Name: a facile and efficient approach for the production of reversible disulfide cross-linked micelles
Uploaded: 2016-12-23T12:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles at JoVE.com

The authors thank Ms.Alisha Knudson for the editorial help. They would also like to acknowledge the financial support from the NIH/NCI (3R01CA115483, to K.S.L.), the DoD PRMRP Award (W81XWH-13-1-0490, to K.S.L.), the NIH/NCI (1R01CA199668, to Y.L.), and the NIH/NICHD (1R01HD086195, to Y.L.).

伦理学声明:雌性无胸腺裸鼠（怒族/怒族株），6-8周龄，购然后根据指引AAALAC无病原体条件下保存，被允许适应环境之前的任何实验至少4天。根据协议号07-13119和09-15584号，由动物使用和注意事项管理咨询委员会在加州大学戴维斯分校的认可符合机构的指导方针所有的动物实验进行和。 1. TD PEG 5K -Cys合成4- -Ebes 8 -CA 8 <ol><li>在一个圆底烧瓶中，在10至20毫升无水二甲基甲酰胺（DMF）和冰浴的溶解的MeO-PEG 5K -NH 2（2克，0.4毫摩尔）。 </li><li>在玻璃烧杯中，溶解3当量。的1-羟基-6-氯 - 苯并三唑三唑（HOBt），3当量。的N，N&#39;-diisopropylcarbodiimide（DIC）和3当量。中基（Fmoc）2赖氨酸-OH在无水D-MF（10-15毫升）。搅拌在磁搅拌板上15-20分钟。 </li><li>该混合物添加到包含的MeO-PEG 5K -NH 2.取下冰浴，在室温下过夜搅拌该反应混合物的反应烧瓶中。 </li><li>确认薄层色谱（TLC）16和Kaiser试验17在反应完成后（黄色表明不存在游离NH 2）。加入大约200毫升冰冷的乙醚中反应烧瓶沉淀出聚合物产品1（的MeO-PEG 5K -Lys（NH-Fmoc基）2）。分离通过离心沉淀的聚合物（在6000 xg离心6分钟，4℃）。 <ol><li>要执行TLC，当场涂硅凝胶TLC板样品。使用二氯甲烷/甲醇（9:1）作为流动相。开发TLC板后观察紫外线灯下点。人们也可以使用茚三酮染色试剂来可视化在热板上胺斑点。 </li><li>启SER的试验，放置在玻璃管含Kaiser的试剂样品的一点点。加热它在100℃下5分钟，并寻找变色（如果溶液的颜色保持黄色，然后将反应完成）。 </li></ol></li><li>重新溶解在无水DMF（10-20毫升）的产物，重复沉淀和离心（参见步骤1.4）。 </li><li>重复步骤1.5，然后洗涤聚合物沉淀三次用冰冷的乙醚。 </li><li>转移聚合物沉淀1到干净的反应烧瓶中并将烧瓶连接到高真空源以除去残留乙醚。 </li><li>制备并加入大约20-30毫升的20％（体积/体积）-4-甲基哌啶在DMF中与聚合物中间体1。搅拌直至完全溶解。运行3小时的反应。 </li><li>执行薄层16和Kaiser试验（蓝色证实游离NH 2存在下 ）17（参见步骤1.4），以确认completio反应的n个。如果反应已完成，则继续进行醚沉淀，如所提到产品的产品1（步骤1.4-1.6）。 </li><li>干燥的聚合物产物2（的MeO-PEG 5K -Lys（NH 2）2）的真空下。 </li><li>执行一个更圆基（Fmoc）2赖氨酸-OH-耦合到中间体2（步骤1.1-1.7）生成产物3（的MeO-PEG 5K -Lys（赖氨酸（NH-Fmoc基）2）2）。脱保护（步骤1.8-1.10）Fmoc基（4，的MeO-PEG 5K -Lys（赖氨酸（NH 2）2）2）和一对（步骤1.1-1.7）的基（Fmoc）赖氨酸（BOC）-OH向产生第三代树枝状聚赖氨酸（5，的MeO-PEG 5K -Lys赖氨酸-2 - （（Fmoc基）赖氨酸（BOC））4）终止对PEG链的一端4的Boc和Fmoc基团。 </li><li>所得聚合物中间体5转移到反应烧瓶中。在如在二氯甲烷（DCM）1（V / V）三氟乙酸（TFA）:eparate反应烧瓶中，准备1。加15-20毫升1:1的TFA / DCM（体积/体积）混合物的聚合物中间体5。搅拌混合物直至聚合物完全溶解。搅拌另外3小时。 </li><li>执行薄层16和Kaiser试验（蓝色证实游离NH 2存在下 ）17（参见步骤1.4），以确认反应完成。如果反应完全，蒸除聚合物在TFA / DCM混合物用空气，直到获得粘稠溶液。继续乙醚沉淀，提到产品1（步骤1.4-1.6）。干燥的聚合物产物6（的MeO-PEG 5K -Lys赖氨酸-2 - （（Fmoc基）赖氨酸（NH 2））4）在真空下。 </li><li>转印聚合物中间体6到反应烧瓶中。使用约40毫升含8当量的无水DMF中。的N，N- -diisopropylethylamiNE（DIEA）以溶解聚合物中间体6。在玻璃烧杯中，溶解12当量。的HOBt，12当量。的DIC和12当量。中基（Fmoc）半胱氨酸（TRT）-OH在20-25毫升无水DMF的。振摇10-15分钟，然后将反应混合物加入到含有6反应烧瓶中。夜间运行的反应。 </li><li>确认在用TLC 16和Kaiser试验反应完成（参见步骤1.4;黄色表示不存在的游离NH 2）17。