDM) and polysorbate 80 (P80), and to determinate the CD and surfactant
quantities enabling the formation of mixed nanoobjects. The non-ionic surfactants were chosen because they
show a number of advantages in terms of nanoparticles stability, biocompatibility, and lower toxicity as
compared to ionic surfactants [4].
Several physico-chemical methods were employed. Solubilization experiments were performed by optical
density measurements (UV-Visible spectrophotometer) upon continuous addition of surfactants to CD
nanosphere suspensions. Quasi-elastic light scattering (QELS) was employed for determination of the
particles sizes and optical microscopy for establishing the colloidal behavior of the systems.
Shiga toxin is produced by E. coli strains that are responsible for dysentery and hemolitic- uremic syndrome. It
is composed of an A-subunit that mediates the toxic activity by inhibiting protein biosynthesis, and a B-subunit
(STxB) that drives in cellular entry of the toxin through binding to the glycosphingolipid globotriaosyl ceramide
(Gb3). Shiga toxin has evolved with its host to pass critical physiological barriers in order to reach its molecular
target, ribosomal RNA. STxB has thus naturally developed properties required for a therapeutic delivery tool:
high stability and resistance to extreme pH, low immunogenicity, and intracellular retrograde transport
preventing its degradation in lysosomes. Gb3 expression is found on two therapeutically relevant cell types:
cancer cells, and antigen presenting cells (dendritic cells). We thus took advantage of these characteristics to
develop a technology based on the conjugation of a genetically modified STxB, bearing an additional cysteine
residue at its C-terminus, to contrast agents as well as nucleic acids for anti-tumor therapy and tumor imaging.
STxB-functionalized ecogenic microbubbles and nanorods are developed in the field of tumor theranostics to
combine detection/localization and treatment of primary tumors and/or metastatic disease, based respectively
on sonoporation or thermal properties of these particles.
The delivery of nucleic acids by STxB is based on the use of STxB-polyethymenimine conjugates; two main
applications are developed. On the one hand, STxB is used to target mRNA encoding antigenic proteins to
dendritic cells in order to promote cytotoxic T cell response against tumors. On the other hand, cDNA encoding
pro-apoptotic proteins are targeted to tumor epithelial cells.
The present poster illustrates proofs of concept for each of these STxB-based biomedical applications.
∗Intervenant
∗Intervenant
-53-
-54-
POSTER A13
POSTER A14
ANTI-PPRV THERAPY BASED ON IN VIVO EFFIENCY OF SIRNA: PROOF OF CONCEPT USING A
SMALL ANIMAL MODEL
1
1
1
1
Cécile Minet∗ , Renata Servan De Almeida , Carine Holz , Renaud Lancelot , Geneviève
1
2
2
3
Libeau , Mattias Hallbrink , Ülo Langel , and Emmanuel Albina
TRIPPARTITE PH-SENSITIVE MICELLES OF POLYETHYLENIMINE AND DOUBLE
HYDROPHILIC BLOCK COPOLYMERS AS GENE DELIVERY SYSTEMS
1
†1
1
1
Naila Mebarek , Anne Aubert , Corine Gerardin , Jean-Marie Devoisselle , and Sylvie Bégu
1
Institut Charles Gerhardt Montpellier, UMR 5253 CNRS-ENSCM-UM2-U – CNRS: UMR5253 – 8 rue de l‘Ecole Normale - 34296 Montpellier, France
1CIRAD UMR CMAEE (CMAEE : contrôle des maladies animales exotiques et émergentes)) – CIRAD, Institut national de la recherche agronomique (INRA) :
UMR1309 – F-34398 Montpellier, France
2Dpt. Neurochemistry, Stockholm University – Dpt. Neurochemistry, Stockholm
UniversityS.Arrheniusv. 21A, SE-106 91 Stockholm, Suède
3CIRAD UMR CMAEE (CMAEE : controle des maladies animales exotiques et émergentes)) – CIRAD, Institut national de la recherche agronomique (INRA) :
UMR1309 – F-97170 Petit-Bourg, Guadeloupe, France
Morbilliviruses are responsible of important diseases in human beings and animals with economical impact in
affected countries. Efficient vaccines exist but they are often used in emergency situation in animals and ten
days are necessary to induce a sufficient immune protection. Co-administration of an antiviral treatment with the
vaccine could limit the disease impact while conferring a long-lasting protection. CIRAD has explored a
biological antiviral therapy based on RNA interference. The discovery of siRNA against morbilliviruses was
patented. The objective of this work was to validate the in vivo efficiency of these siRNAs. To this end, one of
our siRNAs anti-PPRV was assessed in mice, using a double-reporter plasmid expressing two luciferases. The
plasmid is made of a firefly gene placed downstream of our morbillivirus siRNA target sequence and a renilla
gene used as a housekeeping gene. Mice received a co-injection (double reporter plasmid + relevant or
irrelevant si-RNA-PPRV) in the tibialis muscle, followed by an electroporation to promote cellular uptake of DNA.
The firefly and renilla signals were measured daily using a bio-imager. The firefly expression was normalized
using renilla signal. A good correlation in mice injected with the irrelevant siRNA was observed between firefly
and renilla signals. In contrast, mice treated with siRNA-PPRV in one leg showed a strong inhibition of about
99% of the firefly signal. The specificity of siRNA-PPRV activity was demonstrated in the same mice since the
irrelevant siRNA administered in the second leg did not inhibit the firefly signal. This mouse model system is a
proof of concept of in vivo siRNA efficiency and a very useful tool to assess in vivo siRNA delivery systems.
Several candidates for in vivo delivery systems are currently investigated in our laboratory, including
recombinant baculovirus, adenovirus and non viral vehicles. Initial results showed that a cell membrane
penetrating peptide (PF6) could efficiently deliver a siRNA and inhibit the expression of Firefly in tibialis muscle.
The delivery of nucleic acids has great potential for advanced biomedical and therapeutic research. In RNA
interference strategy with small interfering RNAs (siRNAs) and microR- NAs (miRNA) to silence homologous
genes or in vaccination strategy with plasmid DNA (pDNA) to mediate specific expression, nucleic acids are
important tools. However, their use for therapeutic applications is hindered by limited efficacy and delivery
capacity, and safety problems. In order to express their best efficiency, nucleic acids need to reach their
subcellular targets: the nucleus for pDNA and the RISC complex in the cytoplasm for siRNAs. It requires the
crossing of multiple cellular barriers in particular cytoplasmic membrane and endosomal membrane while
resisting to nuclease degradation.
Among the numerous types of vectors, block copolymer micelles have shown encouraging results for siRNA
and pDNA delivery. They possess a core-shell structure, and a narrow size distribution centered on a few tens
of nanometers allowing a stealth facing to the reticulo-endothelial system or a possible accumulation into
inflammatory or cancers tissues. Some polymers were selected for their endosomal escape capacity which was
induced by various mechanisms such as proton sponge effect in the case of the polyethylene imine (PEI), or
membrane interaction in the case of poly(acrylic acid) block copolymers.
Polyion complex micelles were developed with poly(methacrylic acid)-b-poly(ethylene oxide) block copolymers
(PMAA-b-PEO) and polyethyleneimine in order to deliver nucleic acids into cells. The aim of the study was to
prepare a vector able to efficiently encapsulate nucleic acids, and carry them into the cytoplasm; this required
an appropriate cellular uptake, and an efficient endosomal escape. Two different PEI (branched-modified PEI Y
and a linear PEI) were tested with PMAA-b-POE copolymers to assess their capacity to form micelles, with
nanometric size, efficient siRNA and pDNA loading and a high cytotolerance. The PEI Y based micelles showed
better characteristics than the linear PEI based micelles with homogeneous sizes varying from 45 to 200 nm
according to the molecular weight of the used PMAA-b-PEO, and a loading efficiency of 4 nmol/mL for siRNA
and 10 g/mL for pDNA. Finally, PMAA-b-PEO / PEI Y micelles were stable in physiological medium and
presented a pH-sensitivity permitting the delivery of nucleic acids in endosomes and a satisfying cytotolerance
making possible cellular in vitro or in vivo applications.
N. Mebarek, A. Aubert-Pouessel, C. Gerardin, R. Vicente, J.M. Devoisselle, S. Begu, Polymeric micelles based on poly(methacrylic acid)
block-containing copolymers with different membrane destabilizing properties for cellular drug delivery. Int J Pharm (2013).
∗Intervenant
†Auteur correspondant: aaubert@univ-montp1.fr
∗Intervenant
-55-
-56-
POSTER A15
POSTER A16
PH-SENSITIVE POLYION COMPLEX MICELLES AS SIRNA VECTORS FOR DC ENGINNEERING TO
INDUCE TOLERANCE
1
2
†
BIODISTRIBUTION IN VIVO DE FORMULATIONS LIPIDIQUES FLUORESCENTES APRÈS INJECTION
SYSTÉMIQUE
1,2
3
Nawal Belmadi∗
Naila Mebarek , Rita Vicente , Anne Aubert∗ , Christian Jorgensen , Jean-Marie Devoisselle,
3
‡1
Pascale Louis-Plence , and Sylvie Bégu∗
1Institut Charles Gerhardt Montpellier, UMR 5253 CNRS-ENSCM-UM2-U – CNRS: UMR5253 – 8 rue de l‘Ecole Normale - 34296 Montpellier, France
2INSERM, U844, Université Montpellier 1, Hôpital St Eloi – Inserm : U844 – 80 Avenue Augustin Fliche - 34296 Montpellier - France
3INSERM U844 – Inserm : U844 – Hôpital St Eloi, 80 Avenue Augustin Fliche - 34296 Montpellier, France
Dendritic cells (DCs) are professional antigen-presenting cells of the immune system, able to stimulate or to
inhibit immune response according to their maturation state. Due to this contrasting properties DCs have a
dual role in autoimmune diseases (AID). Indeed, mature DCs can induce T cell activation, participating in the
deleterious role of these cells in AID but at immature state can also induce tolerance by inducing regulatory T
cells (Treg). Our goal is to genetically engineer DCs with small interfering RNA (siRNA), targeting
immunogenic molecules to enhance DC tolerogenic properties. In order to do so, we developed pH-sensitive
tripartite polymeric micelles composed of a double-hydrophilic block copolymer (poly methacrylic acid -b poly
ethylene oxide) (PMAA-b-POE) and a poly-counter ion poly-L-lysine (PLL) to be used as a siRNA vector. Four
micelles were developed with different molecular weights copolymers. These micelles present a well-adapted
stability in physiological conditions [1] and are biocompatibility with DCs [2,3]. In order to validate the micelles
potential as siRNA vectors the capacity of endossomal destabilization was studied as well as their capacity to
be endocytosed by DCs and to encapsulate siRNA. Interestingly, even if the four micelles were endocytosed
by DCs, only the low molecular weight micelles presented the ability to destabilize endosomes and to
encapsulate siRNA. siRNA targeting CD86, a DCs co-stimulatory molecule important for T cell activation, was
used as model of targeting gene. A specific silencing of CD86 molecules was observed with both low
molecular weight micelles by Flow Cytometry analysis (FACS).
Our results underscore the potential of low molecular weight pH-sensitive polyion complex micelles to
formulate therapeutic siRNA for DC engineering in AID therapy.
1.
2.
3.
Boudier,A. et al.(2009).”Tripartite siRNA micelles as controlled delivery systems for primary dendritic cells”. Drug Dev Ind
Pharm 35(8): 950-8.
Boudier,A. et al.(2009).”The control of dendritic cell maturation by pH-sensitive polyion complex micelles”. Biomaterials
30(2): 233-41.
Boudier,A. et al.(2011).”Development of tripartite polyion micelles for efficient peptide delivery into dendritic cells without
altering their plasticity”. J Control Release 154(2): 156-63.
∗Intervenant
†Auteur correspondant: aaubert@univ-montp1.fr
‡Auteur correspondant: sylvie.begu@enscm.fr
1 Génétique, Génomique Fonctionnelle et Biotechnologies – Inserm – INSERM U1078 ” Génétique, Génomique Fonctionnelle et Biotechnologies
22 Rue Camille Desmoulins – CS 93837 - 29238 BREST Cedex 3 - FRANCE, France
2Université de Bretagne Occidentale (UBO) – Université de Bretagne Occidentale (UBO), Université de Bretagne Occidentale [UBO] – 3 rue des Archives CS 93837 - F29238 Brest cedex 3, France
Afin de poursuivre le développement et l’optimisation de formulations pour le transfert de gènes dans le cadre
de thérapie génique de la mucoviscidose, des sondes fluorescentes lipidiques porteuses d’une cyanine (Exc :
645nm, Em : 665nm) ont été synthétisées et formulées en présence de lipides cationiques de type
lipophosphoramidate. L’objectif est d’affiner nos connaissances sur le devenir de ces formulations in situ en
fonction de la voie d’administration tout en évaluant leur efficacité et leur tolérance.
Dans le cadre de ce travail, nous avons tout d’abord évalué plusieurs formulations, en présence ou en
l’absence de sonde fluorescente, à la fois sur le plan d’une éventuelle toxicité ainsi que de leur efficacité de
transfection sur différentes lignées cellulaires. Dans un second temps, des études de biodistribution avec l’une
des formulations parmi les plus efficaces, avec ou sans DSPE-PEG2000, ont été menées après injection
systémique sur souris Nude NMRI. Parallèlement à la mesure de l’expression du gène rapporteur par
bioluminescence, les complexes ont été suivis et quantifiés par fluorescence in situ, puis sur organes isolés.
Sur lignées cellulaires, nous avons ainsi montré que l’addition d’une sonde fluorescente (5 ou 10%; M/M) avec
un vecteur de type lipide cationique n’interférait ni sur la capacité de complexation, ni sur l’efficacité du
lipoplexe et n’induisait pas de toxicité additionnelle. La pharmacocinétique des liposomes et des lipoplexes
formulés en présence de DSPE-PEG2000 indiquent également une distribution supérieure de 80% environ
par rapport aux formulations sans PEG. Par ailleurs, les liposomes ont une demi-vie supérieure de 40% aux
lipoplexes dans la circulation systémique. 24h après l’injection, les principaux organes fluorescents sont tout
d’abord les poumons, le foie, la rate, les reins et la vessie, les intestins et le cœur. Aucune toxicité hépatique
in vivo n’est mesurée avec les liposomes alors qu’il existe une cytolyse de 2N, réversible sous 48h à 72h avec
les lipoplexes. Dans tous les cas, l’inflammation est mineure et complètement réversible en quelques heures.
Les perspectives consistent maintenant à évaluer le comportement de ces lipoplexes après administration par
aérosolisation afin d’optimiser au mieux les formulations chimiques et en accroitre l’efficacité. Nous
réaliserons à la suite des études de réadministration de ces molécules par les voies aériennes hautes.
D’autres concentrations de lipides fluorescents seront également étudiées.
∗Intervenant
-57-
-58-
POSTER A17
FORMULATION ET BIODISTRIBUTION IN VIVO DE VECTEURS SYNTHETIQUES POUR LE TRANSFERT
DE GENES DANS LE CADRE DE LA THERAPIE GENIQUE DE LA MUCOVISCIDOSE
POSTER A18
DELIVERY OF SIRNA IN LUNG METASTASIS BY TARGETED HISTIDINYLATED LIPOPOLYPLEXES
1
1
Nawal Belmadi∗
1
Génétique, génomique fonctionnelle et biotechnologies (UMR1078) – Inserm : U1078, Université de Bretagne Occidentale [UBO], EFS –
EFS Bretagne, 46 rue Félix le Dantec - 29218 Brest, France
Depuis des années, l’équipe “Transfert de gènes” de l’UMR1078 INSERM, en collaboration avec l’UMR6562
CNRS, travaille sur le développement et l’optimisation de vecteurs synthétiques pour le transfert de gènes et
notamment dans le cadre d’une thérapie génique de la mucoviscidose.
Dans le cadre de ce projet de thérapie génique de la mucoviscidose, l’objectif est d’atteindre les cellules
épithéliales pulmonaires afin d’y apporter le gène correcteur de la déficience génétique. Or, il a été démontré
que les lipoplexes développés à Brest étaient en mesure de conduire à une expression du gène rapporteur
dans les pneumocytes de type II aussi bien apr`ès injection systémique qu’après administration intratrachéale.
Dans le cadre de ce travail, nous souhaitons réaliser des études de biodistribution et d’élimination de nos
molécules après administration selon une voie générale (injection systémique) ou locale (intratrachéale). Pour
ce faire, nous nous basons sur des vecteurs lipidiques fluorescents développés dans le cadre de cette
collaboration, afin de suivre le chemin de ses nanostructures. Nous souhaitons également tester la possibilité
de réadministration (par voie intratrachéale) de nos composés dans le but de restaurer et maintenir
l’expression du gène d’intérêt à un niveau soutenu. Des études sur la cinétique d’expression et des effets
secondaires potentiels de nos molécules seront réalisées, afin d’optimiser le travail de formulation en fonction
de la voie d’administration. Cette approche sera complétée par des études d’immunohistochimie sur coupes
de tissus.
1
1
1
†1
Marie-Pierre Gosselin∗ , Cristine Goncalves , Safia Ezzine , Virginie Malard , Chantal Pichon ,
and Patrick Midoux‡1
1 Centre de biophysique moléculaire (CBM) – CNRS : UPR4301 – Rue Charles Sadron - 45071 Orléans, France
SiRNA generate great hope for targeted therapies of a wide variety of diseases but clinical applications are
delayed due to the absence of very efficient and safe delivery systems. To face the challenge of siRNA
delivery, we are developing non-viral vectors called lipopolyplex (LPRi). We are using lipophosphoramidatebased liposomes in association with histidinylated polymers to produce siRNA lipopoplyplexes (LPRi)
subsequently used for gene silencing (1). Compared to siRNA formulations made with commercial transfection
reagents, our formulation appears to give the best specific inhibition of gene expression. His-lPEI LPRi100 is
composed of siRNA, histidinylated lPEI (His-lPEI) (2) and liposomes made with lipophosphoramidates
comprising imidazole/imidazolium moieties. We obtained the best inhibition at 10 nM siRNA with low
cytotoxicity. This formulation exhibits a size and a zeta potential of 60 nm and +84 mV, respectively. According
to our previous work, this formulation appears as a versatile formulation for DNA, mRNA and siRNA
transfection.
We are using these new LPRi to deliver siRNA in 4T1 cells, a murine model of tumor and lung metastasis of
breast cancer. Ninety % inhibition of luciferase expression in 4T1- Luc cells stably expressing luciferase were
obtained with 50 nM anti-Luc siRNA His-lPEI LPRi100. For in vivo delivery, LPRi are targeted using two
peptides: uPA peptide specific of 4T1 cells and cyclic RGD which binds to v3 integrins frequently expressed in
cancer cells and tumor microvessels. These peptides are attached to liposomes upon reaction with DSPEPEG2000-maleimide. The endocytosis efficiency of targeted liposomes in 4T1 cells, measured by flow
cytometry, showed that they have a six-time greater endocytosis than non- targeted liposomes. In vivo
biodistribution of intratracheally injected LPRi was analyzed by fluorescence using liposomes and polymer
labeled with fluorophores emitting in the near infrared. We showed that the various components of the LPD
(liposome and polymer) were localized in the lungs until 24 hours post-injection. Biological effect of targeted
LPRi100 will be evaluated in small animals in 4T1-Luc experimental lung metastasis.
(1) Perche F, Benvegnu T, Berchel M, Lebegue L, Pichon C, Jaffrs PA, Midoux P. , Enhancement of dendritic cells transfection in
vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen
messenger RNA, (2011) Nanomedicine: Nanotechnology, Biology and Medicine 7: 445-453.
(2) Bertrand, E., Gonçalves, C., Billiet, L., Gomez, J-P., Pichon, C., Cheradame, H., Midoux, P. and Guégan, P. Histidinylated linear PEI:
a new efficient non-toxic polymer for gene transfer, (2011) Chem. Commun., 47: 12547-12549.
∗Intervenant
†Auteur correspondant:
‡Auteur correspondant:
∗ Intervenant
-59-
-60-
POSTER A19
POSTER A20
DEVELOPMENT AND EVALUATION OF NEW DRUG DELIVERY SYSTEMS OF NITROSOTHIOLS FOR
ORAL ADMINISTRATION
SYNTHESE ET PROPRIETES DE NUCLEOSIDES AMPHIPHILES FLUORES
†1
†1
1
2
1
2
Guilhem Godeau∗ , Laurent Latxague , Christophe Di Giorgio , and Philippe Barthélémy
‡1
Shefaat Ullah Shah∗ , Marie Socha , and Stéphane Gibaud∗
1
1Cibles thérapeutiques, formulation et expertise préclinique du médicament (CITHEFOR) – Université de Lorraine : EA3452 – Faculté de Pharmacie –
5, rue Albert Lebrun - 54000 Nancy, France
The oral delivery of Nitric oxide and the methods to improve its oral bioavailability is being a keen interest for
scientists and researchers. The aim of our work was to develop nitroso thiolated polymers by coupling
glutathione with chitosan and then perform a post nitrosation.
