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.培养细胞系<ol><li>解冻的TE 71（小鼠胸腺上皮细胞），MDA-MB-231（人乳腺癌细胞），A549（人肺癌细胞）和OVCAR3（人类卵巢癌细胞）由保持在温水中冷冻剂瓶为小于2分钟。添加补充有10％FBS的每个细胞系，离心6分钟，在106×g下10 ml的DMEM培养基。 </li><li>吸出悬挂和3毫升媒体添加到沉淀。适当吹打几次的细胞混合。加该细胞溶液到预先温热7 ml，在T75烧瓶补充有10％FBS的DMEM培养基，并保持在95％空气/ 5％CO 2的潮湿气氛中的烧瓶在37℃。标签此瓶为通道0。 </li><li>当细胞的汇合达到80％，通过将它们与0.05％胰蛋白酶10分钟收获细胞。通过加入媒体等量中和胰蛋白酶。离心该溶液6分钟，在106×g下。取下悬挂和中加3毫升的新鲜媒体吧。混合逢L和少量（100微升）转移到含有7毫升媒体培养瓶中。孵育培养瓶中的孵化器。标签的烧瓶通道1。 </li><li>培养细胞系直至通路11或12。 </li></ol> 2.制造纳米粒子<ol><li>的〜10 -6 M（调整后）未稀释的MEH-PPV原液的制备<ol><li>在小瓶中添加1mg的聚[2-甲氧基-5-（2-乙基己氧基）-1,4-亚苯基亚乙烯基]（MEH-PPV）与分子量（平均M n）150,000-250,000克/摩尔和3ml四氢呋喃（THF） 。 2小时搅拌混合物，同时在80℃的热板上加热。 </li><li>使用0.2微米的注射器过滤器过滤在一个新的小瓶上述溶液中。标签这一解决方案为“未稀释的MEH-PPV原液”。该解决方案将参与集中的微调后的纳米颗粒制备如步骤2.1.3和2.1.4描述。 </li><li>加50微升未稀释的MEH-PPV原液入3毫升THF。标签这个解决方案是“稀释MEH-PPV原液”。通过紫外可见光谱转移到1厘米石英试管，并测量吸光度495纳米。 </li><li>如果稀释MEH-PPV原液的吸光度比0.17以上，稀释未稀释的MEH-PPV原液通过一次加入更多的THF 1毫升，并重复步骤2.1.3直到在495nm处测定的吸光度是在范围0.13-0.17。 </li><li>通过使用朗伯 - 比尔定律，如下所示计算稀释MEH-PPV库存溶液的摩尔浓度。此处0.15吸光度用作示例的协议的其余部分（即，基于观察到的吸光度调整所有后面的计算），使用的MEH-PPV消光 ​​系数为10 7 M -1 -1和所使用的路径长度是1厘米。 A =εxbxc（方程1） （ε - 的摩尔消光系数，A - 吸光度，B - 路径长度和c - 溶液的浓度） 0。15 = 10 7 M -1 -1×1cm的XC（方程式2） C = 1.5×10 -8 M（等式3） 使用此浓度找到未稀释的MEH-PPV原液的浓度如下： M 1 -V 1 = M 2 V 2（方程4） 0.15×10 -7的M×3050微升= M 2×50微升（方程5） M 2 = 9.15×10 -7 M（方程6） 这是&#39;调整未稀释的MEH-PPV原液“的浓度。 </li></ol></li><li>计算MEH-PPV的质量在调整未稀释的MEH-PPV原液<ol><li>计算MEH-PPV的质量在1毫升的调整未稀释原液如下所示使用在节2.1.4中获得的摩尔浓度和分子量（M w）的 MEH-PPV的，这是10 6克/摩尔。 M = N / V（N - 没有痣，V - L中量）（方程7） 由此，MEH-PPV在1ml质量调整UNDiluted原液是9.15×10 -4克</li></ol></li><li>在MEH-PPV混合PCBM <ol><li>计算的苯基-C 61 -丁酸甲酯（PCBM）的质量要加入到库存MEH-PPV溶液以使50％（重量）PCBM掺杂MEH-PPV溶液，其中50％（重量）PCBM定义相对于所述质量MEH-PPV（即MEH-PPV的质量的一半）。通过使用MEH-PPV的2.2节所获得的权重。 的PCBM / 9.15×10 -4克MEH-PPV×100％= 50％（重量）PCBM的质量（方程8） 对PCBM = 4.57×10 -4 g质量（方程9） </li><li>重1毫克PCBM在小瓶中并加入500μlTHF中。