Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

Summary

This protocol describes a method for the fabrication of conducting polymer nanoparticles blended with fullerene. These nanoparticles were investigated for their potential use as a next generation photosensitizers for Photodynamic Therapy (PDT).

Abstract

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for Photodynamic Therapy (PDT) is reported. The nanoparticles are formed by hydrophobic interaction of the semiconducting polymer MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) with the fullerene PCBM (phenyl-C₆₁-butyric acid methyl ester) in the presence of a non-compatible solvent. MEH-PPV has a high extinction coefficient that leads to high rates of triplet formation, and efficient charge and energy transfer to the fullerene PCBM. The latter processes enhance the efficiency of the PDT system through fullerene assisted triplet and radical formation, and ultrafast deactivation of MEH-PPV excited stated. The results reported here show that this nanoparticle PDT sensitizing system is highly effective and shows unexpected specificity to cancer cell lines.

Introduction

In Photodynamic Therapy (PDT) photosensitizers are administered to target tissue, and upon exposure to light the photosensitizer generates Reactive Oxygen Species (ROS). ROS species such as singlet oxygen and superoxide can induce oxidative stress and subsequent structural damage to cells and tissue^1-4. Due to its ease of application this method has been actively investigated and clinical trials have taken place^5,6. However, significant issues such as dark toxicity of the sensitizers, patient sensitivity to light (due to non-selective distribution of the sensitizer), and hydrophobicity of the sensitizers (which leads to reduced bioavailability an....

Protocol

1. Culturing Cell Lines

1. Thaw TE 71 (Mouse thymic epithelial cells), MDA-MB-231 (Human breast cancer cells), A549 (Human lung cancer cells) and OVCAR3 (Human ovarian tumor cells) by holding the cryogen vials in warm water for less than 2 min. Add 10 ml DMEM media supplemented with 10% FBS to each cell line and centrifuge for 6 min at 106 x g.
2. Aspirate the suspension and add 3ml media to the pellet. Mix the cells properly by pipetting several times. Add this cell solution to pre-warmed 7 ml DMEM media supplemented with 10% FBS in T75 flasks and keep the flasks in humidified atmosphere of 95% air/ 5% CO₂ at 37 °C. Label this flask as....

Results

Uptake and intrinsic cytotoxicity of nanoparticles

The 50 wt% blended MEH-PPV/PCBM nanoparticles were incubated with TE 71, MDA-MB-231, A549 and OVCAR3 cell lines. The PCBM blending level was chosen as 50 wt% PCBM, which has been shown to provide ideal charge and energy transfer properties between conjugated polymers and fullerenes¹⁴. Fluorescence images of nanoparticle uptake are shown in Figure 1B. Cells were incubated for 24 hr with nanoparticles to ensure nanopa.......

Discussion

To achieve nanoparticle uptake it was necessary to maintain some critical measures while fabricating the nanoparticles. A 10^-6 M MEH-PPV solution (blended with 50 wt% PCBM) in THF was prepared to inject into DI water, as it was observed that the concentration of this solution plays an important role in determining the size of nanoparticles being formed. Concentration was checked by UV-vis spectroscopy. Note that in protocol step 2.1.3 it was necessary to dilute the initially prepared MEH-PPV solution (undilute.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV)	Sigma Aidrich	536512-1G	average Mn 150,000-250,000
[6,6]-Phenyl C61 butyric acid methyl ester (PCBM)	Sigma Aidrich	684449-500MG	>99.5%
Tetrahydrofuran (THF)	EMD	TX0284-6	Drisolv
1 ml syringe	National Scientific Company	37510-1	For filtration of MEH-PPV solution
Syringe filter	VWR	28145-495	25 mm, 0.2 µm, PTFE
1 ml syringe	Hamilton Company	81320	For injection of MEH-PPV solution into water to make nanoparticles
Dulbecco's Modification of Eagle's Medium/Ham's F-12 50/50 Mix (DMEM)	Corning (VWR)	45000-350
Hank's Balanced Salt Solution without phenol red (HBSS)	Quality Biological (VWR)	10128-740
Dulbecco's Phosphate-Buffered Saline, 1x without calcium and magnesium (DPBS)	Corning (VWR)	45000-436
Fetal Bovine Serum, Regular (Heat Inactivated) (FBS)	Corning (VWR)	45000-736
Trypsin EDTA 1x 0.25%	Corning (VWR)	45000-664	Trypsin/2.21 mM EDTA in HBSS without sodium bicarbonate, calcium and magnesium Porcine Parvovirus Tested
16% Paraformaldehyde	Electron Microscopy Sciences	15710	16% paraformaldehyde is diluted to 4% by adding PBS
DAPI	Biotium VWR	89139-054	Nuclear stain
5 ml pipettes	VWR	82050-478
75 cm2 culture flask	VWR	82050-856	for culturing cells
96-well plates	VWR	82050-771	for MTT assays
Tissue Culture Dishes with Vents	Greiner Bio-One (VWR)	82050-538
Propidium iodide	Molecular probes	P3566
Annexin V FITC	Invitrogen	A13199	dye for apoptosis
Celltiter 96 non-R 1000 assays	Promega (VWR)	PAG4000	MTT
CellROX Green Reagent, for oxidative stress detection	Invitrogen	C10444	For ROS detection
UV-vis spectrometer	Agilent 8453
Fluorescence spectrometer	NanoLog HoribaJobin Yvon
Dynamic light scattering	PD2000DLS, Precision detector
Incubator	NuAir DH Autoflow
Confocal microscope	Zeiss Axioskop2		63X oil immersion objective lens
Epiluminescence microscope	Olympus IX71		60X water immersion objective lens, Andor Zyla sCMOS camera
Solar Simulator	Newport 67005 Oriel Instruments
Reference solar cell	Oriel		VLSI Standards Incorporated
Microplate reader	BioTek Ex808
Hemocytometer	Hausser Scientific Partnership	3200	For counting cells