S-nitroso glutathione (GSNO) was synthesized by reacting NaNO2 with glutathione at predetermined
conditions. S-nitroso-glutathione-chitosan (SNOC) was synthesized by covalently attaching chitosan
oligosaccharide to glutathione resulting in Chitosan-glutathione conjugate followed by a post nitrosation step.
The amount of thiol groups (-SH) available for Nitric oxide linkage in chitosan-glutathione conjugate was
assessed by Ellman’s methods and Nitric oxide loading/releasing capacity was evaluated by Griess and
Saville methods. In-vitro stability study of GSNO and permeation studies were performed in the Ussing
Chamber (WPI Europe) through rat intestine (male Wister) at constant temperature and oxygen flow.
The resulting conjugate displayed 216 1 mol reduced thiol groups and 776 9 mol total thiol groups in one gram
of polymer. The amount of disulfide bonds was 560 per gram of polymer. Results from the nitrosation studies
revealed that a total of 175 11 mol/g of NO was linked to the available reduced thiol groups on the polymer.
Stability studies of GSNO and SNOC in the Ussing chamber at a constant temperature and oxygen flow
showed that SNOC was more stable as compared to GSNO. SNOC showed a nearly sustained release as
compared to GSNO exhibiting nearly 6 h of NO holding capacity while that of GSNO was only 4-5 h. Results
from in-vitro studies through rat intestine by the Ussing chamber indicated that a small amount of GSNO
(maximum 10%) could cross the rat intestine as compared to SNOC which was less than 5% permeated.
2
Institut de Chimie de Nice (ICN) – CNRS : UMR7272, Université Nice Sophia Antipolis [UNS] – Faculté des Sciences Parc Valrose
28 Avenue Valrose - 06108 Nice, France
ARN : régulations naturelle et artificielle – Inserm : U869, Université Victor Segalen - Bordeaux II, Institut Européen de Chimie et de Biologie –
Université Victor Segalen 146, Rue Léo Saignat bâtiment 3a - 33076 Bordeaux, France
Les dérivés d’acides nucléiques amphiphiles offrent une plateforme polyvalente pour le développement de
ressources biomédicales. Ils combinent les propriétés propres aux dérivés d’acides nucléiques, telles que
l’organisation structurale et la reconnaissance moléculaire, aux propriétés d’auto organisation des
amphiphiles. Cette association aboutit à une vaste gamme de structures (nucléoside, gluconucléoside et
oligonucléotide). Dans cette famille de composés amphiphiles une attention particulière doit être portée aux
dérivés acides nucléiques amphiphiles fluorés. Les propriétés particulières du fluor en matière d’hydrophilie et
de lipophilie renforcent considérablement les propriétés d’organisation de ces amphiphiles tout en diminuant
leur toxicité de manière très significative.
Dans le cadre de ce projet, nous nous proposons d’étudier la relation structure/comportement chez les
nucléosides amphiphiles fluorés. Pour cela les nucléosides amphiphiles fluorés dérivés des différentes bases
(A, C, G et T) seront préparés en faisant varier la chaîne fluorée.
Les propriétés d’organisation des espèces synthétisées seront observées par microscopie électronique et les
propriétés d’hydro- et organogélation de ces composés seront mises en évidence. Les recherches porteront
sur la capacité des bases à reconnaître leur nucléoside complémentaire et sur la mise en évidence de la
reconnaissance d’un gel nucléoside par le gel de son nucléoside complémentaire.
The resulting polymeric S-nitroso glutathione (SNOC) was having a sustained release invitro as compared to
GSNO and could be a promising oral drug delivery system for ischemic diseases.
∗Intervenant
†Auteur correspondant: shefaat-ullah.shah@univ- lorraine.fr
‡Auteur correspondant: stephane.gibaud@univ- lorraine.fr
∗Intervenant
†Auteur correspondant: guilhem.godeau@unice.fr
-61-
-62-
POSTER A21
POSTER A22
EFFECT OF INFLAMMATORY CYTOKINES ON TRANS-ENDOTHELIAL PASSAGE OF NON-VIRAL GENE
DELIVERY VECTORS IN A SKELETAL MUSCLE ENDOTHELIUM MODEL
LIPOPHOSPHORAMIDYL-CYCLODEXTRIN NANOPARTICLES AS NOVEL DRUG DELIVERY SYSTEMS
1
2
1
1
Mathieu Létévé , Olivier Wattraint∗ , Véronique Bonnet , Florence Djedaïni-Pilard , and Catherine Sarazin
†1
2
2
3
2
4
Jean-Pierre Gomez∗ , Mathieu Berchel , Paul-Alain Jaffres , Hervé Cheradame , Philippe Guegan , Mathieu
5
5
6
6
1
1
Mevel , Bruno Pitard , Tristan Montier , Pierre Lehn , Chantal Pichon , and Patrick Midoux
1 Laboratoire des Glucides (LG) – CNRS : FRE3517, Université de Picardie Jules Verne – 33, rue St Leu - 80039 Amiens, France
2Unité de Génie Enzymatique et Cellulaire (GEC) – CNRS : FRE3580, Université de Picardie Jules Verne – 33, rue St Leu - 80039 Amiens, France
1
2
Centre de biophysique moléculaire (CBM) – CNRS : UPR4301 – Rue Charles Sadron – 45071 Orléans, France
CNRS (CEMCA) – CNRS UMR 6521, IFR148 ScInBioS – Université Européenne de Bretagne, Université de Brest, France
3
CNRS (Polytheragene) – Genopole Evry et LAMBE, CNRS 8587 – Université d’Evry Val d’Essonne, France
4
Laboratoire de Chimie des Polymères – UPMC UMR 7610 – 94200 Ivry sur Seine, France
5
INSERM (Institut du Thorax) – INSERM U915 – Université de Nantes, Faculté de Médecine, France
6
INSERM U613, IFR148ScInBioS – Université de Bretagne Occidentale, Hôpital Morvan - CHU Brest, France
In Duchenne muscular dystrophy (DMD) treatment, vascular delivery of gene encoding dystrophin by synthetic
vectors faces to trans-endothelial passage (TEP) through capillary vascular wall to transfect underlying
skeletal muscle cells. Furthermore, DMD results in aberrant regulation of inflammatory signaling cascades with
the release of cytokines which contribute to amplify the deleterious effects of the pathology and could modify
the endothelium integrity. We have evaluated TEP of DNA complexes with cationic polymers or cationic
liposomes in an in vitro model comprising an endothelium of mouse endothelial cells (MCEC) seeded on a cell
insert and mouse skeletal myoblast (C2C12) cells seeded on the well bottom. Luciferase activity in C2C12
cells was used to measure TEP efficiency of DNA complexes encoding luciferase. Among several synthetic
vectors, polyplexes with histidinylated lPEI (His-lPEI) exhibit the best capacity (24%) to cross endothelium
compared to that (< 2%) of other vectors. In the presence of 1 ng/ml IL-1b, TEP decreases significantly
whatever the vectors used while in the presence of the same quantity of TNF-a, TEP efficiency depends on
the vectors. Nevertheless, polyplexes keep high capacity to pass through MCEC endothelium. TEP is further
reduced in the presence of the two cytokines. It is observed that the cytotoxicity of C2C12 cells is greatly
increased when endothelial cells are stimulated with these cytokines whereas the cytokines do not induce
direct cytotoxicity effect on C2C12 cells. This suggests that cytotoxicity is provided by pro-inflammatory factors
produced by endothelial cells. These results indicate that the microenvironment of DMD skeletal muscles
negatively influences the transfection of myoblasts by synthetic vectors. We find that His- lPEI polyplexes
exhibit good capacity to cross the endothelium under both non-inflammatory and inflammatory conditions.
Nanoparticles prepared from amphiphilic cyclodextrins (CD) constitute attractive drug delivery systems for
their high stability and for wider surface area leading to a higher bioavailability [1]. Recently, amphiphilic CD
have been used to develop drug delivery system for anticancer drugs [2].
The results presented in this poster include both the synthesis [3] of lipophosphoramidyl-b- CDwith various
hydrocarbon chains (C12:0, C14:0, C16:0 et C18:0, C18:1 and C18:2) and the characterization of
nanoparticles (NP) obtained from self-assembly in aqueous media (water, NaCl 0,9 % and glucose 5%). Size
distributions and stability were determined by Photon Correlation Spectroscopy. The results demonstrated that
the particles had diameters ranging from 200 to 600 nm and were stable over several days at room
temperature.
Encapsulation and drug release of a therapeutic agent currently used in the HIV treatment (atanazavir) were
performed. Quantitative assays have revealed that about 130 g of atanazavir were encapsulated in 2 mg of
NP formed from C14:0 acyl chains.
1. Duchêne, D., Ponchel, G., & Wouessidjewe, D. (1999). Cyclodextrins in targeting: Application to nanoparticles. Advanced Drug
Delivery Reviews, 36(1), 29–40
2.
çirpanli,
Y., Bilensoy, E., Dogan, A. L., &C¸ ali¸s, S. (2009). Comparative evaluation of polymeric and amphiphiliccyclodextrin
nanoparticles for effective camptothecin delivery. European Journal of Pharmaceutics and Biopharmaceutics, 73(1), 82–89
3. Gervaise, C., Bonnet, V., Wattraint, O., Aubry, F., Sarazin, C., Jaffrès, P.-A., & Djedaïni- Pilard, F. (2012). Synthesis of
lipophosphoramidyl-cyclodextrins and their supramolecular properties. Biochimie, 94(1), 66–74
∗Intervenant
†Auteur correspondant: jean-pierre.gomez@cnrs-orleans.fr
∗ Intervenant
-63-
-64-
POSTER A23
POLYPLEX MIXING: EVIDENCE OF PDNA EXCHANGE BETWEEN POLYPLEXES
POSTER A24
EFFECTS OF A NOVEL ARCHAEAL TETRAETHER-BASED COLIPID ON THE IN VIVO GENE TRANSFER
ACTIVITY OF TWO CATIONIC AMPHIPHILES
Cristine Gonçalves∗, Lucie Pigeon, Chantal Pichon, and Patrick Midoux†
†1
Centre de Biophysique Moléculaire (CBM) – CNRS : UPR4301 – Centre de Biophysique Moléculaire, CNRS UPR4301, Inserm and University of Orléans,
45071 Orléans cedex 02, France
1
Electrostatic complexes between plasmid DNA (pDNA) and polycationic molecules such as cationic polymers
or lipids are a non-viral alternative for gene therapy. Those interactions induce pDNA condensation with size
reduction from ˜1000 nm to 50 – 100 nm, protection against pDNA degradation by nucleases and
enhancement of the cellular uptake due the positive global charge of the resulting particles. The complexation
of cationic liposomes with negatively charged pDNA leads spontaneously to the formation of dense positively
charged particles rearranged in a multilayer structure with average diameter between 100 nm and 200 nm
termed lipoplexes. The condensation of pDNA by cationic polymers differs from that of cationic liposomes, it
often gives toroidal shape of outer radius between 50 nm and 100 nm termed polyplexes. It has been
determined that several pDNA molecules are present in polyplexes and lipoplexes. It is well known that
lipoplexes and polyplexes can be dissociated in the presence of an excess of polyanions such as dextran
sulphate and heparin sulphate showing the instability of those particles.
Here, we report evidence of pDNA exchange between one FITC-pDNA/polyplex and another Cy5pDNA/polyplex formulated with the same cationic polymer. We investigated complexes formed with four
cationic vectors: linear Polyethylenimine (lPEI), Polylysine (pLK), Histidinylated lPEI (His-lPEI) and
KLN25/MM27 liposome (Lip100). First fluorescence status of single labeled complexes (FITC and Cy5) is
separately analyzed by confocal microscopy and flow cytometry. Secondly, each type of fluorescent
polyplexes or lipoplexes are equally mixed (e.g.: FITC-pDNA/His-lPEI plus Cy5-pDNA/His-lPEI (v/v)) and dual
fluorescence is analyzed.
2
2
3
Gene therapy for treating inherited diseases like cystic fibrosis might be achieved using multi-modular nonviral lipid-based systems. To date, most optimizations have concerned cationic lipids rather than colipids. In
this study, an original archaeal tetraether derivative was used as a colipid in combination with one or the
other of two monocationic amphiphiles. The liposomes obtained, termed archaeosomes, were characterized
regarding lipid self-assembling properties, macroscopic/microscopic structures, DNA condensation/
neutralization/relaxation abilities, and colloidal stability in presence of serum. In addition, gene transfer
experiments were conducted in mice with lipid/DNA complexes being administered via systemic or local
delivery routes. Altogether, the results showed that the tetraether colipid can provide complexes with
different in vivo transfection abilities depending on the lipid combination, the lipid/colipid molar ratio, and the
administration route. This original colipid appears thus as an innovative modular platform endowed with
properties possibly beneficial for fine-tuning of in vivo lipofection and other biomedical applications.
Our results show that polyplex mixing is a new parameter that could be important in the design of efficient
cationic polymer for gene transfer. Moreover polyplex mixing can be helpful to evaluate the number of pDNA
molecules inside one polyplex, providing a new insight in formulation of monomolecular pDNA polyplexes.
∗Intervenant
†Auteur correspondant: tony.legall@univ- brest.fr
‡Auteur correspondant
§Auteur correspondant
-65-
2
Institut National de la Santé et de la Recherche Médicale – Inserm : UMR1078 – Faculté de Médecine - 22, avenue Camille Desmoulins - CS 93837 –
29238 Brest, France
2
Ecole Nationale Supérieure de Chimie de Rennes – CNRS : UMR6226 – UMR 6226 - 11 allée de Beaulieu - CS 50837 - 35708 Rennes, France
3
Université des Sciences de Brest – CNRS : UMR6521 – 3, rue des Archives - CS 93837 - 29238 Brest, France
4
Institut de Physique de Rennes – CNRS : UMR6251 – Campus Beaulieu Bat. 11A - 35042 Rennes, France
5
Gene Medicine Group, Nuffield Division of Clinical Laboratory Sciences – University of Oxford – John Radcliffe Hospital, Royaume-Uni
We show that the phenomenon of pDNA exchange between polyplexes (so called polyplex mixing) is
depending on the nature of the polymer and the N/P ratio. Polyplex mixing can result from the pDNA
accessibility inside the toroid structure of polyplexes. In contrast, no pDNA mixing was observed with
lipoplexes due to their multilayer structure and complete encapsulation of pDNA by lipid preventing its
exchange with other complexes.
∗Intervenant
†Auteur correspondant: patrick.midoux@cnrs- orleans.fr
2
Tony Le Gall∗ , Julie Barbeau , Sylvain Barrier , Mathieu Berchel , Loic Lemiegre , Jelena Jeftic ,
4
4
5
5
1
1
Cristelle Meriadec , Franck Artzner , Deborah Gill , Stephen Hyde , Claude Ferec , Pierre Lehn ,
3
‡2
§1
Paul-Alain Jaffres , Thierry Benvegnu , and Tristan Montier
-66-
POSTER A25
POSTER A26
OPTIMIZING THE FORMULATION PROTOCOL OF MAGNETIC SIRNA NANOVECTORS USING
EXPERIMENTAL DESIGN METHODOLOGY
NEW METHODS OF CHITOSAN-INSULIN NANOPARTICLES FORMULATION: IN VITRO AND IN VIVO
VALIDATION OF STABILITY AND BIOFUNCTIONALITY
El Hadji Mouhamadou Bamba Diop∗, Séverine Sigrist†, Frere Yves, Nathalie Auberval,
and Elisa Pedracini
†
Stéphanie David , Hervé Marchais∗, Didier Bedin, and Igor Chourpa
EA6295 nanomédicaments et nanosondes – Université de Tours – UFR des Sciences Pharmaceutiques - 31 avenue Monge - 37200 Tours, France
Centre Européen d’étude du diabète (CeeD) – Université de Strasbourg: EA7294 – Bld René Leriche - 67200 Strasbourg, France
Short interfering RNAs (siRNAs) appear to be a promising tool to treat various human diseases, such as
cancer. Once in the cell, siRNAs can regulate the protein synthesis via the RNA interference (RNAi)
mechanism. New delivery systems are developed in the aim to prevent the siRNA degradation, to facilitate
their delivery in the cell, which should improve their action and prevent unwanted side effects.
Previously, biocompatible superparamegnetic iron oxide nanoparticles (SPIONs) were developed in our
laboratory to be used for magnetic drug targeting (MDT) and magnet resonant imaging (MRI) (1).
These SPIONs were functionalized and then associated with siRNAs and chitosan to form magnetic siRNA
nanovectors (MSN). Previously, the feasibility of the formulation was demonstrated and experimental design
methodology was used to analyze the influence of different formulation parameters. The charge ratio (CR)
between positive charged chitosan and negative charged siRNA as well as the ionic strength, expressed as
[NaNO3] showed a significant effect on the hydrodynamic diameter (DH) of the MSNs, whereas the mass ratio
(MR) between functionalized SPIONs and siRNA, as well as the siRNA concentration seems to influence the potential of the MSNs (2).
In this work, experimental design was used to improve the formulation protocol and to optimize the formulation
parameters identified previously (MR, CR, [NaNO3], [siRNA]). The aim was to obtain MSNs with DH smaller
than 100nm and -potentials near zero to allow their systemic administration. In the first time, a PlackettBurman design was used to determine the influent parameters on the formulation protocol which resulted in
three different protocols. Afterwards a Box-Behnken design was performed using the two most promising
protocols. The formulation parameters identified in the previous study were used in the aim to obtain a surface
response diagram which will help to optimize the physicochemical characteristics of the MSNs.
1. Hervé K, Douziech-Eyrolles L, Munnier E, Cohen-Jonathan S, Soucé M, Marchais H, et al. The development of stable aqueous
suspensions of PEGylated SPIONs for biomedical applications. Nanotechnology. 2008 Nov 19;19(46):465608.
2. David S, Marchais H, Hervé-Aubert K, Bedin D, Garin A-S, Hoinard C, et al. Use of experimental design methodology for the
development of new magnetic siRNA nanovectors (MSN). Int J Pharm. 2013 Oct 1;454(2):660–7.
The oral route for administration of insulin has been considered to be the most attractive for diabetic patients,
but also the more difficult route of administration (pH, enzymes...). Development of a complex pharmaceutical
carrier composed of an enteric coated capsule and mucoadhesive NPs [1.2.3] has been performed to solve
this issue. NPs are obtained by complex coacervation, with chitosan and insulin. However, insufficient stability
of the system has been observed limiting in vitro and in vivo validation.
The aim of this work was to stabilize NPs using methods as cross linking and/or freeze- drying and to validate
in vitro and in vivo the passage through the intestinal barrier.
For chitosan NPs, we developed two strategies to improve the stability: i) cross linking which consist on the
addition of a solution of sodium tripolyphosphate (0.1mg/ml) in the complexing medium in order to increase the
quality of interaction; ii) freeze-drying consisting on the sublimation of the colloidal suspension, in presence or
not of mannitol (5 mg/ml) as cryoprotectant, to increase the of NPs stability. In vitro validation of intestinal
permeation was performed using flow cytometry. Fluorescent nanoparticles using insulin FITC were incubated
during 4 hours with Caco-2 cell line monolayer or co-culture (75% Caco-2 and 25% revHT29MTX) cultured
during 21 days [1]. On these cells was realized the flow cytometry to quantify the absorption of NPs by the
monolayer cell.
For in vivo validation of NPs biofunctionality, diabetes was induced in Wistar rat by a single dose (75mg/kg) of
streptozotocin by intraperitonal route. Intraperitoneal administration of encapsulated insulin was used as
control of biofunctionality and intraduodenal route selected to validate the bioeffectiveness of NPs formulation
(blood glucose measurement).
Size analysis give a NPs size going from 390 to 330nm after cross-linking. The combination between crosslinking and freeze-drying reduce the size to 270nm when mannitol was used as cryoprotectant. A positive
charge (+34mV) was maintained in all formulations. The flow cytometry analysis showed that NPs are able to
be internalizing significantly by the Caco-2 monolayer cell line and co-culture, reflecting the passage through
the intestinal barrier. Cross-linked and freeze-dryed NPs are most significantly uptakes by cells compared to
others formulations (Geo-mean X % cell FITC (%): 633514 for reticulated and freeze dryed NPs, 333376 for
reticulated and freeze dryed NPs with mannitol, 468250 for unreticulated and not freeze dryed NPs, 464450
for unreticulated and freeze dryed NPs). Biofunctionality of encapsulated insulin was demonstrated after
intraperitoneal administration with a reduction of glyceamia from 6g/L at 0 hours to 1g/L after 3hours
comparable to unencapsulated insulin. Finally, an intraduodenal administration of NPs demonstrated their
ability to cross the intestinal barrier with the best efficiency for the combination freeze-dryed and cross-linked.
This study demonstrated that a combination of freeze-dryed and cross linked of chitosaninsulin nanoparticules
could improve their stability and their efficiency.