如果反应已完成，则继续进行醚沉淀，如所提到产品的产品1（步骤1.4-1.6），以分离产物7（的MeO-PEG 5K -Lys赖氨酸-2 - （（Fmoc基）赖氨酸- （（Fmoc）保护的Cys （TRT）））4。 </li><li> 7执行的Fmoc脱保护，如在步骤1.8中概述，得到产物8（PEG 5K -Lys赖氨酸-2 - （赖氨酸（NH 2） - （半胱氨酸（NH 2）（TRT）））4）。情侣基（Fmoc）-PEG 2 -Suc-OH于聚合物使用上述用于添加HOBt过程中间/ DIC-介导的偶联（"EBES"连接子，24当量），得到中间体9（的MeO-PEG 5K -Lys赖氨酸2 -Lys 4 - （半胱氨酸（TRT） ）4（EBES（NH-Fmoc基））8）。 </li><li>执行一个更圆的Fmoc脱保护的（步骤1.6-1.8），以获得中间10（的MeO-PEG 5K -赖赖氨酸2 -赖4 - （半胱氨酸（TRT））4（EBES（NH 2））8）。 </li><li>转移聚合物中间体10到反应烧瓶中，并添加无水DMF（约30-40毫升）以溶解它。在另一反应烧瓶中，溶解24当量。的CAOSu（根据先前公开的方法制备的）的无水DMF（20-30毫升）18。添加48当量。的N，N-二异丙基乙胺，并让它搅拌10-15分钟。转移内容到含有10反应瓶中，让反应来看Øvernight。 </li><li>确认在用TLC 16和Kaiser试验反应的完成（参见步骤1.4;黄色表示不存在的游离NH 2）17。如果反应已完成，则继续进行醚沉淀，如所提到产品的产品1（步骤1.4-1.6）以分离产物11（的MeO-PEG 5K -Lys赖氨酸2 -Lys 4 - （半胱氨酸（TRT））4 -Ebes 8 -CA 8）。透析它在去离子水和冻干的样品，得到白色粉末。 </li><li>聚合物中间体11放置到反应烧瓶中。准备并加入20毫升TFA / 1,2-乙二硫醇（EDT）/三乙基硅烷（TIS）的/ H 2 O（94 / 2.5 / 1 / 2.5，V / V）混合物到聚合物溶液中。搅拌混合物，直至完全溶解。运行4小时的反应。确认通过TLC 16反应的完成。 </li><li>根据通风柜，吹空气到聚合物-TFA / EDT / TIS / H 2Ø混合物，直到溶液变粘稠。继续到醚沉淀，如所提到产品的产品1（步骤1.4-1.6），以分离出最终产物，12（PEG 5K -Cys 4 -Ebes 8 -CA 8）。溶解于乙腈并冻干它，得到白色粉末。 </li></ol> 2. PTX-胶束的制备<ol><li>准备用标准蒸发方法的PTX-PEG装-半胱氨酸5K 4 -Ebes 8 -CA 8胶束。 <ol><li>溶解20毫克的TD与在1ml氯仿（ 三氯甲烷 ）不同量的PTX（1-9毫克）。使用旋转蒸发器，以得到均匀的，干燥的聚合物薄膜除去溶剂。重构用1ml通过涡旋磷酸盐缓冲盐水（PBS）的薄膜上，然后通过超声处理30分钟，在40千赫兹，如有必要，以允许载药胶束的形成。 </li><li>添加6微升3％（W / W） 的 H 2 O 2（1当量到free值的硫醇基）氧化对TD的硫醇基团。一旦游离巯基的水平保持在恒定的低价值，通过埃尔曼的试验19所示使用作进一步鉴定胶束溶液。 </li><li>筛选具有0.22μm的过滤器消毒的样品的溶液。分析药物的加入9倍乙腈和执行超声处理10分钟释放从胶束的药物后，在微胶粒加载的HPLC 20的系统上的量。使用C18柱与乙腈/水作为流动相HPLC。 </li><li>根据HPLC面积值和药物标准11的浓度之间的校准曲线计算的药物负载。 注:装载效率被定义为药物的装载到胶束到初始药物含量的比率。 </li></ol></li></ol> 3.胶束的表征<ol><li>测量的微米尺寸和尺寸分布icelles用动态光散射（DLS）仪器29。在室温下进行测量，并保持胶束浓度为1毫克/毫升。 注:要执行粒度分析，用PBS为空白，然后记录粒径为实际样品。取一式三份样品的读数，然后取平均读数。 </li><li>使用荧光光谱测量的PEG 5K -Cys 4 -Ebes 8 -CA 8的临界胶束浓度（CMC）之前和芘交联作为疏水荧光探针后，如先前13,21说明。 注意:通常情况下，胶束浓度范围为5×10 -7至5×10 -4 M. </li></ol> 4.胶束的稳定性SDS有或无还原剂<ol><li>制备十二烷基硫酸钠的储备溶液（SDS）溶液（7.5毫克/毫升），并在PBS二硫键交联的胶束（1.5毫克/毫升）。接下来，U唱储备溶液，使溶液混合物，其中最后的SDS浓度为2.5毫克/毫升和胶束浓度为1.0毫克/毫升。 </li><li>测量的尺寸和尺寸分布在有或没有10毫谷胱甘肽（GSH）的存在预定的时间间隔的胶束溶液（如在步骤3.1中提到）。 </li></ol> 5.溶血试验<ol><li>评价PTX加载的，非交联的胶束（PTX-NCMS）的根据从收集的先前公布的程序11，并使用新鲜的柠檬酸化的血液从裸鼠PTX加载交联胶束（PTX-的DCMs）中制备的溶血潜在尾面纱。 <ol><li>收集红血球通过血样（1.0毫升）的离心分离在1000 xg离心10分钟，用PBS洗三次，然后重悬浮，用PBS将细胞沉淀到2％的终浓度。 </li></ol></li><li>混合200μl的红细胞悬浮液用不同浓度（0.2和1.0的毫克PTX-NCMS和PTX-的DCMs的/毫升）分别与在培养箱摇床孵育4小时，在37℃。 </li><li>离心胶束红细胞的混合物在3000×g离心5分钟。转移100μl的所有样本的上清液至96孔板中。使用微板读数器测量游离血红蛋白在上清液的吸光度在540nm处。 注意:用Triton-100（2％）和PBS孵育红细胞将被用作阳性和阴性对照，分别。红细胞的百分比溶血计算公式如下:红细胞溶血=（OD 样品 - OD 阴性对照 ）/（OD 阳性对照 - OD 阴性对照 ）×100％。 </li></ol>

