Name: testing the in vitro and in vivo efficiency of mrna-lipid nanoparticles formulated by microfluidic mixing
Uploaded: 2023-01-20T13:20:00
Description: Watch this Scientific Journal Video about Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing at JoVE.com

M.J.M. anerkender støtte fra en US National Institutes of Health (NIH) Director's New Innovator Award (DP2 TR002776), en Burroughs Wellcome Fund Career Award ved Scientific Interface (CASI), en US National Science Foundation CAREER-pris (CBET-2145491) og yderligere finansiering fra National Institutes of Health (NCI R01 CA241661, NCI R37 CA244911 og NIDDK R01 DK123049).

BEMÆRK: Oprethold altid RNase-frie forhold, når du formulerer mRNA-LNP'er ved at tørre overfladerne og udstyret af med et overfladedekontaminant til RNaser og DNA. Brug kun RNase-fri spidser og reagenser.
Alle dyreforsøg blev udført i overensstemmelse med retningslinjerne for pleje og brug af forsøgsdyr ved University of Pennsylvania og en protokol godkendt af Institutional Animal Care and Use Committee (IACUC) ved University of Pennsylvania.
1. Forbered...

<ol>
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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=Design+of+lipid+nanoparticles+for+in+vitro+and+in+vivo+delivery+of+plasmid+DNA.">Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. 13 (4), 1377-1387 (2017).</li><li>Cheng, X., Lee, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+helper+lipids+in+lipid+nanoparticles+(LNPs)+designed+for+oligonucleotide+delivery.">The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery.</a> Advanced Drug Delivery Reviews. 99, 129-137 (2016).</li><li>Varkouhi, A. K., Scholte, M., Storm, G., Haisma, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomal+escape+pathways+for+delivery+of+biologicals.">Endosomal escape pathways for delivery of biologicals.</a> Journal of Controlled Release. 151 (3), 220-228 (2011).</li><li>Granot, Y., Peer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivering+the+right+message:+Challenges+and+opportunities+in+lipid+nanoparticles-mediated+modified+mRNA+therapeutics-An+innate+immune+system+standpoint.">Delivering the right message: Challenges and opportunities in lipid nanoparticles-mediated modified mRNA therapeutics-An innate immune system standpoint.</a> Seminars in Immunology. 34, 68-77 (2017).</li><li>Gan, Z., 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=Nanoparticles+containing+constrained+phospholipids+deliver+mRNA+to+liver+immune+cells+in+vivo+without+targeting+ligands.">Nanoparticles containing constrained phospholipids deliver mRNA to liver immune cells in vivo without targeting ligands.</a> Bioengineering and Translational Medicine. 5 (3), 10161 (2020).</li><li>Patel, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxycholesterol+substitution+in+ionizable+lipid+nanoparticles+for+mRNA+delivery+to+T+cells.">Hydroxycholesterol substitution in ionizable lipid nanoparticles for mRNA delivery to T cells.</a> Journal of Controlled Release. 347, 521-532 (2022).</li><li>Robinson, E., 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=Lipid+nanoparticle-delivered+chemically+modified+mRNA+restores+chloride+secretion+in+cystic+fibrosis.">Lipid nanoparticle-delivered chemically modified mRNA restores chloride secretion in cystic fibrosis.</a> Molecular Therapy. 26 (8), 2034-2046 (2018).</li><li>Love, K. T., 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=Lipid-like+materials+for+low-dose,+in+vivo+gene+silencing.">Lipid-like materials for low-dose, in vivo gene silencing.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (5), 1864-1869 (2010).</li><li>Kauffman, K. 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=Optimization+of+lipid+nanoparticle+formulations+for+mRNA+delivery+in+vivo+with+fractional+factorial+and+definitive+screening+designs.">Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs.</a> Nano Letters. 15 (11), 7300-7306 (2015).</li><li>Billingsley, M. 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=Ionizable+lipid+nanoparticle-mediated+mRNA+delivery+for+human+CAR+T+cell+engineering.">Ionizable lipid nanoparticle-mediated mRNA delivery for human CAR T cell engineering.</a> Nano Letters. 20 (3), 1578-1589 (2020).</li><li>Ramaswamy, 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=Systemic+delivery+of+factor+IX+messenger+RNA+for+protein+replacement+therapy.">Systemic delivery of factor IX messenger RNA for protein replacement therapy.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (10), 1941-1950 (2017).</li><li>Leung, A. K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+nanoparticles+containing+siRNA+synthesized+by+microfluidic+mixing+exhibit+an+electron-dense+nanostructured+core.">Lipid nanoparticles containing siRNA synthesized by microfluidic mixing exhibit an electron-dense nanostructured core.</a> Journal of Physical Chemistry C. 116 (34), 18440-18450 (2012).</li><li>Billingsley, M. 