使用PCBM的分子量计算PCBM的浓度在该溶液作为910.88克/摩尔 对PCBM溶液= 0.001克/（910.88克/摩尔* 500微升）摩尔浓度（方程10） 对PCBM溶液摩尔浓度= 2.19×10 -9摩尔/微升（方程11） </li><li>计算PCBM溶液的体积需要添加至调整未稀释的急H-PPV原液通过利用在步骤2.3.2中计算的摩尔浓度，以获得在THF中50％（重量）PCBM掺杂MEH-PPV溶液 4.57×10 -4克PCBM×1摩尔/ 910.88 GX 1微升/ 2.19×10 -9摩尔= 229微升（方程12） 加入229微升PCBM溶液加入到1毫升稀释调节MEH-PPV原液拌匀。 </li></ol></li><li>通过再沉淀法纳米粒子的制备<ol><li>转移1毫升共混MEH-PPV / PCBM溶液入1ml注射器与连接到它的针头的。 </li><li>迅速注入1毫升共混MEH-PPV / PCBM溶液加入到4毫升DI水在1,200rpm下搅拌。停止注射后立即搅拌。使用这些纳米粒子没有进一步处理。 </li></ol></li></ol> 3.孵化与纳米粒子细胞系成像注：所有成像实验进行35毫米培养皿中完成<ol><li> nanoparti的吸收在细胞系克莱斯<ol><li>培养细胞系直至12 个通道。在第 12次传代培养的细胞35毫米培养皿。调整加入到35毫米培养皿这种细胞的浓度在24小时后，细胞是40％汇合。 </li><li>在这个阶段中删除添加有从培养皿10％FBS的DMEM培养基，洗涤细胞用1×DPBS两次，并添加100μl的纳米颗粒悬浮液于2ml DMEM中的培养皿。 </li><li> 24小时后除去从培养皿的DMEM /纳米颗粒悬浮液，并用1×DPBS 3次洗细胞。然后，通过用4％多聚甲醛孵育10分钟固定细胞。用DPBS洗两次。通过与染料孵育2分钟将细胞染色用300nM的DAPI染色。用DPBS洗两次。然后，使细胞保持在DPBS成像。 </li></ol></li><li>检测活性氧<ol><li>文化A549和在培养皿中，6％的细胞系，如第3.1.1解释OVCAR3细胞系。标注培养皿的表1中所示。 </li><li>加入100μl的纳米颗粒悬浮液的于2ml DMEM中成三个培养皿所示的表1中，并培育24小时。用于将接收的光的剂量，24小时后洗涤细胞并悬浮细胞在HBSS（Hank氏平衡盐溶液）染料可用媒体的培养皿。 </li><li>预热太阳模拟器的灯15分钟。放置过滤紫外线灯的前方以滤除紫外光。通过调整灯的功率，以获得0.5太阳（50毫瓦/厘米2）强度在培养皿的表面校准与基准太阳能电池的灯。在这种特定的设置该条件是与提供给灯218功率W实现。 </li><li>将培养皿在灯（盖打开）60分钟，这导致了180焦耳/厘米2，如下面的计算的光剂量： 50毫瓦/厘米2×3600秒/ 1000 = 180焦耳/ cm 2的</li><li>除去HBSS和不洗进一步添加补充有10％FBS的对陪替氏培养皿2 ml的DMEM培养基。孵育细胞另外2小时。 </li><li>为阳性对照孵育细胞100μM的过氧化氢​​（H 2 O 2），30分钟。 </li><li>通过在37℃下孵育细胞与染料30分钟将细胞染色在所有的培养皿中以5微米的活性氧检测用试剂的终浓度。 </li><li>通过用4％多聚甲醛孵育10分钟固定细胞。用DPBS洗两次。通过与染料孵育2分钟将细胞染色用300nM的DAPI染色。用DPBS洗两次。然后，使细胞保持在DPBS成像。 </li></ol></li><li>细胞凋亡和坏死PI和Annexin V FITC <ol><li>培养在5培养皿每个细胞系。这三个培养皿将是实验，而其余2培养皿将对照样品。孵育3培养皿的实验，纳米粒子为explained 3.1.2节。对照样品没有孵育纳米颗粒。 </li><li>经过24小时培养遵循的步骤3.2.3。 </li><li>将在3个实验培养皿灯（盖打开）下，并删除一个20分钟，一个在40分钟和一个60分钟。这就产生了暴露于光剂量的60，120和180焦耳/ 平方厘米，分别为（计算第3.2.4节）三个样品。其次，管理一个180焦耳/ 平方厘米的光剂量的控制培养皿之一。这与在不存在纳米颗粒的施加光剂量的控制。不要使用光剂量剩余的对照样品（无纳米粒子和没有光剂量应用）。 </li><li>取代的HBSS用补充有10％FBS的DMEM培养基，并保持培养皿在培养箱4小时。 </li><li>通过孵育细胞与染料15分钟将细胞染色在实验和对照培养皿与20μl膜联蛋白V FITC。用DPBS洗两次。染色的CELLS与300nM的DAPI以及300nM的PI（碘化丙啶）通过与染料2分钟孵育。用DPBS洗两次。