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Pharm. 9, 1727-1735 (2012).</li><li>Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glutathione+synthesis.">Regulation of glutathione synthesis.</a> Mol. Aspects Med. 30, 42-59 (2009).</li><li>Xiao, K., 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="OA02"+peptide+facilitates+the+precise+targeting+of+paclitaxel-loaded+micellar+nanoparticles+to+ovarian+cancer+in+vivo.">"OA02" peptide facilitates the precise targeting of paclitaxel-loaded micellar nanoparticles to ovarian cancer in vivo.</a> Cancer Res. 72, 2100-2110 (2012).</li><li>Koo, A. N., 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=Disulfide-cross-linked+PEG-poly(amino+acid)s+copolymer+micelles+for+glutathione-mediated+intracellular+drug+delivery.">Disulfide-cross-linked PEG-poly(amino acid)s copolymer micelles for glutathione-mediated intracellular drug delivery.</a> Chem. Commun. 28, 6570-6572 (2008).</li><li>McLellan, L. I., Wolf, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+and+glutathione-dependent+enzymes+in+cancer+drug+resistance.">Glutathione and glutathione-dependent enzymes in cancer drug resistance.</a> Drug. Resist. Update. 2, 153-164 (1999).</li><li>Karala, A. R., Lappi, A. K., Saaranen, M. J., Ruddock, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+peroxide-mediated+oxidative+refolding+of+a+protein+at+physiological+pH+and+implications+for+oxidative+folding+in+the+endoplasmic+reticulum.">Efficient peroxide-mediated oxidative refolding of a protein at physiological pH and implications for oxidative folding in the endoplasmic reticulum.</a> Antioxid. Redox Signal. 11, 963-970 (2009).</li><li>Gabizon, A., 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=Cancer+nanomedicines:+closing+the+translational+gap.">Cancer nanomedicines: closing the translational gap.</a> Lancet. 384, 2175-2176 (2014).</li></ol>

