Name: synthesis and characterization of mrna-loaded poly(beta aminoesters) nanoparticles for vaccination purposes
Uploaded: 2021-08-13T14:00:00
Description: Watch this Scientific Journal Video about Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes at JoVE.com

MINECO/FEDER（授予SAF2015-64927-C2-2-R，RTI2018-094734-B-C22和COV20/01100）的财政支持得到确认。CGF承认了她的IQS博士奖学金。

1. pBAE聚合物与末端寡肽（OM-pBAE）的合成
<ol><li>C6-pBAE的聚合<ol>
<li>加入5-氨基-1-戊醇（38毫摩尔;分子量= 103.16 Da）1-己胺（38毫摩尔;MW = 101.19 Da）放入圆底玻璃瓶（100 mL）中。然后，加入1，4-丁二醇二丙烯酸酯（82毫摩尔;MW = 198.22 Da）。</li>
		<li>将硅油浴预热至90°C，将圆底烧瓶放入油浴中，并借助磁力搅拌棒搅拌混合物过夜（〜18小时）。然后，从圆底烧瓶中取出产品并将其置于-20°C的冰箱中。<br...

<ol>
	<li>Chumakov, K., Benn, C. S., Aaby, P., Kottilil, S., Gallo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+existing+live+vaccines+prevent+COVID-19.">Can existing live vaccines prevent COVID-19.</a> Science. 368 (6496), 1187-1188 (2020).</li><li>Zhang, C., Maruggi, G., Shan, H., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+mRNA+vaccines+for+infectious+diseases.">Advances in mRNA vaccines for infectious diseases.</a> Frontiers in Immunology. 10, 1-13 (2019).</li><li>Wherry, E. J., Jaffee, E. M., Warren, N., D'Souza, G., Ribas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+did+we+get+a+COVID-19+vaccine+in+less+than+1+year.">How did we get a COVID-19 vaccine in less than 1 year.</a> Clinical Cancer Research. 27 (8), 2136-2138 (2021).</li><li>Folegatti, P. 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=Safety+and+immunogenicity+of+the+ChAdOx1+nCoV-19+vaccine+against+SARS-CoV-2:+a+preliminary+report+of+a+phase+1/2,+single-blind,+randomised+controlled+trial.">Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial.</a> The Lancet. 396 (10249), 467-478 (2020).</li><li>Geall, A. J., Mandl, C. W., Ulmer, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA:+The+new+revolution+in+nucleic+acid+vaccines.">RNA: The new revolution in nucleic acid vaccines.</a> Seminars in Immunology. 25 (2), 152-159 (2013).</li><li>Ulmer, J. B., Geall, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+innovations+in+mRNA+vaccines.">Recent innovations in mRNA vaccines.</a> Current Opinion in Immunology. 41, 18-22 (2016).</li><li>Kranz, L. 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=Systemic+RNA+delivery+to+dendritic+cells+exploits+antiviral+defence+for+cancer+immunotherapy.">Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy.</a> Nature. 534 (7607), 396-401 (2016).</li><li>Milane, L., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+approval+of+nanotechnology-based+SARS-CoV-2+mRNA+vaccines:+impact+on+translational+nanomedicine.">Clinical approval of nanotechnology-based SARS-CoV-2 mRNA vaccines: impact on translational nanomedicine.</a> Drug Delivery and Translational Research. 1 (4), 3 (2020).</li><li>Green, J. J., Langer, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combinatorial+polymer+library+approach+yields+insight+into+nonviral+gene+delivery.">A combinatorial polymer library approach yields insight into nonviral gene delivery.</a> Accounts of Chemical Research. 41 (6), 749-759 (2008).</li><li>Guerrero-C&#225;zares, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polymeric+nanoparticles+show+high+efficacy+and+specificity+at+DNA+delivery+to+human+glioblastoma+in+vitro+and+in+vivo.">Biodegradable polymeric nanoparticles show high efficacy and specificity at DNA delivery to human glioblastoma in vitro and in vivo.</a> ACS Nano. 8 (5), 5141-5153 (2014).</li><li>Kozielski, K. L., Tzeng, S. Y., Hurtado De Mendoza, B. A., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreducible+cationic+polymer-based+nanoparticles+for+efficient+and+environmentally+triggered+cytoplasmic+siRNA+delivery+to+primary+human+brain+cancer+cells.">Bioreducible cationic polymer-based nanoparticles for efficient and environmentally triggered cytoplasmic siRNA delivery to primary human brain cancer cells.</a> ACS Nano. 8 (4), 3232-3241 (2014).</li><li>Segovia, N., Dosta, P., Cascante, A., Ramos, V., Borr&#243;s, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligopeptide-terminated+poly(&#946;-amino+ester)s+for+highly+efficient+gene+delivery+and+intracellular+localization.">Oligopeptide-terminated poly(&#946;-amino ester)s for highly efficient gene delivery and intracellular localization.