然后，使细胞保持在DPBS成像。 </li></ol></li></ol>纳米四固有细胞毒性<ol><li>在96孔板计数细胞和培养<ol><li>通过除去培养基并用DPBS后跟细胞0.05％胰蛋白酶进行10分钟的温育洗涤细胞两次收获从培养瓶中的细胞。添加补充有10％FBS的向所得细胞溶液2 ml的DMEM培养基。适当地混合该溶液到细胞团分离成单峰。 </li><li>取100微升细胞悬浮液，并加入到补充有10％FBS的900微升DMEM培养基。拌匀。放置10微升此悬浮液到血球。计数使用血球细胞和调节细胞悬浮液的浓度在4.1.1至5×10 4个细胞/ ml。 </li><li>取5 96孔板标记为0小时，24小时R，48小时，72小时和96小时。添加50微升5×10 4个细胞/ ml的细胞溶液（TE 71和A549）插入孔中作为示于表2中，从而接种2500个细胞/孔。执行相同的过程OVCAR3和MDA-MB-231细胞系。 </li><li> 24小时后洗掉的孔用1×DPBS添加增加纳米颗粒引入该井中的浓度如表 2所示的50微升。通过加入20，100，准备不同的纳米颗粒的浓度，和180微升纳米颗粒悬浮液的从2.4.2至DMEM中成获得的2毫升的最终体积，其产生0.4×10 -4毫克/毫升的纳米颗粒的浓度，2.0×10 -4毫克/毫升和3.6×10 -4毫克/毫升。每个细胞系具有一式三份对纳米颗粒的各浓度。 </li></ol></li><li>测量在黑暗的细胞生存力<ol><li>加入纳米粒子后，立即加入10微升MTT到0小时板。将培养板4小时的甲臜crystALS形成。添加50μl的增溶溶液入井。孵育平板6小时以溶解甲臜晶体。 </li><li>通过记录570nm处的吸光度与酶标仪测量0小时板的细胞生存力。 </li><li> 24小时盘，洗涤细胞与1X DPBS，并在每一个好后，24小时孵化与纳米粒子加入50微升的媒体。加入MTT和阅读4.2.1和4.2.2所解释的板块。 </li><li> 48小时，72小时，和96小时板，洗涤细胞用1×DPBS和24小时后孵育纳米颗粒添加50μl的介质插入到孔中。孵育细胞对剩余时间段。 </li><li> 4.2.5）测量细胞存活力在特定时间点在4.2.1和4.2.2说明。 </li><li>重复整个实验进行3次（n = 3） </li></ol></li></ol> 5.测量细胞活性PDT后<ol><li>计数细胞并在96孔板中培养。 <ol><li>标注一组96孔板如表 3所示，无论对于TE 71 + A549板和OVCAR3 + MDA-MB-231板。如图4.1.3所述板的布局将是相同的。 </li><li>种子96孔板如在第4.1节具有相同的布局说明如在4.1.2节示于表2。 </li><li> 24小时后添加纳米颗粒板作为4.1.4说明。 </li><li>另外的纳米颗粒后24小时，洗涤细胞用1×DPBS并添加50微升HBSS到每个孔中。 </li><li>照射与各光剂量的板块如3.2.3解释。 </li><li>取代的HBSS用补充有10％FBS 50微升DMEM培养基孵育所述板对PDT后的各时间段。 </li><li>后每个时间段测量细胞存活力在4.2.1和4.2.2说明。 </li></ol></li></ol> 6.荧光显微镜<ol><li>纳米粒子的吸收<ol><li>打开显微镜的灯第二成像前的激光30分钟。把含有固定的细胞在显微镜的阶段（如在第3.1节阐述）培养皿。 </li><li>收集从纳米颗粒和DAPI通过使用过滤器如表 4中所示的荧光。叠加的相位相反，纳米颗粒和DAPI图像的ImageJ软件。 </li></ol></li><li>检测活性氧<ol><li>打开的成像前的显微镜30分钟的灯。将含有细胞在显微镜的阶段（如第3.2节所述）的培养皿中。 </li><li>收集从纳米颗粒，DAPI，和活性氧检测用试剂通过使用过滤器如表 4中所示的荧光。叠加的相位相反，纳米颗粒，DAPI和ROS检测试剂图像的ImageJ软件。 </li></ol></li><li>细胞凋亡和坏死PI和Annexin V FITC <ol><li>打开的成像前的显微镜30分钟的灯。把含有细胞（如在解释培养皿在显微镜的阶段，第3.3节）。 </li><li>收集从纳米颗粒，DAPI，PI和膜联蛋白V FITC通过使用过滤器如表 4中所示的荧光。叠加的相位相反，纳米颗粒，DAPI，PI和膜联蛋白V FITC图像的ImageJ软件。 </li></ol></li></ol>