Y. Li and K.S.L. are the inventors of a pending patent on reversibly crosslinked micelle systems (US patent application 61/485,774). K.S.L. is the founding scientist of LamnoTherapeutics Inc., which plans to develop the nanotherapeutics described in the manuscript. The remaining authors declare no competing financial interests.

几个纳米颗粒已经研究了其在药物递送的潜在用途。脂质体阿霉素和紫杉醇（PTX）-loaded人血清白蛋白的纳米聚集体用于癌症治疗由FDA批准的纳米治疗剂之中。然而，虽然临床上有效的，这两种纳米治疗剂的在尺寸上相对"大"，他们往往在肝脏和肺蓄积。具有相对较小的颗粒尺寸和较高的药物装载容量高分子胶束正在出现用于药物递送的纳米载体。其独特的核 - 壳结构""溶解"&#39;通过物理包封在含?...

纳米技术是受益了许多医学领域1的快速新兴领域。纳米颗粒提供用于设计和调节是不与其他类型的常规治疗剂的可行性的机会。纳米载体增强药物对生物降解的稳定性，延长药物的循环时间，克服药物的溶解度的问题，并且可以进行微调为靶向药物递送和用于共输送显像剂1,2。基于纳米粒子输送系统持有癌症成像和治疗的承诺。肿瘤脉管系统是泄漏到大分子，并可能导致在通过增强的渗透性和保留（EPR）效果3肿瘤部位循环纳米颗粒的优先积累。间正在积极进行作为抗癌药物载体的几个纳米载体（ 例如，脂质体，水凝胶，和聚合物胶束），聚合物胶束已超过第得到广泛普及Ë过去十年4,5。 聚合物胶束是一个热力学系统，该系统，在静脉内给药，可以潜在地稀释的临界胶束浓度（CMC）以下，从而导致它们的解离成unimers。交联的策略已被用于最小化胶束解离成unimers。但是，过分稳定胶束可以防止药物从靶部位释放，从而降低了整体的治疗效果。几种化学方法已被开发，以使交联降解响应于氧化还原或外界刺激，如还原的二硫键6,7-和pH裂解8或可水解酯键9,10。 我们以前曾报道了设计和由树突胆酸（CA）的块和线性聚乙二醇（PEG）共聚物，胶束纳米颗粒的合成称为末端树枝状聚合物（TD）11-15 </SUP&gt;。这些TD被表示为PEG 了nK -CAy（其中n =分子量以千道尔顿（K），Y =（CA）的单位胆酸数）。他们封装药物的特点是体积小，保质期长，效率高，如疏水核心紫杉醇（PTX）和阿霉素（DOX）。 TD的积木，如PEG，赖氨酸，和CA，是生物相容的，和聚乙二醇电晕的存在可以赋予"隐形"纳米颗粒性质，防止胶束纳米颗粒的非特异性摄取被网状内皮系统。 巯基化线性树枝状聚合物可以很容易地通过引入半胱氨酸到我们的标准TD的树枝状寡聚赖氨酸骨干产生。本文介绍通过引入二硫键交联成阵的疏水性芯（ 图1）用于生产可逆交联的胶束药物递送系统的一个浅显协议。

