5,10,15,20-tetrakis(meso-hydroxyphenyl)porphyrin Loaded

5,10,15,20-tetrakis(meso-hydroxyphenyl)porphyrin Loaded Polymeric Micelles for
Photodynamic Treatment of Head and Neck Cancer Cells
Evan M. Cohen; Huiying Ding, PhD; Chase W. Kessinger, PhD; Chalermchai Khemtong, PhD; Jinming Gao, PhD; Baran D. Sumer, MD
University of Texas Southwestern Medical Center at Dallas
Hydrophobic
mTHPP
was
encapsulated with high loading
efficiency
and
density
into
poly(ethylene glycol)-co-poly(D,Llactic acid) micelles. The resulting
micelle nanoparticles are spherical
in shape, and have a uniform size
distribution. These micelles exhibit
fluorescence. In vitro studies
demonstrate that mTHPP-loaded
micelles produce PDT-mediated
cytotoxicity against head and neck
cancer cells.
Photodynamic therapy (PDT) is an effective treatment modality clinically
used for the treatment of several different types of cancer including
cancers of the head and neck.1 In PDT, a photosensitizer (PS) is
administered and when exposed to light is excited to a triplet state,
which can then subsequently lead to the generation of singlet oxygen
(1O2) or free radicals. These reactive oxygen species (ROS) can lead to
significant cellular damage, destruction of tumor blood vessels and the
stimulation of anti-neoplastic immunity.2 The advantages of PDT include
the ability to target cancer cells to minimize toxicity to normal tissues.
One potential challenge of PDT therapy is that many PS agents are
lipophilic making parenteral administration problematic. Polymeric
micelles are composed of amphiphilic block copolymers that contain
distinguished hydrophobic and hydrophilic segments. This unique
architecture enables the micelle core to serve as a nanoscopic depot for
hydrophobic PS agents, and the hydrophilic shell as a stabilizing corona.
Sterically stabilized micelles have shown prolonged blood circulation,
and passive targeting to solid tumors through porous tumor vasculature,
leading to Phase II clinical trials of several micellar systems in cancer
patientsIn this study, we describe micelle encapsulation of a
representative hydrophobic PS agent, 5,10,15,20-tetrakis(mesohydroxyphenyl)porphyrin (mTHPP) . High drug loading efficiency was
observed that results in effective solubilization. The resulting mTHPPloaded micelles also demonstrated PDT mediated cytotoxicity against
head and neck HSC-3 and HN-5 cells in vitro.
METHODS AND MATERIALS
RESULTS
Poly(ethylene glycol)-b-poly (D,L-lactic acid) (PEG5K-PLA5K) was synthesized
using a ring-opening polymerization procedure. mTHPP micelles were fabricated
using a solvent evaporation method. Micelle size was determined using dynamic
light scattering (DLS) and verified via transmission electron microscopy (TEM).
The drug loading content, defined as the weight percentage of mTHPP over the
total micelle weight was quantified by UV-Vis analysis. Emission spectra are
recorded by fluorescence spectrophotometer. In vitro cell culture using HSC-3 and
HN-5 human head and neck cancer cells was performed to test the photodynamic
therapy efficacy of the mTHPP micelles using confocal microscopy and MTT
assay. PDT was performed with a 532 nm laser (total light dose of 12 J/cm2 at 20
mW/cm2)
Hydrophobic mTHPP was encapsulated with high loading efficiency
(>85%) and density (up to 17%) into poly(ethylene glycol)-co-poly(D,Llactic acid) micelles. Micelle size distribution was 30.6 ± 3.3 nm by
transmission electron microscopy and 30.8 ± 0.6 nm by dynamic light
scattering. Confocal microscopy was used to test the photodynamic
cytotoxicity of mTHPP-loaded micelles in vitro in human squamous
HSC-3 cells. All of the cells within the treated area are nonviable 32
minutes after light exposure. Significant in vitro cytotoxicity was
observed when HSC-3 cells were treated with 10% mTHPP micelles
with 100% cell death within the zone of laser light exposure at 420 nm.
No cellular cytotoxicity was observed for cells treated with mTHPP
micelles alone or cells treated with blank micelles exposed to the 420
nm laser light. Further testing of the cytotoxicity of the mTHPP micelles
was carried out using an MTT assay for cell viability. Further testing of
the cytotoxicity of the mTHPP micelles was carried out using an MTT
assay for cell viability as shown in the dose response curves in Figures
2. Phototoxicity and dark toxicity measured against HSC-3 and HN-5
cells with 5% and 10% loaded mTHPP micelles, demonstrated greater
than 90% cell cytotoxicity with PDT, and less than 10% dark toxicity at a
micelle concentration of 25 µg/ml.