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=Orthogonal+design+of+experiments+for+optimization+of+lipid+nanoparticles+for+mRNA+engineering+of+CAR+T+cells.">Orthogonal design of experiments for optimization of lipid nanoparticles for mRNA engineering of CAR T cells.</a> Nano Letters. 22 (1), 533-542 (2022).</li><li>Khalil, A. 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=Subcutaneous+administration+of+D-luciferin+is+an+effective+alternative+to+intraperitoneal+injection+in+bioluminescence+imaging+of+xenograft+tumors+in+nude+mice.">Subcutaneous administration of D-luciferin is an effective alternative to intraperitoneal injection in bioluminescence imaging of xenograft tumors in nude mice.</a> ISRN Molecular Imaging. 2013, 689279 (2013).</li><li>Qin, 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=RGD+peptide-based+lipids+for+targeted+mRNA+delivery+and+gene+editing+applications.">RGD peptide-based lipids for targeted mRNA delivery and gene editing applications.</a> RSC Advances. 12 (39), 25397-25404 (2022).</li><li>Pardi, 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=Expression+kinetics+of+nucleoside-modified+mRNA+delivered+in+lipid+nanoparticles+to+mice+by+various+routes.">Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes.</a> Journal of Controlled Release. 217, 345-351 (2015).</li><li>Finn, J. 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=A+single+administration+of+CRISPR/Cas9+lipid+nanoparticles+achieves+robust+and+persistent+in+vivo+genome+editing.">A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing.</a> Cell Reports. 22 (9), 2227-2235 (2018).</li><li>Truong, 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=Lipid+nanoparticle-targeted+mRNA+therapy+as+a+treatment+for+the+inherited+metabolic+liver+disorder+arginase+deficiency.">Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (42), 21150-21159 (2019).</li><li>Cheng, Q., 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=Dendrimer-based+lipid+nanoparticles+deliver+therapeutic+FAH+mRNA+to+normalize+liver+function+and+extend+survival+in+a+mouse+model+of+hepatorenal+tyrosinemia+type+I.">Dendrimer-based lipid nanoparticles deliver therapeutic FAH mRNA to normalize liver function and extend survival in a mouse model of hepatorenal tyrosinemia type I.</a> Advanced Materials. 30 (52), 1805308 (2018).</li><li>Sedic, 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+evaluation+of+lipid+nanoparticle-formulated+modified+mRNA+in+the+Sprague-Dawley+rat+and+cynomolgus+monkey.">Safety evaluation of lipid nanoparticle-formulated modified mRNA in the Sprague-Dawley rat and cynomolgus monkey.</a> Veterinary Pathology. 55 (2), 341-354 (2018).</li><li>Veiga, 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=Cell+specific+delivery+of+modified+mRNA+expressing+therapeutic+proteins+to+leukocytes.">Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes.</a> Nature Communications. 9 (1), 4493 (2018).</li><li>Pattipeiluhu, R., 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=Anionic+lipid+nanoparticles+preferentially+deliver+mRNA+to+the+hepatic+reticuloendothelial+system.">Anionic lipid nanoparticles preferentially deliver mRNA to the hepatic reticuloendothelial system.</a> Advanced Materials. 34 (16), 2201095 (2022).</li><li>Rosenblum, 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=CRISPR-Cas9+genome+editing+using+targeted+lipid+nanoparticles+for+cancer+therapy.">CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy.</a> Science Advances. 6 (47), (2020).</li><li>Fenton, O. 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=Bioinspired+alkenyl+amino+alcohol+ionizable+lipid+materials+for+highly+potent+in+vivo+mRNA+delivery.">Bioinspired alkenyl amino alcohol ionizable lipid materials for highly potent in vivo mRNA delivery.</a> Advanced Materials. 28 (15), 2939-2943 (2016).</li><li>Kauffman, K. 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=Optimization+of+lipid+nanoparticle+formulations+for+mRNA+delivery+in+vivo+with+fractional+factorial+and+definitive+screening+designs.">Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs.</a> Nano Letters. 15 (11), 7300-7306 (2015).</li><li>Tomb&#225;cz, I., 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=Highly+efficient+CD4++T+cell+targeting+and+genetic+recombination+using+engineered+CD4++cell-homing+mRNA-LNPs.">Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs.</a> Molecular Therapy. 29 (11), 3293-3304 (2021).</li><li>Kim, 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=Engineering+lipid+nanoparticles+for+enhanced+intracellular+delivery+of+mRNA+through+inhalation.">Engineering lipid nanoparticles for enhanced intracellular delivery of mRNA through inhalation.</a> Nano. 9 (9), 14792-14806 (2022).</li><li>Bevers, 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=mRNA-LNP+vaccines+tuned+for+systemic+immunization+induce+strong+antitumor+immunity+by+engaging+splenic+immune+cells.">mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells.</a> Molecular Therapy. 30 (9), 3078-3094 (2022).</li></ol>

