Name: photodynamic therapy with blended conducting polymer/fullerene nanoparticle photosensitizers
Uploaded: 2015-10-28T14:00:00
Description: Watch this Scientific Journal Video about Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers at JoVE.com

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.

1. 배양 세포주 <ol><li> 이하의 2 분 동안 온수에 냉각제 튜브를 보유함으로써 해동 TE 71 (마우스 흉선 상피 세포), MDA-MB-231 (인간 유방암 세포), A549 (인간 폐암 세포) 및 OVCAR3 (인간 난소 종양 세포) . 106 × g으로 6 분 동안 각각의 세포주를 원심 분리하고, 10 % FBS가 보충 된 10 mL의 DMEM 배지를 추가한다. </li><li> 서스펜션을 기음과 펠렛에 3 ㎖ 미디어를 추가 할 수 있습니다. 적절 수회 피펫 팅하여 세포?...

<ol>
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L., Belichenko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+apoptosis+in+response+to+photodynamic+therapy:+what,+where,+why,+and+how.">The role of apoptosis in response to photodynamic therapy: what, where, why, and how.</a> Photochem Photobiol Sci. 1 (1), 1-21 (2002).</li><li>Ormond, A., Freeman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+Sensitizers+for+Photodynamic+Therapy.">Dye Sensitizers for Photodynamic Therapy.</a> Materials. 6 (3), 817-840 (2013).</li><li>Pass, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+Therapy+in+Oncology+-+Mechanisms+and+Clinical+Use.">Photodynamic Therapy in Oncology - Mechanisms and Clinical Use.</a> J Natl Cancer Inst. 85 (6), 443-456 (1993).</li><li>Sariciftci, N. S., Smilowitz, L., Heeger, A. J., Wudl, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinduced+electron+transfer+from+a+conducting+polymer+to+buckminsterfullerene.">Photoinduced electron transfer from a conducting polymer to buckminsterfullerene.</a> Science. 258 (5087), 1474-1476 (1992).</li><li>Sperandio, F. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinduced+electron-transfer+mechanisms+for+radical-enhanced+photodynamic+therapy+mediated+by+water-soluble+decacationic+C-70+and+C84O2+Fullerene+Derivatives.">Photoinduced electron-transfer mechanisms for radical-enhanced photodynamic therapy mediated by water-soluble decacationic C-70 and C84O2 Fullerene Derivatives.</a> Nanomed-Nanotechnol. 9 (4), 570-579 (2013).</li><li>Fan, J. Q., Fang, G., Zeng, F., Wang, X. D., Wu, S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-Dispersible+Fullerene+Aggregates+as+a+Targeted+Anticancer+Prodrug+with+both+Chemo-+and+Photodynamic+Therapeutic+Actions.">Water-Dispersible Fullerene Aggregates as a Targeted Anticancer Prodrug with both Chemo- and Photodynamic Therapeutic Actions.</a> Small. 9 (4), 613-621 (2013).</li><li>Grynyuk, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoexcited+fullerene+C-60+disturbs+prooxidant-antioxidant+balance+in+leukemic+L1210+cells.">Photoexcited fullerene C-60 disturbs prooxidant-antioxidant balance in leukemic L1210 cells.</a> Materialwiss Werkstofftech. 44 (2-3), 139-143 (2013).</li><li>Liu, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separately+doped+upconversion-C-60+nanoplatform+for+NIR+imaging-guided+photodynamic+therapy+of+cancer+cells.">Separately doped upconversion-C-60 nanoplatform for NIR imaging-guided photodynamic therapy of cancer cells.</a> Chem Commun. 49 (31), 3224-3226 (2013).</li><li>Trpkovic, A., Todorovic-Markovic, B., Trajkovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+pristine+versus+functionalized+fullerenes:+mechanisms+of+cell+damage+and+the+role+of+oxidative+stress.">Toxicity of pristine versus functionalized fullerenes: mechanisms of cell damage and the role of oxidative stress.</a> Arch Toxicol. 86 (12), 1809-1827 (2012).</li><li>Chen, Z. Y., MA, L. J., Liu, Y., Chen, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Functionalized+Fullerenes+in+Tumor+Theranostics.">Applications of Functionalized Fullerenes in Tumor Theranostics.</a> Theranostics. 2 (3), 238-250 (2012).</li><li>Park, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+heterojunction+solar+cells+with+internal+quantum+efficiency+approaching+100%.">Bulk heterojunction solar cells with internal quantum efficiency approaching 100%.</a> Nat Photonics. 3 (5), 297-302 (2009).</li></ol>

