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. 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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&quot; 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&quot; 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&quot; Bore A Diameter, 1/4&quot; Bore B Diameter, 1&quot; OD, 1-1/4&quot; 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&#039;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

Universidad de Cartagena UDEC

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

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 ...

科研

生物工程

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

免疫与感染

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

化学

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

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 ...

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 ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were composed of entire or fractions of the infectious agent. However, challenges remain, and new vaccine technologies are mandatory. In this context, the use of mRNA for immunizing purposes has shown an enhanced performance, as demonstrated by the speedy approval of two mRNA vaccines preventing SARS-CoV-2 infection. Beyond success in preventing viral infections, mRNA vaccines can also be used for therapeutic cancer applications.
Nevertheless, the instability of mRNA and its fast clearance from the body due to the presence of nucleases makes its naked delivery not possible. In this context, nanomedicines, and specifically polymeric nanoparticles, are critical mRNA delivery systems. Thus, the aim of this article is to describe the protocol for the formulation and test of an mRNA vaccine candidate based on the proprietary polymeric nanoparticles. The synthesis and chemical characterization of the poly(beta aminoesters) polymers used, their complexation with mRNA to form nanoparticles, and their lyophilization methodology will be discussed here. This is a crucial step for decreasing storage and distribution costs. Finally, the required tests to demonstrate their capacity to in vitro transfect and mature model dendritic cells will be indicated. This protocol will benefit the scientific community working on vaccination because of its high versatility that enables these vaccines to prevent or cure a wide variety of diseases.

Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes

Here, a simple protocol is presented for producing mRNA nanoparticles based on poly(beta aminoester) polymers, easy to be tailored by changing the encapsulated mRNA. The workflow for synthesizing the polymers, the nanoparticles, and their in vitro essential characterization are also described. A proof-of-concept regarding immunization is also added.

用于疫苗接种目的的mRNA负载的聚（β-氨基酯）纳米颗粒的合成和表征

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were ...

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp&#160;encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp&#160;is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp&#160;is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp&#160;nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

使用肽-泊洛沙明纳米颗粒在培养细胞中高效转染 体外 转录的mRNA

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. ...

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

摘要

探索更多视频

此视频中的章节

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

使用水性一步交联和共纳米聚合合成刺激响应纳米凝胶

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

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

用于疫苗接种目的的mRNA负载的聚（β-氨基酯）纳米颗粒的合成和表征

使用肽-泊洛沙明纳米颗粒在培养细胞中高效转染体外转录的mRNA

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

摘要

探索更多视频

此视频中的章节

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

使用水性一步交联和共纳米聚合合成刺激响应纳米凝胶

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

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

用于疫苗接种目的的mRNA负载的聚（β-氨基酯）纳米颗粒的合成和表征

使用肽-泊洛沙明纳米颗粒在培养细胞中高效转染 体外 转录的mRNA

使用肽-泊洛沙明纳米颗粒在培养细胞中高效转染体外转录的mRNA