The nano delivery systems and applications of mRNA

If appropriate nano delivery systems (lipid nanoparticles, liposomes, polymer complexes, micelles, cationic peptides) and chemical modifications are applied to mRNA, then mRNA can overcome its instability and delivery barriers, enabling a wide range of therapeutic applications including infectious disease vaccines, cancer immunotherapy, cardiovascular disease treatment, genetic disease therapy, and regenerative medicine.

• Secondary Aims:
• To review nano delivery systems for mRNA, including lipid nanoparticles (LNPs), liposomes, polymer complexes, micelles, and cationic peptides.
• To discuss the similarities and differences between LNPs and liposomes.
• To present applications of mRNA beyond COVID-19 vaccines, including infectious diseases, tumors, cardiovascular disease, genetic disease, fetal immune system immaturity, neurological diseases, and iPSC technology.
• To discuss the outlook and remaining challenges for mRNA therapeutics.

Component: mRNA Payloads; Examples Discussed: S-mRNA (SARS-CoV-2 spike protein), self-amplifying mRNA (sa-mRNA), mRNA encoding viral antigens (influenza, rabies, Zika, CMV, chikungunya, neonatal herpes), tumor antigens (neoantigens, KRAS, survivin-T34A, cytokeratin), VEGF-A mRNA, Cas9 mRNA (CRISPR), reprogramming transcription factors (iPSC)

Component: Lipid Nanoparticles (LNPs); Examples Discussed: SM-102, ALC-0315, DSPC, cholesterol, DMG-PEG2000, ALC-0159; ionizable cationic lipids (DOTAP, DODAP, DOBAQ, MC3 amino-lipid, L608 amino-lipid, IBL0713); helper lipids; PEG-lipids

Component: Liposomes; Examples Discussed: Cationic liposomes (DOTAP/Chol/DSPE-PEG, DC-cholesterol/DOPE); modified liposomes (mannose-modified, DP7 peptide-modified); liposome/protamine-mRNA complexes

Component: Polymer Complexes; Examples Discussed: Polyethylenimine (PEI), chitosan, hyaluronic acid, graphene oxide (GO)-PEI complexes, poly(β-amino ester) (PBAE)

Component: Micelles; Examples Discussed: PEG-polyaspartamide nanomicelles with aminoethylene side chains (DET, TET, TEP)

Component: Cationic Peptides; Examples Discussed: Cell-penetrating peptides (CPPs), protamine, PEG12KL4 peptide, anionic peptides, GALA peptides

Component: Routes of Administration; Examples Discussed: Intramuscular, subcutaneous, intradermal, intravenous, intranasal, intracerebroventricular (ICV), intraperitoneal, epicardial, in utero

• Covers mRNA delivery systems from 1978 (first liposome-mediated mRNA delivery) to 2021 (COVID-19 mRNA vaccines).
• Discusses in vitro studies (cell lines including Jurkat, HepG2, DCs, fibroblasts) and in vivo animal models (mice, rats, ferrets, pigs, non-human primates).
• Reviews clinical trials for mRNA vaccines and therapeutics (Table 3, Table 6).
• Compares LNP and liposome formulations (Table 5).
• Summarizes lipid materials used for mRNA delivery (Table 4) and mRNA-LNP products in development or on the market (Table 3).

• mRNA chemical modification: Nucleoside modification (e.g., N1-methylpseudouridine) to increase stability and reduce immunogenicity.
• Microfluidic preparation: Ethanol dilution for LNP formulation.
• Thin film dispersion, solvent injection, freeze drying, pH gradient methods: Liposome preparation.
• Electrostatic complexation: Polymer-mRNA nanoparticle formation.
• Reporter gene assays: Luciferase, GFP expression.
• Immunogenicity assays: IgG1, IgG2A, neutralizing antibody titers, T cell responses.
• Clinical trial endpoints: Safety, tolerability, efficacy (COVID-19 prevention rates).

• No primary experimental data; conclusions are synthesized from existing literature.
• No systematic search strategy or meta-analysis.
• Focus is primarily on mRNA delivery systems; other nucleic acid modalities (siRNA, pDNA) are discussed but not exhaustively.
• Limited discussion of long-term safety and regulatory challenges.

Limitations of the field highlighted by the authors:

• Inefficient delivery: Only ~0.01% of mRNA reaches the cytoplasm; high-dose administration is still normal and brings side effects.
• Endosomal escape bottleneck: Limited endosomal escape of nano delivery systems remains a major hurdle.
• Stability and storage: Poor stability makes mRNA-LNP expensive to transport and store; long-term storage is underexplored.
• Production complexity: Liposome preparation and mRNA encapsulation processes are more complex than LNPs.
• Unwanted immunogenicity: mRNA can activate innate immune responses; delivery systems must transfect cells without causing toxicity or unwanted immunogenicity.
• Polymer toxicity: PEI and other cationic polymers can be cytotoxic; modification is required to improve transfection efficiency and stability.
• Limited clinical data for non-COVID applications: Most mRNA therapeutics for cancer, cardiovascular disease, and genetic disorders are still in preclinical or early clinical stages.