我们承认西北大学结构生物学设施的工作人员和仪器支持。右侧西北大学综合癌症中心和西北大学结构生物学设施的支持。加坦 K2 直接电子探测器购买了由芝加哥生物医学财团提供的资金在芝加哥社区信托基金的支持。我们还感谢西北大学的下列设施: 凯克跨学科表面科学设施、结构生物学设施、生物成像设施、高级分子成像中心和分析Bionanotechnology 设备核心。这项研究得到1453576国家科学基金会资助, 国家卫生研究院主任的新创新奖 1DP2HL132390-01, 再生纳米医学催化剂奖和2014麦考克催化剂奖的中心。全国卫生研究院 predoctoral 生物技术培训补助金 T32GM008449 部分支助了该基金。

<ol>
	<li>Stano, A., Scott, E. A., Dane, K. Y., Swartz, M. A., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+T+cell+immunity+towards+a+protein+antigen+using+polymersomes+vs.+solid-core+nanoparticles.">Tunable T cell immunity towards a protein antigen using polymersomes vs. solid-core nanoparticles.</a> Biomaterials. 34 (17), 4339-4346 (2013).</li><li>Discher, B. 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=Polymersomes:+tough+vesicles+made+from+diblock+copolymers.">Polymersomes: tough vesicles made from diblock copolymers.</a> Science. 284 (5417), 1143-1146 (1999).</li><li>Vasdekis, A. E., Scott, E. A., O'Neil, C. P., Psaltis, D., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+intracellular+delivery+based+on+optofluidic+polymersome+rupture.">Precision intracellular delivery based on optofluidic polymersome rupture.</a> ACS Nano. 6 (9), 7850-7857 (2012).</li><li>Yi, S., 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=Tailoring+Nanostructure+Morphology+for+Enhanced+Targeting+of+Dendritic+Cells+in+Atherosclerosis.">Tailoring Nanostructure Morphology for Enhanced Targeting of Dendritic Cells in Atherosclerosis.</a> ACS Nano. 10 (12), 11290-11303 (2016).</li><li>Shum, H. C., Kim, J. W., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+fabrication+of+monodisperse+biocompatible+and+biodegradable+polymersomes+with+controlled+permeability.">Microfluidic fabrication of monodisperse biocompatible and biodegradable polymersomes with controlled permeability.</a> Journal of the American Chemical Society. 130 (29), 9543-9549 (2008).</li><li>Pessi, J., 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=Microfluidics-assisted+engineering+of+polymeric+microcapsules+with+high+encapsulation+efficiency+for+protein+drug+delivery.">Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery.</a> International Journal of Pharmaceutics. 472 (1-2), 82-87 (2014).</li><li>O'Neil, C. P., Suzuki, T., Demurtas, D., Finka, A., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+encapsulation+of+biomolecules+into+polymersomes+via+direct+hydration.">A novel method for the encapsulation of biomolecules into polymersomes via direct hydration.</a> Langmuir. 25 (16), 9025-9029 (2009).</li><li>Saad, W. S., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+nanoparticle+formation+by+flash+nanoprecipitation.">Principles of nanoparticle formation by flash nanoprecipitation.</a> Nano Today. 11 (2), 212-227 (2016).</li><li>Johnson, B. K., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+rapid+self-assembly+of+block+copolymer+nanoparticles.">Mechanism for rapid self-assembly of block copolymer nanoparticles.</a> Physical Review Letters. 91 (11), 118302 (2003).</li><li>Han, J., 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=A+simple+confined+impingement+jets+mixer+for+flash+nanoprecipitation.">A simple confined impingement jets mixer for flash nanoprecipitation.</a> Journal of Pharmaceutical Sciences. 101 (10), 4018-4023 (2012).</li><li>Allen, S., Osorio, O., Liu, Y. G., Scott, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+assembly+and+loading+of+theranostic+polymersomes+via+multi-impingement+flash+nanoprecipitation.">Facile assembly and loading of theranostic polymersomes via multi-impingement flash nanoprecipitation.</a> Journal of Controlled Release. 262, 91-103 (2017).</li><li>Bobbala, S., Allen, S. D., Scott, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+nanoprecipitation+permits+versatile+assembly+and+loading+of+polymeric+bicontinuous+cubic+nanospheres.">Flash nanoprecipitation permits versatile assembly and loading of polymeric bicontinuous cubic nanospheres.</a> Nanoscale. 10 (11), 5078-5088 (2018).</li><li>Allen, S. D., 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=Polymersomes+scalably+fabricated+via+flash+nanoprecipitation+are+non-toxic+in+non-human+primates+and+associate+with+leukocytes+in+the+spleen+and+kidney+following+intravenous+administration.">Polymersomes scalably fabricated via flash nanoprecipitation are non-toxic in non-human primates and associate with leukocytes in the spleen and kidney following intravenous administration.</a> Nano Research. , (2018).</li><li>Karabin, N. B., 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=Sustained+micellar+delivery+via+inducible+transitions+in+nanostructure+morphology.">Sustained micellar delivery via inducible transitions in nanostructure morphology.</a> Nature Communications. 9 (1), 624 (2018).</li><li>Mascoli, C. C., Weary, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limulus+amebocyte+lysate+(LAL)+test+for+detecting+pyrogens+in+parenteral+injectable+products+and+medical+devices:+advantages+to+manufacturers+and+regulatory+officials.">Limulus amebocyte lysate (LAL) test for detecting pyrogens in parenteral injectable products and medical devices: advantages to manufacturers and regulatory officials.</a> Journal of the Parenteral Drug Association. 33 (2), 81-95 (1979).</li><li>Pustulka, K. 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=Flash+nanoprecipitation:+particle+structure+and+stability.">Flash nanoprecipitation: particle structure and stability.</a> Molecular Pharmaceutics. 10 (11), 4367-4377 (2013).</li><li>Tang, C., Amin, D., Messersmith, P. B., Anthony, J. E., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+directed+self-assembly+of+pH-responsive+antioxidant+nanoparticles.">Polymer directed self-assembly of pH-responsive antioxidant nanoparticles.</a> Langmuir. 31 (12), 3612-3620 (2015).</li><li>Gindy, M. E., Panagiotopoulos, A. Z., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+block+copolymer+stabilized+nanoparticles:+simultaneous+encapsulation+of+organic+actives+and+inorganic+nanostructures.">Composite block copolymer stabilized nanoparticles: simultaneous encapsulation of organic actives and inorganic nanostructures.</a> Langmuir. 24 (1), 83-90 (2008).</li></ol>