</a> Acta Biomaterialia. 10 (5), 2147-2158 (2014).</li><li>Dosta, P., Segovia, N., Cascante, A., Ramos, V., Borr&#243;s, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+charge+tunability+as+a+powerful+strategy+to+control+electrostatic+interaction+for+high+efficiency+silencing,+using+tailored+oligopeptide-+modified+poly+(beta-amino+ester)s+(PBAEs).">Surface charge tunability as a powerful strategy to control electrostatic interaction for high efficiency silencing, using tailored oligopeptide- modified poly (beta-amino ester)s (PBAEs).</a> Acta Biomaterialia. 20, 82-93 (2015).</li><li>Fornaguera, C., 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=mRNA+delivery+system+for+targeting+antigen-presenting+cells+in+vivo.">mRNA delivery system for targeting antigen-presenting cells in vivo.</a> Advanced Healthcare Materials. 7 (17), 180033 (2018).</li><li>Fornaguera, C., Castells-Sala, C., L&#225;zaro, M. &. #. 1. 9. 3. ;., Cascante, A., Borr&#243;s, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+optimized+freeze-drying+protocol+for+OM-PBAE+nucleic+acid+polyplexes.">Development of an optimized freeze-drying protocol for OM-PBAE nucleic acid polyplexes.</a> International Journal Pharmaceutics. 569, (2019).</li><li>Fornaguera, C., Solans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+methods+to+characterize+and+purify+polymeric+nanoparticles.">Analytical methods to characterize and purify polymeric nanoparticles.</a> International Journal of Polymer Science. , (2018).</li><li>Fornaguera, C., Solans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+polymeric+nanoparticle+dispersions+for+biomedical+applications:+size,+surface+charge+and+stability.">Characterization of polymeric nanoparticle dispersions for biomedical applications: size, surface charge and stability.</a> Pharmaceutical Nanotechnology. 6 (3), 147-164 (2018).</li><li>Sahin, U., Karik&#243;, K., T&#252;reci, &. #. 2. 1. 4. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRNA-based+therapeutics-developing+a+new+class+of+drugs.">MRNA-based therapeutics-developing a new class of drugs.</a> Nature Reviews Drug Discovery. 13 (10), 759-780 (2014).</li><li>Fan, Y. 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=Cationic+lipid-assisted+nanoparticles+for+delivery+of+mRNA+cancer+vaccine.">Cationic lipid-assisted nanoparticles for delivery of mRNA cancer vaccine.</a> Biomaterials Science. 6 (11), 3009-3018 (2018).</li><li>Le Moignic, 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=Preclinical+evaluation+of+mRNA+trimannosylated+lipopolyplexes+as+therapeutic+cancer+vaccines+targeting+dendritic+cells.">Preclinical evaluation of mRNA trimannosylated lipopolyplexes as therapeutic cancer vaccines targeting dendritic cells.</a> Journal of Controlled Release. 278, 110-121 (2018).</li><li>Banerji, 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=mRNA+vaccines+to+prevent+COVID-19+disease+and+reported+allergic+reactions:+Current+evidence+and+approach.">mRNA vaccines to prevent COVID-19 disease and reported allergic reactions: Current evidence and approach.</a> Journal of Allergy and Clinical Immunology: In Practice. 9 (4), 1423-1437 (2021).</li><li>Kaczmarek, J. C., Kowalski, P. S., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+delivery+of+RNA+therapeutics:+from+concept+to+clinical+reality.">Advances in the delivery of RNA therapeutics: from concept to clinical reality.</a> Genome Medicine. 9 (1), 1-16 (2017).</li><li>Dosta, P., 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=Delivery+of+anti-microRNA-712+to+inflamed+endothelial+cells+using+poly(&#946;-amino+ester)+nanoparticles+conjugated+with+VCAM-1+targeting+peptide.">Delivery of anti-microRNA-712 to inflamed endothelial cells using poly(&#946;-amino ester) nanoparticles conjugated with VCAM-1 targeting peptide.</a> Advanced Healthcare Materials. , 1-11 (2021).</li><li>Segovia, 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=Hydrogel+doped+with+nanoparticles+for+local+sustained+release+of+siRNA+in+breast+cancer.">Hydrogel doped with nanoparticles for local sustained release of siRNA in breast cancer.</a> Advanced Healthcare Materials. 4 (2), 271-280 (2015).</li></ol>