Med denne arbejdsgang kan en række mRNA-LNP'er formuleres og testes for deres in vitro- og in vivo-effektivitet. Ioniserbare lipider og hjælpestoffer kan udskiftes og kombineres ved forskellige molære forhold og forskellige ioniserbare lipid til mRNA-vægtforhold for at producere mRNA-LNP'er med forskellige fysisk-kemiske egenskaber22. Her formulerede vi C12-200 mRNA-LNP'er med et molært forhold på 35/16/46,5/2,5 (ioniserbart lipid:hjælperlipid:kolesterol:lipid-PEG) ved et ...

Lipidnanopartikler (LNP'er) har vist sig meget lovende i de senere år inden for ikke-viral genterapi. I 2018 godkendte United States Food and Drug Administration (FDA) den første RNA-interferens (RNAi) terapeutiske, Onpattro af Alnylam, til behandling af arvelig transthyretin amyloidose 1,2,3,4. Dette var et vigtigt skridt fremad for lipidnanopartikler og RNA-baserede terapier. For nylig modtog Moderna og Pfizer/BioNTech FDA-godkendelser for deres mRNA-LNP-vacciner mod SARS-CoV-2 4,5. I hver af disse LNP-baserede nukleinsyreterapier tjener LNP til at beskytte sin last mod nedbrydning af nukleaser og lette potent intracellulær levering 6,7. Mens LNP'er har set succes i RNAi-terapier og vaccineapplikationer, er mRNA-LNP'er også blevet undersøgt til brug i proteinerstatningsterapier8 samt til co-levering af Cas9 mRNA og guide-RNA til levering af CRISPR-Cas9-systemet til genredigering9. Der er imidlertid ingen specifik formulering, der er velegnet til alle applikationer, og subtile ændringer i LNP-formuleringsparametrene kan i høj grad påvirke styrken og biodistributionen in vivo 8,10,11. Således skal individuelle mRNA-LNP'er udvikles og evalueres for at bestemme den optimale formulering for hver LNP-baseret terapi.
LNP'er formuleres almindeligvis med fire lipidkomponenter: et ioniserbart lipid, et phospholipid, kolesterol og et lipidforankret polyethylenglycol (PEG) konjugat (lipid-PEG)11,12,13. Den potente intracellulære levering lettet af LNP'er er delvis afhængig af den ioniserbare lipidkomponent12. Denne komponent er neutral ved fysiologisk pH, men bliver positivt ladet i det sure miljø i endosomet11. Denne ændring i ionisk ladning menes at være en vigtig bidragyder til endosomal flugt12,14,15. Ud over det ioniserbare lipid forbedrer phospholipidkomponenten (hjælperlipid) indkapslingen af lasten og hjælper med endosomal flugt, kolesterolet giver stabilitet og forbedrer membranfusion, og lipid-PEG minimerer LNP-aggregering og opsonisering i omløb10,11,14,16. For at formulere LNP kombineres disse lipidkomponenter i en organisk fase, typisk ethanol, og blandes med en vandig fase indeholdende nukleinsyrelasten. LNP-formuleringsprocessen er meget alsidig, idet den gør det muligt let at erstatte og kombinere forskellige komponenter i forskellige molære forhold for at formulere mange LNP-formuleringer med en lang række fysisk-kemiske egenskaber10,17. Men når man udforsker dette store udvalg af LNP'er, er det afgørende, at hver formulering evalueres ved hjælp af en standardiseret procedure for nøjagtigt at måle forskellene i karakterisering og ydeevne.
Her skitseres den komplette arbejdsgang til formulering af mRNA-LNP'er og vurderingen af deres ydeevne i celler og dyr.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 M Hydrochloric Acid</td>
			<td>Sigma</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m Syringe Filters</td>
			<td>Genesee</td>
			<td>25-243</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL BD Slip Tip Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000)</td>
			<td>Avanti Polar Lipids</td>
			<td>880150P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)</td>
			<td>Avanti Polar Lipids</td>
			<td>850725P</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>23G Needles</td>