∗Intervenant
∗ Intervenant
†Auteur correspondant: s.sigrist0@ceed- diabete.org
† Auteur correspondant: stephanie.david@univ-tours.fr
-67-
-68-
POSTER A27
POSTER A28
LUNG CANCER TREATMENT BY TARGETING CD44 RECEPTORS: SIRNA DELIVERY BY HYALURONIC
ACIDACID-MODIFIED LIPOPLEXES
STUDY OF NFKB CONTRIBUTION TO TRANSGENE EXPRESSION MEDIATED BY ELECTROTRANSFER
IN MOUSE TIBIAL CRANIAL MUSCLE
*†1
1
1
2
1
Thais Leite Nascimento , Hervé Hillaireau , Magali Noiray , Delphine Courilleau , Myriam Taverna , Silvia
3
1
Arpicco , and Elias Fattal
2
1
Institut Galien Paris Sud – Université Paris Sud - Paris XI – France
Criblage, Interface Biologie-chimie et Laboratoire Opérationnel de Transfert, IFR 141 – Université Paris Sud - Paris XI – France
3
Dipartemento di Scienza e Tecnologia del Farmaco (Università degli Studi di Torino) – Italie
Background: Small interfering RNAs (siRNAs) are potent molecules capable of blocking gene expression after
entering cell cytoplasm. However, they need to be carried by nanoscale delivery systems that can protect
them against degradation in biological fluids, increase their cellular uptake and favor their subcellular
distribution.
Purpose: Explore the potential of cationic liposomes as siRNA carriers and the targeting of CD44 receptor on
lung cancer cells by hyaluronic acid (HA), and to better understand the aspects regarding the preparation of
hyaluronic acid-bearing cationic liposomes and their interaction with siRNA to form lipoplexes.
Methods: Liposomes of L-alpha-dioleylphosphatidylethanolamine (DOPE) and the cationic lipid [2-(23didodecyloxypropyl)hydroxyethyl]ammonium bromide (DE) were prepared containing different amounts of
hyaluronic acid by the ethanolic injection method. They were complexed with luc-siRNA, and characterized in
terms of lipid-siRNA and HA-DOPE conjugate- liposome interactions by ITC and capillary electrophoresis.
Conjugation rate between liposomes and siRNA was determined after labelling siRNA with 33P-ATP and
separating free from bound siRNA. Luciferase-expressing A549 lung cancer cells were used to study
lipoplexes uptake by flow citometry and siRNA delivery by luciferase inhibition.
Results: Lipoplexes showed negative zeta potential and mean diameter around 260 nm, depending on the
amount of HA-DOPE conjugate added and the (+/-) charge ratio. The addition of the HA-DOPE conjugate did
not compromise siRNA binding. Cytotoxicity was not modified by the presence of HA-DOPE and was
decreased upon complexation with siRNA. Lipoplexes internalization and luciferase inhibition were shown to
be efficient and CD44-mediated.
Conclusions: HA-DOPE-containing cationic liposomes show promising results as carriers of siRNA into CD44expressing lung cancer cells.
1
1
1
1
2
Saysouda Mahindhoratep , Hamid Ait Bouda , Nelly El Shafey , Daniel Scherman , Antoine Kichler , Chantal
3
3
1
*†1
Pichon , Patrick Midoux , Nathalie Mignet , and Michel Francis Bureau
1
UPCGI, Faculté de Pharmacie, Paris, France (Unité de Pharmacologie Chimique et Génétique d’imagerie) – CNRS : UMR8151, Inserm : U1022 – France
2
V-SAT, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France – CNRS : UMR7199 – France
3
Centre de Biophysique Moléculaire, rue Charles Sadron, Orléans, France – CNRS : UPR4301 – France
Activation of the NF-kB pathway induces the transcription of genes involved in inflammatory process, immune
response and cell growth. When activated NF-kB can also favour nuclear import and transcription of
exogenous DNA possessing consensus sequences for NF- kB such as DNA of some virus or plasmid DNA.
Our question was: Is NF-kB involved in DNA electrotransfer?
We transfected the mouse tibial cranial muscle with plasmid encoding luciferase gene, bearing or not NF-kB
consensus sequences (p3NF-luc, p3NF-luc-3NF and pTAL- luc). Transgene expression was evaluated noninvasively by luminescence imaging after injection of the luciferase substrate at day two after transfection
experiment. A methodological originality of our study was to evaluate the transgene expression and number of
pDNA copies in paired experiments on the same muscles. Q-PCR to evaluate pDNA copies number was
performed on muscles withdrawn the day after evaluation of luciferase expression. At this time, it was
assumed that pDNA in extracellular space and in cell cytoplasm was totally degraded. It was thus possible to
obtain the expression per nuclear pDNA copy that indicates the efficiency of nuclear pDNA transcription and or
mRNA translation. RT-PCR of HsP70 mRNA was done to evidence the cell stress. Lastly, Western blot of
phosphorylated Ikb alpha gave an index of the NF-kB activation.
Electrotransfer increased significantly the total transgene expression per muscle and the expression per pDNA
copy whatever the pDNA tested. It means that even with pTAL-luc which bears the weakest promoter, the
pDNA transcription and/or mRNA translation was stimulated by the delivery of electric pulses, independently of
NF-kB. But, it is also clear that NF-kB consensus sequences improved greatly pDNA total expression per
muscle and the expression per DNA copy with or without electrotransfer. Electrotransfer induced a cell stress
which was evidenced by HsP70 m RNA increase. However, relatively to non-treated muscle phosphorylated
IkB alpha was slightly increased by simple pDNA injection and a bit more with electrotransfer. We also
observed a basal level of phosphorylated IkB alpha and thus of free NF-kB without any stimulation. Our
hypothesis is that this basal amount of free NF-kB could be used by plasmid with NF-kB consensus
sequences for their expression.
We evidence the usefulness of NF-kB consensus sequence to improve pDNA expression in muscle with or
without electrotransfer. In this tissue the activation of NF-kB does not seems necessary to improve the
expression of these pDNA as free NF-kB is available. We also showed that electrotransfer can increase
efficiency of pDNA expression independently of NF-kB.
∗Intervenant
∗Intervenant
†Auteur correspondant
†Auteur correspondant: thais.leite-nascimento@u-psud.fr
-69-
-70-
POSTER A29
POSTER A30
POLYMERS WITH PH-SENSITIVE DISSOLUTION SWITCH FOR INTRACYTOSOLIC DELIVERY OF
NUCLEIC ACIDS AND PROTEINS: RECENT ADVANCES
DEVELOPMENT AND EVALUATION OF NEW DRUG DELIVERY SYSTEMS OF NITROSOTHIOLS FOR
ORAL ADMINISTRATION
Guy Zuber
Shefaat Ullah Shah∗ , Marie Socha, and Stéphane Gibaud∗
UMR7199 (LCAMB) – Université de Strasbourg, CNRS : UMR7199 – VSAT, Faculté de Pharmacie, 74, Route du rhin 67400 Illkirsch, France
Cibles thérapeutiques, formulation et expertise préclinique du médicament (CITHEFOR) – Université de Lorraine : EA3452 –
Faculté de Pharmacie - 5, rue Albert Lebrun - 54000 Nancy, France
Synthetic oligonucleotides are highly specific modulators of the genetic information and may provide huge
benefits in medicine. The oligonucleotides have unfortunately poor pharmacological properties and rely on
multifunctional carriers for protection in biological fluids but also for cell anchorage and for crossing the cell
membrane to reach their molecular target. In parallel, advances in biotechnology have led to therapeutic
monoclonal antibodies able to mediate a benefit to patients by targeting extracellular receptors. Expending the
therapeutic scope of antibodies will depend on our ability to develop specialized delivery systems that can
successfully shuttle them into the cytoplasm for targeting intracellular pathology-mediating proteins. In here,
we report our most recent results on intracytosolic protein and siRNA delivery ability using self-assembling
PEIs which were prepared to possess a pH-dissolution at endosomal pHs.
†
‡
The oral delivery of Nitric oxide and the methods to improve its oral bioavailability is being a keen interest for
scientists and researchers. The aim of our work was to develop nitroso thiolated polymers by coupling
glutathione with chitosan and then perform a post nitrosation.
S-nitroso glutathione (GSNO) was synthesized by reacting NaNO2 with glutathione at predetermined
conditions. S-nitroso-glutathione-chitosan (SNOC) was synthesized by covalently attaching chitosan
oligosaccharide to glutathione resulting in Chitosan-glutathione conjugate followed by a post nitrosation step.
The amount of thiol groups (-SH) available for Nitric oxide linkage in chitosan-glutathione conjugate was
assessed by Ellman’s methods and Nitric oxide loading/releasing capacity was evaluated by Griess and
Saville methods. In-vitro stability study of GSNO and permeation studies were performed in the Ussing
Chamber (WPI Europe) through rat intestine (male Wister) at constant temperature and oxygen flow.
The resulting conjugate displayed 216 1 mol reduced thiol groups and 776 9 mol total thiol groups in one gram
of polymer. The amount of disulfide bonds was 560 per gram of polymer. Results from the nitrosation studies
revealed that a total of 175 11 mol/g of NO was linked to the available reduced thiol groups on the polymer.
Stability studies of GSNO and SNOC in the Ussing chamber at a constant temperature and oxygen flow
showed that SNOC was more stable as compared to GSNO. SNOC showed a nearly sustained release as
compared to GSNO exhibiting nearly 6 h of NO holding capacity while that of GSNO was only 4-5 h. Results
from in-vitro studies through rat intestine by the Ussing chamber indicated that a small amount of GSNO
(maximum 10%) could cross the rat intestine as compared to SNOC which was less than 5% permeated.
The resulting polymeric S-nitroso glutathione (SNOC) was having a sustained release in- vitro as compared to
GSNO and could be a promising oral drug delivery system for ischemic diseases.
∗Intervenant
†Auteur correspondant: shefaat-ullah.shah@univ- lorraine.fr
‡Auteur correspondant: stephane.gibaud@univ- lorraine.fr
-71-
-72-
POSTER A31
POSTER A32
CHEMICAL GELS OF LIPID NANOPARTICLES FOR THE CONTROLLED DELIVERY OF BIOLOGICAL
AND LIPOPHILIC DRUGS
CHITOSAN-BASED NANOPARTICLES: A STRATEGY FOR THE INTRACELLULAR DELIVERY OF
NUCLEOTIDE ANALOGS
Ismail Cheibani*1, Antoine Hoang1, Marie Escudé1, Eric Bayma2, Rachel Auzély2, and Isabelle Texier-Nogues1
Giovanna Giacalone*, Hervé Hillaireau, and Elias Fattal
1
Laboratoire d’Electronique et des Technologies de l’Information (LETI) – CEA – MINATEC 17, rue des Martyrs, 38054, Grenoble, France
2
Centre de recherches sur les macromolécules végétales (CERMAV) – CNRS : UPR5301, Université
Joseph Fourier - Grenoble I – 601 Rue de la Chimie 38400 Saint Martin D’heres, France
Proteins, antibodies, DNA and their derivatives have opened the way for new promising treatments, but these
molecules are fragile, unstable in vivo and degraded before reaching their site of action. Therefore, new
materials adapted to the efficient encapsulation of these therapeutic agents are required for their controlled
delivery.
Institut Galien Paris-Sud – Université de Paris-Sud Orsay – France
Nucleotide analogues such as Azidothymidine-triphosphate (AZT-TP), the active form of Zidovudine, display
important pharmacological activity for the treatment of HIV. The administration of these nucleotide analogues
would bypass the intracellular phosphorylation which can be a metabolic bottleneck. This possibility is however
limited by their instability in physiological conditions, and furthermore their hydrophilicity restricts their access to
the target cells.
We have developped new nanostructured gel materials, based on lipid nanoparticles which can act as
reservoirs for lipophilic drugs and can be surface-modified to vectorize biologicals. The lipid nanoparticles are
composed of an oily core, and a shell of phospholipids and PEG surfactants. These nanoparticles are
colloidally stable, biocompatible, non-immunogenic, and obtained from a simple solvent-free process. The
particles can be self-assembled by hydrogen bonds to form physical gels displaying tunable rheological
properties, or by covalent bonds to form chemical gels.
Several nanocarriers have been proposed so far for the encapsulation of these molecules, but their applications
are limited due to the low drug loading achieved.
We will here present examples of nanostructured lipid gels with photo triggered assembling or photo cleaving
properties. These lipid gels constitute two-compartment materials: an oily phase for trapping lipophilic drugs,
and an aqueous phase that can encapsulate biomolecules such as therapeutic proteins or DNA.
Different molar ratios between chitosan and AZT-TP (or ATP, used here as a model drug) have been tested in
order to study the formation of nanoparticles; some selected ratios have been then evaluated for their size,
surface properties, drug encapsulation and loading. In vitro cell uptake experiments were performed on a cell
line of mouse macrophages (J774A.1), using the free molecule as a negative control. The intracellular
distribution of the delivered molecules was further investigated using confocal laser microscopy.
Assembly of the gel material can be obtained using different strategies: 1) By formation of covalent bonds
between thiol functionalized nanoparticles and maleimide functionalized nanoparticles; 2) By formation of
disulfide bonds between thiol bearing nanoparticles (upon photo-activation); 3) By crosslinking, in the
presence of glutaraldehyde, particles bearing-ONHPOC functions which, upon 365 nm irradiation, are
deprotected to expose oxyamine groups. The nanostructured gel materials have been characterized and resist
to dilution.
When a photoclivable group is inserted in the covalent bond linking 2 particles, light ex posure of the material
destroys the gel and releases the nanovectors. The nanoparticles retain their integrity during the gel assembly
and disassembly. The rheology and physicalchemical properties of the materials were investigated. The
results obtained with different materials will be presented.
Our strategy proposes the use of chitosan, a biocompatible polysaccharide which is known to form
nanoparticles through complexation with TPP[1]; in contrast with previous methods, in our case the drug itself
will be the driving force for the formation of nanoparticles[2].
Colloidal suspensions have been obtained from chitosan and AZT-TP at N/P 1; nanoparticles present a minimal
size about 200 nm with a zeta potential above +20 mV. An encapsulation efficiency of 70% can be reached,
allowing loading rates as high as 44% w/w. A cell viability of 80% has been found for particle concentrations up
to 0.6 mg/mL. The cellular uptake is at least 2-fold higher when molecules are delivered as nanoparticles,
compared to the free molecules. The delivery in the cell cytoplasm was further confirmed using confocal laser
microscopy.
An original method is proposed to design nanoparticles, which allows high loading rates; this lowers the
amounts of excipients needed, thus limiting the toxicity concerns. These nanosystems permit an efficient in vitro
intracellular delivery of nucleotide analogues, namely AZT-TP. Further in vivo studies will be worth being
investigated to confirm the potential of these nanocarriers.
1.
2.
∗
Calvo, P.; RemunanLopez, C.; VilaJato, J. L.; Alonso, M. J., Novel hydrophilic chitosan- polyethylene oxide nanoparticles as protein carriers. Journal of
Applied Polymer Science 1997, 63, (1), 125-132.
Giacalone, G.; Bochot, A.; Fattal, E.; Hillaireau, H., Drug-induced nanocarrier assembly as a strategy for the cellular delivery of nucleotides and nucleotide
analogues. Biomacromolecules 2013, 14, (3), 737-42.
∗Intervenant
Intervenant
-73-
-74-
POSTER A33
POSTERS – SESSION B
THE INTRACELLULAR FATE OF POLYCARBONATE POLYPLEXES MODULATES THE EFFICACY OF
SIRNA
†1
2
3
1
2
B1
ULTRASOUND-INDUCED BIOORTHOGONAL CHEMISTRY IN-SITU USING COMPOSITE
DROPLETS
MARINE BEZAGU, CLAUDIA ERRICO, VICTOR CHAULOT-TALMON, FABRICE MONTI, STELLIOS
ARSENIYADIS, OLIVIER COUTURE, MICKAEL TANTER, JANINE COSSY , AND PATRICK TABELING
B2
CONVENTIONAL VERSUS STEALTH LIPID NANOCAPSULES: IN VIVO FATE PREDICTION
THROUGH FRET MONITORING
ANNE-LAURE LAINÉ, MAXIME HENRY, JÉROME BEJAUD, JEAN-PIERRE BENOIT, JEAN-LUC COLL, AND
CATHERINE PASSIRANI
B3
EFFECTS OF LN-DO3A-PIB DERIVATIVES ON THE AMYLOID PEPTIDES: SELFASSEMBLY AND IN VITRO INTERACTIONS
ANDRÉ MARTINS, DAVID DIAS, JEAN-FRANCOIS MORFIN, DOUGLAS LAURENTS, EVA TOTH, AND
CARLOS GERALDES
B4
IN SITU IMPLANTS FOR MYOCARDIAL REGENERATION AFTER INFARCTION
MARIANNE PARENT, CAROLINE GAUCHER, NGUYEN TRAN, MAREK RYCHTER, PIERRE LEROY, AND
ARIANE BOUDIER
B5
POLYMERIC COMPOSITE BEADS FOR PER OS ADMINISTRATION OF S
NITROSOGLUTATHIONE.
WEN W U, CAROLINE GAUCHER, SONIA DARELLE TOUKAM, ISABELLE FRIES, PIERRE LEROY, XIANMING HU, PHILIPPE MAINCENT, AND ANNE SAPIN-MINET
B6
LACTOSYL ALBUMIN AS IMAGING AGENT OF THE LIVER FUNCTION
JOHANNE SEGUIN, MICHEL BESSODES, ZAHRA AIT RAMDANE, KATHIA LEMDANI, PHILIPPE CHAUMETRIFFAUD, DANIEL SCHERMAN, AND NATHALIE MIGNET
B7
USE OF F-TAC OLIGOMERS FOR THERANOSTICS DEVELOPMENT
STÉPHANE DESGRANGES, KSENIA ASTAFYEVA , LUCIE SOMAGLINO, NICOLAS TAULIER, W LADIMIR
URBACH, ANGE POLIDORI, AND CHRISTIANE CONTINO-PEPIN
B8
5-AMINOSALICYLIC ACID RELEASE PROFILE FROM XYLAN AND XYLAN/EUDRAGIT R
S100 MICROPARTICLES PRODUCED BY CHEMICAL AND PHYSICOMECHANICAL
APPROACHES
BARTOLOMEU SOUZA, KAREN SILVA, FRANCISCO ALEXANDRINO-JÚNIOR , ANDRÉ SILVA , HENRIQUE
MARCELINO AND ERYVALDO EGITO
B9
DEVELOPMENT OF NANOEMULSIONS CONTAINING 8-METHOXYSALEN FOR TOPICAL
TREATMENT OF VITILIGO
THAIS BARRADAS, ANDRÉ SILVA, KATTYA SILVA , JÚLIA CONCEICAO , AND CLAUDIA MANSUR
B10
ANTI-ABETA ANTIBODY DECORATED NANOPARTICLES FOR ALZHEIMER DISEASE: IN
VIVO PROOF OF CONCEPT
KARINE ANDRIEUX
B11
ONE-SHOT
PROCEDURE
FOR
DEXTRAN,
PULLULAN
MICROASSEMBLY AND NANOASSEMBLY FORMATION
MARTINA BOMBARDI, NARIMANE ZEGHBIB, AND KAWTHAR BOUCHEMAL
2
Antoine Frère∗ , Michal Kawalec , Paul Peixoto , Brigitte Evrard , Philippe Dubois , Laetitia Mespouille ,
Denis Mottet3, and Géraldine Piel‡1
1
Laboratory of Pharmaceutical Technology and Biopharmacy - CIRM, University of Liège, Liège, Belgium – Belgique
2
Laboratory of Polymeric and Composite Materials, University of Mons, Mons, Belgium – Belgique
3
Metastasis Research Laboratory – GIGA Cancer, University of Liège, Liège, Belgium – Belgique
Vectors used in this work are polyplexes formed by the self-assembly of biodegradable polycarbonate
polymers and siRNA specifically targeted against HDAC7. The specific inhibition of HDAC7 disturbs the
angiogenic process, making it an attractive target for an anti-angiogenic therapy. The delivery of siRNA into
the cytoplasm of target cells to exert their effects remains a significant challenge. To be effective in vivo,
polyplexes must meet several physico-chemical characteristics. In this study, the main characteristics
evaluated are the incorporation of the siRNA into the polyplexes (determined by agarose gel electrophoresis
or by the Quant-iTTM RiboGreen R kit), the size (measured by dynamic light scattering), the charge (zeta
potential measured by laser Doppler velocimetry) and the buffering capacity (measured by titration). The
transfection capacity of polyplexes with good physicochemical characteristics has been examined in HeLa
cells (determined by flow cytometry and microscopy). A western blot has been performed to assess the
expression level of HDAC7 protein in treated cells compared to a control.
Different architectures of biodegradable polycarbonate polymers at different ratios (N/P, polymer/siRNA) have
been tested. Most of them show a complete incorporation of the siRNA at N/P above 10. At these N/P ratios,
size measurements show an average diameter between 200 and 500 nm, ideal to avoid renal excretion, to
escape the monomolecular phagocytic system and to allow accumulation of nanoparticles in the tumor due to
the enhanced permeability and retention(EPR)effect. Zeta potential is slightly positive, around +5mV, positive
enough to interact with cell membranes but lower than 10 mV to avoid a too high toxicity. In vitro, flow
cytometry shows a high transfection level for most selected polyplexes (up to 90% of transfected cells).
Unfortunately, whatever the architecture of the polymer, no decrease of the expression of HDAC7 has been
observed by western blot.
The two main hypotheses to explain the lack of efficacy of our new polyplexes are either a too high affinity
between the polymer and siRNA that prevents the release of the siRNAin the cytoplasm or either the use of an
endocytosis pathway without vesicle acidification. This acidification allows the endosome bursting using the
buffering capacity of the polymer through the “proton sponge effect”.