在去年Covid-19大流行爆发后，疫苗在传染病控制方面的重要性已表现为关键组成部分8。全世界科学家的努力使许多疫苗得以投放市场。历史上第一次，mRNA疫苗已经证明了他们以前假设的成功，这要归功于他们的快速设计，因为他们有能力在几个月内适应任何新的抗原5，6，21。mRNA或信使核糖核酸是指从DNA中的...

传染病对全球数百万人构成严重威胁，仍然是一些发展中国家死亡的主要原因之一。预防性疫苗接种一直是现代社会预防和控制传染病最有效的干预措施之一1，2。这些科学在20世纪相关性中的关键里程碑已经通过最近由SARS-CoV-2病毒3引起的全球Covid-19大流行来表达。认识到拥有有效疫苗以遏制疾病传播的重要性，所有生物医学界的合作努力已成功导致许多预防性疫苗在不到一年的时间内进入市场4。
传统上，疫苗由减毒（活的，降低的毒力）或灭活的（死亡颗粒）病毒组成。然而，对于一些没有安全误差余地的疾病，病毒颗粒是不可能的，而是使用蛋白质亚基。然而，亚基通常不能实现一种以上表位/抗原的组合，并且需要佐剂来增强疫苗接种效力5，6。因此，对新型疫苗类型的需求是显而易见的。
正如在当前大流行期间所表明的那样，基于核酸的新型候选疫苗在避免漫长的开发过程和提供高多功能性方面可能是有利的，同时产生重要的患者免疫接种。mRNA疫苗就是这种情况，它最初被设计为实验性癌症疫苗。由于它们产生抗原特异性T细胞反应的天然能力3，5，6，7。由于mRNA是编码抗原蛋白的分子，只是改变相同，疫苗可以快速定制以免疫同一微生物的其他变体，不同的菌株，其他传染性微生物，甚至成为癌症免疫治疗。此外，它们在大规模生产成本方面具有优势。然而，mRNA有一个阻碍其裸施用的重大障碍：其稳定性和完整性在充满核酸酶的生理介质中受到损害。因此，需要使用纳米载体来保护它并将mRNA矢量化到抗原呈递细胞2，8。
在这种情况下，聚（β-氨基酯）（pBAE）是一类生物相容性和可生物降解的聚合物，由于其阳离子电荷9，10，11，它们表现出在纳米颗粒中复合mRNA的显着能力。这些聚合物由酯键组成，这使得它们在生理条件下容易被酯酶降解。在pBAE候选文库中，那些用末端阳离子寡肽功能化的pBAE文库显示出更高的能力，可以形成小纳米颗粒，通过内吞作用有效地穿透细胞并转染包封的基因材料。此外，由于它们的缓冲能力，内体室的酸化允许内体逃逸12，13。即，一种特定类型的pBAE，包括其骨架上的疏水部分（所谓的C6 pBAE）以增强其稳定性和终寡肽组合（用三赖氨酸修饰的60%聚合物和40%的聚合物用三组氨酸修饰），其在肠胃外给药后选择性地转染抗原呈递细胞并产生mRNA编码的抗原呈递，然后进行小鼠免疫接种，最近已发表14.此外，还证明这些配方可以规避纳米医学配方的主要瓶颈步骤之一：在不失去功能的情况下冷冻干燥它们的可能性，这使得在软干燥环境中具有长期稳定性15。
在这种情况下，当前方案的目标是通过描述方案中的关键步骤并使用于传染病预防和肿瘤治疗应用的有效疫苗能够生产，使mRNA纳米颗粒的形成程序可供科学界使用。
以下方案描述了合成寡肽末端修饰聚（β-氨基酯） - OM-pBAE聚合物的完整锻炼，这些聚合物将进一步用于纳米颗粒合成。在该协议中，还包括纳米颗粒制剂。此外，还提供了程序成功的关键步骤和代表性结果，以确保所得配方完成所需的质量控制表征特征，以定义阳性或阴性结果。该协议总结在 图1中。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-butanediol diacrylate</td>
			<td>Sigma Aldrich</td>
			<td>123048</td>
			<td></td>
		</tr>
		<tr>
			<td>1-hexylamine</td>
			<td>Sigma Aldrich</td>
			<td>219703</td>
			<td></td>
		</tr>
		<tr>
			<td>5-amino-1-pentanol</td>
			<td>Sigma Aldrich</td>
			<td>411744</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Panreac</td>
			<td>141007</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b antibody</td>
			<td>BD</td>
			<td>550993</td>
			<td></td>
		</tr>
		<tr>
			<td>CD86 antibody</td>
			<td>Bioligend</td>
			<td>105007</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlor hydroxhyde</td>
			<td>Panreac</td>
			<td>181023</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform-d</td>
			<td>Sigma Aldrich</td>
