Next-Generation Vaccines: Nanoparticle-Mediated DNA and mRNA Delivery

Purpose: Nucleic acid vaccines (DNA and mRNA) offer rapid design, adaptability to changing pathogen strains, and stimulation of both humoral and cellular immunity, but their clinical translation is limited by inefficient delivery—nucleic acids are rapidly degraded by endogenous nucleases, have poor cellular uptake due to their negative charge, and must cross cellular barriers (cytoplasm for mRNA, nucleus for DNA). Nanoparticles (NPs) are promising non-viral vectors to overcome these delivery challenges.

Hypothesis: No formal experimental hypothesis. Central thesis: nanoparticle-mediated delivery can protect DNA/mRNA payloads from degradation, facilitate cellular uptake and endosomal escape, enable targeting to antigen-presenting cells and lymph nodes, and co-deliver multiple vaccine components, thereby eliciting effective immune responses and enabling next-generation nucleic acid vaccines for infectious diseases and cancer.

Aims: Summarize the advantages and challenges of DNA and mRNA vaccines. - Review different NP formulations for DNA and mRNA vaccine delivery, including liposomal, polymeric, inorganic, and peptide-based systems. - Provide an overview of NP vaccine clinical trials. - Discuss future perspectives and challenges for NP-mediated nucleic acid vaccines.

Delivery system: Liposomal nanoparticles: ionizable lipids (SM-102, ALC-0315, TT3, C12-200), phospholipids (DSPC, DOPE), cholesterol, PEGylated lipids (PEG2000-DMG, ALC-0159); cationic lipid-assisted nanoparticles (CLANs); cationic nanoemulsions (CNE) with DOTAP and MF59; lipopolyplexes; mannose-mimicking shikimoylated cationic amphiphiles for DC targeting. - Polymeric nanoparticles: PAMAM dendrimers, polyethylenimine (PEI), PLGA, PLGA-PLL/γ-PGA, polyanhydrides (CPTEG:CPH), polysulfenamides; natural polymers: chitosan (N-2-HACC-CMC), hyaluronic acid (HA-PEI); microneedle (MN) delivery systems. - Inorganic nanoparticles: gold nanoparticles (AuNPs) conjugated with mannose-mimicking shikimoyl ligands; mesoporous silica nanoparticles (MSNs), including rambutan-like MSNs modified with PEI. - Peptide-based nanoparticles: cell-penetrating peptides (RALA, LAH4, LAH4-L1), virus-like particles (VLPs, CMP-001). - Payloads: plasmid DNA (pDNA), mRNA, self-amplifying RNA (saRNA), CpG oligodeoxynucleotides, antigens (OVA, MART1, TRP2, gp100, SARS-CoV-2 spike, RSV F, HIV, Ebola, influenza). - Targeting/functionalization: mannose receptor targeting, CD44 targeting (HA), lymph node targeting, DC targeting, PEGylation, cationic lipid/mRNA electrostatic complexation. - Routes: intramuscular, intradermal, subcutaneous, intravenous, intranasal, microneedle patch.

Approach: Review of preclinical and clinical literature; no primary experiments. In vitro systems include dendritic cells, macrophages, J774A.1, RAW264.7, and various cancer cell lines. In vivo models include mice (B16F10 melanoma, E.G7-OVA lymphoma, SARS-CoV-2, H1N1 influenza, Ebola, Toxoplasma gondii, RSV, HIV), pigs (swine influenza), rats, rabbits, and nonhuman primates. Clinical trials include mRNA-1273 (phase III, 30,000+ participants, 95% effective), BNT162b2 (phase III, 95% effective), CMP-001 (phase II), and various other NP vaccine trials.

Key methods: Nanoparticle characterization: size (DLS), morphology (TEM, cryo-TEM), zeta potential, nitrogen sorption isotherm. - Immune response: antigen-specific antibody titers (IgG, IgA), neutralizing antibody titers, CD4+ and CD8+ T cell responses, IFN-γ secretion, cytokine production (IL-2, IL-4), lymphoproliferative responses. - Cellular uptake and transfection: flow cytometry, fluorescence microscopy, luciferase expression. - In vivo efficacy: tumor growth inhibition, survival, viral challenge protection, viral shedding, lung virus titers. - Safety: toxicity, body weight, histopathology.

