Optimization of DOTAP/chol Cationic Lipid Nanoparticles for mRNA, pDNA, and Oligonucleotide Delivery

Abstract

Lipid nanoparticles (LNPs) can be used as delivery vehicles for nucleic acid biotherapeutics. In fact, LNPs are currently being used in the Pfizer/BioNTech and Moderna COVID-19 vaccines. Cationic LNPs composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (chol) LNPs have been classified as one of the most efficient gene delivery systems and are being tested in numerous clinical trials. The objective of this study was to examine the effect of the molar ratio of DOTAP/chol, PEGylation, and lipid to mRNA ratio on mRNA transfection, and explore the applications of DOTAP/chol LNPs in pDNA and oligonucleotide transfection. Here we showed that PEGylation significantly decreased mRNA transfection efficiency of DOTAP/chol LNPs. Among non-PEGylated LNP formulations, 1:3 molar ratio of DOTAP/chol in DOTAP/chol LNPs showed the highest mRNA transfection efficiency. Furthermore, the optimal ratio of DOTAP/chol LNPs to mRNA was tested to be 62.5 µM lipid to 1 μg mRNA. More importantly, these mRNA-loaded nanoparticles were stable for 60 days at 4 °C storage without showing reduction in transfection efficacy. We further found that DOTAP/chol LNPs were able to transfect pDNA and oligonucleotides, demonstrating the ability of these LNPs to transport the cargo into the cell nucleus. The influence of various factors in the formulation of DOTAP/chol cationic LNPs is thus described and will help improve drug delivery of nucleic acid-based vaccines and therapies.

Keywords: DOTAP/cholesterol; LNP-mRNA; lipid nanoparticles; mRNA delivery; transfection efficiency.

Fig. 1

The size (a), PDI (b), and zeta potential (c) of non-PEGylated and PEGylated DOTAP/chol LNPs and LNPs/mRNA complexes; the size (d), PDI (e), and zeta potential (f) of non-PEGylated and PEGylated DOTAP/chol LNPs and LNPs/pDNA complexes. Data are presented as mean ± SD (n = 4)

Fig. 2

mRNA gel retardation assays of a non-PEGylated DOTAP/chol lipoplexes and b PEGylated DOTAP/chol lipoplexes. DOTAP/chol LNPs were complexed with mRNA at various lipid concentrations, and then run through a 2% agarose gel. The mobility of mRNA was visualized by ethidium bromide staining. The amount of mRNA was fixed at 1 μg

Fig. 3

Cell viability assays. SK-OV-3 cells were treated with DOTAP/chol LNPs (blue bars) or PEGylated DOTAP/chol LNPs (orange bars) at five DOTAP/chol molar ratios (2:1, 1:1, 1:2, 1:3, and 1:4) and three lipid concentrations (31.25, 62.5, and 125 µM). After a 24-h incubation, MTS assays were performed to test the viability of the cells. Data are presented as mean ± SD (n = 4)

Fig. 4

mRNA transfection assays. Non-PEGylated DOTAP/chol LNPs (blue bars) or PEGylated DOTAP/chol LNPs (orange bars) at five DOTAP/chol molar ratios (2:1, 1:1, 1:2, 1:3, and 1:4) and three lipid concentrations (31.25, 62.5, and 125 µM) were complexed with GFP-expressing mRNA (a fixed amount of 1 μg). The transfection efficiency was determined by the percentage of GFP-expressing cells against all cells counted using flow cytometry. Data are presented as mean ± SD (n = 3)

Fig. 5

Stimulated Raman scattering (SRS) and fluorescence images of SK-OV-3 cells treated with 62.5 µM non-PEGylated and PEGylated DOTAP/chol LNPs at five DOTAP/chol molar ratios (2:1, 1:1, 1:2, 1:3, and 1:4) complexed with a fixed amount of 1 μg mRNA. Naked mRNA and JetPRIME were used to treat the cells as a negative control and a positive control, respectively. The images show overlap of the cell protein (blue) and GFP fluorescence (green) distribution in the cells. Scale bar: 100 µm

Fig. 6

a pDNA transfection assays. Non-PEGylated or PEGylated DOTAP/chol lipoplexes containing 0.3 μg pDNA were added to SK-OV-3 cells and analyzed by flow cytometry after a 48-h treatment. b Oligonucleotide (oligo) transfection assays. SK-OV-3 cells were treated with non-PEGylated and PEGylated DOTAP/chol lipoplexes containing 1 μg FAM-labeled oligonucleotide. Data are presented as mean ± SD (n = 3)

Fig. 7

The stability of non-PEGylated 1:3 DOTAP/chol and PEGylated 1:2 DOTAP/chol lipoplexes. a The size and PDI changes of non-PEGylated 1:3 and PEGylated 1:2 DOTAP/chol lipoplexes with different storage time at 4 °C. b mRNA transfection efficiency in SK-OV-3 cells after transfection with fresh or stored 62.5 µM non-PEGylated 1:3 or PEGylated 1:2 DOTAP/chol lipoplexes containing the same amount of mRNA (1 μg). Data are shown as mean ± SD (n = 3)