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-2^4,5. In each of these LNP-based nucleic acid therapies, the LNP serves to protect its cargo from degradation by nucleases and facilitate potent intracellular delivery^6,7. 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 vivo^8,10,11. 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 escape^12,14,15. 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 circulation^10,11,14,16. 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 with a multitude of physicochemical properties^10,17. 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.

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.

1. Pre-formulation preparation

1. Fill a clean 4 L beaker with 200-300 mL of fresh 10x phosphate-buffered saline (PBS).
   1. Dilute the 10x PBS in the beaker 10-fold with ultrapure water to obtain a beaker of 1x PBS. Ensure that the final volume of the 1x PBS is between 2-3 L.
   2. Place dialysis cassettes (20 kDa molecular weight cutoff [MWCO]) of the appropriate capacity for the LNP formulation into the filled beaker to hydrate them.
      NOTE: Dialysis cassettes require a minimum of 2 min to hydrate before use.
   3. Place a stir bar into the beaker, and cover the beaker with aluminum foil.
   4. Place the covered beaker onto a magnetic stir plate. Turn on the stir plate, and set it to spin at 300-400 rpm.
2. Dilute a 100 mM citric acid buffer stock (pH 3) 10-fold with ultrapure water to create the 10 mM citric acid buffer used for mRNA dilution.
3. Make C12-200 ionizable lipid, helper lipid, cholesterol, and lipid-anchored PEG (lipid-PEG) stock solutions by dissolving each lipid in 100% ethanol. The concentrations of the lipid components are detailed in Table 1.
   1. Heat and mix the stock solutions at 37 °C to ensure that they are fully dissolved.

2. Preparation of the lipid and nucleic acid mixes

1. Calculate the required volumes of ionizable lipid, helper lipid, cholesterol, and lipid-PEG based on the desired molar ratio and ionizable lipid to mRNA weight ratio (Table 1). Prepare 10% extra volume of the organic phase to account for dead volume in the syringes during formulation.
   1. Combine the calculated volumes of the different lipid components in a conical tube.
   2. Dilute the lipids with 100% ethanol to a final volume, V, which represents 25% of the final volume of LNP.
2. Calculate the amount of mRNA required for formulation based on the ionizable lipid to mRNA weight ratio and the total volume of mRNA-LNP needed (Table 1).
   1. Thaw the firefly luciferase-encoding mRNA on ice.
   2. Dilute the mRNA to a final volume, 3V, three times the volume of the organic phase, in 10 mM citric acid buffer.
      ​NOTE: Every lipid volume, V, must contain enough ionizable lipid for the mRNA diluted in 3V. This corresponds to the desired ionizable lipid to mRNA weight ratio.

3. Microfluidic formulation of mRNA-LNPs

1. Spray a delicate task wipe with a surface decontaminant for RNases and DNA, and wipe the interior of the microfluidic instrument thoroughly.
   1. Open a new microfluidic cartridge, and insert it with the inlet channels facing down and away.
   2. Ensure that the conical tube arm holds two 15 mL conical tubes and is in the position furthest to the right.
   3. Configure two sterile 15 mL conical tubes on the holder with the caps off.
2. Fill a 5 mL syringe with the mRNA solution that contains mRNA diluted in 10 mM citric acid buffer.
   1. Fill a 3 mL syringe with the lipid solution that contains the lipids diluted in pure 100% ethanol.
   2. Flip up the cartridge holder on the microfluidic device.
   3. Insert the 5 mL syringe, without the needle, that contains mRNA into the left inlet of the cartridge.
   4. Insert the 3 mL syringe, without the needle, that contains lipids into the right inlet of the cartridge.
   5. Flip the cartridge holder back down, and close the lid of the microfluidic instrument.
3. Turn on the microfluidic instrument by using the switch located on the back of the instrument.
   1. Select Quick Run.
   2. Select the correct syringe types for the 5 mL and 3 mL syringes inserted.
   3. Input the parameters for mRNA-LNP formulation at a 3:1 aqueous to organic flow rate ratio as described in Table 2.
   4. Press the Next button to go to the next screen.
   5. Confirm that the correct parameters have been inputted.
   6. Press the Start button to formulate the mRNA-LNPs.

