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. Graphical abstract

Liposomal Delivery of MIW815 (ADU-S100) for Potentiated STING Activation

“…By dissolving the drug in the aqueous buffer used to hydrate the thin lipid film containing the DOTAP and helper lipids (cholesterol, HSPC, DSPE-PEG 2000 ), ADU-S100 can be encapsulated in PEGylated DOTAP/cholesterol liposomes (N/P ratio ≥ 10) that remain partially stable in serum for days. The optimization of the N/P ratio also needs to be balanced with safety considerations because DOTAP, as a cationic lipid, can non-specifically bind to anionic plasma membranes and cause dose-dependent cytotoxicity [ 41 , 42 ]. Given the nearly identical encapsulation and bioactivity profiles observed for liposomal ADU-S100 with 45 mol% (N/P ratio = 20) and 34 mol% DOTAP (N/P ratio = 15), we chose to focus on the later formulation to minimize potential cytotoxicity.…”

A Pilot Study Exploiting the Industrialization Potential of Solid Lipid Nanoparticle-Based Metered-Dose Inhalers

“…The reason is the presence of CTAB in SLN(+). Various cationic materials has been reported to be toxic, even including the most commonly used cationic lecithin for cationic liposome preparation, i.e., 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) [ 25 ]. The underlying mechanism was the strong interaction between cationic materials and anionic cell membrane which disturbs the physiological structure and function of the latter [ 26 ].…”

A biomimetic camouflaged metal organic framework for enhanced siRNA delivery in the tumor environment

“…This electrostatic interaction not only facilitates the formation of lipoplexes but also offers a dual advantage: on the one hand, it provides a protective shield to siRNA, safeguarding it from rapid enzymatic degradation in the bloodstream; on the other, it bolsters cellular uptake, primarily due to the enhanced permeability and retention (EPR) effect. 72 Furthermore, once inside the cell, these lipid formulations play a crucial role in mediating endosomal escape, a pivotal step that ensures the siRNA's release into the cytoplasm where it can exert its gene-silencing effect. This multifaceted approach offered by LNPs underscores their potential in advancing siRNA-based therapeutics to clinical applications.…”