Name: ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs
Uploaded: 2013-03-09T14:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

的承认通过国防部的资助，国会导演医学研究计划（＃W81XWH-10-1-0445），美国国立卫生研究院（NIH R21 HL110056），和美国心脏协会（＃11SDG4890030）。

1。缓冲器和测试代理的制备和消毒<ol><li> 150mM NaCl的缓冲液：溶解在500毫升水nanopure4.383克NaCl的晶体。 </li><li> pH缓冲液：准备在pH为5.6，6.2，6.8和7.4的磷酸盐缓冲液混合适量的一元和二元磷酸钠。如果样品是在较低的pH值（ 即pH &lt;5.6），然后一个更合适的缓冲液，如柠檬酸盐缓冲液进行测试，应该被使用。缓冲液配方都是现成的，这里提供了一个例子参考。 </li><li>消毒瓶顶真空?...

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C., Guo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unsaturated+Cationic+Ortho+Esters+for+Endosome+Permeation+in+Gene+Delivery.">Unsaturated Cationic Ortho Esters for Endosome Permeation in Gene Delivery.</a> Journal of Medicinal Chemistry. 50, 4269-4278 (2007).</li><li>Roth, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Measurements+and+Rational+Materials+Design+for+Intracellular+Delivery+of+Oligonucleotides.">Quantitative Measurements and Rational Materials Design for Intracellular Delivery of Oligonucleotides.</a> Biotechnology Progress. 24, 23-28 (2008).</li><li>Blumenthal, R., Seth, P., Willingham, M. C., Pastan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-dependent+lysis+of+liposomes+by+adenovirus.">pH-dependent lysis of liposomes by adenovirus.</a> Biochemistry. 25, 2231-2237 (1986).</li><li>Moore, N. M., Sheppard, C. L., Barbour, T. R., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+endosomal+escape+peptides+on+in+vitro+gene+delivery+of+polyethylene+glycol-based+vehicles.">The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol-based vehicles.</a> The Journal of Gene Medicine. 10, 1134-1149 (2008).</li><li>Panyam, J., Zhou, W. Z., Prabha, S., Sahoo, S. 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的pH响应性聚合物或其他代理设计用于endosomolytic功能可以迅速和有效地筛选的基础上裂解过程中遇到的内体，在pH值的红血细胞（ 图1，pH为6.8 -早 ​​期内体，pH值6.2 -晚期内含体，pH为5.6 -溶酶体）14-17 pH依赖性溶血已用于筛选进行调解内体释放的生物大分子的治疗药物（ 例如肽，的siRNA，反义寡核苷酸，蛋白质）的载流子的能力，并且该测定的结果，可以预测的性能?...

虽然有许多潜在的高影响力的治疗靶点在细胞内，细胞内输送的代理商带来了显着的挑战。通常，药物，尤其是生物制剂，被细胞内化，贩卖到囊泡，要么导致它们的内容通过内溶酶体途径降解，或穿梭出细胞通过胞吐作用。1在后者的方法中，内部的pH值囊泡酸化至约5-6，这是活性的酶的最佳pH值，在此室的功能，如溶菌酶2 最近，一些材料经过特别设计，以充分利用酸化胞浆内吞体，以方便他们的货物交付。这种方法的一个示例中使用合成的聚合物胶束的纳米粒子，其核心是在生理pH值（ 即 7.4），两性离子和电荷中性。然而，在pH 6.0- 6.5，聚合物成为质子化并购入的净正电荷，破坏了胶束的核心，和暴露的聚合物链段相互作用和破坏核内体膜。这项活动已经表明，以促进核内体逃逸的多肽和核酸为基础的疗法，让他们来访问他们的胞浆目标。3,4调解胞内逃生，破坏黏膜屏障的方法的例子包括“膜融合肽或介导膜融合或瞬态孔形成的蛋白质，可以在磷脂双分子层中。5均聚物的阴离子的烷基丙烯酸，如聚（丙基丙烯酸酸）是另一种被充分研究的方法，并且在这些聚合物中，羧酸侧基的质子化状态的决定过渡为疏水性，膜中断状态内溶酶体的pH值范围6,7。 一个有用的模型系统，为筛选endosomolytic行为是电子x体内pH-依赖的溶血试验。8在该模型系统中，红细胞膜的脂双层膜的替代品，包围内溶酶体囊泡作为。此一般化模型已被用于别人，到评估的endosomolytic的行为的细胞穿透肽和其它聚合物的基因传递系统。8-11在本实验中，在模仿在定义的pH值的缓冲液共孵育的人的红血细胞和试验材料胞外（7.4），早期内体（6.8），和晚期内溶酶体（&lt;6.8）的环境中。潜伏期过程中释放的血红蛋白的量进行定量的红血细胞溶解，其被归一化到在阳性对照样品用洗涤剂裂解释放血红蛋白量作为衡量。 从一个小型图书馆的潜在的endosomolytic测试材料筛选，可以推断出，不产生溶血的样品，在pH值7.4，但显着升高下摆olysis在pH &lt;6.5时，将是最有效的和细胞相容性的候选人胞浆药物递送。预计将保持惰性的，而不是不分青红皂白地破坏脂质双分子层膜（ 即可能导致细胞毒性），直到被暴露后的pH值下降到溶酶体内舱的内符合这些条件的材料。 在这个协议中，红细胞从人供体中分离，并在pH为5.6，6.2，6.8或7.4与实验endosomolytic药物输送剂共同孵育。完整的红细胞沉淀，上清液（含血红蛋白释放溶解红细胞） 经由板读数器（ 图1）为特征的血红蛋白的吸光度分析。