Key results: mRNA-1273 (Moderna): phase III trial with 30,000+ participants showed 95% efficacy in preventing COVID-19; phase I showed dose-dependent antibody responses (day 57 GMT: 299,751, 782,719, and 1,192,154 for 25, 100, and 250 µg doses, respectively). - BNT162b2 (Pfizer/BioNTech): phase III trial showed 95% efficacy with no safety concerns. - TT3-formulated NASAR mRNA SARS-CoV-2 vaccine induced 300-fold more anti-S1 antibodies than MC3 (FDA-approved lipid delivery vehicle); intramuscular injection induced 5-fold more antigen-specific antibodies than subcutaneous injection. - LNP-formulated saRNA SARS-CoV-2 vaccine induced high, dose-dependent neutralizing antibody titers higher than convalescent COVID-19 patient sera. - MDNP saRNA vaccine protected mice against lethal H1N1 influenza and Ebola virus; multiplexed vaccine protected against lethal Toxoplasma gondii; production timeline from DNA sequence to milligram-scale injection-ready vaccine was only 7 days. - AuNP-SL/pCMV-MART1 DC vaccine induced long-lasting (200 days) anti-tumor immune response in mice challenged with lethal melanoma. - Rambutan-like MSN/PEI/pDNA-OVA vaccine enhanced antigen-specific IgG, CD80/CD86 expression, IFN-γ production, and CD8+ T cell activation, outperforming in vivo-jetPEI. - RALA-mRNA nanocomplexes induced potent cytolytic T cell responses and outperformed liposomal mRNA formulations (DOTAP/DOPE). - CMP-001 (VLP-encapsulated CpG-A ODN) enhanced tumor response to anti-PD1 therapy and activated pDCs and NK cells in HPV+ tumor model; currently in clinical trials.

Interpretation: Nanoparticle-mediated delivery of DNA and mRNA vaccines offers a promising approach to modernize vaccination, enabling rapid adaptation to emerging pathogens and cost-effective manufacturing. NPs protect nucleic acids, enhance cellular uptake and endosomal escape, enable APC/lymph node targeting, and allow co-delivery of multiple vaccine components. The success of LNP-based COVID-19 mRNA vaccines (mRNA-1273, BNT162b2) demonstrates the clinical potential of this platform. The authors conclude that further optimization of NP formulations and nucleic acid design, along with improved understanding of immune responses, will unlock the full potential of NP-mediated nucleic acid vaccines.

Limitations: Review article; no primary data. - No nucleic acid vaccine had received FDA approval for human use at the time of writing (though mRNA-1273 and BNT162b2 were in phase III trials with emergency use authorizations pending). - Poor immunogenicity of naked DNA/mRNA; 95–98% of intramuscularly injected plasmid DNA remains in the interfibrilar space. - Cationic lipid/polymer toxicity (membrane disruption, vacuolization, cell lysis, necrosis) and reduced transfection efficiency when cationic charge is reduced. - Inorganic NPs: potential cytotoxicity from cationic ligands, endosomal entrapment, limited large-animal validation, proof-of-concept only. - Peptide-based NPs: limited effective compound libraries, nephrotoxicity of arginine-rich CPPs, VLP stability and phagocyte-mediated clearance. - Polymeric NPs: relatively low transfection efficiency, potential cytotoxicity, limited understanding of protein corona interactions, complex synthesis for scale-up. - Most clinical trials are liposomal-based; minority are nucleic acid-based. - Differences between animal and human immune systems require comprehensive evaluation of NP formulations, doses, and administration routes. - Need for detailed mechanistic understanding of endosomal escape (mRNA) and nuclear transport (DNA) to improve transfection efficiency.