18 - ETP Nanomedicine

Pulmonary delivery of tobramycin-loaded nanoparticles
for the treatment of Pseudomonas aeruginosa infection in
cystic fibrosis patients
M. Moreno-Sastre*, JL Pedraz**, A. Esquisabel**, M. Pastor***, E. Gainza***
•NanoBioCel Group, Laboratory of Pharmaceutics, University of Basque Country (UPV/EHU), School of Pharmacy, Paseo de la Universidad 7, Vitoria-Gasteiz, 01006, Spain. Maria.moreno@ehu.es
** NanoBioCel Group, Laboratory of Pharmaceutics, University of Basque Country (UPV/EHU), School of Pharmacy, Paseo de la Universidad 7, Vitoria-Gasteiz, 01006, Spain. Joseluis.pedraz@ehu.es, Amaia.esquisabel@ehu.es
*** Grupo Praxis. Parque tecnológico de Álava , Hermanos Lumiere 5, 01510, Vitoria-Gasteiz . Mpastor@praxisph.com , Egainza@praxisph.com
Abstract
Pseudomonas aeruginosa is the main pathogen that affect the
respiratory tract of cystic fibrosis (CF) patients. As a way to fight
against this infection, nanotechnology has emerged over the last
decades as a promising alternative to overcome the resistance in
infectious diseases. The aim of this work was to elaborate and
characterize lipid nanoparticles for pulmonary delivery of
tobramycin. Tobramycin-loaded nanostructured lipid carriers (TbNLCs) showed to be active against clinically isolated P. aeruginosa
displaying a MIC (minimum inhibitory concentration) of 0.5 µg/mL.
Moreover , in an in vivo study after an intratracheal administration
in mice , nanoparticles presented a wide distribution in lungs.
Therefore, Tb-NLCs could represent an alternative drug delivery
system for pulmonary infection treatment.
Results
Tb-NLCs displayed a mean diameter size around 250 nm and a
zeta potential of -23 mV. Likewise, IR-labeled NLCs displayed a
similar size and charge, around 289 nm and -26 mV. Tobramycin
and IR were successfully loaded achieving an encapsulation
efficiency up to 93% and 99%, respectively (Figure 1A). TEM
images revealed that nanoparticles had a regular homogeneous
spherical shape (Figure 1B). Both types of NLCs displayed a
sustained release lasting 92 hours (Figure 1C).
Fig. 1. Characterization of NLCs
A
#Tobramycin #Nanoparticles #Pulmonary administration
Introduction
Methods
Two NPs loaded with tobramycin were elaborated by a
hot melt homogenization technique using Precirol®
ATO5 (Tb-NLC P) or mixture of Precirol and Compritol®
ATO 888 (Tb-NLC PC) and Miglyol as core materials. Infrared-labeled NLCs (IR-NLC) were prepared
for the
biodistribution assays. In both cases, the nanoparticles
were washed by centrifugal filtration units (Amicon®)
followed by freeze-drying using trehalose (15%, w/w) as
cryoprotectant (3). Size and zeta potential were
estimated using Zetasizer Nano ZS. Encapsulation
efficiency (EE) and release profile were assessed by UVVIS spectrophotometer (2) and NPs morphology was
examined by transmission electron microscope (TEM).
The minimum inhibitory concentration (MIC) of
nanoparticles and free antibiotic were tested against P.
aeruginosa strains isolated from sputum of cystic fibrosis
patients
(mucoid
and
non-mucoid)
by
broth
microdilution method in 96-well microplates. P.
aeruginosa ATCC was used as control strain.
For an in vivo biodistribution study in BALB/c OlaHsd
mice , 1 mg of IR-NLCs was administered intratracheally
to each mouse by a MicroSprayer™aerosolizer (Penn
Century® Liquid ). The mice were placed in an intubation
platform and the trachea and epiglottis of the animals
were visualized by using a small animal laryngoscope. At
pre-established time points, mice were sacrificed and
lungs and other organs were removed and analyzed by LICOR Pearl® impulse small animal imaging system.
B
Mean size (nm)
PDI
Z Potential (mV)
EE (%)
Tb-NLC P
254.05 ± 14.50
0.311 ± 0.01
- 23.03 ± 2.76
93.15 ± 0.65
Tb-NLC PC
278.66 ± 20.48
0.371 ± 0.01
- 22.25 ± 0.49
94.03 ± 0.22
IR-NLC P
283.93 ± 5.79
0.368 ± 0.03
-25.73 ± 0.25
99.50 ± 0.02
IR-NLC PC
295.16 ± 17.35
0.304 ± 0.06
-26.30 ± 0.41
99.35 ± 0.09
Tb-NLC P
Tb-NLC PC
C
Tb released (%)
Cystic fibrosis (CF) is a genetic disorder that affects nearly 70,000
patients worldwide. Pseudomonas aeruginosa is the most frequent
pathogen identified in CF patients. Over the last decades, antibioticresistant strains have increased due to the misuse and overuse of antiinfectious drugs. In this regard, nanotechnology has emerged as a new
alternative to drug encapsulation in order to overcome the limitations
of conventional drugs. Nanoparticles (NPs) are currently being
extensively investigated for antibiotic inhalation therapy. Pulmonary
drug delivery has gained much attention as a non-invasive route for the
delivery of high amounts of therapeutic agents directly to the desired
site of action minimizing systemic exposure and adverse effects (1).