作者声明他们没有竞争的财政利益。

我们提供了详细的指示, 以快速制造 polymersomes 使用 PEG17-bl-PPS35-SH 作为两嵌段共聚物。水泡 polymersomes 是在亲水性 PEG 和疏水性 PPS 块分子量的比值下组装的主要骨料形态。当撞击多次, 他们有一个直径和多分散性匹配 polymersomes 挤出通过200纳米膜后,通过薄膜水化形成。因此, 该协议消除了在制造单分散 polymersome nanocarriers 过程中需要额外的挤出步骤。Polymersomes 通过 <...

Nanocarriers 允许控制交付小和大分子货物, 包括活跃实体, 如果不封装, 将是高度降解和/或太疏水性, 以管理在体内。nanocarrier 的形态通常是捏造的, 类似于脂质体 (也称为 polymersomes) 的聚合物泡, 提供了同时负载亲水性和疏水货物1,2的能力。尽管他们有很好的优势, 但 polymersomes 在临床应用方面仍不多见, 部分原因是他们的制造业面临一些关键挑战。对于临床使用, polymersome 配方需要在大规模, 不育, 和一致的批次。
一些技术可以用来形成 polymersomes 从两嵌段共聚物, 如聚乙二醇-聚 (丙烯硫化物) (PEG-苯硫醚), 包括溶剂分散3,薄膜补液1,4, 微流体5,6, 直接水化7。溶剂分散在有机溶剂的存在下需要长时间的潜伏期, 这可能变性一些生物活性有效载荷, 如蛋白质。薄膜补液不提供对形成的 polymersomes 的多分散性的控制, 通常需要昂贵和耗时的挤出技术, 以达到可接受的单分散性。此外, 微流体和直接水化都很难扩大到较大的生产量。在不同的 nanocarrier 制造方法中, 闪光 nanoprecipitation (FNP) 提供了大规模和可再生配方8,9,10的能力。虽然 FNP 以前是为制备固体核纳米粒子而预留的, 但我们的实验室最近扩大了 FNP 的使用范围, 包括了各种不同的 PEG-苯硫醚纳米结构形貌的一致形成11, 12, 包括 polymersomes11和 bicontinuous 球12。我们发现, FNP 是能够形成单分散配方的 polymersomes 不需要挤出, 导致优越的多分散性指数值与非挤压 polymersomes 形成的薄膜补液和溶剂分散11. 尽管在许多溶剂条件下形成了 FNP12, 但 Bicontinuous 球的大疏水域却无法通过薄膜补液形成。
在这里, 我们提供了一个详细的描述, 以合成的 PEG-bl-PPS 两嵌段共聚物用于 polymersome 的形成, 密闭撞击射流 (CIJ) 混合器用于 FNP, FNP 协议本身, 并实施一个自动化系统, 以减少用户的可变性。包括关于如何充分消毒系统以产生体内使用的无内毒素制剂的信息, 以及关于 FNP 形成的 polymersomes 的代表性数据。有了这些信息, 有兴趣的读者在体外和体内工作中使用 polymersomes 将能够制造出他们自己的无菌, 单分散配方。具有 nanocarrier 配方和高分子合成专业知识经验的读者将能够使用 FNP 作为现有配方技术的潜在替代品, 快速测试自己的聚合物系统。此外, 本文所描述的协议可作为 nanocarriers 在纳米技术实验室课程中的应用的教育工具。

