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. acknowledges support from a US National Institutes of Health (NIH) Director&#8217;s New Innovator Award (DP2 TR002776), a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a US National Science Foundation CAREER award (CBET-2145491), and additional funding from the National Institutes of Health (NCI R01 CA241661, NCI R37 CA244911, and NIDDK R01 DK123049).

NOTE: Always maintain RNase-free conditions when formulating mRNA-LNPs by wiping the surfaces and equipment with a surface decontaminant for RNases and DNA. Use only RNase-free tips and reagents.
All the animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at the University of Pennsylvania and a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania.
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<ol>
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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. 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With this workflow, a variety of mRNA-LNPs can be formulated and tested for their in vitro and in vivo efficiency. Ionizable lipids and excipients can be swapped out and combined at different molar ratios and different ionizable lipid to mRNA weight ratios to produce mRNA-LNPs with differing physicochemical properties22. Here, we formulated C12-200 mRNA-LNPs with a molar ratio of 35/16/46.5/2.5 (ionizable lipid:helper lipid:cholesterol:lipid-PEG) at a 10:1 ionizable lipid to mRNA...

Lipid nanoparticles (LNPs) have demonstrated great promise in recent years in the field of non-viral gene therapy. In 2018, the United States Food and Drug Administration (FDA) approved the first-ever RNA interference (RNAi) therapeutic, Onpattro by Alnylam, for the treatment of hereditary transthyretin amyloidosis1,2,3,4. This was an important step forward for lipid nanoparticles and RNA-based therapies. More recently, Moderna and Pfizer/BioNTech received FDA approvals for their mRNA-LNP vaccines against SARS-CoV-24,5. In each of these LNP-based nucleic acid therapies, the LNP serves to protect its cargo from degradation by nucleases and facilitate potent intracellular delivery6,7. While LNPs have seen success in RNAi therapies and vaccine applications, mRNA-LNPs have also been explored for use in protein replacement therapies8 as well as for the co-delivery of Cas9 mRNA and guide RNA for the delivery of the CRISPR-Cas9 system for gene editing9. However, there is no one specific formulation that is well-suited for all applications, and subtle changes in the LNP formulation parameters can greatly affect the potency and biodistribution in vivo8,10,11. Thus, individual mRNA-LNPs must be developed and evaluated to determine the optimal formulation for each LNP-based therapy.
LNPs are commonly formulated with four lipid components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene-glycol (PEG) conjugate (lipid-PEG)11,12,13. The potent intracellular delivery facilitated by LNPs relies, in part, on the ionizable lipid component12. This component is neutral at physiological pH but becomes positively charged in the acidic environment of the endosome11. This change in ionic charge is thought to be a key contributor to endosomal escape12,14,15. In addition to the ionizable lipid, the phospholipid (helper lipid) component improves the encapsulation of the cargo and aids in endosomal escape, the cholesterol offers stability and enhances membrane fusion, and the lipid-PEG minimizes LNP aggregation and opsonization in circulation10,11,14,16. To formulate the LNP, these lipid components are combined in an organic phase, typically ethanol, and mixed with an aqueous phase containing the nucleic acid cargo. The LNP formulation process is very versatile in that it allows for different components to be easily substituted and combined at different molar ratios in order to formulate many LNP formulations&#160;with a multitude of physicochemical properties10,17. However, when exploring this vast variety of LNPs, it is crucial that each formulation is evaluated using a standardized procedure to accurately measure the differences in characterization and performance.
Here, the complete workflow for the formulation of mRNA-LNPs and the assessment of their performance in cells and animals is outlined.

<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

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

mRNA-LNPs were formulated using a microfluidic instrument that possessed an average hydrodynamic diameter of 76.16 nm and a polydispersity index of 0.098. The pKa of the mRNA-LNPs was found to be 5.75 by performing a TNS assay18. The encapsulation efficiency for these mRNA-LNPs was calculated to be 92.3% by using the modified fluorescence assay and equation 4.4. The overall RNA concentration that was used for the cell treatment and animal dosing was 40.24 ng/&#...

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

Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

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

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

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JoVE Journal

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