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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>

The authors have nothing to disclose.

为了实现纳米粒子吸收，有必要保持一定的临界措施而制造该纳米颗粒。甲10 -6 M的 MEH-PPV溶液（混合有50重量％PCBM）在THF准备注入DI水，因为据观察，这种溶液的浓度在确定纳米粒子的尺寸起重要作用而形成。浓度通过紫外可见光谱检查。需要注意的是在协议步骤2.1.3，有必要首先在服药前的UV-vis光谱，因为该溶液稀释最初制备的MEH-PPV溶液（未稀释MEH-PPV原液）具有吸光度大于1喷射的速度?...

在光动力疗法（PDT）光敏剂施用给靶组织，并在接触光光敏剂产生活性氧（ROS）。活性氧物种如单线态氧和超氧化物可以诱导氧化应激和细胞和组织1-4后续结构损坏。因为它易于应用的这种方法已被积极地研究和临床试验已经发生5,6-。然而，显著的问题，如敏化剂的暗毒性，患者感光度（由于敏化剂的非选择性分布），以及疏水性的敏化剂（其导致生物利用度降低和潜在的急性毒性）保持。 这里，我们报告了一种用于制造和体外进行配合富勒烯作为下一代光敏剂用于光动力聚合物纳米颗粒的评价。纳米颗粒通过自聚集形成所述高分子半导体MEH-PPV（聚[2-甲氧基-5-（2-乙基己氧基）-1,4-亚苯基亚乙烯基]）与富勒烯PCBM（苯基-C 61 -丁酸甲酯）当溶解在相容的这些材料溶剂迅速注入到非相容的溶剂（图1A）。 MEH-PPV作为宿主聚合物的选择是由它的高消光系数，导致的激发三重态的形成率高激励，既高效又超快电荷和能量转移到富勒烯PCBM 7。这些特性非常适合单线态氧和超氧形成PDT的致敏。 富勒烯事实上已经在PDT中被应用在两个分子和纳米颗粒形式8-13。然而，严重的毒性阻碍了进一步的发展12。在这里，我们表明，在MEH-PPV的基质包封富勒烯，得到复合MEH-PPV / PCBM的纳米颗粒的结果在一个PDT敏化材料，我不是本质的细胞毒性，表明向癌细胞由于纳米颗粒尺寸和表面电荷，和产量高效PDT治疗在低光剂量的特异性由于上述光物理特性。

<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.

吸收和纳米粒子的固有细胞毒性的50重量％的共混MEH-PPV / PCBM的纳米颗粒孵育用TE 71，MDA-MB-231，A549和OVCAR3细胞系。的PCBM共混水平被选为50重量％PCBM，这已被证明是提供共轭聚合物和富勒烯14之间理想电荷和能量转移性能。纳米颗粒摄取的荧光图像示于图1B。细胞孵育24小时，用纳米粒子，以确保纳米颗粒摄取。然后将细胞用4％多聚甲醛在成像之前，以及为?...

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).

光动力疗法与混合导电高分子/富勒烯纳米颗粒光敏剂

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

photodynamic-therapy-with-blended-conducting-polymerfullerene

Research

JoVE Journal

Chemistry

8.4K 次观看.  University of Central Florida. 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). 