			<td>Fisher Scientific</td>
			<td>14-826-6C</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL BD Disposable Syringes with Luer-Lok tips</td>
			<td>Fisher Scientific</td>
			<td>14-823-435</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL Dialysis Cassettes</td>
			<td>Thermo Scientific</td>
			<td>A52976</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Black Wall Black Bottom Plate</td>
			<td>Fisher Scientific</td>
			<td>07-000-135</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well White/Clear Bottom Plate, TC Surface</td>
			<td>Thermo Scientific</td>
			<td>165306</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate, 1 Kilogram</td>
			<td>Research Products International</td>
			<td>&#160;631-61-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Citrate dibasic</td>
			<td>SIgma</td>
			<td>3012-65-5</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Luer-Lok Syringe sterile, single use, 5 mL</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>C12-200 Ionizable Lipid</td>
			<td>Cayman Chemical</td>
			<td>36699</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>57-88-5</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap FLuc mRNA (5moU)</td>
			<td>TriLink Biotechnologies</td>
			<td>L-7202</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable cuvettes</td>
			<td>Fisher Scientific</td>
			<td>14955129</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt</td>
			<td>Thermo Scientific</td>
			<td>L2916</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Thermofisher Scientific</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Exel Insulin Syringes - 0.5 mL</td>
			<td>Fisher Scientific</td>
			<td>1484132</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Hep G2 [HEPG2]</td>
			<td>ATCC</td>
			<td>HB-8065</td>
			<td></td>
		</tr>
		<tr>
			<td>HyPure Molecular Biology Grade Water</td>
			<td>Cytiva</td>
			<td>SH30538.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite 200 PRO Plate Reader</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>06-666-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase Assay Kit</td>
			<td>Promega</td>
			<td>E4550</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoAssemblr Ignite Cartridges - Classic - 100 Pack</td>
			<td>Precision Nanosystems</td>
			<td>NIN0065</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoAssemblr Ignite Instrument</td>
			<td>Precision Nanosystems</td>
			<td>NIN0001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS - Phosphate-Buffered Saline (10x) pH 7.4, RNase-free</td>
			<td>Thermo Scientific</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermofisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>QB Citrate Buffer, (Citrate 100 mM) pH 3.0</td>
			<td>Teknova</td>
			<td>Q2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-it RiboGreen RNA Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr>
			<td>Reporter Lysis 5x Buffer</td>
			<td>Promega</td>
			<td>E3971</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Away Surface Decontaminant</td>
			<td>Thermofisher Scientific</td>
			<td>7000TS1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate</td>
			<td>Sigma</td>
			<td>7601-54-9</td>
			<td></td>
		</tr>
		<tr>
			<td>TNS reagent (6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt)</td>
			<td>Sigma</td>
			<td>T9792</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9036-19-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Panalytical</td>
			<td>NanoZS</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing the in vitro and in vivo efficiency of mrna-lipid nanoparticles formulated by microfluidic mixing