			<td>151823</td>
			<td></td>
		</tr>
		<tr>
			<td>Cys-His-His-His peptide</td>
			<td>Ontores</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Cys-Lys-Lys-Lys peptide</td>
			<td>Ontores</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>D2O</td>
			<td>Sigma Aldrich</td>
			<td>151882</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC reagent for Rnase free water</td>
			<td>Sigma Aldrich</td>
			<td>D5758</td>
			<td>This reagent is important to treat MilliQ water to remove any RNases of the buffers</td>
		</tr>
		<tr>
			<td>Diethyl eter</td>
			<td>Panreac</td>
			<td>212770</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>mRNA EGFP</td>
			<td>TriLink Technologies</td>
			<td>L-7601</td>
			<td></td>
		</tr>
		<tr>
			<td>mRNA OVA</td>
			<td>TriLink Technologies</td>
			<td>L-7610</td>
			<td></td>
		</tr>
		<tr>
			<td>RiboGreen kit</td>
			<td>ThermoFisher</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium acetate</td>
			<td>Sigma Aldrich</td>
			<td>71196</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Fisher Scientific</td>
			<td>11570626</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-mouse AlexaFluor488 antibody</td>
			<td>Abcam</td>
			<td>Ab450105</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticle Tracking Analyzer</td>
			<td>Malvern Panalytical</td>
			<td>NanoSight NS300</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Magnetic Ressonance Spectrometer</td>
			<td>Varian</td>
			<td>400 MHz</td>
			<td></td>
		</tr>
		<tr>
			<td>ZetaSizer</td>
			<td>Malvern Panalytical</td>
			<td>Nano ZS</td>
			<td>For zeta potential and hydrodynamic size determination</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NTA software</td>
			<td>Malvern Panalytical</td>
			<td>MAN0515-02-EN-00</td>
			<td></td>
		</tr>
		<tr>
			<td>NovoExpress Software</td>
			<td>Agilent</td>
			<td>Not specified</td>
			<td></td>
		</tr>
		<tr>
			<td>ZetaSizer software</td>
			<td>Malvern Panalytical</td>
			<td>DTS Application</td>
			<td>To analyze surface charge and hydrodynamic sizes</td>
		</tr>
	</tbody>
</table>

synthesis and characterization of mrna-loaded poly(beta aminoesters) nanoparticles for vaccination purposes

