Lipid‐Polymer Hybrid “Particle‐in‐Particle” Nanostructure Gene Delivery Platform Explored for Lyophilizable DNA and mRNA COVID‐19 Vaccines

Abstract

SARS‐CoV‐2 has led to a worldwide pandemic, catastrophically impacting public health and the global economy. Herein, a new class of lipid‐modified polymer poly (β‐amino esters) (L‐PBAEs) is developed via enzyme‐catalyzed esterification and further formulation of the L‐PBAEs with poly( d , l ‐lactide‐coglycolide)‐ b ‐poly(ethylene glycol) (PLGA‐PEG) leads to self‐assembly into a “particle‐in‐particle” (PNP) nanostructure for gene delivery. Out of 24 PNP candidates, the top‐performing PNP/C12‐PBAE nanoparticles efficiently deliver both DNA and mRNA in vitro and in vivo, presenting enhanced transfection efficacy, sustained gene release behavior, and excellent stability for at least 12 months of storage at −20 °C after lyophilization without loss of transfection efficacy. Encapsulated with spike encoded plasmid DNA and mRNA, the lipid‐modified polymeric PNP COVID‐19 vaccines successfully elicit spike‐specific antibodies and Th1‐biased T cell immune responses in immunized mice even after 12 months of lyophilized storage at −20 °C. This newly developed lipid‐polymer hybrid PNP nanoparticle system demonstrates a new strategy for both plasmid DNA and mRNA delivery with the capability of long‐term lyophilized storage.

Enzyme-Catalyzed One-Step Synthesis of Ionizable Cationic Lipids for Lipid Nanoparticle-Based mRNA COVID-19 Vaccines

“…We then explored the long-term storage capability of AA3-DLin LNPs under different storage conditions. Sucrose was formulated into AA3-DLin LNP as the cryoprotectant to stabilize LNP structure during storage. , Previous reports showed that sucrose was the most effective cryoprotectant with high viscosity and low molecular mobility, and serves as a stabilizer to minimize the van der Waals interaction and preserve the spacing between the nanoparticles during frozen storage. ,, A sucrose solution was added to LNPs with final concentrations of 0–40 wt % and the resulting LNPs were further lyophilized as dry powder or stored at 4 °C and −20 °C with a timeline of 12 months. The transfection efficacy of stored LNPs was characterized in vitro and in vivo with the fresh LNPs as the positive control.…”

“…A CALB enzyme-catalyzed esterification was utilized to synthesize ionizable cationic lipids between hydroxyl groups (−OH) of amino alcohol and carboxylic acid groups (−COOH) of lipid acids. , Briefly, amino alcohol was dissolved in THF using two-neck round-bottom flask, and then 0.5 g of amino alcohol was added and dissolved in 5 mL of THF followed by the addition of 2 molar ratio excess of lipid acid and 0.5 g of CALB. The reaction lasted for 72 h with the protection of nitrogen at 60 °C.…”

“…Enzyme-assisted chemical reactions have been widely employed in laboratory and industrial processes since enzymes are non-toxic, eco-friendly, recyclable biocatalysts; in particular, Candida antarctica Lipase B (CALB) is a frequently used high-efficiency biocatalyst in organic syntheses such as esterification and transesterification. − Herein, we have utilized a chemical and biological tool, the enzyme catalyst Candida antarctica Lipase B, to synthesize a chemical library of degradable ionizable cationic lipids using amino alcohols and lipid acids, resulting in 144 lipid-like materials with high chemical yield and purity. Notably, this method provides a clean and eco-friendly chemical reaction avoiding corrosive catalysts used in conventional esterification, and the enzyme can be recycled for repeated use with minimized pollution to fulfill the principles of “Green Chemistry”.…”

Novel bionic inspired nanosystem construction for precise delivery of mRNA

“…The prepared SiO 2 nanoparticles were immediately modified with PEI (V-SiO 2 -P), mRNA loading followed by the addition of a complete LB coating (V-SiO 2 -P/LB; The thickness of LB is 7-10 nm; Red arrow). The two steps of PEI and LB modifications could improve the mRNA binding efficiency, facilitate lysosomal escape, and maintain colloidal stability that is capable of effectively avoiding mRNA degradation (Wang et al, 2018) (Noureddine et al, 2020) (Xia et al, 2009;Wang et al, 2018) ( Noureddine et al, 2020;Gao et al, 2021;Riley et al, 2021;Li et al, 2022). At present, there are few studies on mRNA delivery by inorganic nanomaterials, and LNPs have been an effective mRNA delivery (Meng et al, 2015).…”

“…Second, their biomimetic morphology enhances gene delivery efficiency both in vitro and in vivo. Finally, a supported LB coating maintains colloidal stability and effectively prevents mRNA degradation (Noureddine et al, 2020;Gao et al, 2021;Riley et al, 2021;Li et al, 2022). Using green fluorescence protein (GFP)-expressing mRNA as a model, the intracellular delivery and transfection efficiency of m@V-SiO 2 -P/LB nanoparticles were demonstrated to be much higher than that of the benchmark LNPs loaded with mRNA (m@LNPs).…”