나노 입자를 제조하는 동안 나노 입자의 흡수를 달성하기 위해 그것은 몇 가지 중요한 조치를 유지하기 위해 필요한이었다. 이 용액의 농도가 형성되는 나노 입자의 크기를 결정하는데 중요한 역할을하는 것으로 관찰되었다로 THF에 (50 중량 % PCBM 혼합) × 10-6 M MEH-PPV 용액을 DI 물 내로 주입하도록 준비 하였다. 농도는 자외선 - 마주 분광에 의해 확인되었다. 프로토콜 단계에서 그 처음이 ?...

광 역학 요법 (PDT) 감광제는 조직을 대상으로 투여하고, 감광제를 노광시에 활성 산소 종 (ROS)를 생성한다. 이러한 중항 산소와 슈퍼 옥사이드 등의 활성 산소 종은 산화 스트레스 및 세포 및 조직 1-4 이후에 구조적 손상을 유도 할 수 있습니다. 때문에 응용 프로그램의 용이성이 방법을 적극적으로 조사되었으며, 임상 시험 장소 5,6를 촬영했다. 그러나, (감소 생체 이용률 및 잠재적 인 급성 독성에 이르게) 증감의 어두운 증감의 독성, 빛 (으로 인한 증감의 비 선택적 분포) 환자 감도 및 소수성과 같은 중요한 문제가 남아있다. 여기에서 우리는 제작과 시험관 PDT의 차세대 감광제로 풀러렌과 혼합 고분자 나노 입자를 실시하고 평가하는 방법을보고합니다. 나노 입자의자가 응집에 의해 형성된다반도체 성 중합체 MEH-PPV (폴리 [2- 메 톡시 -5- (2- 에틸 헥 실옥시) -1,4- 페닐 렌])이 물질에 용해 호환 플러렌 PCBM (C-61 페닐 - 부티르산 메틸 에스터)와 용매를 신속하게 호환되지 않는 용매 (그림 1A)에 주입된다. 호스트 중합체로서 MEH-PPV의 선택은 삼중 형성 높은 속도에 이르게 높은 흡광 계수에 의해 동기 및 플러렌 PCBM 7 효율적인 초고속 충전 및 에너지 전달을 모두한다. 이러한 속성은 단일 산소와 PDT의 과산화물 형성의 증감에 이상적입니다. 플러렌은 실제로 두 분자 및 나노 입자 형태로 8-13 PDT에 적용되어왔다. 그러나 심각한 독성은 추가 개발 (12) 방해했다. 여기에서 우리는 MEH-PPV의 호스트 매트릭스에 풀러렌을 캡슐화하는 PDT 증감 재료 복합 MEH-PPV / PCBM 나노 입자의 결과를 얻을 것을 보여 그 전본질적으로 세포 독성이 아니다 인해 상기 광 물리 특성에 암 나노 입자의 크기 및 표면 전하에 의한 세포 및 낮은 조명 용량에서 수율이 매우 효과적인 PDT 치료 대한 특이성을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV)</td>
			<td>Sigma Aidrich</td>
			<td>536512-1G</td>
			<td>average Mn&#160;150,000-250,000</td>
		</tr>
		<tr>
			<td>[6,6]-Phenyl C61&#160;butyric acid methyl ester (PCBM)</td>
			<td>Sigma Aidrich</td>
			<td>684449-500MG</td>
			<td>&#62; 99.5%</td>
		</tr>
		<tr>
			<td>Tetrahydrofuran (THF)</td>
			<td>EMD</td>
			<td>TX0284-6</td>
			<td>Drisolv</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>National Scientific Company</td>
			<td>37510-1</td>
			<td>For filtration of MEH-PPV solution</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>VWR</td>
			<td>28145-495</td>
			<td>25 mm, 0.2 &#181;m, PTFE</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Hamilton Company</td>
			<td>81320</td>
			<td>For injection of MEH-PPV solution into water to make nanoparticles</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium/Ham&#39;s F-12 50/50 Mix (DMEM)</td>
			<td>Corning (VWR)</td>
			<td>45000-350</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution without phenol red (HBSS)</td>
			<td>Quality Biological (VWR)</td>
			<td>10128-740</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, 1X without calcium and magnesium (DPBS)</td>
			<td>Corning (VWR)</td>
			<td>45000-436</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Regular (Heat Inactivated) (FBS)</td>
			<td>Corning (VWR)</td>
			<td>45000-736</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA 1X 0.25%</td>
			<td>Corning (VWR)</td>
			<td>45000-664</td>
			<td>Trypsin/2.21 mM EDTA in HBSS without sodium bicarbonate, calcium and magnesium Porcine Parvovirus Tested</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences&#160;</td>
			<td>15710</td>
			<td>16% paraformaldehyde is diluted to 4% by adding PBS</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>&#160;Biotium VWR</td>