疫苗接种是现代社会的重大成功之一，在控制和预防疾病方面是不可或缺的。传统疫苗由全部或部分感染因子组成。然而，挑战依然存在，新的疫苗技术是强制性的。在这种情况下，将mRNA用于免疫目的已显示出增强的性能，正如迅速批准两种预防SARS-CoV-2感染的mRNA疫苗所证明的那样。除了成功预防病毒感染外，mRNA疫苗还可用于治疗性癌症应用。
然而，由于核酸酶的存在，mRNA的不稳定性及其从体内的快速清除使得其裸递送是不可能的。在这种情况下，纳米药物，特别是聚合物纳米颗粒，是关键的mRNA递送系统。因此，本文的目的是描述基于专有聚合物纳米颗粒的mRNA候选疫苗的配方和测试方案。本文将讨论所用聚（β-氨基酯）聚合物的合成和化学表征，它们与mRNA的络合形成纳米颗粒，以及它们的冻干方法。这是降低存储和配送成本的关键步骤。最后，将进行必要的测试，以证明其 体外 转染和成熟模型树突状细胞的能力。该协议将使从事疫苗接种工作的科学界受益，因为它具有高度的多功能性，使这些疫苗能够预防或治愈各种疾病。

聚合物合成和表征 OM-pBAE合成过程如图 2所示。如图 2A 所示，获得OM-pBAE的第一步是通过将胺（1-己胺和5-氨基-1-戊醇，比例为1：1）添加到二丙烯酸酯（1，4-丁二醇二丙烯酸酯）中来合成C6-pBAE。该反应在90°C下进行20小时，并不断搅拌。之后，将寡肽溶液加入到从先前反应中获得的C6聚合物溶液中（图2B）。最后，使?...

Watch this Scientific Journal Video about Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes at JoVE.com

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

在这里，提出了一种基于聚（β-氨基酯）聚合物的mRNA纳米颗粒的简单方案，易于通过改变封装的mRNA进行定制。还描述了合成聚合物，纳米颗粒及其 体外 基本表征的工作流程。还增加了关于免疫接种的概念验证。

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 ...