Lipidnanopartikler (LNP'er) har tiltrukket sig stor opmærksomhed for nylig med den vellykkede udvikling af COVID-19 mRNA-vaccinerne af Moderna og Pfizer/BioNTech. Disse vacciner har vist effektiviteten af mRNA-LNP-terapi og åbnet døren for fremtidige kliniske anvendelser. I mRNA-LNP-systemer fungerer LNP'erne som leveringsplatforme, der beskytter mRNA-lasten mod nedbrydning af nukleaser og formidler deres intracellulære levering. LNP'erne består typisk af fire komponenter: et ioniserbart lipid, et phospholipid, kolesterol og et lipidforankret polyethylenglycol (PEG) konjugat (lipid-PEG). Her formuleres LNP'er, der indkapsler mRNA-kodende firefly luciferase ved mikrofluidisk blanding af den organiske fase indeholdende LNP-lipidkomponenter og den vandige fase indeholdende mRNA. Disse mRNA-LNP'er testes derefter in vitro for at evaluere deres transfektionseffektivitet i HepG2-celler ved hjælp af et bioluminescerende pladebaseret assay. Derudover evalueres mRNA-LNP'er in vivo i C57BL/6-mus efter en intravenøs injektion via den laterale halevene. Helkropsbioluminescensbilleddannelse udføres ved hjælp af et in vivo-billeddannelsessystem. Repræsentative resultater vises for mRNA-LNP-egenskaberne, deres transfektionseffektivitet i HepG2-celler og den totale selvlysende flux i C57BL/6-mus.

mRNA-LNP'er blev formuleret under anvendelse af et mikrofluidisk instrument, der havde en gennemsnitlig hydrodynamisk diameter på 76, 16 nm og et polydispersitetsindeks på 0, 098. PKa af mRNA-LNP'erne viste sig at være 5,75 ved at udføre et TNS-assay18. Indkapslingseffektiviteten for disse mRNA-LNP'er blev beregnet til at være 92,3% ved anvendelse af det modificerede fluorescensassay og ligning 4,4. Den samlede RNA-koncentration, der blev anvendt til celle...

Watch this Scientific Journal Video about Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing at JoVE.com

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Her præsenteres en protokol til formulering af lipidnanopartikler (LNP'er), der indkapsler mRNA-kodning af ildflueluciferase. Disse LNP'er blev testet for deres styrke in vitro i HepG2-celler og in vivo i C57BL/6-mus.

Test af in vitro - og in vivo-effektiviteten af mRNA-lipidnanopartikler formuleret ved mikrofluidisk blanding