We first studied the effect of pharmacological inhibitors of endocytic pathways. Chlorpromazine is used to
inhibit the clathrin-mediated endocytosis. This pathway leads to endosome acidification and fusion with
lysosome, enabling polyplexes to use the “proton sponge effect” to escape from the endosome. Another
inhibitor, filipin, is used to block caveolae-mediated endocytosis. In contrast, vesicles formed in this pathway
do not lead to acidification and can sequester polyplexes. The first results show that chlorpromazine inhibits
partially cellular internalization of polyplexes while filipin shows no effect on it. These results tend to confirm
that part of the polyplexes population reached the cytosplasm using the clathrin-mediated endocytosis. In the
next future, we will confirm these results and try to evaluate if the lack of efficacy of our polyplexes may be
explained by a too high affinity between the polymer and siRNA.
∗Intervenant
†Auteur correspondant: antoine.frere@ulg.ac.be
‡Auteur correspondant: geraldine.piel@ulg.ac.be
-75-
-76-
AND
AMYLOPECTIN
B12
AUTO-ASSOCIATIVE ASSEMBLIES: A BIOMIMETIC PLATFORM AGAINST THE HEPARAN
SULFATE-DEPENDENT VIRUSES HSV-1, HSV-2 AND HPV-16
DAVID LEMBO, MANUELA DONALISIO, CLAIRE LAINE, VALERIA CAGNO, ANDREA CIVRA, NARIMANE
ZEGHBIB, AND KAWTHAR BOUCHEMAL
B24
SUPERPARAMAGNETIC NANOPARTICLES FOR RMI BRAIN TUMORS MOLECULAR
IMAGING
SOPHIE RICHARD, AMAURY HERBET, YOANN LALATONNE, SÉBASTIEN MÉRIAUX, FRÉDÉRICDUCONGÉ,
DIDIER BOQUET , AND LAURENCE MOTTE
B13
SYNTHESIS OF AMPHIPHILIC COPOLYMERS TO FORMULATE NO-GRAFTED MICELLAR
NANO-OB JECTS
CÉCILE NOUVEL, ARIANE BOUDIER, MAÉVA CHAPON, FERJI KHALID, AND JEAN-LUC SIX
B25
PASSIVE,
ACTIVE
AND
MAGNETIC
TARGETING
OF
MULTIFUNCTIONAL
NANOPARTICLES FOR THERANOSTIC APPLICATIONS
NATHALIE SCHLEICH, JULIE MAGAT , CHRYSTELLE PO, VINCENT POURCELLE, JACQUELINE MARCHAND,
BERNARD GALLEZ, VERONIQUE PREAT, AND FABIENNE DANHIER
B14
NCAPSULATION OF HSP90 INHIBITOR LOADED NANOLIPOSOMES; EVALUATION OF
THE CYTOTOXIC EFFECT IN VITRO ON BREAST AND PROSTATE CANCER CELLS
FÉLIX SAUVAGE
B26
FORMULATION OF SELF-EMULSIFYING MICROBUBBLES FOR DRUG DELIVERY
ASSISTED BY ULTRASOUNDS
CAROLINE THEBAULT, S YLVIE CRAUSTE-MANCIET, FREDÉRIC ROSA, SIMONA MANTA, YOHANN
CORVIS, PHILIPPE ESPEAU, GILLES RENAULT, MICHEL FRANCIS BUREAU, NATHALIE MIGNET, AND
MICHEL BESSODES
B27
THERMAL ANALYSIS TOOLS FOR PHYSICO-CHEMICAL CHARACTERIZATION OF
MICROBUBBLES FOR IMAGING
FREDÉRIC ROSA, YOHANN CORVIS , NATHALIE MIGNET, DENIS BROSSARD, SYLVIE CRAUSTEMANCIET, AND PHILIPPE ESPEAU
B28
FORMULATION OF HSP90 INHIBITOR LOADED NANOLIPOSOMES : EVALUATION OF THE
CYTOTOXIC EFFECT IN VITRO ON BREAST AND PROSTATE CANCER CELLS
FÉLIX SAUVAGE, SILVIA FRANZE, SAMIR MESSAOUDI, STÉPHANIE DENIS, BAPTISTE MARTIN, JEANDANIEL BRION, MOUAD ALAMI, GILLIAN BARRATT, AND JULIETTE VERGNAUD-GAUDUCHON
B29
PACLITAXEL-LOADED MICELLES ENHANCE THE VASCULAR PERMEABILITY AND
RETENTION OF NANOMEDICINES IN TUMORS
FABIENNE DANHIER, PIERRE DANHIER, CHRISTOPHE DE SAEDELEER, NATHALIE SCHLEICH, BERNARD
UCAKAR, PIERRE SONVEAUX, BERNARD GALLEZ, AND VÉRONIQUE PRÉAT
B30
PHOTOACTIVE POROUS SILICON NANOVECTORS FOR PHOTODYNAMIC THERAPY
FREDERIQUE CUNIN, EMILIE SECRET, MARIE MAYNADIER, ARNAUD CHAIX, MAGALI GARY-BOBO,
MARCEL GARCIA, PHILIPPE MAILLARD, FRANCESCO DI RENZO, MICHAEL SAILOR, AND JEAN-OLIVIER
DURAND
B31
PLGA NANOPARTICLES INCORPORATING LIPOPHILIC GD CONTRAST AGENTS
GUILLAUME RIGAUX, VALÉRIE GAËLLE ROULLIN, CYRIL CADIOU, GAËLLE BOEUF, NICOLAE-BOGDAN
BERCU, MARIE-CHRISTINE ANDRY, MICHAËL MOLINARI, CHRISTOPHE PORTEFAI, CHRISTINE HOEFFEL,
LUCE VANDER ELST, SOPHIE LAURENT, AND FRANÇOISE CHUBURU
B32
DEVELOPMENT OF MULTIFUNCTIONAL PARTICLES FOR DIAGNOSTIC & TARGETED
DELIVERY IN CANCER THERAPEUTICS
TAO JIA, JEREMIE CICCIONE, MICHAEL CLARON, DIDIER BOTURYN, GILLES SUBRA, AND JEAN-LUC
COLL
B33
VECTORIZATION OF MULTIMODAL GD-NANOPARTICLES FOR AMYLOIDOSIS
DIAGNOSTIC.
MARIE PLISSONNEAU, PIERRE MOWAT, NATHALIE STRANSKY-HEILKRON, CHRISTEL MARQUETTE,
FRANCOIS LUX, ERIC ALLÉMANN,CÉDRIC LOUIS, VINCENT FORGE, VINCENT MONTET, MIREILLE
DUMOULIN, AND OLIVIER TILLEMENT
B34
GLYCOPOLYPEPTIDE-BASED NANOCARRIERS FOR LUNG CANCER TREATMENT
SILVIA MAZZAFERRO, VICTOR JEANNOT, CHRISTOPHE SCHATZ, AMANDINE HURBIN, JEAN-LUC
COLL, AND SEBASTIEN LECOMMANDOUX
B15
TAZAROTENE ENCAPSULATION IN POLYLACTIC-ACID (PLA) NANOPARTICLES FOR
PSORIASIS TREATMENT, IN VITRO AND EX VIVO ASSAYS
ANNE-SOPHIE BOISGARD, VINCENT LAHAYE, CARINE MAINZER, CELINE TERRAT, FIORENZA RANCAN,
ANNIKA VOGT, AND BERNARD VERRIER
B16
POLYMERIC MICELLE NANOCARRIERS FOR THE TARGETED TOPICAL CUTANEOUS
DELIVERY OF TACROLIMUS
MARIA LAPTEVA, KARINE MONDON, MICHAEL MÖLLER , AND YOGESHVAR KALIA
B17
FORMULATION OF ALGINATE-BASED NANOCAPSULES FOR TOPICAL DELIVERY
HOANG TRUC PHUONG NGUYEN, EMILIE MUNNIER, XAVIER PERSE, SIMONE COHEN-JONATHAN, AND
IGOR CHOURPA
B18
CURCUMIN NANOCARRIERS DESIGNED FOR A TOPICAL USE AND THEIR STABILITY IN
HYDROGELS
HOANG TRUC PHUONG NGUYEN, EMILIE MUNNIER, XAVIER PERSE, MARTIN SOUCE, IGOR CHOURPA,
AND SIMONE COHEN-JONATHAN
B19
POLYMERIC COMPOSITE BEADS FOR PER OS ADMINISTRATION OF SNITROSOGLUTATHIONE
WU WEN, CAROLINE GAUCHER, SONIA TOUKAM, ISABELLE FRIES, PIERRE LEROY, MING HU, PHILIPPE
MAINCENT, AND ANNE MINET
B20
NANOHYBRID BASED ON ZN(FE)O PULLULAN GRAFTED NANOPARTICLES AS
POTENTIAL LUMINESCENT AND MAGNETIC BIMODAL IMAGING PROBES: SYNTHESIS,
CHARACTERIZATION, MODIFICATION AND CYTOXICITY STUDY
IMEN BALTI
B21
ZINC RESPONSIVE CONTRAST AGENTS FOR MRI
CÉLIA BONNET, FABIEN CAILLÉ, AGNES PALLIER, FRANCK SUZENET, AND EVA TOTH
B22
DESIGN AND EVALUATION OF BIODEGRADABLE NANOPARTICLES FOR
TRANSPORT OF ACTIVE MOLECULES
SOPHIE LEGAZ, AGNÈS BOREL, VINCENT LAHAYE, BARBARA VIGINIER-MONOURY, CHRISTOPHE
TERZIAN, AND BERNARD VERRIER
B23
THE
POLYMERIC MICRO/NANOCAPSULES OF PERFLUOROHEXANE AS CANDIDATES FOR
ULTRASOUND CONTRAST AGENTS
LUDIVINE MOUSNIER, ELIAS FATTAL, AND NICOLAS TSAPIS
-77-
-78-
POSTER B1
POSTER B2
ULTRASOUND-INDUCED BIOORTHOGONAL CHEMISTRY IN-SITU USING COMPOSITE DROPLETS
†1
2
3
CONVENTIONAL VERSUS STEALTH LIPID NANOCAPSULES: IN VIVO FATE PREDICTION THROUGH
FRET MONITORING
3
Marine Bezagu∗ , Claudia Errico , Victor Chaulot-Talmon , Fabrice Monti , Stellios
Arseniyadis1, Olivier Couture2, Mickael Tanter2, Janine Cossy1, and Patrick Tabeling3
2
1
Laboratoire de Chimie Organique - ESPCI ParisTech (LCO) – CNRS : UMR7084, ESPCI ParisTech – 10 rue Vauquelin 75005 Paris, France
Institut Langevin ”ondes et images” – Université Pierre et Marie Curie (UPMC) - Paris VI, ESPCI ParisTech, CNRS : UMR7587, Université Paris VII –
Paris Diderot – 10 Rue Vauquelin - 75231 Paris, France
3
Laboratoire de Microfluidique, MEMs et Nanostructures - ESPCI ParisTech (MMN) – CNRS : UMR7083, ESPCI ParisTech – 10 rue Vauquelin
75005 Paris, France
Localized delivery of active agents in the vicinity of a diseased tissue could improve their efficacy and reduce
their side effects. In this context, we recently established that perfluorocarbon composite droplets comprised of
a nanoemulsion of water within a perfluorohexane matrix (4 m in diameter) loaded with fluorescein could
release their content by acoustic vaporization (Couture et al. Medical Physics, 2011, 2012). Here, we wish to
report our efforts to control both temporally and spatially a spontaneous chemical reaction, using droplets to
isolate two reactants (A and B) from one another. Our technology would allow the formation of the reactive
species in a zone of interest upon vaporization of the perfluorocarbon matrix and the in situ generation of a
new product (C) (Figure 1). The steep threshold of release (1.7 MPa PNP at 5 MHz) of these droplets allows
the triggering of a chemical reaction with a high spatial (mm) and temporal resolution (ms).
The demonstration was achieved by encapsulating a solution of A (azidocoumarin) in DMSO into composite
droplets of perfluorohexane and releasing the content into the external flow containing B (reactive alkyne).
Most importantly, the product of the reaction of A and B is fluorescent (ex: 350 nm, em: 430 nm). The two
solutions (A encapulsated + B in solution) were injected in a microfluidic channel where a 2.25 MHz
transducer was focused. A single pulse of 30 cycles was emitted while a camera (10 fps) mounted on a
fluorescent microscope (10X, DAPI) recorded the fluorescence induced during the reaction.
Within the microfluidic channel, a single acoustic pulse was able to vaporize several droplets, leading to the
release of their content in the surrounding medium. As shown below, in the following 100 ms upon release, the
fluorescence intensity within the focus zone was multiplied by a factor of 2.9 (A + B). As the fluorescence is
specific to the reaction product, this demonstrates the subsequent formation of the latter after ultrasound
release.
In summary, we managed to remotely induce a chemical reaction with ultrasounds by releasing an
encapsulated reactant locally into a flow containing its reactive partner. This reaction was limited temporally,
following the 10 ms pulse, and spatially, within the focus of the transducer. We expect that such targeted
chemistry could lead to the localized release of prodrugs or to the localized production of drugs in-vivo that are
either too toxic or unstable to be injected.
1
2
1
1
Micro et nanomédecines biomimétiques (MINT) – Inserm : U1066, Université d’Angers – Université d’Angers, 10 rue André Boquel 49100 Angers, France
2
INSERM/UJF U823, Grenoble, France – Add this new organization – France
Lipid nanocapsules (LNCs) have been taking an active part in the nanomedicine landscape for the past
decade [1]. Today LNCs have reached an advanced level in term of preparation as well as characterization
and showed promising performance as drug delivery systems. Before any translation from laboratory to
clinical trials, the determination of the nanocarrier fate in preclinical models is required. Modern
fluorescent imaging techniques have gained considerable advances becoming a powerful technology for
non-invasive visualization in living subject. Among them, Forster (fluorescence) resonance energy transfer
(FRET) is a particular fluorescence imaging which involves energy transfer between 2 fluorophores in a
distance-dependent manner [2]. Considering this feature, the encapsulation of an acceptor/donor pair in
LNCs allowed the carrier integrity to be tracked. Accordingly, we used this FRET technique to evaluate
their in vitro /in vivo behavior. Two types of LNCs were studied, conventional LNCs and stealth LNCs
obtained through a newly designed “one- step process” in contrast to the standard post-insertion
method [3]. We firstly assessed in vitro guest exchange dynamics and release kinetics and secondly in vivo
biodistribution of both LNCs. Results showed enhanced stability of encapsulation in stealth LNCs in
comparison to conventional LNCs. Additionally, the presence of the long PEG chains on the LNC surface
modified the biodistribution pattern. Besides showing extended blood circulation time, stealth LNCs
mostly accumulated in the skin at 24 h whereas a residual FRET signal coming from conventional
LNCs could be observed in bones. Both LNC types showed great structure stability over several hours.
Further studies have been planned in order to assess the biodistribution of these LNCs in a
subcutaneous glioma model.
1.
2.
3.
Huynh N.T., Passirani C., Saulnier P., Benoît J.P: Lipid nanocapsules: a new platform for nanomedicine. International Journal of
Pharmaceutics 2009, 379:201–209.
Jares-Erijman E.A., Jovin T.M.: FRET imaging. Nature Biotechnology 2003, 21:1387–1395.
Perrier T., Saulnier P., Fouchet F., Lautram N., Benoît J.P: Post-insertion into Lipid NanoCapsules (LNCs): From experimental
aspects to mechanisms. International Journal of Pharmaceutics 2010, 396:204–209.
∗Intervenant
†Auteur correspondant: marine.bezagu@gmail.com
∗Intervenant
‡Auteur correspondant: Jean-Luc.Coll@ujf-grenoble.fr
-79-
†1
Anne-Laure Lainé∗ , Maxime Henry , Jérome Bejaud , Jean-Pierre Benoit , Jean-Luc
Coll‡2, and Catherine Passirani1
-80-
POSTER B3
POSTER B4
EFFECTS OF LN-DO3A-PIB DERIVATIVES ON THE AMYLOID PEPTIDES: SELF-ASSEMBLY AND
IN VITRO INTERACTIONS
IN SITU IMPLANTS FOR MYOCARDIAL REGENERATION AFTER INFARCTION
André Martins1,2, David Dias3, Jean-Francois Morfin∗1, Douglas Laurents4, Eva Toth1,
Marianne Parent∗, Caroline Gaucher, Nguyen Tran, Marek Rychter, Pierre Leroy, and Ariane Boudier∗†
and Carlos Geraldes
2
1 CITHEFOR (EA 3452) – Université de Lorraine : EA3452 – Faculté de Pharmacie, Nancy, France
2
1
Centre de biophysique moléculaire (CBM) – CNRS : UPR4301 – Rue Charles Sadron - 45071 Orléans, France
Department of Life Sciences, Center of Neurosciences and Cell Biology (CNC) and Coimbra Chemistry Center, University of Coimbra – Coimbra,
Portugal
3
Department of Chemistry, University of Cambridge – Cambridge, Royaume-Uni
4
Instituto de Química Física “Rocasolano – Madrid, Espagne
As a contribution towards the visualization of b-amyloid plaques by in vivo imaging techniques for early
detection of Alzheimer’s disease (AD), we have recently reported a promising candidate to be used as a
metal-based multimodal imaging probe for the detection of such plaques (L1). These are stable, noncharged M3+ complexes of a DO3A-monoamide derivative of Pittsburgh compound B (PiB), a wellestablished marker of Ab amyloid plaque [1,2]. (L1) coordinated with Gd3+ showed a good relaxivity (r1),
reasonable binding properties to Ab1-40 and also to human serum albumin (HSA), excellent specificity to
ex vivo amyloid deposits and moderate in vivo brain uptake of its radioactive 111In3+ labeled probe in
normal Swiss mice [3]. Here we report on the synthesis, characterization, and the in vitro interaction with
the amyloid peptide Ab1-40, of Gd3+ complexes formed by another DO3A-monoamide PiB derivatives
(L2) that differ from the original one in the nature and size of the spacer linking the macrocyclic reporter
probe and the PiB targeting moiety.
In this work we describe studies of the interaction of these Ln3+ complexes with the Ab1-40 peptide in the
aggregated or monomeric form by Surface Plasmon Resonance (SPR) and Saturation Transfer
Difference (STD) NMR [4]. The group epitope mapping (GEM) for the corresponding La3+ complexes,
obtained by STD NMR, shows that the complexes interact with immobilized Ab1-40 mainly through the
benzothiazole ring and the attached methoxy group. Their mode of interaction with the 15N-labeled Ab140 peptide monomer was studied at the atomic level using 1H15N Heteronuclear Single Quantum
Coherence (HSQC) NMR. The assessment of their effect on the secondary structure and aggregation
process of Ab1-40 was studied by Circular Dichroism (CD), ThT Fluorescence, Dynamic Light Scattering
(DLS) and Transmission Emission Microscopy (TEM). We show in this study that the complexes interact
weakly with the Ab1-40 peptide monomer, but much more strongly with b1-40 aggregates. They do not
affect its self-association in the same way. They promote the early formation of -helical or b-sheet ordered
structures, depending on their nature and concentration. As a consequence, they show inhibition or
promotion of the formation of amyloid fibrils [57]. These studies also give important clues to improve the
targeted specificity and affinity of this type of multimodal imaging probes.
1. Mathis, C. A.; Bacskai, B. J.; Kajdasz, S.T. et. al. W.E. Bioorg. Med. Chem. Lett. 2002, 12, 295-298.
2. Vandenberghe, R.; Van Laere, K.; Ivanoiu, A. et. al. Ann. Neurol. 2010, 68, 319-29.
3. Martins, A.F., Morfin J.-F., Kub´ıˇckov´a A, et al. ACS Med. Chem. Lett. 2013, 5,436–440.
4. Martins, A.F., Morfin J.-F., Geraldes C. F.G.C., et al. J. Biol. Inorg. Chem.,submitted
5. Lendel, C.; Bolognesi, B.; Wahlstrom, A. et. al. Biochemistry 2010, 49, 1358–1360.
6. Jarvet, J.; Danielsson, J.; Damberg, P.; et. al. J. Biomol. NMR 2007, 39, 63–72.
7. Abelein, A.; Bolognesi, B.; Dobson C.M. et.al. Biochemistry 2012, 51, 126–137.
Myocardial infarction corresponds to an ischemic necrosis of the myocardium as a result of coronary
occlusion. Beyond recurrence prevention and morbidity reduction, research is now focusing on regeneration of
the ischemic area as well as avoidance of left ventricular remodeling and heart failure. For such a purpose, we
studied the feasibility of a polymeric solution directly injected into the myocardium. Formulation will solidify in
situ, fill tissue deficit and offer a scaffold for cell recruitment. Moreover, this formulation is able to release in a
sustain manner a nitric oxide donor, i.e. S-nitrosoglutathione (GSNO), which showed previously benefits in
ischemic pathologies. Rational of GSNO administration in myocardial infarction includes vasodilation, antiplatelet effect and progenitor cells recruitment.