该协议具有重要意义，因为它演示了如何在一个简单的步骤中制备封装的聚合物纳米颗粒。该技术的主要优点是多功能性，因为它可以应用于不同的核酸和细胞类型。使用简单的程序进行颗粒制备，例如上下移液，以及通过冻干延长纳米颗粒和滚筒条件的稳定性的可能性。演示该程序的将是Laura Olmo，Coral Garcia-Fernandez，Maria Stampa Lopez-Pinto和Maria Navalon-Lopez，我们实验室的博士生以及我们实验室的博士后Marta Diaz-Caballero。对于多链形成，解冻先前制备的聚合物，C6肽，聚β氨基酯，并涡旋溶液。上下移液聚合物混合物后，在乙酸钠中制备12.5毫摩尔溶液。涡旋溶液并等待10分钟。接下来，以每毫升0.5毫克的速度制备mRNA，并通过移液混合。再次涡旋聚合物溶液，然后将聚合物溶液中的遗传物质溶液在微量离心管中以一比一的比例混合，并将管在25摄氏度下在热块中孵育30分钟。孵育后，通过将样品加入含有水的微量离心管中，用一到两个RNase游离水沉淀混合物，然后包括赋形剂，加入与mRNA和聚β氨基酯混合物相同体积的20毫摩尔堆和4%蔗糖溶液，并通过移液混合。为了在零下80摄氏度下冷冻多孔溶液一小时后冻干，进行初级干燥，然后立即将多聚物储存在零下20摄氏度以避免再水化。对于多链重悬，从零下20摄氏度的冰箱中除去冻干的纳米颗粒，并迅速加入相应量的深层氢化水以重新分散固体并达到所需的浓度。轻轻移液直至完全重悬，溶解后，上下大力移液，避免气泡。转染24小时后，通过荧光显微镜进行定性评估，将含有转染Hela细胞的96孔板放在显微镜上，然后开始使用10倍物镜进行可视化。首先，应用白平衡为软件创建背景参考，然后在分析过程中获取图像以将其与荧光图像叠加。将显微镜模式更改为反射模式，并将滤光片轮移动到蓝色激光器以可视化EGFP。然后使用相同的曝光时间获取所有条件或孔的图像。对于流式细胞术的定量评估，使用每孔100微升PBS在96孔板中洗涤转染的Hela细胞。将PBS以每孔25微升胰蛋白酶吸出并在37摄氏度下孵育5分钟后。一旦细胞分离，加入先前回收的培养基以灭活胰蛋白酶，然后通过每孔添加31.25微升10%福尔马林并孵育20分钟来固定细胞。接下来打开流式细胞仪和软件，然后为实验设置适当的条件，包括板样品体积的类型和其他参数，例如样品之间的振荡和漂洗。设置适当的参数来量化阳性转染细胞的百分比，方法是首先查看前向散射光与侧向散射光的流动数据，以区分细胞和碎片。然后绘制另一个散点图，比较振幅与高度到门并区分单个单元格。转染前一天，将每孔板10， 000个JAWS II细胞放入96孔板中过夜孵育。第二天，用每孔0.6微克mRNA转染细胞，并将板在37摄氏度和5%二氧化碳的干燥空气培养箱中孵育24小时。第二天，用25微升PBS洗涤留在孔中的细胞。在25微升胰蛋白酶下吸入PBS并在37摄氏度下孵育板五分钟后分离细胞。通过将先前恢复的培养基添加到相应的孔中来停止胰蛋白酶反应。然后离心板，吸出培养基，并通过每孔添加50微升PBS和2.5%福尔马林来固定细胞，然后将板在4摄氏度下孵育20分钟，离心板。然后每孔加入50微升PBS和3%BSA，并在4摄氏度下培养30分钟。孵育后再次离心板，然后在PBS和3%BSA中加入50微升小鼠卵清蛋白抗体，并在4摄氏度下培养30分钟。重复离心步骤，然后用50微升PBS洗涤细胞。吸出PBS后，加入二抗并在四摄氏度下孵育一小时。孵育后，重复离心和洗涤步骤。然后将细胞和100微升PBS和2.5%福尔马林重悬，用于流式细胞术分析。新鲜和冻干GFP mRNA包封纳米颗粒的物理化学表征在尺寸和聚分散度指数方面没有显着差异。多相束的透射电子显微镜证实了直径约50纳米的球形和单分散纳米颗粒的形成。乐高肽和修饰的聚β-氨基酯多链体包封GFP或卵清蛋白的包封效率数据根据mRNA的类型没有显着差异。这里显示了JAWS II细胞系中EGFP表达的定性分析，交易后24小时。定量比较阴性对照。GFP阳性细胞百分比显着更高，与使用经典转染试剂进行的阳性对照相当。卵清蛋白mRNA负载的寡肽和修饰的聚β氨基酸纳米颗粒可以激活树突状细胞，如转染细胞中CD11b和CD86膜标志物的表达明显高于非转染细胞。我们基于使用mRNA作为活性原理的疫苗接种平台，以及我们专有的聚合物可以适应于预防和癌症治疗。我们可以利用我们的技术来突出对癌症免疫治疗的贡献。因为我们可以将肿瘤相关抗原封装在我们的纳米颗粒中，并在癌症治疗中做出转变。

Research

JoVE Journal

Bioengineering

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

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

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

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

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 ...

Viral Nanoparticles for In vivo Tumor Imaging

Viral Nanoparticles for In vivo Tumor Imaging

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Millifluidics的化学合成和时间分辨机理研究

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Harmonic Nanoparticles for Regenerative Research

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

细胞标记和超顺磁性氧化铁纳米粒子靶向

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

液体中脉冲激光烧蚀制备金属纳米粒子：研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...