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

Summary

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.

Abstract

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.

Introduction

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 amyloidosis^1,2,3,4. 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<....

Protocol

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.

Discussion

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.......

Materials

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