<table>
	<tbody>
		<tr>
			<td>CanaKit Raspberry Pi 3 Ultimate Starter Kit - 32 GB Edition</td>
			<td>CanaKit</td>
			<td>UPC 682710991511</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Bearing Platform (Small) - 8mm Diameter</td>
			<td>Adafruit</td>
			<td>1179</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Motion 8 mm Shaft, 330 mm Length, Chrome Plated, Case Hardened, Metric</td>
			<td>VXB</td>
			<td>kit11868</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Rail Shaft Guide/Support - 8 mm Diameter</td>
			<td>Adafruit</td>
			<td>1182</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual-Position Precision Slide 4.5&#34; Stroke, 15 lb load capacity</td>
			<td>McMaster-Carr</td>
			<td>5236A16</td>
			<td></td>
		</tr>
		<tr>
			<td>MTPM-P10-1JK43 Iron Horse DC motor</td>
			<td>Iron Horse</td>
			<td>MTPM-P10-1JK43</td>
			<td></td>
		</tr>
		<tr>
			<td>Official Raspberry Pi Foundation 7&#34; Touchscreen LCD Display</td>
			<td>Raspberry Pi</td>
			<td>B0153R2A9I (ASIN)</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoBorg Reverse - Advanced motor control for Raspberry Pi</td>
			<td>PiBorg</td>
			<td>BURN-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Pololu Carrier with Sharp GP2Y0D810Z0F Digital Distance Sensor 10cm</td>
			<td>Pololu</td>
			<td>1134</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruland PSR16-5-4-A Set Screw Beam Coupling, Polished Aluminum, Inch, 5/16&#34; Bore A Diameter, 1/4&#34; Bore B Diameter, 1&#34; OD, 1-1/4&#34; Length, 44 lb-in Nominal Torque</td>
			<td>Ruland</td>
			<td>PSR16-5-4-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol monomethyl ether</td>
			<td>Sigma Aldrich</td>
			<td>202495</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanesulfonyl chloride</td>
			<td>Sigma Aldrich</td>
			<td>471259</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma Aldrich</td>
			<td>179418</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene, Anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>244511</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Celite 545 (Diatomaceous Earth)</td>
			<td>Sigma Aldrich</td>
			<td>419931</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>320269</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma Aldrich</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide, anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Sigma Aldrich</td>
			<td>791776</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T30805</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran</td>
			<td>Sigma Aldrich</td>
			<td>360589</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum oxide, neutral, activated, Brockmann I</td>
			<td>Sigma Aldrich</td>
			<td>199974</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium methoxide solution, 0.5 M in methanol</td>
			<td>Sigma Aldrich</td>
			<td>403067</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene sulfide</td>
			<td>Sigma Aldrich</td>
			<td>P53209</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>320390</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution 1.0 N</td>
			<td>Sigma Aldrich</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free water</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30529.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper pH strips</td>
			<td>Fisher Scientific</td>
			<td>13-640-508</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free Dulbecco&#39;s PBS</td>
			<td>Sigma Aldrich</td>
			<td>TMS-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>03-337-4</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL all-plastic syringe</td>
			<td>Thermo Scientific</td>
			<td>S75101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sepharose CL-6B</td>
			<td>Sigma Aldrich</td>
			<td>CL6B200</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid chromatography column</td>
			<td>Sigma Aldrich</td>
			<td>C4169</td>
			<td></td>
		</tr>
		<tr>
			<td>CIJ mixer, HDPE</td>
			<td>Custom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma Aldrich</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-Blue hTLR4</td>
			<td>InvivoGen</td>
			<td>hkb-htlr4</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW-Blue Cells</td>
			<td>InvivoGen</td>
			<td>raw-sp</td>
			<td></td>
		</tr>
		<tr>
			<td>QUANTI-Blue</td>
			<td>InvivoGen</td>
			<td>rep-qb1</td>
			<td></td>
		</tr>
		<tr>
			<td>PYROGENT Gel Clot LAL Assays</td>
			<td>Lonza</td>
			<td>N183-125</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid, scalable assembly and loading of bioactive proteins and immunostimulants into diverse synthetic nanocarriers via flash nanoprecipitation