1.0
0.8
5% loading -dark
10% loading -dark
5% loading -light
10% loading -light
HSC-3 cell line
1.00
0.6
0.75
0.4
Cell Viability
INTRODUCTION
Cell Viability
ABSTRACT
0.50
0.25
0.2
0.00
0
0.0
4
8
12
16
20
50
100
150
CONCLUSIONS
200
mTHPP micelle Concentration (μg/ml)
1.0
0.6
0.75
0.4
0.50
REFERENCES
0.25
0.2
0.00
0
0.0
Poster Design & Printing by Genigraphics® - 800.790.4001
This study shows that polymeric micelles are effective carriers able to
solubilize mTHPP, a hydrophobic photosensitizer, with high loading
efficiency and loading density. The resulting micelle nanoparticles are
spherical in shape, and have a uniform size distribution. In vitro studies
demonstrate that mTHPP-loaded micelles produce PDT-mediated
cytotoxicity against head and neck cancer cells.
5% loading-dark
10% loading-dark
5% loading-light
10% loading-light
HN5 cell line
1.00
Cell Viability
Cell Viability
0.8
Baran D. Sumer, MD
University of Texas Southwestern Medical Center
Department of Otolaryngology, head and Neck
Surgery
5323 Harry Hines Blvd.
Dallas, TX 75390-9035
Fax: 214 648-2246
Telephone: 214 648-3102
Baran.Sumer@UTSouthwestern.edu
The objective of the present study was to develop polymeric micelles
that are able to efficiently encapsulate mTHPP, a porphyrin-based PS
agent. The solvent evaporation method proved to be very efficient at
encapsulating mTHPP into the PEG-PLA micelle formulation with
minimal drug loss and uniform micelle size. Fluorescence properties of
micelle samples with lower theoretical mTHPP loading were
investigated, and the results showed greater fluorescence on a per dye
molecule basis (1% theoretical loading). This is likely due to fluorescent
quenching of the mTHPP molecules when local concentration inside the
micelle core is extremely high.
Confocal microscopy was used to test the photodynamic cytotoxicity of
mTHPP-loaded micelles in vitro in human squamous HSC-3 cells.
Figure 3A shows that the green fluorescence in HSC-3 cells from
Calcein was lost and replaced by the red fluorescence of PI in a time
dependent manner in the area treated with light at 420 nm after the cells
had been incubated with mTHPP micelles. All of the cells within the
treated area are nonviable 32 minutes after light exposure. It is also
evident that cell damage is only induced in the area where cells are
exposed to light. The area outside of the dashed yellow circles remains
green indicating that mTHPP micelles alone are not toxic. The images
in Figure 3B further demonstrate that the combination of mTHPP-loaded
micelles and light is required for cell death in vitro and that neither alone
is toxic to the HSC-3 cells. The mTHPP micelles exhibited significant
cytotoxicity in the presence of light against both HSC-3 and HN-5 cells
even at concentrations as low as 2 µg/ml, with almost no toxicity
observed for the dark experiments, confirming the photosensitizing
effect of the mTHPP.
mTHPP micelle Concentration(μg/ml)
0
CONTACT
DISCUSSION
4
8
12
16
20
mTHPP Micelle Concentration (μg/ml)
0
50
100
150
1. Biel MA. Photodynamic therapy in head and neck cancer. Curr Oncol Rep
2002;4:87-96.
200
mTHPP Micelle Concentration (μg/ml)
Figure 1. (A) TEM image of mTHPP
micelles with 2% PTA counterstain. (B)
Histogram depicting mTHPP micelle size
distribution determined by DLS analysis.
Figure 2. The response of HSC-3 and
HN5 cells to mTHPP micelle mediated
photo and dark toxicity by MTT assay.
2. Juarranz A, Jaen P, Sanz-Rodriguez F, et al. Photodynamic therapy of
cancer. Basic principles and applications. Clin Transl Oncol 2008;10:148-54.
Figure 3. Confocal images of HSC-3 cells after treatment with a 0.18
mg/mL solution of 10% theoretically loaded mTHPP micelles. (A) Images
were captured at a wavelength of 488 nm for Calcein and 568 nm for
Propidium Iodide at the labeled time points. The approximate area of the
cells exposed to 420 nm light is outlined in the hatched yellow circles. (B)
Images captured at 488 nm for Calcein, 568 nm for Propidium Iodide,
and overlay images taken at 32 min after light exposure of four separate
dishes of cells under different experimental conditions.
ACKNOWLEDGMENTS
This work was supported by the American Academy of Otolaryngology-Head and
Neck Surgery Foundation, (AAO-HNSF) through the Percy Memorial Research
Award to BDS. C. Khemtong was supported by a Multidisciplinary Postdoctoral
Award (W81XWH-06-1-0751) from the Department of Defense Breast Cancer
Research Program. The authors wish to acknowledge Dr. Michael Story Ph.D.
for his contribution of the HN5 cell line. This work was published in the journal
Otolaryngology-HNS July 2010.