<table><tbody><tr><td>MeO-PEG&lt;sup&gt;5K&lt;/sup&gt;-NH&lt;sub&gt;2&lt;/sub&gt;</td><td>Rapp Polymere</td><td>125000-2</td><td></td></tr><tr><td>Fmoc-Lys(Fmoc)-OH</td><td>Aaptec</td><td>AFK107</td><td></td></tr><tr><td>Fmoc-Lys(Boc)-OH</td><td>Anaspec</td><td>AS-20132</td><td></td></tr><tr><td>Fmoc-Cys(Trt)-OH</td><td>Aapptec</td><td>AAC105</td><td></td></tr><tr><td>Dimethylformamide</td><td>Fisher Scientific</td><td>BP1160-4</td><td></td></tr><tr><td>Ethyl ether</td><td>Fisher Scientific</td><td>E134-20</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Diisopropylethylamine</td><td>Sigma Aldrich</td><td>D125806</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma Aldrich</td><td>T6508</td><td>Corrosive, handle with care</td></tr><tr><td>4-methyl piperidine</td><td>Alfa-Aesar</td><td>L-02709</td><td></td></tr><tr><td>Ebes linker</td><td>Anaspec</td><td>AS-61924</td><td></td></tr><tr><td>Cholic acid</td><td>Sigma Aldrich</td><td>C1129</td><td></td></tr><tr><td>1,2-Ethanedithiol</td><td>Sigma Aldrich</td><td>02390</td><td>Handle inside fume hood. Bleach gloves after usage.</td></tr><tr><td>Triisopropylsilane</td><td>Sigma Aldrich</td><td>233781</td><td></td></tr><tr><td>Chloroform (anhydrous)</td><td>Sigma Aldrich</td><td>288306</td><td></td></tr><tr><td>Hydrogen peroxide solution 30%</td><td>Aaron Industries</td><td>NA</td><td></td></tr><tr><td>HoBt-Cl</td><td>Aaptec</td><td>CXZ096</td><td></td></tr><tr><td>DIC</td><td>Sigma Aldrich</td><td>D125407</td><td></td></tr><tr><td>Female athymic nude mice (Nu/Nu strain), 6&amp;ndash;8 weeks age</td><td>Harlan (Livermore, CA)</td><td></td><td></td></tr></tbody></table>

a facile and efficient approach for the production of reversible disulfide cross-linked micelles

纳米是治疗，运用是在尺度纳米生物医学应用的颗粒的独特性能的一种新兴的形式。提高药物输送到最大限度的治疗效果，减少药物的副作用是一些当今纳米的基石。特别是纳米颗粒已经发现在癌症治疗中具有广泛的应用。纳米颗粒提供了高度的设计，应用，以及基于所述肿瘤微环境的生产灵活性预计为与快速翻译到临床实践更有效。聚合物胶束纳米载体药物输送应用的热门选择。 在本文中，我们描述了用于合成根据一个明确定义的两亲性线性树枝状共聚物（telodendrimer，TD）的自组装载药，二硫化交联的胶束一个简单而有效的协议。 TD是由聚乙烯GL的ycol（PEG）作为亲水性链段和硫醇化胆酸群集作为使用基于溶液的肽化学芯成形疏水部分附着逐步到胺封端的聚乙二醇。化疗药物，如紫杉醇（PTX），可以使用标准溶剂蒸发方法被加载。将O 2介导的氧化先前用于形成从上阵的游离巯基帧内胶束二硫化物交联。然而，反应缓慢，对于大规模生产是不可行的。最近，H 2 O 2介导的氧化方法进行了探讨作为一种更可行和有效的方法，它比以前报道的方法快96倍。使用这种方法，将50克的PTX加载，二硫化物交联的纳米颗粒已成功地生产具有窄粒度分布和高载药量的效率。将所得的胶束溶液的稳定性使用扰乱条件如共孵育瓦特分析了第i洗涤剂，十二烷基硫酸钠，有或没有还原剂。相比，他们的非交联的对应时，载药，二硫化交联的胶束显示出较少的溶血活性。