<table border="1"><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> BD真空采血管 - K2EDTA Vacutainer管</td><td> Fisher Scientific则</td><td> 22-253-145 </td><td>对于血液采集</td></tr><tr><td> BD真空采血管采血针，20.5号</td><td> Fisher Scientific则</td><td> 02-665-31 </td><td>对于血液采集</td></tr><tr><td> BD真空管座/针适配器</td><td> Fisher Scientific则</td><td> 22-289-953 </td><td>对于血液采集</td></tr><tr><td> BD品牌异丙基酒精棉签</td><td> Fisher Scientific则</td><td> 13-680-63 </td><td>对于血液采集</td></tr><tr><td> BD真空采无乳胶止血带</td><td> Fisher Scientific则</td><td> 02-657-6 </td><td>对于血液采集</TD&gt; </tr><tr><td>盐酸（浓） </td><td> Fisher Scientific则</td><td> A144-500 </td><td>为了调整pH值的D-PBS。 </td></tr><tr><td>的Triton X-100的</td><td> Sigma-Aldrich公司</td><td> T8787 </td><td>阳性对照</td></tr><tr><td>的Dulbecco PBS </td><td> Invitrogen公司</td><td> 14190 </td><td></td></tr><tr><td> NALGENE MF75一次性使用无菌瓶顶过滤单元与SFCA膜</td><td> Fisher Scientific则</td><td> 09-740-44A </td><td></td></tr><tr><td> BD 96孔板，平底，组织培养处理的聚苯乙烯</td><td> Fisher Scientific则</td><td> 08-772-2C </td><td>对于板读取的测定结束时。 </td></tr><tr><td> BD 96孔板，圆底，组织培养处理的聚苯乙烯</td><td> Fisher Scientific则</td><td> 08-772-17 </td><td>孵化的红血细胞与实验药物。 </td></tr></表&gt;

ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs

内溶酶体膜泡的磷脂双分子层构成，可以构成一道屏障，细胞内的目标提供生物药物。为了克服这个障碍，已设计了一些合成的药物载体积极破坏核内体膜，并提供货物进入细胞质。这里，我们描述的溶血测定，这可以用作快速，高通量筛选的细胞相容性和endosomolytic活性的细胞内的药物输送系统。 溶血试验，人的红血细胞和试验材料共同模仿细胞外的，早期内体，和晚期溶酶体内环境的定义的pH值的缓冲液中温育。颗粒完整的红血细胞的离心步骤之后，释放到培养基中的血红蛋白量的分光光度测定（405nm的为最佳的动态范围）。 ％的红血细胞破碎，然后相对于阳性对照SAMP量化LES用洗涤剂溶解。在该模型系统中，在红细胞膜的脂双层膜的替代品，包围内溶酶体囊泡作为。期望的结果是可以忽略不计的在生理pH（7.4）和pH约为5-6.8的溶酶体内pH值范围从鲁棒溶血溶血。

<html>通常情况下，表现出理想的pH-依赖的溶血行为的代理具有胞质交付药物，核酸，或其它生物活性分子的最高电位。描绘在图2中，在pH = 7.4，显示出最小的溶血，这是举例说明由Agent＃1，但在核内体内的pH值范围（&lt;6.5）的溶血行为急剧增加。有些剂可以表现出溶血行为的显着水平的在生理pH值范围（代理＃2（40微克/毫升）， 图2），这表明，这些药物可能不是血液相?...

Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

溶血试验可作为一种快速，高通量筛选，药物传递系统的细胞相容性和内货物配送endosomolytic活动的。该试验测量作为环境pH值的函数的红细胞膜的破坏。

体外红血细胞溶血含量的生物大分子药物的细胞内交货的评价的pH响应Endosomolytic代理

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

Research

JoVE Journal

Immunology and Infection

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

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CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

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