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

Denne protokol kan bruges til at formulere konsistente lipidnanopartikler, der indkapsler MRA. Formuleringsparametre kan indstilles, og de forskellige lipidnanopartikler kan evalueres for deres styrke og biodistribution. En standardiseret protokol for lipid nanopartikel formulering og in vitro og in vivo test kan anvendes til at formulere konsistente lipid nanopartikler, der kan sammenlignes med hinanden.Der er mange små detaljer at huske på, når man formulerer og evaluerer nanopartikelstyrke, men hvilke detaljer tegnede sig for? Forberedelse af formuleringen bør ikke være svært. Begynd med at fylde et rent, fire liters bægerglas med 200 til 300 ml frisk 10X PBS og fortynd det til 1X med ultrarent vand.Sørg for, at det endelige volumen af PBS er mellem to og tre liter. Dialysekassetter med passende kapacitet til lipidnanopartiklen eller LNP-formuleringen anbringes i feltbægeret. Foretag den tidobbelte fortynding af en 100 millimolær citronsyrebufferbestand ved hjælp af ultrarent vand for at skabe en 10 millimolær citronsyrebuffer til mRNA-fortynding.Derefter fremstilles C 12 200 ioniserbare lipid-, hjælperlipid-, kolesterol- og lipidforankrede PEG- eller lipid-PEG-stamopløsninger ved at opløse hvert lipid i 100% ethanol. Sørg for, at stamopløsningerne er helt opløst ved opvarmning og blanding ved 37 grader Celsius. Kombiner de beregnede volumener af de forskellige lipidkomponenter i et konisk rør.Fortynd lipiderne med 100% ethanol til slutvolumen af V, hvilket repræsenterer 25% af det endelige volumen af LNP. Beregn mængden af mRNA, der kræves til formulering baseret på det ioniserbare lipid til mRNA-vægtforhold og det totale volumen af mRNA-LNP. Optø derefter ildfluen luciferase, der koder for mRNA på is.Fortynd derefter mRNA'et til et endeligt volumen tre gange den organiske fase ved anvendelse af den 10 millimolære citronsyrebuffer. Sprøjt en delikat opgavetørring med en overfladedekontaminering for RNA'er og DNA, og tør det indre af det mikrofluidiske instrument grundigt. Åbn derefter en ny mikrofluidisk patron og indsæt den med indløbskanalerne nedad og væk.Anbring to sterile 15 ml koniske rør på holderen med hætterne af. Når du er færdig, skal du fylde en fem milliliter sprøjte med mRNA-opløsningen indeholdende mRNA fortyndet i 10 millimolær citronsyrebuffer. Fyld derefter en tre milliliter sprøjte med lipidopløsningen indeholdende lipiderne fortyndet i 100% ethanol.Sørg for at fjerne luft fra sprøjterne. Indsæt derefter den fem milliliter sprøjte, der indeholder mRNA-opløsningen, i patronens venstre indgang uden nålen, og indsæt sprøjten på tre milliliter indeholdende lipidopløsningen i patronens højre indløb. Tænd for det mikrofluidiske instrument, og vælg hurtig kørsel.Når du har valgt de korrekte sprøjtetyper til de fem og tre milliliter sprøjter, der er indsat, skal du indtaste parametrene for LNP-formulering ved et tre til et vandigt til organisk strømningsforhold, før du trykker på startknappen for at formulere mRNA-LNP'erne. Læg mRNA-LNP'erne opsamlet i det højre koniske rør i de tidligere hydrerede dialysekassetter ved hjælp af en sprøjte uden at punktere eller røre kassettemembranen, læg dialysekassetterne indeholdende mRNA-LNP'erne tilbage i bægerglasset, der indeholder PBS, og lad dem dialysere i mindst to timer. Efter dialyse bringes dialysekassetterne indeholdende mRNA-LNP'erne ind i et sterilt biosikkerhedsskab og fjernes mRNA-LNP'erne ved hjælp af en sprøjte med en nål.Sterilt filtrer mRNA-LNP'erne ved hjælp af et 0,22 mikrometer sprøjtefilter og saml dem i et sterilt konisk rør. Anbring derefter 65 mikroliter af mRNA-LNP-opløsningen i et 1,5 ml mikrorør til karakterisering. Plade 5.000 HepG2-celler i en hvid væg, klar bund, 96-brøndplade i 100 mikroliter komplet cellekulturmedium og inkuberer cellerne natten over i en kontrolleret inkubator ved 37 grader Celsius indeholdende 5% kuldioxid.Før behandling af celler med mRNA-LNP'er fjernes det komplette medium fra brøndene. Derefter behandles cellerne med enten 100 mikroliter mRNA-LNP'er fortyndet i komplet cellekulturmedium ved de ønskede doser