纳米材料提供了多种选择, 用于自定义用于治疗和成像应用的单一和组合分子有效载荷的控制交付。这种增加的特异性可能具有重要的临床意义, 包括减少副作用和较低剂量的功效。此外, 对特定细胞亚群的原位靶向和控制调制可以提高体外和体内对基本生物学现象和探针细胞功能的研究。不幸的是, 在纳米科学、化学和工程方面所需的专门知识往往禁止在这些领域没有经验的实验室从制造和定制纳米材料作为其调查或车辆的工具, 以供其治疗策略。在这里, 我们提供了合成和可伸缩组装的协议, 一个多功能的无毒嵌段共聚物系统, 适于简便地形成和加载纳米车辆的生物医学应用。闪光 nanoprecipitation 是一种快速制造多种 nanocarriers 的方法, 从聚乙二醇-bl-聚丙烯 (丙烯硫化物) 共聚物。这些协议将允许实验室具有广泛的专业知识和资源, 方便和重现性制造先进的 nanocarrier 交付系统的应用。设计和建造一个自动化的仪器, 使用高速注射器泵, 以促进闪光 nanoprecipitation 过程, 并允许加强控制的均匀性, 大小, 形态和负载的 polymersome nanocarriers 是描述。

在这里, 我们提出了一个简单的协议, 以制定 nanocarriers 能够装载亲水性和疏水的货物, 是安全的在体内鼠标和非人类灵长类管理11,13。我们还包括了在我们的代表性结果中使用的聚合物合成的详细协议, 以及为 CIJ 搅拌机中的机械控制的解决方案制造一个定制仪器的描述。图 1提供了用于生产 PEG17</...

Watch this Scientific Journal Video about Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation at JoVE.com

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

纳米材料为基础科学和平移应用提供了可控制的治疗付的多种机制 , 但它们的制造往往需要在大多数生物医学实验室中不可用的专业知识。在这里, 我们提出了可伸缩的制造和治疗负载的各种自组装 nanocarriers 使用闪光 nanoprecipitation 的协议。

快速, 可伸缩的组装和负载的生物活性蛋白和免疫增强剂到不同的合成 Nanocarriers 通过闪光 Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

rapid-scalable-assembly-loading-bioactive-proteins-immunostimulants

Research

JoVE Journal

Bioengineering

7.8K 次观看.  Northwestern University. 纳米材料为基础科学和平移应用提供了可控制的治疗付的多种机制 , 但它们的制造往往需要在大多数生物医学实验室中不可用的专业知识。在这里, 我们提出了可伸缩的制造和治疗负载的各种自组装 nanocarriers 使用闪光 nanoprecipitation 的协议。

视频: Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

固相Submonomer Peptoid聚合物及其自组装成高度有序的纳米片的合成

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

科研

生物工程

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

通过反应挤出、微流体、自组装和电纺丝制备多长尺度可降解 Thermoresponsive 水凝胶

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生物活性蛋白或多肽在水凝胶上的应用

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

闪速纳米沉淀法包聚物纳米粒子中疏水亲水化合物的作用

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

生物活性亲水球状蛋白的纳米脂质体制备与表征

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

将伽马改性肽核酸自组装成有机溶剂混合物中复杂的纳米结构

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...