4. Post-formulation processing and characterization of the mRNA-LNPs

1. Load the mRNA-LNPs collected in the right conical tube into the previously hydrated dialysis cassettes.
   NOTE: Do not puncture or damage the membrane of the cassette when loading the mRNA-LNPs. If the membrane is damaged, mRNA-LNPs will be lost upon loading.
   1. Place the dialysis cassettes containing the mRNA-LNPs back into the beaker containing 1x PBS, and leave them to dialyze for a minimum of 2 h.
   2. 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.
   3. Sterile-filter the mRNA-LNPs using a 0.22 µm syringe filter, and collect them in a sterile conical tube.
   4. Place 65 µL of the mRNA-LNP solution into a 1.5 mL microtube for characterization purposes.
2. Dilute 10 µL of mRNA-LNPs 1:100 in 990 µL of 1x PBS in a cuvette for the measurement of the hydrodynamic size and polydispersity index using dynamic light scattering.
3. Assess the concentration and encapsulation efficiency of the mRNA-LNPs using a fluorescence assay (i.e., RiboGreen).
   1. Prepare a stock solution of 1x TE buffer by diluting 20x TE buffer with molecular biology water.
   2. Prepare a stock solution of 1% Triton X-100 buffer by diluting Triton X-100 with 1x TE buffer.
   3. Prepare the fluorescent reagent by diluting the reagent stock 1:200 with 1x TE buffer.
   4. Dilute the mRNA-LNPs 1:100 in 1x TE buffer and 1% Triton X-100 buffer in a black-wall black-bottom 96-well plate in 100 µL.
   5. Prepare a low-range standard curve by diluting the RNA standard as per the manufacturer's instructions, and plate the standard curve in the 96-well plate.
   6. Add 100 µL of diluted fluorescent reagent to each well containing diluted mRNA-LNP or diluted standard.
   7. Place the 96-well plate inside a plate reader, and shake for 1 min, followed by a 4 min wait.
   8. Measure the fluorescence intensity of each well according to the manufacturer's protocol at an excitation/emission of 480 nm/520 nm.
4. Use the standard curve to convert the fluorescence values to concentrations.
   1. Calculate the encapsulation efficiency by subtracting the value obtained from diluting LNPs in TE buffer (unencapsulated mRNA) from the value obtained from diluting LNPs in 1% Triton X-100 (total mRNA) and dividing by the value obtained from diluting LNPs in 1% Triton X-100, according to equation 4.4.
   2. Calculate the mRNA-LNP concentration used for cell and animal studies by subtracting the value obtained from diluting LNPs in TE buffer (unencapsulated mRNA) from the value obtained from diluting LNPs in 1% Triton X-100 (total mRNA).
5. Measure the pK_a of the mRNA-LNPs by performing a 6-(p-toluidino)-2-naphthalenesulfonyl chloride (TNS) assay.
   1. Prepare the TNS reagent by creating a 0.16 mM solution of TNS in ultrapure water.
      NOTE: When using the reagent, be sure to keep it on ice and away from light to avoid degradation.
   2. Prepare buffered solutions of 150 mM sodium chloride, 20 mM sodium phosphate, 25 mM ammonium citrate, and 20 mM ammonium acetate adjusted to different pH values ranging from 2 to 12 in increments of 0.5.
   3. Add 2.5 µL of the LNP to each pH-adjusted buffer solution in a black-wall black-bottom 96-well plate.
   4. Add TNS reagent to each well so that the final TNS concentration is 6 µM.
   5. Incubate the 96-well plate in a dark location for 5 min.
   6. Measure the fluorescence at an excitation/emission of 322 nm/431 nm using a fluorescence plate reader.
   7. Fit the data to a sigmoidal curve, and calculate the pK_a by using the fitted equation to find the pH at which 50% of the maximum intensity was observed.
6. Representative results for the mRNA-LNP hydrodynamic size, PDI, encapsulation efficiency, and pK_a are shown in Table 3.