eller 100 mikroliter komplet medium før en 24 timers inkubation. Efter inkubation fjernes cellekulturmediet fra 96-brøndpladen, tilsættes 20 mikroliter lysisbuffer til hver brønd efterfulgt af 100 mikroliter luciferase-assayreagenser.Beskyt pladen mod lys, og anbring pladen i en pladelæser for at måle det bioluminescerende signal. Fortynd mRNA-LNP-opløsningen ved hjælp af steril PBS, således at to mikrogram total mRNA er til stede i 100 mikroliter haleveneinjektionsvolumen. Fyld en 29 gauge insulinsprøjte med enten 100 mikroliter mRNA-LNP eller 100 mikroliter PBS, og fjern eventuelle bobler fra sprøjten.Indsæt nålen med skråningen opad i musens laterale halevene og injicer langsomt de 100 mikroliter mRNA-LNP eller PBS. Seks timer efter injektionen fremstilles en 15 mg pr. milliliter stamopløsning af D-Luciferinkaliumsalt i steril PBS. I billedbehandlingssoftwaren til in vivo-billeddannelsessystemet eller IVIS skal du markere feltet ud for luminescens, før du trykker på initialiser for at forberede instrumentet til dataindsamling.Derefter administreres 200 mikroliter D-Luciferin intraperitonealy til de tidligere behandlede mus og vent i 10 minutter, mens luciferasesignalet stabiliseres, før musene bedøves. Overfør de bedøvede mus til næsekeglerne inde i in vivo-billeddannelsessystemet, og sørg for, at de er på ryggen med deres underliv udsat, før du lukker kammerdøren. I billedbehandlingssoftwaren skal du sikre dig, at det korrekte synsfelt er valgt, og indstille eksponeringstiden til auto, klikke på knappen Hent i softwaren for at få bioluminescensbilleder.Indkapslingseffektiviteten af mRNA-LNP'erne blev bestemt til 92,3% ved anvendelse af det modificerede fluorescensassay og ligning vist her. Stigende bioluminescens blev observeret ved behandling af 5.000 HepG2-celler med stigende doser af mRNA-LNP. Luciferaseekspression blev let detekteret ved denne undersøgelses laveste dosis på fem nanogram pr. Brønd.Et stærkt bioluminescenssignal blev observeret i den øvre del af maven hos de mus, der blev behandlet med formulerede LNP'er. Områder af interesse blev trukket omkring leversignalerne for at beregne den samlede selvlysende flux. Den samlede selvlysende flux var cirka fem gange 10 til den 9.Oprethold altid det ioniserbare lipid til nukleinsyrevægtforhold, når du formulerer LNP'er. Hvert volumen af den organiske fase skal indeholde ioniserbart lipid svarende til mRNA i tre volumener af den vandige fase.

Research

JoVE Journal

Bioengineering

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Viral Nanoparticles for In vivo Tumor Imaging

Viral Nanoparticles for In vivo Tumor Imaging

Virale Nanopartikler for<em&gt; In vivo</em&gt; Tumor Imaging

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

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

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Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Kvantificering af Global diastolisk funktion ved Kinematisk Modeling-baseret analyse af Transmitral Flow via parametriserede diastolisk Filling Formalisme

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

Analyserer Blanding Inhomogeniteter i en mikrofluidapparat af Individuel Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

Øjenirritation Test (EIT) for Hazard Identifikation af Eye Irriterer Kemikalier hjælp rekonstrueret human Cornea-lignende Epithelial (RHCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Layer-by-lags kollagenaflejring i mikrofluidapparater for Microtissue Stabilisering

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

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

Fremstilling af metal nanopartikler ved pulserende laserablation i væsker: Et værktøj til at studere de antibakterielle egenskaber ved nanopartikler

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Mikrofluid Bioprinting for Engineering vaskulariserede væv og Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

Stive indlejring af faste og plettet, hele, Millimeter-skala prøver for sektion-gratis 3D histologi ved mikro-beregnet tomografi

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...