Formulation consisted of biocompatible polymer (poly(lactide-co-glycolide) PLGA 20% (m/m) dissolved in Nmethyl-2-pyrrolidone. GSNO 5% (m/m) was extemporaneously added to this solution. Precipitation of the
polymeric matrix occurs upon contact with water or body fluids. In previous experiments, sustained release of
GSNO was demonstrated both in vitro (102 6% after 24h, n = 3) and in vivo. Subcutaneous injection of GSNO
formulations led indeed to a prolonged but mild reduction of Wistar rats arterial pressure (30h, i.e. a 18-fold
increase in duration compared to GSNO alone, n = 7, telemetry system). Furthermore, scanning electron
microscopy of implants showed surface and internal porosities allowing cell colonization.
To evaluate this latter property, smooth muscle cells (A-10) were seeded onto implants previously formed in
vitro (2 x 105 cells/implant). Viable cells were found on both unloaded and GSNO-loaded matrices, 24 or 48h
after seeding. Cell proliferation at 24h was increased by 4.0 1.5% with GSNO (n = 3). Values were maintained
after 48h (two-way Anova + Bonferoni, pinteraction ns, ptime and pdrug < 5%), despite the reduction of
matrices volume observed within time (-60% after 24h, -85% after 48h vs initial volume).
As the formulation showed a sustained release of GSNO and cytocompatibility, a feasibility study was
conducted in a rat model of myocardial infarction (permanent coronary ligation). One month after surgery, rats
received directly into the infarct area: unloaded formulation (n
= 2), GSNO solution (n = 2; 5 mg/kg), or GSNO-loaded implant (n = 3; 5 mg GSNO/kg). Heart perfusion and
function (ejection fraction EF) were assessed two weeks before (t-2) and after (t+2) injection by positron
emission tomography. For all animals, at t-2, infarction was validated by a decrease of EF from -3% to -40%.
Results indicated that GSNO solution did not improve heart perfusion or function. Unloaded formulations
limited heart function worsening (e.g. EF 50% at t-2, 49% at t+2). As regards GSNO-loaded implants, heart
function was degraded for one rat (EF 42% at t-2, 37% at t+2), conserved for the second one (47%, 47%) and
improved for the third one (37%, 46%). Heart perfusion followed the same trends. These preliminary results
are encouraging and will be confirmed by ongoing experiments.
∗Intervenant
†Auteur correspondant: ariane.boudier@univ-lorraine.fr
-81-
-82-
POSTER B5
POSTER B6
POLYMERIC COMPOSITE BEADS FOR PER OS ADMINISTRATION OF S-NITROSOGLUTATHIONE
LACTOSYL ALBUMIN AS IMAGING AGENT OF THE LIVER FUNCTION
1
1
1
1
1
2
Wen Wu , Caroline Gaucher , Sonia Darelle Toukam , Isabelle Fries , Pierre Leroy , Xian-Ming Hu , Philippe
1
1
Maincent , and Anne Sapin-Minet∗
†1
‡1
Johanne Seguin∗ , Michel Bessodes , Zahra Ait Ramdane, Kathia Lemdani, Philippe Chaumet-Riffaud, Daniel
1
§1
Scherman , and Nathalie Mignet
1
Unité de pharmacologie chimique et génétique et d’imagerie – Inserm, CNRS : UMR8151, Université Paris V - Paris Descartes,
Ecole Nationale Supérieure de Chimie de Paris – Paris, France
1
2
EA3452 “Drug targets, formulation and preclinical assessment” (CITHEFOR) – Université de Lorraine : EA3452 – Faculté de Pharmacie Université de
Lorraine 5, rue Albert Lebrun - 5400 Nancy, France
State Key Laboratory of Virology, Ministry of Education Key Laboratory of Combinatorial Biosynthesis and Drug Discovery – Wuhan University School of
Pharmaceutical Sciences - 430071Wuhan, Chine
The benefic role of nitric oxide (NO) in the treatment of cardiovascular diseases and as a chemical messenger
on cell signaling is already known[1]. Unfortunately, the low stability of this radical and its derivatives NO
donors such as S-nitrosothiols, constitute a limitation for its therapeutic administration and chronic treatment
establishment [2]. Pharmaceutical design and drug delivery systems, such as polymeric particles, can protect
and control the release of various active compounds. The present study proposes the development of
composite polymeric particles for oral delivery of S-nitrosoglutathione (GSNO). This composite form is
constituted by two different polymers adapted to oral route [3-5]: poly(methyl)metacrylate GSNO nanoparticles
(obtained by double emulsion method) are encapsulated into calcium alginate beads (obtained by ionic
gelation process). The integrity and biodisponibility of encapsulated GSNO into nanoparticles was verified
following protein S-nitrosation (i.e. NO transfer to the protein thiol group) in intestinal cell line model (Caco-2
cells). This protein post-translational modification is crucial for cell signaling pathways and NO physiological
functions. During 24 h of exposure, the intracellular protein S-nitrosation induces by GSNO released by
nanoparticles was maintained at a constant level (around 100 nmol/mg protein). In parallel, an optimization of
the gelation process was performed to define to the most adapted parameters (needle size equal to 21 G and
sodium alginate concentration equal to 1% (w/w)) for encapsulation of the GSNO nanosuspension into
alginate beads. The obtained GSNO nanoparticles and composite beads presented a size equal to 3367 nm
and around 1 mm, respectively. The in vitro release study was then performed during 24 h and showed an
increase in the sustained release of GSNO by composite particles when compared to GSNO nanoparticles,
certainly due to a larger diffusion distance (composite particles and nanoparticles released 30% and 100% of
encapsulated GSNO within 6 h, respectively). Therefore, protection and sustained release of NO in the
gastrointestinal tract could be achieved by this new composite form associating NO donor nanoparticles and
alginate bead.
Assessment of the liver function is not directly accessible, but instead it has been routinely evaluated by indirect
set of biochemical measurement and/or by hepatic biliary clearance of indocyanine green. In Corea and Japan
however, the liver function is measured directly thanks to a dedicated imaging agent targeting the hepatocytes
[1]. Few years ago, we obtained a similar agent based on an albumin support [2]. Our novelty was the process
which in one short step, allowed to obtain a targeted and labelled protein devoided of undesirable by-products,
while maintaining the number of charges of the protein.
In this work, we wanted to investigate an optical agent of the liver function for animal imaging.
Addition of fluorophores to a protein often induces aggregation and non-covalent binding. We addressed these
points by varying the ratio between cyanin and albumin. Mass spectrometry analysis by Maldi and
spectrofluorescence experiments were performed. We grafted a lactosyl maleimide derivative as targeting
ligand. The imaging agent was then injected intravenously to healthy mice and the results compared to our
previous data obtained by scintigraphy.
We found that more than two fluorophores was damaging as the level of fluorescence diminished with the
amount of fluorophores linked to the protein. Optimising the conditions of coupling the fluorophores also allowed
obtaining only covalently linked derivatives. The number of lactosyl derivatives was found to be 16 to 20
derivatives per protein. The lactosyl albumin accumulated into the liver in few minutes while the non-targeted
albumin taken as a control continued to circulate in whole organs, which was consistent with what we had
obtained by scintigraphy. An optical imaging agent of the liver function was developed. This agent was validated
by comparison with the data obtained with its radioactive counterpart labeled with 99mTc.
1.
2.
Jeong JM, Hong MK, Lee J, Son M, So Y, Lee DS, Chung JK, Lee MC. 99mTc-neolactosylated human serum albumin for imaging the
hepatic asialoglycoprotein receptor. Bioconjug Chem. 2004;15(4):850-5.
[2] Chaumet-Riffaud P, Martinez-Duncker I, Marty AL, Richard C, Prigent A, Moati F, Sarda-Mantel L, Scherman D, Bessodes M,
Mignet N. Synthesis and application of lactosylated, 99mTc chelating albumin for measurement of liver function. Bioconjug Chem.
2010;21(4):589-96.
1. Heinrich TA., da Silva RS, Miranda KM, Switzer CH, Wink DA, Fukuto JM, (2013) Biological nitric oxide signalling: chemistry and
terminology British Journal of Pharmacology, 169, 1417-1429 .
2. Gaucher C, Boudier A, Dahboul F, Parent M, Leroy P, (2013) S-nitrosation/Denitrosation in Cardiovascular Pathologies: Facts
and Concepts for the Rational Design of S-nitrosothiols. Current Pharmaceutical Design, 19, 458-472.
3. Viehof A, Javot L, Beduneau A, Pellequer Y, Lamprecht A, (2013) Oral insulin delivery in rats by nanoparticles prepared with
non-toxic solvents. International Journal of Pharmaceutics, 443, 169-174.
4. Collnot EM, Ali H, Lehr CM, (2012) Nano and microparticulate drug carriers for targeting of the inflamed intestinal mucosa
Journal of Controlled Release, 161, 235-246.
5. [5] Wong T W, (2011) Alginate graft copolymers and alginate-co-excipient physical mixture in oral drug delivery, Journal of
Pharmacy and Pharmacology, 63, 1497-1512.
∗Intervenant
†Auteur correspondant: johanne.seguin@parisdescartes.fr
‡Auteur correspondant: michel.bessodes@parisdescartes.fr
§Auteur correspondant: nathalie.mignet@parisdescartes.fr
∗Intervenant
-83-
-84-
POSTER B7
POSTER B8
USE OF F-TAC OLIGOMERS FOR THERANOSTICS DEVELOPMENT
1
2
2
2
5-AMINOSALICYLIC ACID RELEASE PROFILE FROM XYLAN AND XYLAN/EUDRAGIT R S100
MICROPARTICLES PRODUCED BY CHEMICAL AND PHYSICOMECHANICAL APPROACHES
2
Stéphane Desgranges , Ksenia Astafyeva , Lucie Somaglino , Nicolas Taulier , Wladimir Urbach ,
Ange Polidori1, and Christiane Contino-Pepin∗†1
1
1,2
Institut des Biomolécules Max Mousseron (IBMM) – CNRS : UMR5247, Université Montpellier I, Université Montpellier II - Sciences et techniques Faculté de
pharmacie 15, Avenue Charles Flahault- 34060 Montpellier, France
2
Laboratoire d’Imagerie Paramétrique (LIP) – IFR58, CNRS : UMR7623, Université Pierre et Marie Curie (UPMC) - Paris VI –
15 Rue de l’école de Médecine - 75006 Paris, France
Cancer remains one of the leading causes of mortality worldwide. Early detection of tumor development plays a
key role in therapy success; moreover, the efficiency of many chemotherapeutic agents is hindered by their low
therapeutic window, hence there is a growing need for controlled drug delivery systems.1 In this context, the
development of sophisticated tools called “theranostics” combining therapeutic and diagnostic capabilities into a
single construct appears to be a promising strategy for the future of cancer treatment.2
By this way, we focused our work on the development of stable nano-droplets of liquid perfluorocarbon
dispersed in aqueous solution thanks to a shell resulting from the self-assembling properties of amphiphilic
fluorinated oligomers called “F-TAC telomers”.3 When exposed to variable intensities of incident ultrasound
waves, these nano-emulsions can be used as ultrasound-mediated theranostics with “two in one” medical
applications: (i) low intensity ultrasound waves provide echogenicity according to the difference in acoustic
impedance between the perfluorocarbon encapsulated within the shell and the surrounding tissues, (ii) highpower ultrasound waves focused at a specific site like a solid tumor or its surrounding vasculature trigger the
disruption of the nano-emulsion. If a hydrophobic cytotoxic agent is encapsulated into the liquid core, it can
hence be selectively delivered at its site of action.4
We will report herein the synthesis and structural characterization of a series of F-TAC telomers considered as
promising tools for ultrasonic theranostics development. Some preliminary physicochemical results of the
resulting nano-emulsions (size distribution and stability, acoustic echogenicity, encapsulation ability ...) will also
be described.
1.
2.
3.
P.S. Sheeran, S.H. Luois, L.B. Mullin et al. Biomaterials. 2012; 33, 3262-9. R. Duncan, R. Gaspar. Mol. Pharmaceutics 2011, 8, 21012141.
C. Contino-Pépin, J. C. Maurizis, B. Pucci. Curr. Med. Chem. 2002, 2, 645-665.
R. Diaz-Lopez, N. Tsapis, D. Libong, P. Chaminade, C. Connan, M.M.Chehimi, R. Berti, N. Taulier, W. Urbach, V. Nicolas, E. Fattal.
Biomaterials 2009, 30, 1462-1472.
1,2
2,3
1
-85-
2,5
Universidade Federal do Rio Grande do Norte (UFRN) – R. Gal. Cordeiro de Farias, Departamento de Farmacia. Petropolis, Natal-RN. 59012-570, Brésil
2
Laboratorio de Sistemas Dispersos - UFRN (LaSiD) – Brésil
3
Programa de Pos Graduac¸ao em Ciências Farmacˆeuticas (PPGCF) – Brésil
4
Rede Nordeste de Biotecnologia (RENORBIO) – Brésil
5
Programa de Pos Graduacão em Ciências da Saúde (PPGCSA) – Brésil
Polymeric drug carriers have been described as able to modify the bioavailability of encapsulated or entrapped
drugs. Such ability is intrinsically related to the control of the drug release from the polymeric matrix. Both the
complex interaction among the formulation materials and the methods used to produce the polymeric drug
carrier plays an important role on the drug release profile, which is an essential and mandatory step for its future
use on clinical trials. The aim of this work was to evaluate the influence of three different manufacturing
approaches on the release of 5-aminosalicylic acid (5-ASA) from microparticle based carriers. Xylan and xylanEudragit ® S-100 (ES100) were used as polymeric sources. The approaches used to the production of the
microparticles were (i) interfacial crosslinking (B1); (ii) interfacial crosslinking followed by spray-drying (B2); and
(iii) one-step spray-drying with xylan: ES100 (ratios 1:1 and 1:3) for B3 and B4 formulation, respectively. Fifteen
milligrams of 5-ASA were added to all formulations. The in vitro drug release assay, for the formulations B2, B3
and B4, was performed by the addition of 40mg of microparticles into a beaker with 30 mL of phosphate buffer
pH = 7.4, under magnetic stirring. For the batch B1 distilled water media was used. Aliquots were withdrew at
time 0.5, 1, 2, 3, 4, 5 and 24 h, centrifuged at 2,300g for 3 min, and analyzed by spectrophotometry at 328 nm
(the withdraw volume was replaced to maintain sink conditions). The experiments were perfomed in triplicate.
Higuchi model was able to fit data obtained from B1, in which almost all drug content was released after around
40h. On the other hand, the release profile for B2, B3 and B4 showed an unexpected profile, in which all the
drug content was released as soon as the powder was added into the media. The probable reason for this
behavior is the presence of remains of NaOH used to dissolve the polymers. In fact, pH measurements of the
release media after the assay, for these formulations, revealed that the pH were around 9.0, which would
completely dissolve the microparticles structure due to the polymer dissolution. Besides the unexpected results
revealed by three formulations, the approach B1 seems to be a promising one to produce 5-ASA loading drug
carriers to future use.
∗Intervenant
†Auteur correspondant: socratesegito@gmail.com
∗Intervenant
†Auteur correspondant: christine.pepin@univ-avignon.fr
2,4
Bartolomeu Souza∗ , Karen Silva , Francisco Alexandrino-Júnior , André Silva∗ , Henrique Marcelino ,
†1,2,3,4,5
and Eryvaldo Egito
-86-
POSTER B9
DEVELOPMENT OF NANOEMULSIONS CONTAINING 8-METHOXYSALEN FOR TOPICAL TREATMENT
OF VITILIGO
POSTER B10
ANTI-ABETA ANTIBODY DECORATED NANOPARTICLES FOR ALZHEIMER DISEASE: IN VIVO
PROOF OF CONCEPT
Karine Andrieux∗
Thaís
1
Barradas∗ ,
†2
André Silva , Kattya Silva, Júlia Conceicão, and Cláudia Mansur
Institut Galien Paris-Sud UMR CNRS 8612 – Université Paris Sud - Paris XI – 5, rue Jean-Baptiste Clément - 92290 Chatenay-Malabry, France
1 Federal University of Rio de Janeiro (UFRJ) – Rio de Janeiro, P.O. Box 68525, 21945-970, Brésil
2Federal University of Rio Grande do Norte (UFRN) – LaSiD - DFAR. R. Gal. Cordeiro de Faria, Petropolis. Natal/RN. 59012-570, Brésil
The aim of this work was to synthetize nanoparticles (NPs) able to interact with the A peptide 1-42 in order to
increase its elimination and to correct the memory defect observed in Alzheimer Disease.
Vitiligo is the most frequent depigmenting disorder affecting 0.5-2% of the population worldwide and is
cosmetically and psychologically devastating. There are two main goals of any vitiligo treatment: (i) to stop the
arrest of further depigmentation and (ii) to induce repigmentation1. The main topical treatment for vitiligo is
based on a therapy with a psolaren such as 8-methoxalen (8-MOP) associated with ultraviolet A radiation. It is
currently approved in different dosage forms for topical use. However, this approach has presented some
disadvantages, such as frequent phototoxicity and slow response2. The effective treatment requires prolonged
use of topical of 8-MOP. Nanostructured systems are able to promote controlled release and favor the follicular
penetration, which cannot be achieved with conventional dosage forms3;4. In this work we developed and
characterized of nanoemulsions (NE) for prolonged release of 8-MOP. Oil/water nanoemulsions were prepared
by high pressure homogenizer (HPH). All systems were characterized: the hydrodynamic size and
polydispersion index (PdI) were determined by dynamic light scattering, in a Zetasizer Nano ZS (Malvern
Instruments, UK); the structure and morphology of samples were studied by Transmission Electron Microscopy
and the rheological behavior was performed in a RS 600 Rheometer, Haake, using a cone/plate accessory, with
cone diameter 35 and angle of 1°. In vitro drug release studies were carried on a vertical diffusion system. For
selecting the appropriate NE components, solubility of 8-MOP was estimated in various oils and it presented
highest solubility in clove oil. The NE presented droplet sizes of about 100 nm, PdI ranging from 0,094 to 0,116.
Additionally, it was observed that some formulations were stable depending on the number of processing cicles
on HPH. The globule size of NE should be as small as possible to penetrate into the deeper layers of skin. All
the samples presented shear thinning rheological behavior. The results indicate that the formulations developed
in this study represent a promising approach for the topical treatment of vitiligo. Furthermore, the composition of
NE showing highest solubility of drug would probably result into its reduced partitioning into the skin layers, and
majority of the drug will be retained in the vehicle after application of the formulation.
1.
2.
3.
4.
Biotin-functionalized NPs were prepared by nanoprecipitation. Streptavidin- fluorescein isothiocyanate
(SavFITC) was coupled with anti-A monoclonal antibody (anti-A mAb). Biotin-NPs and SavFITC – anti-A mAb
conjugate were linked in a nanoconstruct complex purified by ultracentrifugation and verified by
spectrofluorimetry. The affinity of nanoparticles for Abeta peptide was in vitro evaluated by SPR. The
biodistribution of radiolabelled nanocarriers was determined by one IV injection in mice. Finally, anti-A mAb NPs have been evaluated in vivo on transgenic mice models (Tg2576 mice, 15 months old) receiving
treatment (100 L) by IV 3 days by week during 3 weeks.
Biotin- NPs showed a size around 100 nm, a good stability, a biotin amount of 9.6•1014 molecules by sample.
The decorated NPs with anti-A mAb have a similar size and are stable in water. These nanocarriers have a
very high affinity for monomers (Kd=700pM) and fibrils (Kd=300pM) of Abeta peptide as shown by SPR. Long
circulation in plasma (PEG effect). Their in vivo biodistribution revealed a higher capture by spleen than by
liver and a passage in brain of antiA- NPs. The object recognition memory test evidenced a complete
correction of the memory defect on transgenic mice.
Anti-A mAb decorated NPs are promising for Alzheimer disease treatment. Their mechanism of action would
be evidenced in further experiments.
Alikhan, A. et al. Journal of the American Academy of Dermatology, 65(3), p. 473-491, 2011.
Wolff, K. British Journal of Dermatology, 122, p. 117-125, 1990.
Fang J.-Y. et al. European Journal of Pharmaceutics and Biopharmaceutics, 70(2), p. 633-640, 2008.
Lu, Y.; PARK, K. International Journal of Pharmaceutics, n.65, p.473-491, 2012.
