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 ...

This protocol can be used to formulate consistent lipid nanoparticles encapsulating MRA. Formulation parameters can be tuned and the various lipid nanoparticles can be evaluated for their potency and biodistribution. A standardized protocol for lipid nanoparticle formulation and in vitro and in vivo testing can be used to formulate consistent lipid nanoparticles that can be compared with each other.There are a lot of small details to keep in mind when formulating and evaluating nanoparticle potency, but which details accounted for? Preparing the formulation should not be difficult. Begin by filling a clean, four liter beaker with 200 to 300 milliliters of fresh 10X PBS, and diluting it to 1X with ultrapure water.Ensure the final volume of PBS is between two and three liters. Place dialysis cassettes of the appropriate capacity for the lipid nanoparticle, or LNP formulation, into the field beaker. Make the ten-fold dilution of a 100 millimolar citric acid buffer stock using ultrapure water to create a 10 millimolar citric acid buffer for mRNA dilution.Next, make C 12 200 ionizable lipid, helper lipid, cholesterol and lipid anchored PEG or lipid PEG stock solutions by dissolving each lipid in 100%ethanol. Ensure the stock solutions are fully dissolved by heating and mixing them at 37 degrees Celsius. Combine the calculated volumes of the different lipid components in a conical tube.Dilute the lipids with 100%ethanol to final volume of V, which represents 25%of the final volume of LNP. Calculate the amount of mRNA required for formulation based on the ionizable lipid to mRNA weight ratio and the total volume of mRNA-LNP. Next, thaw the firefly luciferase encoding mRNA on ice.Then dilute the mRNA to a final volume three times the organic phase using the 10 millimolar citric acid buffer. Spray a delicate task wipe with a surface decontaminate for RNAs and DNA, and wipe the interior of the microfluidic instrument thoroughly. Next, open a new microfluidic cartridge and insert it with the inlet channels facing down and away.Arrange two sterile 15 milliliter conical tubes on the holder with the caps off. Once done, fill a five milliliter syringe with the mRNA solution containing mRNA diluted in 10 millimolar citric acid buffer. Then fill a three milliliter syringe with the lipid solution containing the lipids diluted in 100%ethanol.Ensure to remove any air from the syringes. Then without the needle, insert the five milliliter syringe containing the mRNA solution into the left inlet of the cartridge, and insert the three milliliter syringe containing the lipid solution into the right inlet of the cartridge. Turn on the microfluidic instrument and select quick run.After selecting the correct syringe types for the five and three milliliter syringes inserted, input the parameters for LNP formulation at a three to one aqueous to organic flow rate ratio, before pressing the start button to formulate the mRNA-LNPs. Load the mRNA-LNPs collected in the right conical tube into the previously hydrated dialysis cassettes using a syringe without puncturing or touching the cassette membrane, place the dialysis cassettes containing the mRNA-LNPs back into the beaker containing PBS and let them dialyze for at least two hours. After dialysis, bring the dialysis cassettes containing the mRNA-LNPs into a sterile biosafety cabinet and remove the mRNA-LNPs using a syringe with a needle.Sterile filter the mRNA-LNPs using a 0.22 micrometer syringe filter and collect them in a sterile conical tube. Then place 65 microliters of the mRNA-LNP solution into a 1.5 milliliter microtube for characterization. Plate 5, 000 HepG2 cells in a white wall, clear bottom, 96-well plate in 100 microliters of complete cell culture medium and incubate the cells overnight in a controlled incubator at 37 degrees Celsius containing 5%carbon dioxide.Before treatment of cells with mRNA-LNPs, remove the complete medium from the wells. Next, treat the cells with either 100 microliters of mRNA-LNPs diluted in complete cell culture medium at the desired doses, or 100 microliters of complete medium before a 24 hour incubation. After incubation, remove the cell culture medium from the 96-well plate, add 20 microliters of lysis buffer to each well followed by 100 microliters of the luciferase assay reagents.Protect the plate from light and place the plate into a plate reader to measure the bioluminescent signal. Dilute the mRNA-LNP solution using sterile PBS so that two micrograms of total mRNA is present in 100 microliters of the tail vein injection volume. Fill a 29 gauge insulin syringe with either 100 microliters of mRNA-LNP or 100 microliters of PBS and remove any bubbles from the syringe.Insert the needle with the bevel facing up into the mouse's lateral tail vein and slowly inject the 100 microliters of mRNA-LNP or PBS. Six hours post injection, prepare a 15 milligram per milliliter stock solution of D-Luciferin potassium salt in sterile PBS. In the imaging software for the in vivo imaging system, or IVIS, select the box next to luminescence before pressing initialize to prepare the instrument for data acquisition.Next, administer 200 microliters of D-Luciferin intraperitonealy to the previously treated mice and wait for 10 minutes while the luciferase signal stabilizes before anesthetizing the mice. Transfer the anesthetized mice to the nose cones inside the in vivo imaging system, ensuring that they're on their backs with their abdomens exposed, before closing the chamber door. In the imaging software, ensure that the correct field of view is selected and set the exposure time to auto, click the acquire button in the software to obtain bioluminescence images.The encapsulation efficiency of the mRNA-LNPs was determined as 92.3%using the modified fluorescence assay and equation shown here. Increasing bioluminescence was observed upon treatment of 5, 000 HepG2 cells with increasing doses of mRNA-LNP. Luciferase expression was easily detected at this study's lowest dose of five nanograms per well.A strong bioluminescence signal was observed in the upper abdomen of the mice treated with formulated LNPs. Regions of interest were drawn around the liver signals to calculate the total luminescent flux. The total luminescent flux was approximately five times 10 to the 9th.Always maintain the ionizable lipid to nucleic acid weight ratio when formulating LNPs. Every volume of the organic phase should contain ionizable lipid corresponding to mRNA in three volumes of the aqueous phase.

Research

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Bioengineering

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