制备与表征载药，二硫化交联胶束 两亲性聚合物的PEG 5K -Cys 4 -Ebes 8 -CA 8能够形成用于癌症的药物递送二硫化物交联的胶束体系的树枝状聚合物。在结构上，它被定义为通过一个支链聚（赖氨酸半胱氨酸EBES）主链相连的线性PEG分子（亲水域，分子量5K）的一端胆酸（疏水性结构域）的树枝状低聚物。存在使...

Watch this Scientific Journal Video about A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles at JoVE.com

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

a-facile-efficient-approach-for-production-reversible-disulfide-cross

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.8K 次观看.  University of California, Davis. To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

视频: A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

生产二硫键稳定的跨膜肽复合物的结构研究

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

科研

化学

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

使用Photoredox催化剂蠕虫状胶束的简便合成可见光介导的分散聚合

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

半胱氨酸残基的溶剂可及性分析<em&gt;玉米rayado菲诺病毒</em病毒样颗粒<em&gt;烟草</em植物和交联的肽类病毒颗粒

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

免疫与感染

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

结合电喷雾、自组装和溶剂型结构控制制备球形和蜗杆状胶束纳米晶

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a post-translational modification. Thus, it is important to be able to study this modification in living cells. A simple method to analyze these cysteine-stabilized multimeric complexes is through a two-step method of non-reducing SDS-PAGE analysis and formaldehyde cross-linking. This two-step method is advantageous as the first step to uncovering multimeric complexes stabilized by disulfide linkages due to its technical ease and low cost of operation. Here, the human bone osteosarcoma cell line U-2 OS is used to illustrate this method by specifically analyzing the nuclear isoform of dUTPase.

Disulfide linkages have long been known to stabilize the structure of many proteins. A simple method to analyze multimeric complexes stabilized by these linkages is through non-reducing SDS-PAGE analysis. Here, this method is illustrated by analyzing the nuclear isoform of dUTPase from the human bone osteosarcoma cell line U-2 OS.

非还原 SDS-PAGE 分析与化学交联相结合, 检测培养基细胞中的多物质配合物, 以确认培养中的多物质配合

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a ...

发育生物学

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

生物工程

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

使用水性一步交联和共纳米聚合合成刺激响应纳米凝胶

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

含脂脂的微流体生产-温度敏感脂质体

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of ...

开发用于生物医学的海藻酸钠水凝胶微球

Preparation of Cross-Linked Sodium Alginate Microspheres with Different Metal Ions Using the Microfluidic Electrospray Technology

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within polymers. In the field of biomedicine, hydrogel microspheres have been extensively studied for their application as drug carriers owing to their ability to reduce the frequency of drug administration, minimize side effects, and improve patient compliance. Sodium alginate (ALG) is a naturally occurring linear polysaccharide with three backbone glycosidic linkages. There are two auxiliary hydroxyl groups present in each of the moieties of the polymer, which have the characteristics of an alcohol hydroxyl moiety. The synthetic ALG units can undergo chemical cross-linking reactions with metal ions, forming a cross-linked network structure of polymer stacks, ultimately forming a hydrogel. Hydrogel microspheres can be prepared using a simple process involving the ionic cross-linking properties of ALG. In this study, we prepared ALG-based hydrogel microspheres (ALGMS) using a microfluidic electrodeposition strategy. The prepared hydrogel microspheres were uniformly sized and well-dispersed, owing to accurate control of the microfluidic electrospray flow. ALGMS cross-linked with different metal ions were prepared using a microfluidic electrospray technique combining microfluidic and high electric field, and its antimicrobial properties, slow drug release ability, and biocompatibility were investigated. This technology holds promise for application in advanced drug development and production.

作者聚焦：使用生物相容性海藻酸钠水凝胶微球推进治疗

The protocol describes the preparation of sodium alginate microspheres cross-linked with different metal ions using a microfluidic device for drug carrier design. The antimicrobial properties and slow drug release of these microspheres were also investigated.

利用微流控电喷雾技术制备具有不同金属离子的交联海藻酸钠微球

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within ...