∗Auteur correspondant: thaisbarradas@ima.ufrj.br
†Intervenant
∗Intervenant
-87-
-88-
POSTER B11
POSTER B12
ONE-SHOT PROCEDURE FOR DEXTRAN, PULLULAN AND AMYLOPECTIN MICROASSEMBLY AND
NANOASSEMBLY FORMATION
AUTO-ASSOCIATIVE ASSEMBLIES: A BIOMIMETIC PLATFORM AGAINST THE HEPARAN-SULFATEDEPENDENT VIRUSES HSV-1, HSV-2 AND HPV-16
Martina Bombardi∗, Narimane Zeghbib, and Kawthar Bouchemal
David Lembo , Manuela Donalisio , Claire Laine , Valeria Cagno , Andrea Civra , Narimane Zeghbib , and
†2
Kawthar Bouchemal∗
1
1
2
1
1
2
1 Institut Galien Paris Sud (UMR CNRS 8612) – Université Paris Sud – 5, rue Jean-Baptiste Clément - 92296 Chatenay-Malabry, France
2
Although the large number of publications in the field of drug delivery systems, the current methods to produce
microparticles and nanoparticles, have, unfortunately, important limitations due to the complexity of the
processes used to manufacture the drug delivery device and thus the difficulty for the scaling-up of the
production to the industrial sector. A novel method to produce polysaccharide microassemblies and
nanoassemblies was described in the present work. They were obtained by the self-association in aqueous
medium of and -cyclodextrin and O-palmitoyl-dextran, O-palmitoyl-pullulan or Opalmitoyl-amylopectin. The
particles are formed at room temperature, without using surfactants, solvents or pH modification and without
heating or purification steps.Attenuated total reflectance-Fourier transform infrared (ATR-FTIR)
spectroscopyand high-resolution solid-state 13C-NMR were used to confirm that palmitoyl moieties are
covalently bonded to dextran, pullulan or amylopectin. Assembly-mean hydrodynamic diameter was
modulated from 200 nm to 3 m by modifying the ration between the O-palmitoyl-saccharide and-cyclodextrin.
Whatever the size of the assemblies, well-structured crystalline hexagonal assemblies were observed by
transmission electron microscopy.
1
Department of Clinical and Biological Sciences – Regione Gonzole 10 - 10043 Torino, Italie
Institut Galien Paris Sud (UMR CNRS 8612) – Université Paris Sud – Faculté de Pharmacie, 5, rue Jean-Baptiste Clément, 92296 Chatenay-Malabry, France
Heparan-sulfate (HS) dependent viruses such as HSV-1, HSV-2, HPV-16 and RSV interact with HSproteoglycan receptors on the epithelial cells facilitating thus their initial attachment, cellular entry and
infection. In the present work we propose a new approach to design locally-administrated HS-mimetic
formulations able to specifically target the viruses and avoid their attachment to the mucosal surfaces. A new
method was thus developed for the manufacturing of heparin assemblies active against the heparan-sulfatedependent viruses HSV-1, HSV-2, RSV and HPV-16. These assemblies were obtained by the autoassociation of O-palmitoyl-heparin and -cyclodextrin. The synthesized O-palmitoyl-heparin derivatives mixed
with -cyclodextrin resulted in the formation of crystalline hexagonal assemblies as observed by transmission
electron microscopy. The assembly mean hydrodynamic diameters were modulated from 340 to 3175nm
depending on the type and the initial concentration of O-palmitoyl-heparin or -cyclodextrin. The concentration
of the components did not affect the antiviral activity of the assemblies towards HSV-1, HSV-2, RSV and HPV16.
∗Intervenant
†Auteur correspondant: kawthar.bouchemal@u-psud.fr
∗Intervenant
-89-
-90-
POSTER B13
POSTER B14
SYNTHESIS OF AMPHIPHILIC COPOLYMERS TO FORMULATE NO-GRAFTED MICELLAR NANOOBJECTS
ENCAPSULATION OF HSP90 INHIBITOR LOADED NANOLIPOSOMES ; EVALUATION OF THE
CYTOTOXIC EFFECT IN VITRO ON BREAST AND PROSTATE CANCER CELLS
†1
‡2
3
1
1
1
1Laboratoire de chimie physique macromoléculaire (LCPM FRE3564) – Université de Lorraine : FRE3564, CNRS : FRE3564 – Nancy, France
2CITHEFOR (EA3452) – Université de Lorraine : EA3452 – Faculté de Pharmacie, Université de Lorraine, BP 80403 - 54001 Nancy, France,
2
HSP 90 is a heat shock protein involved in folding and maintaining the active conformation of proteins under
stress (thermic stress, pH variation, hypoxia...). Among HSP 90’s client proteins, many are involved in
oncogenic processes like metastatic phenomena, invasion and immune escape which contribute greatly to the
complexity cancer diseases. Thus, HSP 90 is an interesting target for the treatment of cancer since, on one
hand, its inhibition allows a multilateral approach and, on the other hand, it has a higher affinity for proteins and
inhibitors in cancer cells compared with normal ones, (1). Recently, several HSP 90 inhibitors like
geldanamycine have been synthesized, some of which are undergoing clinical trials. 6Br- CaQ is an inhibitor
synthesized by the team of Chimie thérapeutique (collaboration with Dr. Samir Messaoudi, BioCIS UMR 8076)
at the Faculty of Pharmacy in Chatenay-Malabry (University Paris-Sud) (2). This inhibitor binds the C-terminal
domain of the protein, thus avoiding a ”rebound” after the inhibition stops. 6BrCaQ was observed to induce a
G2/M arrest in breast and prostate cancer cell lines (MDA-MB-231 and PC-3, respectively) after 24 hours of
treatment by a high concentration of inhibitor (50 M) and after 72 hours by a lower concentration (10 M). At the
highest concentration, PARP-1 and caspase-3 cleavage were detected by Western Blot, indicating that
apoptosis was induced). The encapsulation of 6BrCaQ in stealth liposomes overcomes its low water solubility
and contributes to a better effect of the molecule at low concentrations. Several methods were used to evaluate
the cellular effect of 6BrCaQ: metabolic activity assay (MTS), cell counting after Trypan Blue dead cell exclusion
and cell cycle analysis (Flow cytometry). The cytostatic effect (G2/M arrest) was significant at 5 and 10M after
24h treatment although apoptosis was not observed. The migration and invasion properties of breast and
prostate cancer cells contribute to tumor resistance. Therefore 6BrCaQ-loaded liposomes are currently being
tested in a wound healing assay in which the migration is induced by a chemokine, MCP-1. In a metabolic
activity assay, a combination of the two free molecules exerted a higher cytotoxic effect than each molecule
alone. The good anti-tumor activity of 6BrCaQ combined with the tumor targeting properties of liposomes
strongly encourage the development of this molecule as a new cancer therapy.
Kamal, 2004
Audisio, 2011
∗Intervenant
∗Intervenant
-91-
1
Univ Paris-Sud, Institut Galien Paris-Sud UMR-CNRS 8612, LabEx LERMIT, Faculté de Pharmacie, 5, rue Jean-Baptiste Clément –
92296 Chatenay-Malabry, France
Univ Paris-Sud, CNRS, BioCIS-UMR 8076, LabEx LERMIT, Laboratoire de Chimie Thérapeutique, Faculté de Pharmacie, 5, rue Jean-Baptiste Clément 92296 Chatenay-Malabry, France
(1)
(2)
†Auteur correspondant: cecile.nouvel@univ-lorraine.fr
‡Auteur correspondant: Ariane.Boudier@univ- lorraine.fr
1
1
2
Recently several S-nitrosothiols (RSNO) have shown high potential for nitric oxide (NO) delivery in
cardiovascular field. Unfortunately they lack of stability. This communication will introduce the first works on
the synthesis of a new amphiphilic block copolymers to formulate NO-grafted micelles. The nano-objects will
be cross-linked to enhance stability in blood stream. The cross-linking and the NO attachment via sulfurnitrogen bond will be redox-sensitive to obtain a sustained release at the targeted site (near muscle cells of
vessel).
1
Félix Sauvage* , Silvia Franze , Samir Messaoudi , Stéphanie Denis , Baptiste Martin ,
2
2
1
1
Jean-Daniel Brion , Mouad Alami , Gillian Barratt and Juliette Vergnaud-Gauduchon
Cécile Nouvel∗ , Ariane Boudier , Maéva Chapon , Ferji Khalid , and Jean-Luc Six
-92-
POSTER B15
POSTER B16
TAZAROTENE ENCAPSULATION IN POLYLACTIC-ACID (PLA) NANOPARTICLES FOR PSORIASIS
TREATMENT, IN VITRO AND EX VIVO ASSAYS
†1
1
1
1
Anne-Sophie Boisgard∗ , Vincent Lahaye , Carine Mainzer , Celine Terrat , Fiorenza
2
2
‡1
Rancan , Annika Vogt , and Bernard Verrier
POLYMERIC MICELLE NANOCARRIERS FOR THE TARGETED TOPICAL CUTANEOUS DELIVERY OF
TACROLIMUS
Maria Lapteva∗, Karine Mondon, Michael Möller, and Yogeshvar Kalia
†
School of Pharmaceutical Sciences, University of Geneva University of Lausanne – 30 Quai Ansermet - 1211 Geneva, Switzerland
1
Biologie Tissulaire et Ingénierie Thérapeutique (LBTI) – CNRS : UMR5305 – 7 Passage du Vercors - 69367 Lyon, France
2
Department of Dermatology and Allergy, Clinical Research Center for Hair and Skin Science (CRC) – Charité – Universitätsmedizin Berlin, Allemagne
Psoriasis is a common skin pathological condition, affecting around 2-3% of the worldwide population. It is a
chronic, immune-mediated inflammatory disease, with a familial predisposition, and has a predilection for
specific body areas such as elbows, knees and scalp. Tazarotene belongs to the pharmaceutical category of
acetylenic retinoids, developed for the topical treatment of psoriasis. As vitamin A analogs, retinoids have a
wide range of biological activities on cellular growth, differentiation, immune functions and malignant
transformation. Tazarotene has a selective affinity for retinoic acid receptors RARb and RARg, liganddependent transcription factors able to induce or down regulate gene expressions. Tazarotene also has an upregulating action on three genes: TIG-1 (Tazarotene-induced gene-1), TIG-2 and TIG-3, which results in an antiproliferative effect. During this work, we encapsulated Tazarotene in Poly(Lactic Acid) (PLA) nanoparticles.
These particles have previously been investigated, and are suitable for local dermatotherapy. They have a
tendency to accumulate preferentially into hair follicles, thus improving the therapeutic index for drug in
dermatotherapy. Our goals are both to improve Tazarotene’s skin distribution and penetration in epidermis and
dermis, and to reduce the adverse events related to its administration.
Tazarotene is encapsulated during the nanoprecipitation process used for manufacturing PLA nanoparticles,
after dissolution in an organic phase (acetone). The encapsulation rates of Tazarotene were measured by
HPLC and drug release study was performed in different buffers, using HPLC as a quantification method.
Expression of TIG-1 was evaluated in vitro on keratinocytes by Immunocytochemistry (ICC) and ex vivo on
human skin explants by immuno-staining. Skin penetration of fluorescent nanoparticles (Coumarin-6) was
investigated using fluorescence microscopy.
We achieved efficient and reproducible encapsulation of Tazarotene in PLA nanoparticles, with a loading
efficiency above 60%. In vitro drug release study showed that encapsulation of Tazarotene remained stable in
physiological pH environment for 12 hours. Fluorescence microscopy revealed a more important concentration
of encapsulated agents in stratum corneum and epidermis compared with the same agents in solution. This
suggests that PLA nanoparticles are able to limit the diffusion of the encapsulated drugs and to target
specifically the epidermis and dermis. ICC and immuno-staining assay of TIG-1 showed that the gene
expression is activated by both Tazarotene in solution and encapsulated in PLA nanoparticles. This suggests
that Tazarotene encapsulation in PLA nanoparticles induces no alteration of its biological activity.
Psoriasis is an auto-immune mediated skin disorder where the mitotic rate of the basal keratinocytes is
abnormally high, resulting in inflamed lesions covered by silvery- white scales. Many inflammatory cells
including dendritic cells, macrophages and T cells are present in the dermis. Unlike in healthy skin, some T
cells can also be found in the epidermis in psoriatic skin. Tacrolimus (TAC) is a potent macrolide
immunosuppressant that inhibits the T-cell mediated immune response and is used off-label for the topical
treatment of psoriasis. However, it suffers from poor topical bioavailability when administered using
conventional vehicles.
The objective of this study was to formulate and to characterize TAC-loaded polymeric micelles made using
the biodegradable and biocompatible MPEG-dihexPLA diblock copolymer and to then evaluate their potential
for the targeted delivery of TAC into the skin.
TAC-loaded micelles were prepared by the solvent evaporation method. TAC was quantified by an in-house
HPLC-UV method. Micelles were characterized in terms of drug content and stability. Size and morphology
were investigated by DLS and by TEM. The micelle formulation was optimized so as to reach a TAC content of
0.1% in order to enable direct comparison with the Protopic ® ointment (0.1 %). In vitro skin transport
experiments were performed first using porcine ear and then human abdominal skin. TAC delivery to skin and
its biodistribution as a function of depth (in the epidermis and upper dermis) was quantified by using a
sensitive UPLC MS/MS method developed in-house to quantify the drug in skin samples.
Spherical micelles with diameters of 10-50 nm and drug loadings ranging from 44.72 0.16 to 233.21 0.64
mgTAC/gcopolymer were formulated. An optimal 0.1% micelle formulation was developed and proved to be
stable over a period of 7 months at 4C. Delivery of TAC into the skin from the optimal 0.1% micelle formulation
was statistically superior to that from Protopic® in both porcine (0.47 0.2 and 4.03 0.88 g/cm2, respectively)
and human skin (0.36 0.05 and 1.50 0.59 g/cm2, respectively). No TAC was detected in the receiver
compartment. The skin biodistribution profile of TAC was also improved using the micelle formulation,
significantly increasing the drug deposited in the stratum corneum (250.7 84.8 ng/cm2) and viable epidermis
(527.9 148.4 ng/cm2) in comparison to Protopic® (68.1 22.2 and 260.4 54.22 ng/cm2, respectively).
MPEG-dihexPLA micelles are extremely efficient nanocarriers that are able to incorporate significant amounts
of TAC, deliver it more efficiently into the skin than Protopic® , and importantly, target the viable epidermis,
where the T cells reside.
This preliminary study showed that PLA nanoparticles lead to an accumulation of Tazarotene in epidermis. The
particles have no negative impact on the biological activity of Tazarotene. For these reasons, PLA nanoparticles
appear to be good candidates for topical delivery of Tazarotene.
∗Intervenant
†Auteur correspondant: boisgard.as@gmail.com
‡Auteur correspondant: bernard.verrier@ibcp.fr
∗Intervenant
†Auteur correspondant: Yogi.kalia@unige.ch
-93-
-94-
POSTER B17
POSTER B18
FORMULATION OF ALGINATE-BASED NANOCAPSULES FOR TOPICAL DELIVERY
1
1
1
Hoang Truc Phuong Nguyen , Emilie Munnier∗ , Xavier Perse , Simone
Cohen-Jonathan1 , and Igor Chourpa1
CURCUMIN NANOCARRIERS DESIGNED FOR A TOPICAL USE AND THEIR STABILITY IN HYDROGELS
∗ †1
Hoang Truc Phuong Nguyen, Emilie Munnier , Xavier Perse, Martin Souce, Igor
Chourpa, and Simone Cohen-Jonathan
1 EA 6295 Nanomédicaments et Nanosondes-Université de Tours (EA 6295 NMNS) –UFR des Sciences Pharmaceutiques 31 avenue Monge 37 Tours, France
1 EA 6295 Nanomédicaments et Nanosondes-Université de Tours (EA 6295 NMNS) –UFR des Sciences Pharmaceutiques 31 avenue Monge 37 Tours, France
Some active pharmaceutical or cosmetic active ingredients could have their performances increased if they
were included into entities that would help them reaching a particular layer of the skin, or that would increase
their physico-chemical stability inside the final galenic form. Natural polysaccharides constitute a very potent
material to create nanodevices allowing the preservation of an API in a formula and the release of this one into
the skin. In the frame of the NANO’derm project, we chose to develop new nano-objects from alginates, a wellknow ingredient currently used as a gelifying agent. Alginates have the capacity to reticulate in presence of
divalent cations. We used a preparation technique based on a nanoemulsion obtained with ultrasounds
without any organic solvent. The calibrated nanoemulsion is solidified with an alginate envelope and contains
curcumin as a model of active ingredient. This molecule is an interesting model because of its optical
properties and its antioxidant activity. A design of experiment methodology was used to analyse the influence
of the formulation parameters on the fundamental physicochemical characteristics of the nano-objects. The
optimized nano-objects were then characterized with different techniques to evaluate their potential: their DH
is close to 140 nm and their -potential close to -30 mV. The shape was observed by TEM, and the loading and
release capacity were studied. Their ability to lyophilisation was also explored. Those nano-objects were
included then into different galenic formula to study their stability in complex media, and their release
properties in these conditions.
Curcumin is a natural molecule extracted from Curcuma longa roots. It exhibits numerous properties that
makes it of interest in dermatology and in cosmetics. Nevertheless, the poor skin penetration and water
solubility of curcumin limit its topical use. The encapsulation of curcumin in nanocarriers could facilitate its
incorporation in hydrogels and increase its skin penetration. Therefore, we encapsulated curcumin lipidic
nanocapsules that could be o potent tool for the skin penetration of active molecules. Spectroscopic methods
and zetametry were used to characterize curcumin loaded LNC (LNC-C). The antioxidant activity of the
encapsulated curcumin was measured with two complementary techniques, testifying that it does not lose its
pharmacological properties during the process. LNC-C were then dispersed in hydrogels currently used in
pharmaceutical products and cosmetics. The physical stability and the rheological behavior of the LNC –
enriched gels was studied in different storage conditions over 6 months (RT,4C and 30C). The size of the geldispersed LNC was followed by DLS and showed that they were not destroyed in the process or in the storage
conditions, and the analysis of the fluorescence spectrum of curcumin showed if it remained encapsulated in
the nanocarriers. We highlighted a hydrogel that seems a good candidate to vehicle curcumin-loaded LNC.
We tested this galenic form on a skin in vitro model, Episkin.
∗Intervenant
†Auteur correspondant: emilie.munnier@univ- tours.fr
∗Intervenant
-95-
-96-
POSTER B19
POSTER B20
POLYMERIC COMPOSITE BEADS FOR PER OS ADMINISTRATION OF S-NITROSOGLUTATHIONE.
NANOHYBRID BASED ON ZN(FE)O PULLULAN GRAFTED NANOPARTICLES AS POTENTIAL
LUMINESCENT AND MAGNETIC BIMODAL IMAGING PROBES: SYNTHESIS, CHARACTERIZATION,
MODIFICATION AND CYTOXICITY STUDY
Wu Wen, Caroline Gaucher, Sonia Toukam, Isabelle Fries, Pierre Leroy, Ming Hu, Philippe Maincent, and
Anne Minet
Imen Balti1
Université Paris 13 – France
“Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie (CITHEFOR EA3452) – Université de Lorraine – France
The benefic role of nitric oxide (NO) in the treatment of cardiovascular diseases and as a chemical messenger
on cell signaling is already known [1]. Unfortunately, the low stability of this radical and its derivatives NO
donors such as S-nitrosothiols, constitute a limitation for its therapeutic administration and chronic treatment
establishment [2]. Pharmaceutical design and drug delivery systems, such as polymeric particles, can protect
and control the release of various active compounds. The present study proposes the development of
composite polymeric particles for oral delivery of S-nitrosoglutathione (GSNO). This composite form is
constituted by two different polymers adapted to oral route [3-5]: poly(methyl)metacrylate GSNO nanoparticles
(obtained by double emulsion method) are encapsulated into calcium alginate beads (obtained by ionic gelation
process). The integrity and biodisponibility of encapsulated GSNO into nanoparticles was verified following
protein S-nitrosation (i.e. NO transfer to the protein thiol group) in intestinal cell line model (Caco-2 cells). This
protein post-translational modification is crucial for cell signaling pathways and NO physiological functions.
During 24 h of exposure, the intracellular protein S-nitrosation induces by GSNO released by nanoparticles was
maintained at a constant level (around 100 nmol/mg protein). In parallel, an optimization of the gelation process
was performed to define to the most adapted parameters (needle size equal to 21 G and sodium alginate
concentration equal to 1% (w/w)) for encapsulation of the GSNO nanosuspension into alginate beads. The
obtained GSNO nanoparticles and composite beads presented a size equal to 3367 nm and around 1 mm,
respectively. The in vitro release study was then performed during 24 h and showed an increase in the
sustained release of GSNO by composite particles when compared to GSNO nanoparticles, certainly due to a
larger diffusion distance (composite particles and nanoparticles released 30% and 100% of encapsulated
GSNO within 6 h, respectively). Therefore, protection and sustained release of NO in the gastrointestinal tract
could be achieved by this new composite form associating NO donor nanoparticles and alginate bead.
During the past decades, the development of multifunctional nano-tools able to perform, at the same time,
optical and magnetic tasks, has gained a significant attraction (for instance as probes for theranostic
applications), where they simultaneously act as luminescent probes for optical imaging and contrast agents for
magnetic resonance imaging (MRI). Bioconjugated QDs, because of their photoluminescence, have been
already used for ultrasensitive biological labeling, multicolor optical detection. In parallel, Fe cations have been
already considered, in the form of either superparamagnetic, as efficient positive contrast agents to improve
MRI sensitivity in medicine. They accelerate the relaxation of the nuclear magnetic moment of water protons in
their proximity, thereby greatly increasing the contrast between specific tissues or organs [1].
∗Intervenant
∗Intervenant
-97-
We propose to produce Zn(Fe)O nanocrystals (see Figure 1) by the polyol method, and to evaluate their optical
and magnetic properties for in vivo multimodal imaging. The as prepared nanoparticles were coated with the
polysaccharide pullulan functionalized with carboxymethyl groups, to improve their compatibility with biological
media and to allow further modifications by coupling the polysaccharidic shell with ligands of interest. Finally,
the cytoxicity of bare and coated nanoparticles was assessed on vascular cells.
