Fotodynamisk terapi med Blended ledende polymer / fulleren Nanopartikel Fotosensibilisatorer
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
I Fotodynamisk terapi (PDT) fotosensibilisatorer indgives til målvæv, og ved udsættelse for lys fotosensibilisatoren genererer reaktive oxygenarter (ROS). ROS arter såsom singlet oxygen og superoxid kan inducere oxidativt stress og efterfølgende strukturel skade på celler og væv 1-4. På grund af sin lette anvendelsen af denne metode har været aktivt undersøgt og kliniske forsøg har fundet sted 5,6. Der er dog væsentlige emner som mørketoksicitet af sensibilisatorer, patientens følsomhed over for lys (som følge af ikke-selektiv fordeling af sensibilisator), og hydrofobicitet sensibilisatorer (som fører til nedsat biotilgængelighed og potentiel akut toksicitet) forbliver.
Her rapporterer vi en fremgangsmåde til fremstilling og in vitro evaluering af ledende polymer nanopartikler blandet med fulleren som den næste generation fotosensibilisatorer til PDT. Nanopartiklerne dannes ved selvaggregering afhalvledende polymer MEH-PPV (poly [2-methoxy-5- (2-ethylhexyloxy) -1,4-phenylenevinylene]) med fulleren PCBM (phenyl-C 61 -smørsyremethylester), når disse materialer opløses i en kompatibel Opløsningsmidlet hurtigt injiceres i et ikke-kompatibelt opløsningsmiddel (figur 1A). Valget af MEH-PPV som vært polymer er motiveret af sin høje ekstinktionskoefficient, der fører til høje triplet dannelse, og både effektiv og ultrahurtig ladning og energi overførsel til fulleren PCBM 7. Disse egenskaber er ideelle til sensibilisering af singlet oxygen og superoxid dannelse i PDT.
Fulleren faktisk er blevet anvendt i PDT i både molekylære og nanopartikel formular 8-13. Imidlertid har alvorlige cytotoksicitet hæmmet videreudvikling 12. Her viser vi, at indkapsle fulleren i et væld matrix af MEH-PPV at give sammensatte MEH-PPV / PCBM nanopartikler resulterer i en PDT sensibiliserende materiale, som jeger ikke uløseligt cytotoksisk, viser specificitet mod kræftceller grund nanopartikel størrelse og overfladeladning, og udbyttet meget effektiv PDT behandling ved doser svagt lys på grund af de førnævnte fotofysiske egenskaber.
Protocol
Representative Results
Discussion
For at opnå nanopartikel optagelse var det nødvendigt at opretholde nogle kritiske foranstaltninger samtidig fremstilling af nanopartikler. En 10 -6 M MEH-PPV løsning (blandet med 50 vægt-% PCBM) i THF var indstillet på at tilføre DI vand, da det blev observeret, at koncentrationen af denne løsning spiller en vigtig rolle i fastsættelsen af størrelsen af nanopartikler dannes. Koncentration blev kontrolleret ved UV-vis spektroskopi. Bemærk, at i protokol trin 2.1.3 det var nødvendigt at fortynde det…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors gratefully acknowledge the National Science Foundation (NSF) for financial support of this work through a CAREER award (CBET-0746210) and through award CBET-1159500. We would like to thank Dr. Turkson (Univ. of Hawaii Cancer Center) and Dr. Altomare (Univ. of Central Florida College of Medicine) for assistance with cell culture.
Materials
| 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 |
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