Taking the above into account, the goal of this work was to elaborate
and characterize tobramycin-loaded nanostructured lipid carriers (TbNLCs) for pulmonary delivery for the treatment of lung infectious
diseases; in particular CF. Two different solid lipids were selected as
core agents for the NLCs (Precirol® ATO 5 and Compritol® ATO 888) and
the antimicrobial activity against P. aeruginosa was investigated. Finally,
the NPs biodistribution was analyzed after intratracheal administration
in mice.
Formulation
100
90
80
70
60
50
40
30
20
10
0
Fig. 3. A) Biodistribution of IR-NLC after intratracheal
administration to mice. IR-intensity image of selected
organ excised (upper row: heart, trachea and lungs,
lower row: gallbladder, liver, kidneys and spleen) B)
Biodistribution of IR-NLCs in the lungs.
Conclusions
Tb-NLC P
Tb-NLC PC
0
10
20
30
40
50
60
70
80
90
100
Time (hours)
Fig. 2. A) Images of Pseudomonas aeruginosa clinical isolates. B) MIC
values of Tb-NLCs and free drug against strains of clinically isolated
P. aeruginosa samples. M, mucoid clinical strain and NM, nonmucoid clinical strain.
A
B
MIC
(µg/mL)
PA
ATCC
27853
PA 852
(NM)
PA 056
(NM)
PA 760
(NM)
PA458
(M)
PA 428
(M)
PA 086
(M)
Free tobramycin
0.5
0.5
1
1
0.5
4
2
Tb-NLC P
0.5
0.5
0.5
0.5
0.5
2
1
Tb-NLC PC
0.5
0.5
0.5
0.5
0.5
4
1
Both types of Tb-NLCs showed to be active against clinically
isolated Pseudomonas aeruginosa displaying a MIC of 0.5 µg/mL in
most of the placktonic bacteria tested. In the same experimental
conditions, free tobramycin displayed the same or higher MIC
indicating that the encapsulation of the drug did not affect the
antimicrobial activity (Figure 2B).
IR emission associated with nanoparticles was detected at
different levels of the pulmonary tree, suggesting a wide
distribution in the lungs until 48 hours (Figure 3A). Immediately
after the administration, a high concentration of IR-NLCs in the
lungs was observed according to the intense red color of the
images (Figure 3B). After two hours of administration, a systemic
absorption of NLCs could be detected in other organs such as liver
and kidney, and less intensively in spleen. At 24 and 48 hours, the
nanoparticles remained in the lungs with less or no signal in other
organs. Both formulations had a similar behavior in vivo. The only
difference was the absence of signal in the liver at 48 hours in the
case of NLCs PC. No IR emission was detected in mice before the
administration of the formulation. These data showed that
nanoparticles can deposit with high efficiency and for long period
in the respiratory tract allowing drug release.
Tb-NLCs demonstrated efficacy against P. aeruginosa
in vitro and large pulmonary distribution and
retention in the in vivo studies. Tb-NLCs (both TbNLC P and Tb-NLC PC) can provide the advantage of a
sustained drug release in the target site, resulting in
reduced-dose schedule and improved patient
compliance. Other clear advantages of these
nanoparticles are the use of biocompatible and
biodegradable lipids and the avoidance of organic
solvents during their preparation leading to
economic efficiency and an environmental friendly
process. Therefore, Tb-NLCs could represent an
alternative drug delivery system for pulmonary
infection treatment. Yet, the results presented in this
study are not sufficient to predict the effectiveness
of the lipid-based nanosystem in CF patients
although the features of the developed formulation
so far examined could be considered promising in a
perspective of an efficacious CF inhalable therapy.
Bibliography
(1) Moreno-Sastre M, Pastor M, Salomon CJ, Esquisabel A,
Pedraz JL. Pulmonary drug delivery: A review on
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Chemother. 2015. doi: dkv192 [pii].
(2) Ungaro F, d'Angelo I, Coletta C, et al. Dry powders based on
PLGA nanoparticles for pulmonary delivery of antibiotics:
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doi:10.1016/j.jconrel.2011.08.010
(3) Pastor M, Moreno-Sastre M, Esquisabel A, et al. Sodium
colistimethate loaded lipid nanocarriers for the treatment
of Pseudomonas aeruginosa infections associated with
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10.1016/j.ijpharm.2014.10.048
Acknowledgments
This work was supported by the TERFIQEC Project, IPT2011-1402-900000 (funded by the Ministry of Economy
and Competitiveness MINECO, Spain). The authors
gratefully acknowledge the support of University of the
Basque Country UPV/EHU (UFI11/32), University of
Barcelona (UB), UIB and CSIC-FISIB Caubet-Cimera.
María Moreno thanks UPV/EHU for the ZabaldUz
fellowship grant. Technical and human support provided by
SGIker (UPV/EHU) is gratefully acknowledged.