			<td>89139-054</td>
			<td>Nuclear stain</td>
		</tr>
		<tr>
			<td>5 ml pipettes</td>
			<td>VWR</td>
			<td>82050-478</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 culture flask</td>
			<td>VWR</td>
			<td>82050-856</td>
			<td>for culturing cells</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>VWR</td>
			<td>82050-771</td>
			<td>for MTT assays</td>
		</tr>
		<tr>
			<td>Tissue Culture Dishes with Vents</td>
			<td>Greiner Bio-One (VWR)</td>
			<td>82050-538</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Molecular probes</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V FITC</td>
			<td>Invitrogen</td>
			<td>A13199</td>
			<td>dye for apoptosis</td>
		</tr>
		<tr>
			<td>Celltiter 96 non-R 1000 assays</td>
			<td>Promega (VWR)</td>
			<td><a href="https://www.vwrsp.com/catalog/product/index.cgi?catalog_number=PAG4000">PAG4000</a></td>
			<td>MTT</td>
		</tr>
		<tr>
			<td>CellROX Green Reagent, for oxidative stress detection</td>
			<td>Invitrogen</td>
			<td>C10444</td>
			<td>For ROS detection</td>
		</tr>
		<tr>
			<td>UV-vis spectrometer</td>
			<td>Agilent 8453</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence spectrometer</td>
			<td>NanoLog HoribaJobin Yvon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic light scattering</td>
			<td>PD2000DLS, Precision detector</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>NuAir DH Autoflow</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss Axioskop2</td>
			<td></td>
			<td>63X oil immersion objective lens</td>
		</tr>
		<tr>
			<td>Epiluminescence microscope</td>
			<td>Olympus IX71</td>
			<td></td>
			<td>60X water immersion objective lens, Andor Zyla sCMOS camera</td>
		</tr>
		<tr>
			<td>Solar Simulator</td>
			<td>Newport 67005 Oriel Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reference solar cell</td>
			<td>Oriel&#160;</td>
			<td></td>
			<td>VLSI Standards Incorporated</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek Ex808</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Hausser Scientific Partnership</td>
			<td>3200</td>
			<td>For counting cells</td>
		</tr>
	</tbody>
</table>

photodynamic therapy with blended conducting polymer/fullerene nanoparticle photosensitizers

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.

통풍 및 나노 입자의 고유 세포 독성 50 중량 % 혼합 MEH-PPV / PCBM 나노 입자 TE (71)와 함께 배양하고, MDA-MB-231, A549 및 OVCAR3 세포주. PCBM 혼합 수준은 공액 폴리머 및 풀러렌 (14) 사이의 최적의 충전 및 에너지 전달 특성을 제공하는 것으로 나타났다 50 중량 % PCBM로서 선택되었다. 나노 입자의 흡수 형광 이미지는도 1b에 도시되어있다. 나노 입자는 세포 흡수를 ?...

Watch this Scientific Journal Video about Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers at JoVE.com

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

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).

혼합 폴리머 / 풀러렌 나노 입자 감광제를 실시와 광 역학 치료

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next ...

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