1. J. Roger, J. Pons, R. Massart, A. Halbreich and J. Bacri. Eur. Phys. J.: Appl. Phys., 5 (1999) 321.
-98-
POSTER B21
POSTER B22
ZINC RESPONSIVE CONTRAST AGENTS FOR MRI
DESIGN AND EVALUATION OF BIODEGRADABLE NANOPARTICLES FOR THE TRANSPORT OF ACTIVE
MOLECULES
*1
2
1
2
Célia Bonnet , Fabien Caillé , Agnès Pallier , Franck Suzenet , and Eva Toth
2
*1
Magnetic Resonance Imaging has been devoted for a long time to obtaining anatomical and functional images.
Recently emerging applications in molecular imaging seek information at the molecular level, looking at the
biochemical or physiological abnormalities underlying the disease. This allows for a better understanding and
earlier diagnosis. Unlike anatomic imaging, molecular imaging always requires an imaging probe that is
selectively responsive to the parameter to detect. Gd3+-based contrast agents are particularly well-adapted for
this purpose and most often the changes on the efficacy (relaxivity) are based on changes of the hydration
number and/or rotational dynamics of the complexes; these two parameters being the easiest to be tailored by
the chemist[1].
Endogenous cations are known to play vital role in many fundamental biological processes. Their concentration
is tightly regulated by the cell, and misregulation of these ions is connected to different pathologies. Zinc is the
second most abundant transition metal ion in humans, and it plays a central role in controlling gene transcription
and metalloenzyme function. Exposure to high zinc concentration can lead to neuronal death. It is also an
important signalling ion in the brain, which is implicated in neurodegenerative diseases like Alzheimer’s
Disease.
We have recently developed zinc responsive contrast agents based on a pyridine unit already used for Gd3+
complexation [2], to which a zinc complexing unit has been added through a linker. Potentiometric studies have
shown that the presence of the amide is necessary for the stability of Gd3+ complexes in the presence of zinc.
GdL2 and GdL3 show a relaxivity response to zinc, and analyses of the 17O NMR, and the NMRD profiles
prove that changes of the rotational correlation time of the complexes are responsible for this behaviour. Finally
the selectivity of the zinc complexing unit has been studied by relaxometry.
2.
2
1
Centre de biophysique moléculaire (CBM) – CNRS : UPR4301 – Rue Charles Sadron – 45071 Orléans, France
Institut de Chimie Organique et Analytique (ICOA) – CNRS : UMR7311, Université d’Orléans – UFR Sciences Rue de Chartres - 45067 Orléans, France
1
1.
1
Sophie Legaz , Agnès Borel, Vincent Lahaye , Barbara Viginier-Monoury ,
ChristopheTerzian2, and Bernard Verrier†1
1
C.S. Bonnet, L. Tei, M. Botta, E. Toth, Responsive Probes in “The Chemistry of Contrast Agents in Medical Magnetic Resonance
Imaging”, 2013, John Wiley & Sons, Chichester, 343-385.
C.S. Bonnet, F. Buron et al., Chem. Eur. J. 2012, 18, 1419-1431.
Biologie Tissulaire et Ingénierie Thérapeutique (LBTI) – CNRS : UMR5305 – 7 Passage du Vercors - 69367 Lyon, France
2
SFR BioSciences Gerland - Lyon Sud – CNRS : UMR754 – 50 Avenue Tony Garnier - 69366 Lyon, France
Poly(lactic acid) (PLA) nanoparticles (NPs) are promising tools for vaccination according to their size close to
virus (180nm) and their biodegradability. One or more molecules can be adsorbed on their surface and/or
encapsulated inside NPs. Many in vivo studies (mouse, rabbit, monkey) showed an equivalent adjuvant
potential to Alum or MF59 which are referent adjuvant used in humans. However in optical fluorescence
microscopy, the nanoparticles become undetectable once in deep tissues. In order to monitor such particles
trafficking we have developed specific tools using drosophila as model system
We have designed PLA particles of 180 nm size, fluorescents or not, loaded with gal4min protein (composed of
amino acids 1 to 100 and 840 to 881), a minimal form of the yeast transcription activator proteinGal4. As
recipient organism, we used transgenic UAS-RFP Drosophila melanogaster strain. In these flies, RFP gene
expression is under control of UAS promoter which could be switch on upon binding by gal4min. Fruit flies are
fed by CAFE technique (Capillary Feeder) with a nutrient solution containing Coumarin-6 PLA NPs (green) or
with the same NPs which gal4min adsorbed. Fluorescence microscopy is then performed on cryo-sections for
observations to identify cells that have up take NPs (RFP positive cells).
After feeding, PLA nanoparticles are mainly found in the digestive system and in the hemolymph of drosophila
When monitoring RFP positive cells, we could identified the presence of particles in fat body and salivary
glands, initiator site of the immune response in Drosophila.
By using PLA nanoparticles loaded with Gal4min, we were able to track the fate of particles in whole organism
of drosophila, after in vivo administration through feeding. We identify a specific uptake through intestinal
mucosa and spreading to the circulatory system before reaching specific organs, involved in immune
responses. This approach could be further extended to study antiviral delivery or tissue targeting by PLA
particles.
∗Intervenant
†Auteur correspondant: bernard.verrier@ibcp.fr
∗Intervenant
-99-
-100-
POSTER B23
POSTER B24
SUPERPARAMAGNETIC NANOPARTICLES FOR RMI BRAIN TUMORS MOLECULAR IMAGING
POLYMERIC MICRO/NANOCAPSULES OF PERFLUOROHEXANE AS CANDIDATES FOR ULTRASOUND
CONTRAST AGENTS
*1
2
1
3
4
Sophie Richard , Amaury Herbet , Yoann Lalatonne , Sébastien Mériaux , FrédéricDucongé ,
2
1
Didier Boquet , and Laurence Motte
Ludivine Mousnier*1, Elias Fattal2, and Nicolas Tsapis1
1
UMR 7244 CNRS – Université Paris-Nord - Paris XIII – 74 rue Marcel Cachin 93017 Bobigny, France
Laboratoire d’Ingénierie des Anticorps pour la Santé (LIAS) – CEA – CEA de Saclay DSV/ iBiTec-S/ SPI/ LIAS - 91191 Gif-sur-Yvette , France
3
Laboratoire d’Imagerie et de Spectroscopie RMN, CEA/DSV/I2BM/NeuroSpin (CEA/DSV/I2BM/NeuroSpin/LRMN) – CEA – CEA/DSV/I2BM/NeuroSpin, CEA
Centre de Saclay, Bâtiment 145 - Point Courrier 156 - 91191 Gif-sur-Yvette, France
4
CEA, Institut d’Imagerie Biomédicale, Service Hospitalier Frédéric Joliot (CEA-DSV-I2BM-SHFJ) – 4 place du Général Leclerc - 91400 Orsay, France
2
1 Univ Paris-Sud, Institut Galien Paris-Sud, LabEx LERMIT, Faculté de Pharmacie, 5, rue Jean-Baptiste Clément, 92296 Chatenay-Malabry, France.
2 CNRS UMR 8612, Institut Galien Paris-Sud, LabEx LERMIT, 5, rue Jean-Baptiste Clément - 92296 Chatenay-Malabry, France.
3BioCIS, Univ Paris-Sud, UMR CNRS 8076, 5, rue Jean-Baptiste Clément - 92296 Chatenay-Malabry, France.
During the last decade, a new type of ultrasound contrast agents (UCAs) composed of a perfluorocarbon core
surrounded by a polymer shell have shown their echogenicity as well as their stability. In a strategy to optimize
the ultrasound properties of these systems, encapsulating a liquid PFC with a low boiling point such as
perfluorohexane (PFH) was suggested. The ultimate aim would be to induce phase- transition of the liquid PFH
into gas by Acoustic Droplet Vaporization (ADV) to further increase the UCA acoustic response. These new
bubbles, highly echogenic, could also trigger a possible drug release by degrading the polymer shell.
Micro- and nanocapsules with a perfluorohexane core have been developped by an emulsion- evaporation
process. For the formulation of microcapsules, three biodegradable polymers were used: PLGA and PLA with
acid (PLA-COOH) and ester (PLA-COOR) terminations. Despite their rather similar properties, these polymers
were found to strongly influence the final microcapsule morphology. While PLGA was able to form nice coreshell microcapsules, the use of PLA-COOH led to decentered microcapsules and big “eye” morphologies and
PLA- COOR induced the formation of “acorn” morphologies. To understand these morphologies we have
focused on the interfacial behaviour of the different polymers at the dichloromethane- water and the PFHdichloromethane interfaces. One can conclude that the core-shell structure is the result of a significant
adsorption of the polymer at the dichloromethane-water interface together with a good stability of the PFH
droplet within the emulsion globule. Previous work has shown that the capsule thickness-to-radius ratio
can be controlled by easily varying the polymer to perfluorocarbon proportions [1]. This versatility was
confirmed for PFH microcapsules with PLA-COOH and PLGA shells. Regarding the formulation of
nanocapsules, using PEGylated copolymers (PLGA-PEG and PLA-PEG-COOH) was also investigated
in a strategy of modifying the surface morphology. This hydrophilic PEG chains coating ensures a better
plasmatic half-life of nanocapsules, and prevents their recognition and elimination by the mononuclear
phagocyte system. Finally, the encapsulation efficiency of PFH of all types of capsules was assessed by
relating the T/R ratio to the volume fraction of PFH and by 19F NMR [2].
1.
2.
Pisani, E., et al., Polymeric nano/microcapsules of liquid perfluorocarbons for ultrasonic imaging: physical characterization.
Langmuir, 2006. 22(9): p. 4397-402.
Diou, O., et al., Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19FMRI. Biomaterials, 2012. 33(22):
p. 5593-602.
Gliomas are the most common primary brain tumour in humans, affecting around 25000 people each year
in Europe. To date, the only treatment of care consists of surgical removal of the tumor bulk, irradiation
and chemotherapy, with finally a very poor prognosis especially for high grade gliomas (i.e. glioblastoma)
for which overall mean survival is of 15-18 months. There are several explanations of the limited efficacy of
current glioma surgery treatments. First, neurosurgeons encounter great difficulty in delimiting the required
extent of tumour resection [1]. Second, gliomas are heterogeneous tumors and a major recent advance in
the field has been the identification of a subpopulation of cells in high-grade gliomas that have high
tumorigenic potential, being thus strongly responsible for tumours resurgence in the case of incomplete
surgery resection [2]. Such cells, referred to as human brain cancer stem cells (hBCSCs) or glioblastoma
stem cells (hGSCs) are multipotent and highly chemo- and radio-resistant [3]. Litterature and our
personal works demonstrate that endothelin receptor B, EDNRB, is much more expressed in glioma stem
cell cultures than in conventional glioma cell lines [4]. EDNRB antagonists had already administrated to
patients during clinical assays for glioma treatment [5]. All together, these results underline the significance
to target EDNRB for imaging and/or eradicating glioma stem cells. Moreover, superparamgnetic
nanoparticles (MNPs) are excellent MRI contrast agents because of their transverse relaxation T2*
property. MNPs can be also used for drug delivery coating the nanoparticle surface with therapeutic
molecules. Hence, this hybrid nanosystem combines both MR (diagnostic) and therapeutic applications.
This strategy is referred to theranostic strategy [6, 7].
The aim of this project is the experimental development of targeted multimodal medical imaging capable
of achieving a high resolution, specificity and sensitivity in vivo MRI imaging through T2 contrast agent
properties of iron oxide nanoparticles with a high specificity and sensitivity in vivo IR fluorescent imaging
for guided surgery. To achieve this goal, we will develop the first antibodies-based dual-modality
Fluorescent-MRI contrast agents, by combining iron oxide nanoparticles SPIOs with fluorescent
nanoprobes with the first available antibodies directed and validated on EDNRB hBCSCs [8].
1.
2.
3.
4.
5.
6.
7.
8.
Van den Bent MJ et al., Eur. J. Cancer Suppl, 2005
Chen J, Li Y, Yu TS, McKay RM, Burns DK, Kernie SG, Parada LF. Nature. 2012 Aug 23;488(7412):522-6
Mannino M & Chalmers AJ, Mol. Oncology, 2011
Paolillo M, Barbieri A, Zanassi P, Schinelli S (2006) Journal of Neuro-Oncology 79 : 1-7
Asundi J, Reed C, Arca J, McCutcheon K, Ferrando R, Clark S, Luis E, Tien J, Firestein R, Polakis P (2011) Clin Cancer
Res.;17(5):965-75
Lalatonne, Y.; Paris, C.; Serfaty, J. M.; Weinmann, P.; Lecouvey, M.; Motte, L., Chemical Communications 2008, (22), 25532555
Benyettou, F.; Lalatonne, Y.; Sainte-Catherine, O.; Monteil, M.; Motte, L., International Journal of Pharmaceutics 2009,
379, (2), 324-327
Ducancel, F., Boquet, D., Couraud, J.Y., Wijkhuisen, A.; Patent WO/2012/045776. International Application n
PCT/EP2011/067390 antagonist antibody of endothelin receptor subtype b, and uses thereof
∗Intervenant
∗Intervenant
-101-
-102-
POSTER B25
POSTER B26
PASSIVE, ACTIVE AND MAGNETIC TARGETING OF MULTIFUNCTIONAL NANOPARTICLES FOR
THERANOSTIC APPLICATIONS
*1
2
2
3
3
FORMULATION OF SELF-EMULSIFYING MICROBUBBLES FOR DRUG DELIVERY ASSISTED BY
ULTRASOUNDS
2
*†1
Nathalie Schleich , Julie Magat , Chrystelle Pô , Vincent Pourcelle , Jacqueline Marchand , Bernard Gallez ,
1
1
Véronique Préat , and Fabienne Danhier
1
1
Université catholique de Louvain - Louvain Drug Research Institute (LDRI) – Pharmaceutics and Drug Delivery - Avenue E. Mounier, 73 B1.73.12.
1200 Bruxelles, Belgique
2
Université catholique de Louvain – Louvain Drug Research Institute – Biomedical Magnetic Resonance – Avenue E. Mounier, 73 B1.73.08
1200 Bruxelles
3
Université catholique de Louvain - Molecules, Solids and Reactivity - Place Louis Pasteur 1 bte L4.01.02 – 1348 Louvain-la-Neuve
Nanoparticles can target tumors either by passive targeting taking advantages of the particular tumor
microenvironment (EPR effect) or by active targeting grafting specific ligand on their surface. A newly
emerging approach is to address multifunctionnal nanoparticles to the tumor tissue using magnetic
guidance. Previously, we developed PTX- loaded PEGylated PLGA-based nanoparticles showing a lower
IC50 in vitro and improved in vivo anti-tumor efficacy when compared to Taxol® , due to the EPR effect
(Danhier et al., 2009a). We also previously developed RGD-grafted PTX-loaded PEGylated PLGA-based
nanoparticles showing an effective v3 targeting of the tumor endothelium (Danhier et al., 2009b). Recently,
we developed PTX/SPIO loaded nanoparticles that could be used as potential tumor-targeting MRI
contrast agents, thanks to the high uptake of nanoparticles by cells as well as their magnetic
characteristics (Schleich et al., 2013). In the present study we compared the three targeting strategies
mentioned above in terms of accumulation in tumor tissue.
In the present study we compared the three targeting strategies mentioned above in terms of
accumulation in tumor tissue.
Nanoparticles presented a spherical morphology and a size of 240 nm. The PTX loading was 4.5-fold
higher compared to previously described nanoparticles. Ex vivo biodistribution (iron dose injected:
15.5mg/kg) showed an iron accumulation in tumor of 3-fold higher for RGD-grafted PTX-loaded
nanoparticles compared with non-targeted nanoparticles. This increase rose to 5-fold higher when using
magnetic guidance and even more when combining active strategy and magnetic guidance. Those results
were confirmed using a 11.7T MRI. The in vivo anti-tumor efficacy showed that all treatments (nontargeted PTX/SPIO co-loaded nanoparticles and RGD-grafted PTX-loaded nanoparticles with or without
magnetic targeting) induced a higher regrowth delay compared with the control group. RGD-grafted PTXloaded nanoparticles and magnetically guided non-targeted PTX- loaded nanoparticles (PTX dose of 5
mg/kg) induced a higher regrowth delay of CT26-tumor bearing mice, compared to the non-targeted PTXloaded nanoparticles. This regrowth delay was even more impressive by combining active (RGD) and
magnetic guidance.
1
1
DNA delivery in vivo in targeted area is facilitated by sonoporation. Indeed, ultrasounds can be focused in a
particular tissue, including deep ones, without altered the environing tissues. A co-injection of microbubbles and
DNA has already allowed protein expression with this method.
Develop a self-emulsifying nanoemulsion of perfluorohexane in water that allows a targeted drug delivery by it
specific acoustic response to ultrasounds.Method: The microbubbles formulation was optimized observing the
concentration, the mean size and the stability of the microbubbles. They were measured using an optical
microscope and counting the microbubbles of the 5 pictures taken using Image J. Thermogravimetry analyze
between 25 and 200C was used to determinate the encapsulation rate of perfluorohexane into the microbubbles
: the results were compared with the ones of a formulation in which perfluorohexane is substituted by medium
chain triglyceride oil. The acoustic responses to ultrasounds of the microbubbles were tested in vitro with an
applied power of the ultrasounds between 300 and 1400 kPa. Preliminary tests of transfection were made coinjecting microbubbles and DNA into the liver of mice.
The optimized formulation of microbubbles consists on a blend of perfluorohexane (16%), surfactants (21%) and
water (63%). It forms around 1x1010 microbubbles per milliliter with a mean diameter around 2m, stable for
several hours, with an encapsulation rate of perfluorohexane closed to 86%. The tests in vitro show a non-linear
response, characteristic of the microbubbles oscillations, from around 450kPa and microbubbles destruction
from around 1000kPa. Preliminary tests in vivo show encouraging results on the mice.
∗Intervenant
-103-
2
Unité de pharmacologie chimique et génétique et d’imagerie (UPCGI) – Inserm : U1022, CNRS : UMR8151, Université Paris V - Paris Descartes, Ecole
Nationale Supérieure de Chimie de Paris – Paris, France
2
Physico-Chimie Industrielle du Médicament, Université Paris Descartes (EA4066) – Université Paris Descartes - France
Although active targeting using RGD-grafted PLGA-based nanoparticle showed interesting results,
combining active targeting and magnetic guidance seems to be the most promising approach
∗Intervenant
†Auteur correspondant: fabienne.danhier@uclouvain.be
†Auteur correspondant: veronqiue.preat@uclouvain.be
1
Caroline Thebault , Sylvie Crauste-Manciet , Fredéric Rosa , Simona Manta , Yohann Corvis , Philippe
Espeau2, Gilles Renault, Michel Francis Bureau1, Nathalie Mignet1, and Michel Bessodes1
-104-
POSTER B27
POSTER B28
THERMAL ANALYSIS TOOLS FOR PHYSICO-CHEMICAL CHARACTERIZATION OF MICROBUBBLES
FOR IMAGING
FORMULATION OF HSP90 INHIBITOR LOADED NANOLIPOSOMES : EVALUATION OF THE CYTOTOXIC
EFFECT IN VITRO ON BREAST AND PROSTATE CANCER CELLS
1
1
1
2
1
1
Physico-chimie industrielle du médicament – Université Paris V - Paris Descartes – Faculté des sciences pharmaceutiques et biologiques –
4 Avenue de l’Observatoire - 75270 Paris, France
2
Unité de pharmacologie chimique et génétique et d’imagerie (UPCGI) – Inserm, CNRS : UMR8151, Université Paris V - Paris Descartes,
Ecole Nationale Supérieure de Chimie de Paris – Paris, France
The objective of this study was to use thermal analyses to improve the formulation of self-emulsifying
nanoemulsions (O/W) with regards to surfactant composition. These nanoemulsions were specially designed
to improve the contrast of ultrasound images by inclusion of perfluorocarbon gas. Nanoemulsions were
prepared using perfluorohexane (C6F14) or perfluoropentane (C5F12), two liquid gases at room temperature
with boiling temperature of 59 and 28 C, respectively. The nanoemulsions consist of a blend of gas (20%),
surfactants (50%) and water (30%). The surfactants mixture included polysorbate-85, span-8 Lutro®, glycerol
and Zonyl®. Zonyl® is a polyethylene glycol with a fluorinated chainused to improve the capture of the gas
within the emulsion. The effect of the Zonyl® concentration on the rate of gas included in the emulsions was
assessed between 0.5 and 3%.The number of droplets per milliliter in the emulsion was determined by optical
microscopy and Image J treatment. A mean value of 7.2x107 droplets/mL and 8.9x107droplets/mL was found
respectively for perfluorohexane and perfluoropentane, with mean droplet sizes equal to 2.12 0.24 nm and
1.97 0.09 nm. With regards to stability, 50% of the droplets was still present after 72 hours for the
perfluorohexane and 54 hours for the perfluoropentane.
