Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Summary

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Abstract

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Introduction

Drug carriers are used to protect and deliver a therapeutic with typical favorable properties including low cytotoxicity, increased bioavailability, and improved stability^1,2,3. Polymeric nanoparticles, micelles, and lipid-based particles have previously been explored for nucleic acid encapsulation and delivery^4,5,6,7. Lipids have been used in different types of nanocarrier systems, including liposomes, and lipid nanoparticles, as they are biocompat....

Protocol

A schematic of the overall process is provided in Figure 1.

1. Preparation of buffers

NOTE: Sterile filtering of the buffers is highly suggested here to remove any particulates which may impact the nucleic acid and LNP quality.

1. Phosphate Buffered Saline (PBS)
   1. Prepare 1x PBS using 8 mM Na₂HPO₄, 2 mM KH₂PO_4, 137 mM NaCl, and 2.7 mM KCl in nuclease free water and adjust the pH to 7.4.
   2. Sterilize by vacuum filtration using a 0.22 µm pore-size filter.
2. Citrate Buffer
   1. Prepare ci....

Results

Multiple batches of LNPs with the same lipid formulation and N/P ratio of 6 were developed on separate days to demonstrate reproducibility of the technique. Batch 1 and 2 resulted in overlapping size distributions with similar polydispersity (Figure 2A) No significant difference was observed in the size or encapsulation efficiency between the two different batches (Figure 2B). The encapsulation efficiency was high for each batch .......

Discussion

Reproducibility, speed, and low volume screening are significant advantages of using microfluidic mixing to form LNPs compared to other existing methods (e.g., lipid film hydration and ethanol injection). We have demonstrated the reproducibility of this method with no impact on encapsulation efficiency or particle size observed with different LNP batches. This is an essential criterion for any therapeutic, including LNPs, to become clinically available.

The technique described here employs sta.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (C-14 PEG)	Avanti Polar Lipids	880151P
10 µl Graduated Filter Tips  (RNase-,DNase-, DNA-free)	USA Scientific	1121-3810
1000 µl Graduated Filter Tips (RNase-,DNase-, DNA-free)	USA Scientific	1111-2831
20 µl Beveled Filter Tips (RNase-,DNase-, DNA-free)	USA Scientific	1120-1810
200 µl Graudated Filter Tips (RNase-,DNase-, DNA-free)	USA Scientific	1120-8810
3β-Hydroxy-5-cholestene, 5-Cholesten-3β-ol (Cholesterol)	Sigma-Aldrich	C8667
BD Slip Tip Sterile Syringes (1 ml syringe)	Thermo Fisher Scientific	14-823-434
BD Slip Tip Sterile Syringes (3 ml syringe)	Thermo Fisher Scientific	14-823-436
BD Vacutainer General Use Syringe Needles (BD Blunt Fill Needle 18G)	Thermo Fisher Scientific	23-021-020
Benchtop Centrifuge	Beckman coulter
Black 96 well plates	Thermo Fisher Scientific	14-245-177
BrandTech BRAND BIO-CERT RNase-, DNase-, DNA-free microcentrifuge tubes (1.5mL)	Thermo Fisher Scientific	14-380-813
Citric Acid	Fisher Scientific	02-002-611
Corning 500ml Vacuum Filter/Storage Bottle System, 0.22 um pore	Corning	430769
Disposable folded capillary cells	Malvern	DTS1070
Ethyl Alcohol, Pure 200 proof	Sigma-Aldrich	459844
Fisher Brand Semi-Micro Cuvette	Thermo Fisher Scientific	14955127
Invitrogen Conical Tubes (15 mL) (DNase-RNase-free)	Thermo Fisher Scientific	AM12500
MilliporeSigma Amicon Ultra Centrifugal Filter Units	Thermo Fisher Scientific	UFC901024
NanoAssemblr Benchtop	Precision Nanyosystems
Nuclease-free water	Thermo Fisher Scientific	AM9930
Phosphate Buffered Saline (PBS)	Thermo Fisher Scientific	AM9624
Quant-iT PicoGreen dsDNA Assay Kit	Thermo Fisher Scientific	P7589
Quant-iT RiboGreen RNA Assay Kit	Thermo Fisher Scientific	R11490
Sodium Chloride	Fisher Scientific	02-004-036
Sodium Citrate, Dihydrate, granular	Fisher Scientific	02-004-056
SpectraMax i3x	Molecular Devices
Zetasizer Nano	Malvern