5. In vitro transfection of HepG2 cells

NOTE: Various other cell lines, such as HeLa cells or HEK-293T cells, may be used for assessing the transfection efficiency of LNPs in vitro. All cells should test negative for mycoplasma prior to the LNP transfection studies.

1. Plate 5,000 HepG2 cells in each well of a white-wall clear-bottom 96-well plate in 100 µL of complete cell culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin)
   1. Leave the cells to adhere overnight in a controlled CO₂ incubator at 37 °C and 5% CO₂.
2. Dilute the mRNA-LNPs in complete cell culture medium so that the total dose is delivered in 100 µL.
   1. Remove the complete medium from the wells.
   2. Treat the cells with either mRNA-LNPs diluted in complete cell culture medium at the desired doses or complete medium (control).
   3. Place the 96-well plate containing the treated cells back into the controlled CO₂ incubator for 24 hours.
3. Assess the transfection efficiency by performing a luciferase assay.
   1. Prepare the luciferase reagent and 1x cell lysis buffer as per the manufacturer's instructions.
   2. Take out the 96-well plate containing the cells from the incubator, and bring it into a biosafety cabinet.
   3. Remove the cell culture medium from every well of the 96-well plate.
   4. Add 20 µL of 1x lysis buffer to each well, followed by 100 µL of the previously prepared luciferase assay reagent.
   5. Protect the plate from light, and place the plate into a plate reader to measure the bioluminescent signal of each well.
      ​NOTE: Representative results demonstrating the treatment of HepG2 cells with previously formulated C12-200 mRNA-LNPs are shown in Figure 1.

6. In vivo evaluation of mRNA-LNPs in mice following tail vein injection

1. Dilute the mRNA-LNP solution with sterile 1x PBS so that 2 µg of total mRNA is present in a 100 µL tail vein injection volume, which corresponds to a body weight dose of approximately 0.1 mg/kg for a 20 g mouse.
2. Restrain each mouse using an approved method, and wipe the tail with a pad moistened with 70% alcohol.
   1. Fill a 29 G insulin syringe with either 100 µL of mRNA-LNP (2 µg) or 100 µL of 1x PBS. Remove any bubbles from the syringe.
   2. Insert the needle with the bevel facing up into the mouse's lateral tail vein, and slowly inject the 100 µL of mRNA-LNP or 1x PBS.
   3. Remove the needle and apply pressure until hemostasis is achieved.
3. Prepare a stock solution of 15 mg/mL d-luciferin potassium salt in sterile 1x PBS 6 hours post injection.
   1. Turn on the in vivo imaging system (IVIS), and open the imaging software.
   2. Press Initialize to prepare the instrument for data acquisition.
   3. Check the box next to luminescence, and set the exposure time to auto. Ensure that the correct field of view is selected for the number of mice being imaged.
   4. Administer 200 µL of d-luciferin intraperitoneally (150 mg of luciferin per kg body weight) to the previously treated mice.
   5. Place the mice into an anesthesia chamber set to 2.5% isoflurane and an oxygen flowrate of 2.0 L/min.
   6. Wait 10 min for the luciferase signal to stabilize before imaging the mRNA-LNP-treated mice.
   7. Ensure that the mice are fully anesthetized, and redirect the anesthetic line to administer isoflurane via nose cones inside the imaging chamber.
   8. Transfer the mice to the nose cones, and ensure that they are on their backs with their abdomens exposed.
   9. Close the chamber door, and click the Acquire button in the software to obtain bioluminescence images.
      NOTE: Representative results for mouse whole-body imaging following mRNA-LNP or 1X PBS treatment are shown in Figure 2.

Results

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 pK_a 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/&#...

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