视频: Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

Conducting Miller-Urey Experiments

In 1953, Stanley Miller reported the production of biomolecules from simple gaseous starting materials, using an apparatus constructed to simulate the primordial Earth&#39;s atmosphere-ocean system. Miller introduced 200 ml of water, 100 mmHg of H2, 200 mmHg of CH4, and 200 mmHg of NH3 into the apparatus, then subjected this mixture, under reflux, to an electric discharge for a week, while the water was simultaneously heated. The purpose of this manuscript is to provide the reader with a general experimental protocol that can be used to conduct a Miller-Urey type spark discharge experiment, using a simplified 3 L reaction flask. Since the experiment involves exposing inflammable gases to a high voltage electric discharge, it is worth highlighting important steps that reduce the risk of explosion. The general procedures described in this work can be extrapolated to design and conduct a wide variety of electric discharge experiments simulating primitive planetary environments.

The Miller-Urey experiment was a pioneering study regarding the abiotic synthesis of organic compounds with possible relevance to the origins of life. Simple gases were introduced into a glass apparatus and subjected to an electric discharge, simulating the effects of lightning in the primordial Earth&#8217;s atmosphere-ocean system. The experiment was conducted for one week, after which, the samples collected from it were analyzed for the chemical building blocks of life.

在进行米勒 - 尤列实验

In 1953, Stanley Miller reported the production of biomolecules from simple gaseous starting materials, using an apparatus constructed to simulate the ...

科研

化学

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

一种技术，功能化和自组装纳米宏观 - 配体单层膜模板上无基材

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

Synthesis and Testing of Supported Pt-Cu Solid Solution Nanoparticle Catalysts for Propane Dehydrogenation

A convenient method for the synthesis of bimetallic Pt-Cu catalysts and performance tests for propane dehydrogenation and characterization are demonstrated here. The catalyst forms a substitutional solid solution structure, with a small and uniform particle size around 2 nm. This is realized by careful control over the impregnation, calcination, and reduction steps during catalyst preparation and is identified by advanced in situ synchrotron techniques. The catalyst propane dehydrogenation performance continuously improves with increasing Cu:Pt atomic ratio.

A convenient method for the synthesis of 2 nm supported bimetallic nanoparticle Pt-Cu catalysts for propane dehydrogenation is reported here. In situ synchrotron X-ray techniques allow for the determination of the catalyst structure, which is typically unobtainable using laboratory instruments.

用于丙烷脱氢的负载型Pt-Cu固溶体纳米粒子催化剂的合成与测试

A convenient method for the synthesis of bimetallic Pt-Cu catalysts and performance tests for propane dehydrogenation and characterization are ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

用于跟踪纳米粒子自组装的液体细胞透射电子显微镜

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

原位金纳米粒子合成快速 Nanoprobe 信号增强

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This work presents the preparation of methionine functionalized biocompatible block copolymers via RAFT polymerization. Firstly, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-aminopropyl)methacrylamide (BNHEMA-b-APMA, BA) was synthesized via RAFT polymerization using 4,4&#8217;-azobis(4-cyanovaleric acid) (ACVA) as an initiating agent and 4-cyanopentanoic acid dithiobenzoate (CTP) as the chain transfer agent. Subsequently, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-guanidinopropyl)methacrylamide (methionine grafted BNHEMA-b-GPMA, mBG) was prepared by modifying amine groups in APMA with methionine and guanidine groups. Three kinds of block polymers, mBG1, mBG2, and mBG3, were synthesized for comparison. A ninhydrin reaction was used to quantify the APMA content; mBG1, mBG2, and mBG3 had 21%, 37%, and 52% of APMA, respectively. Gel permeation chromatography (GPC) results showed that BA copolymers possess molecular weights of 16,200 (BA1), 20,900(BA2), and 27,200(BA3) g/mol. The plasmid DNA (pDNA) complexing ability of the obtained block copolymer gene carriers was also investigated. The charge ratios (N/P) were 8, 16, and 4 when pDNA was complexed completely with mBG1, mBG2, mBG3, respectively. When the N/P ratio of mBG/pDNA polyplexes was higher than 1, the Zeta potential of mBG was positive. At an N/P ratio between 16 and 32, the average particle size of mBG/pDNA polyplexes was between 100-200 nm. Overall, this work illustrates a simple and convenient protocol for the block copolymer carrier synthesis.

This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.

用于靶向质粒DNA输送的美聚氨酸功能化生物相容性块共聚物

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

基因治疗诱导聚化涂层保护 dna 折纸纳米结构

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

用于增强神经化学检测的金纳米粒子改性碳纤维微电极

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

通过实验室间比较，对液态介质中的金纳米粒子进行纳米粒子跟踪分析

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.