The physico-chemical behaviour of the different components of the nanoemulsion, such as the interaction
between surfactants, the amount of gas encapsulated as a function of the amount of zonyl® included in the
emulsions and the influence on the temperature of evaporation of the gas were characterized using thermal
analyses techniques such as thermogravimetry analysis (TGA) and differential scanning calorimetry (DSC) in
the -60 to 200 C temperature range.
We found that: i/ the amount of gas encapsulated tendentiously increases with the amount of zonyl,ii/ the
evaporation temperature of the gas decreases when the percentage of Zonyl® increases; Thermal analysis
was a helpful method to optimize the formulation of the nanoemulsion containing perfluorocarbon gaz. This
allowed us to show the interest of using the fluorinated surfactant on the one hand, and by choosing the
appropriated concentration for the best gas encapsulation rate, on the other hand.
1
2
1
2
Université Paris-Sud, CNRS, BioCIS-UMR 8076, LabEx LERMIT, Laboratoire de Chimie Thérapeutique, Faculté de Pharmacie, 5 rue Jean-Baptiste
Clément, 92296 Chatenay-Malabry, France
HSP 90 is a heat shock protein involved in folding and maintaining the active conformation of proteins
under stress (thermic stress, pH variation, hypoxia...). Among HSP 90’s client proteins, many are involved
in oncogenic processes like metastatic phenomena, invasion and immune escape which contribute
greatly to the complexity cancer diseases. Thus, HSP 90 is an interesting target for the treatment of
cancer since, on one hand, its inhibition allows a multilateral approach and, on the other hand, it has a
higher affinity for proteins and inhibitors in cancer cells compared with normal ones, (1). Recently, several
HSP 90 inhibitors like geldanamycine have been synthesized, some of which are undergoing clinical trials.
6Br- CaQ is an inhibitor synthesized by the team of Chimie thérapeutique (collaboration with Dr. Samir
Messaoudi, BioCIS UMR 8076) at the Faculty of Pharmacy in Chatenay-Malabry (University Paris-Sud)
(2). This inhibitor binds the C-terminal domain of the protein, thus avoiding a ”rebound” after the inhibition
stops. 6BrCaQ was observed to induce a G2/M arrest in breast and prostate cancer cell lines (MDA-MB-231
and PC-3, respectively) after
24 hours of treatment by a high concentration of inhibitor (50 M) and after 72 hours by a lower
concentration (10 M). At the highest concentration, PARP-1 and caspase-3 cleavage were detected by
Western Blot, indicating that apoptosis was induced). The encapsulation of 6BrCaQ in stealth liposomes
overcomes its low water solubility and contributes to a better effect of the molecule at low concentrations.
Several methods were used to evaluate the cellular effect of 6BrCaQ: metabolic activity assay (MTS), cell
counting after Trypan Blue dead cell exclusion and cell cycle analysis (Flow cytometry). The cytostatic
effect (G2/M arrest) was significant at 5 and 10M after 24h treatment although apoptosis was not
observed. The migration and invasion properties of breast and prostate cancer cells contribute to tumor
resistance. Therefore 6BrCaQ-loaded liposomes are currently being tested in a wound healing assay in
which the migration is induced by a chemokine, MCP-1. In a metabolic activity assay, a combination of
the two free molecules exerted a higher cytotoxic effect than each molecule alone. The good anti-tumor
activity of 6BrCaQ combined with the tumor targeting properties of liposomes strongly encourage the
development of this molecule as a new cancer therapy.
∗Intervenant
-105-
2
Université Paris-Sud, Institut Galien Paris-Sud UMR-CNRS 8612, LabEx LERMIT, Faculté de Pharmacie, 5 Rue Jean-Baptiste Clément, 92296
Chatenay-Malabry, France
1. Kamal, 2004
2. Audisio, 2011
∗Intervenant
1
Félix Sauvage , Silvia Franze , Samir Messaoudi , Stéphanie Denis , Baptiste Martin , Jean-Daniel Brion ,
2
1
1
Mouad Alami , Gillian Barratt and Juliette Vergnaud-Gauduchon
2
Fredéric Rosa∗ , Yohann Corvis , Nathalie Mignet , Denis Brossard , Sylvie Crauste-Manciet∗2,
and Philippe Espeau1
-106-
POSTER B30
POSTER B29
PACLITAXEL-LOADED MICELLES ENHANCE THE VASCULAR PERMEABILITY AND RETENTION OF
NANOMEDICINES IN TUMORS
1
2
3
1
1
PHOTOACTIVE POROUS SILICON NANOVECTORS FOR PHOTODYNAMIC THERAPY
Frederique Cunin∗1, Emilie Secret1, Marie Maynadier2, Arnaud Chaix3, Magali Gary-Bobo2, Marcel Garcia2,
Philippe Maillard4, Francesco Di Renzo3, Michael Sailor5, and Jean-Olivier Durand1
Fabienne Danhier∗ , Pierre Danhier , Christophe De Saedeleer , Nathalie Schleich , Bernard Ucakar ,
Pierre Sonveaux3, Bernard Gallez2 , and Véronique Préat†1
1ICGM – CNRS : UMR5253 – France
2 IBMM – Inserm : UMR5247 – France
3 ICGM – CNRS – France
4iNSTITU cURIE – Université Paris VI - Pierre et Marie Curie – France
5 UCSD – Etats-Unis
1
2
Université catholique de Louvain – Louvain Drug Research Institute – Pharmaceutics & Drug Delivery – Av. E. Mounier, 73 bte B1.7312 – 1200 Bruxelles
Université catholique de Louvain – Louvain Drug Research Institute – Biomedical Magnetic Resonance – Av.E. Mounier, 73 bte B1.7308 – 1200 Bruxelles
3
Université catholique de Louvain - Institut de Recherche Expérimentale et Clinique FATH – Av. Mounier 53 bte B1.53.09, 1200 Bruxelles– Belgique
Paclitaxel (PTX)-loaded PEG750-p(CL-co-TMC) polymeric micelles (M-PTX) have been shown to enhance the
oxygenation and blood flow of tumors 24 h after treatment. In the present study, we hypothesized that (i) these
changes in tumor microenvironment could lead to an enhancement of the EPR (Enhanced Permeability and
Retention) effect, allowing a more effective delivery of PTX or other anti-cancer nanomedicine into the tumor
and (ii) that the underlying mechanisms include both the cytotoxic effet of PTX but also an anti-angiogenic effect
of PTX (vascular normalization).
Using two tumor models, Miles assay, Electron Spin Resonance spectroscopy and Intravital microscopy have
been used to demonstrate an increased accumulation of particles after PTX treatment. The proof of concept has
been validated by a regrowth delay assay. Different parameters of the underlying mechanisms were studied: (i)
architecture of the vasculature using immunohistochemistry (CD-31, a-SMA) and whole mount staining (CD-31
and DAPI), (ii) the perfusion using DCE-MRI and (iii) the interstitial fluid pressure (IFP) using the wick-in-needle
technique.
Photodynamic therapy (PDT) has arisen as an alternative to chemo and radio therapy for non-invasive selective
destruction of tumours. PDT involves the use of a photosensitizer which, in the presence of oxygen, upon
irradiation at specific wavelengths, leads to the generation of cytotoxic species and consequently to irreversible
cell damaging. PDT has been in clinical use for over a decade but, despite the advantages of the therapy itself,
photosensitizers in use today induce a long photosensitivity of the patient that limits their use. To overcome this
issue, encapsulation of the photosensitizers into nanoparticles is a promising new approach. In this work, we
studied the use of porous silicon nanoparticles (pSinp) functionalized with both a photosensitizer and a targeting
agent. Porous silicon is a biocompatible and biodegradable material which has been shown to be degraded into
non-toxic silicic acid byproducts in vivo. The multi-functionalized pSiNP studied here were able to target, image
and kill cancer cells in vitro by photodynamic therapy mechanisms with one and two photon excitations.
The Miles assay demonstrated that tumors were more permeable to macromolecules when treated with M-PTX,
whereas ESR spectroscopy showed a higher accumulation of iron oxide particles (30 nm) used as tracers in
tumors. Intravital microscopy also showed that the extravasation of fluorescent FITC-dextran from abnormal
tumor blood vessels was significantly enhanced in tumors treated with M-PTX. Two consecutive treatments with
M-PTX were more efficient than the bolus delivery of the full M-PTX dose. A re-organization of the tumor
vasculature (normalization) as well as a decrease of the IFP was observed when tumors were treated with MPTX, demonstrating an anti-angiogenic activity of PTX
Together these experiments demonstrate an enhanced EPR effect after M-PTX treatment, as pre-treatment,
which could lead to a better availability and an enhanced efficacy of a subsequent treatment. This enhanced
EPR effect can be explained by a decrease of the IFP and a normalization of the tumor vasculature.
∗Intervenant
†Auteur correspondant:veronique.preat@uclouvain.be
∗Intervenant
-107-
-108-
POSTER B31
POSTER B32
PLGA NANOPARTICLES INCORPORATING LIPOPHILIC GD CONTRAST AGENTS
1
Rigaux∗ ,
1
1
1,2
2
Guillaume
Valérie Gaëlle Roullin , Cyril Cadiou , Gaëlle Boeuf , Nicolae-Bogdan Bercu , Marie1
2
3
3
4
Christine Andry , Michaël Molinari , Christophe Portefai , Christine Hoeffel , Luce Vander Elst , Sophie
4
1
Laurent , and Françoise Chuburu
DEVELOPMENT OF MULTIFUNCTIONAL PARTICLES FOR DIAGNOSTIC & TARGETED DELIVERY IN
CANCER THERAPEUTICS
1
To this end, the non-covalent incorporation of a lipophilic gadolinium complex into PLGA nanoparticles
was performed. First, the lipophilic gadolinium complex was synthesized in three steps2 by alkylation of a
DO3A(tBu)3 backbone with a C12 alkyl chain followed by tBu deprotection. Subsequent Gd(III)
complexation led to a GdDO3AC12 complex. Then, the incorporation of this complex into a PLGA matrix
was performed by a modified nanoprecipitation technique.3 Spherical and well-individualized nanoparticles
(max. 150-200 nm), as observed by DLS/ELS and AFM, were formed with a 95% production yield.
Nanoparticle Gd loading was evidenced by AFM / NanoInfrared absorption coupled measurements and
quantified by ICP-OES. The relaxivity of the gadolinium-loaded nanoparticles was determined at 40 MHz
(BrukerMinispec mq40) and their 1H NMRD profile was acquired at 37C and 5C between 0.01 to 300 MHz
(Stelar Spinmaster FFC fast field cycling NMR relaxometer). These results were compared to ones obtained
for the free GdDO3AC12 complex. The comparison indicated that entrapment of GdDO3AC12 complex in
the polymer matrix provoked an important enhancement of the relaxivity of 104% and 126% for r1 and r2
respectively. Imaging of nanoparticle suspension phantoms was performed by using a 3.0 T MRI device
(Achieva, Philips Medical Systems Sense head 8 coil). The images clearly indicated that the gain in Gd
relaxation was translated into a contrast enhancement capability at 3T. Finally, the cytotoxicity
measurements indicated that GdDO3AC12 loaded nanoparticles are not toxic towards fibroblast cells below
1mg/mL.
3
3
3.
∗Intervenant
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1
Tumor targeting depends strongly on the capacity of nanoparticles (NPs) or molecules to cross several barriers
from the injection site to the targeted cells. Thus, it is tempting to develop targeted NPs or molecules that will
recognize receptors overexpressed on both type of tissues: the tumor endothelium and the tumor cells. Integrins
(especially integrin avß3) and Neuropilin-1, a co-receptor of VEGF (NRP) are over-expressed in the tumor
vasculature and tumor cells, and their expression has been correlated with angiogenesis and progression.
Importantly, integrin avß3 and NRP1 can interact physically and functionally. Dual targeting of these two
receptors may thus be useful for development of tumor targeted nanomedicines. In the present study, we
generated bifunctional scaffolds or particles (RAFT-[cRGD]n-[anti NRP1]m, internalized RGD (iRGD) and
RGD/[anti NRP1] NPs) targeting integrin and NRP1 simultaneously and explored their activity. By Western
Boltting and Flow cytometry (FACS) we characterized the expression of the receptors (VEGFR1/2, NRP1/2) in
five different tumor cell lines, and choose MDA-MB-231 and H358 cells for in vitro experimental model. By
introducing different ratio of RGD / [anti NRP1] peptides and several amount of peptides (1132, 113 and 11
peptides) on the surface, we generated 15 batches of NPs. In parallel, we also incorporated [anti NRP1] peptide
or RAFT-[anti NRP1] to RAFT-[cRGD] platform with varying lengths of spacers. Finally, a CendR tumor
penetrating peptide, iRGD, was also synthesized. In conclusion, our study suggests that bifunctional RAFT[cRGD]n-[anti NRP1]m molecules and RGD/[anti NRP1] NPs would be of great potential for cancer targeted
drug delivery and therapy.
P. Hermann, J. Kotek, V. Kubicek, I. Lukes.Dalton Trans. 23, 2008, 3027-3047.
N. Raghunand, G. P. Guntle, V. Gokhale, G. S. Nichol, E. A. Mash, B. Jagadish. J. Med. Chem. 53,
2010, 6747–6757.
H. Fessi, F. Puisieux, J. P. Devissaguet, N. Ammoury, S. Benita. Int. J. Pharm. 55, 1989, R1-R4; M. Callewaert, S. Dukic, L. V.
Gulick, M. Vittier, V. Gafa, M. C. Andry, M. Molinari, V. G. Roullin. J Biomed Mater Res A. 101, 2013, 1319-1327.
∗Intervenant
2
INSERM/UJF U823, Grenoble, France – Add this new organization – France
2
UMR CNRS 5247, Montpellier, France – Add this new organization – France
DCM, UMR CNRS 5250, ICMG FR2607, Grenoble, France – Add this new organization – France
These results are promising for the development of new biocompatible and efficient contrast agents for MRI
applications.
1.
2.
3
1
1Institut de Chimie Moléculaire de Reims (ICMR) – Université de Reims - Champagne Ardenne, CNRS : UMR7312 – UFR Sciences, BP1039, 51687 Reims
Cedex 2, France
2 Nanosciences laboratory (LRN) – Ministère de l’Enseignement Supérieur et de la Recherche Scientifique – Reims, France
3
Hopital Maison Blanche – CHU Reims – 45 rue Cognacq Jay ; 51100 Reims, France
4
Université de Mons (UMons) – 20, place du Parc, B7000 Mons, Belgique
Gadolinium-based contrast agents (GdCAs) are widely used to enhance image contrast in magnetic
resonance imaging (MRI) procedures. To obtain a good contrast image, injection of high GdCA doses are
required (for example the recommended dose for Dotarem® is 0,2mL/kg). Recently, it has been proposed
that dechelation of GdCAs could be responsible for the development of a new disease, namely
nephrogenic systemic fibrosis (NSF), in patients suffering from severe renal failure. Consequently, EU and
US health authorities have expressed some reservations for using and developing new GdCAs. To improve
the performance of existing contrast agents, one of the proposed strategies consists in confining them into
synthetic nanoparticles. Indeed, the Solomon Bloembergen Morgan (SBM) theory assumes that minimizing
the Gd3+ complex rotation, but also locally increasing their concentration, should result in an
improvement in their effectiveness.[1]
2
Tao Jia∗ , Jerémie Ciccione , Michael Claron , Didier Boturyn , Gilles Subra , and Jean-Luc Coll
-110-
POSTER B33
POSTER B34
VECTORIZATION OF MULTIMODAL GD-NANOPARTICLES FOR AMYLOIDOSIS DIAGNOSTIC.
GLYCOPOLYPEPTIDE-BASED NANOCARRIERS FOR LUNG CANCER TREATMENT
Marie Plissonneau∗1, Pierre Mowat2, Nathalie Stransky-Heilkron3, Christel Marquette2, Francois Lux1, Eric
3
4
2
3
5
1
Allémann , Cédric Louis , Vincent Forge , Vincent Montet , Mireille Dumoulin , and Olivier Tillement
Silvia Mazzaferro∗1, Victor Jeannot2, Christophe Schatz1, Amandine Hurbin2, Jean-Luc Coll2,
and Sebastien Lecommandoux1
1
1
Institut Lumière Matière (ILM) – CNRS : UMR5306 – Université Claude Bernard Lyon 1 Domaine Scientifique de La Doua Batiment Kastler,
10 rue Ada Byron 69622 Villeurbanne, France,
2
Laboratoire de Chimie et Biologie des Métaux (LCBM) – CEA, Université Joseph Fourier - Grenoble
I, CNRS : UMR5249 – 17, rue des Martyrs 38054 Grenoble, France
3
Université de Genève (Section des sciences pharmaceutiques) – 24 rue du Général-Dufour CH – 1211 Genève 4, Suisse
4
Nano-H S.A.S. (Nano-H S.A.S.) – Entreprise – 38070 Saint Quentin-Fallavier, France, France
5
Centre d’Ingénierie des Protéines (CIP) – Université de Liège, 4000, Liège, Belgique
The aim of this project is to develop multimodal imaging nanoparticles for amyloidosis diagnosis.
Amyloidosis is a group of disease in which proteins (e.g. Islet Amyloid PolyPeptide IAPP, Amyloid A or
TransThyRetin TTR) form insoluble aggregates (or fibrils) in extracellular tissues. Depending of the protein
nature and its deposition area, this accumulation could lead to different pathologies (e.g. Alzheimer
disease, Neuropathies and Type II diabetes). We propose two different strategies to target the fibrils:
- Camelid heavy-chain antibody fragment (or Nanobody), which shows affinity to any amyloidosis form.[1]
- Peptides of few amino acid derived from the protein responsible of the aggregate, which are specific only
to one form of fibrils.[2]
In our group, we have developed ultra-small nanoparticles (AGuIX R ) with multimodal imaging properties [3]
such as MRI, Scintigraphy and Optical Imaging. Theses nanoparticles are composed of a polysiloxane matrix
holding DOTA-Gd3+ chelates and free DOTA ligands that can chelate radio-isotope. For optical imaging, near
infrared fluorophores can be covalently grafted to the inorganic matrix.
Laboratoire de Chimie des polymères organiques (LCPO) – ENSCPB, CNRS : UMR5629, Université
Sciences et Technologies - Bordeaux I – 16, Avenue Pey Berland 33607 Pessac, France
2
INSERM/UJF U823, Grenoble, France – Add this new organization – France
Because lung cancer is one of the most fatal diseases, there is an urgent need to propose new therapeutic
approaches in addition to current radiotherapy and chemotherapy. In this context, this work aims to
develop a new polysaccharide-block-polypeptide copolymer based nanocarrier and to evaluate its
efficacy to target specifically tumour cells. The amphiphilic copolymers used here are combining
Hyaluronan (Hya), a natural polysaccharide well known for its relative affinity towards CD44 receptors
upregulated on cancer cells, and poly(g-benzyl-L-glutamate) (PBLG), a biodegradable polypeptide
characterized by an ordered secondary structure (-helices). The two blocks were conjugated using the
efficient and versatile azide-alkyne Huisgen cycloaddition (refereed as “click” chemistry) and nanoparticles
were formulated by solvent-displacement technique. By varying the hydrophilic/hydrophobic volume ratio
of the copolymer, it was possible to obtain different nanoparticle (NPs) sizes and shapes in order to
modulate the in vivo biodistribution. The ability of NPs to target the CD44 receptors was studied in vitro
using different lung cancer cell lines (H358, H460, A549), and in vivo using an orthotopic lung cancer
model investigating two routes of administration: systemic route (i.v. injections) and pulmonary route
(nebulization). Non-invasive optical imaging techniques (2D and 3D fluorescence) allowed the real time
followup of the nanoparticles in mice. The results of this study will allow us to select the best formulation
to load the anticancer drugs and evaluate their antitumor activity in an orthotopic lung cancer model in
mice.
By grafting the targeting agents mentioned above on its surface, the particle could accumulate in fibrils area
and lead to a specific and early diagnosis of amyloidosis. The first part of the study consists in optimizing the
number of grafting site on the nanoparticle surface by increasing the number of carboxylic function at the
surface. Indeed the targeting agents are linked to the Np via peptide coupling between their primary amine and
a carboxylic acid function on the nanoparticle. Two different strategies have been tested: (i) addition of other
DOTA on the surface. (ii) grafting of PEG chains ending by carboxylic functions. Grafting of the peptides and
the nanobodies has then been performed on the two types of particles and characterized by granulometry,
relaxometry, HPLC, fluorescence...
First in vivo test on healthy mice demonstrate a rapid renal elimination with no apparent toxicity. The
biodistribution is very closed to the one of the AGuIX nanoparticles without targeting species. The in vitro tests
on IAPP fibrils are encouraging and show an active targeting.
[1] M. Dumoulin and C. M. Dobson. (2004) Probing the origins, diagnosis and treatment of amyloid diseases using antibodies. Biochimie
86, 589-600 (Review).
[2] A. Chenal et al. (2011) Amyloid fibril formed by the programmed cell death regulator Bcl- xL. J Mol Biol 415, 594-99
[3] F. Lux et al. (2011) Ultrasmall Rigid Particles as Multimodal Probes for Medical Applications. Angewandte Chemistry International
Edition 50, 12299-12303.
∗Intervenant
∗Intervenant
-111-
-112-