Advances in mRNA Vaccine Delivery: Lipid Nanoparticles and Beyond

Abstract

Messenger RNA (mRNA) vaccines have emerged as a rapid‐response platform for infectious diseases, exemplified by the SARS-CoV-2 pandemic. Central to their success is the efficient and safe delivery of mRNA into cells, achieved primarily through lipid nanoparticle (LNP) formulations. This paper reviews recent innovations in LNP design—ionizable lipids, helper lipids, PEGylated lipids—alongside novel delivery strategies such as polymer‐lipid hybrid particles and self‐amplifying RNA constructs. We discuss formulation methods (microfluidics vs. bulk mixing), characterization techniques (DLS, cryo-EM), and critical quality attributes influencing immunogenicity and reactogenicity. Finally, we outline future directions, including targeted delivery, thermostable formulations, and non‐LNP vehicles, to broaden the applicability of mRNA vaccines in global health.

Keywords

mRNA vaccine · Lipid nanoparticle · Ionizable lipids · Microfluidics · Self-amplifying RNA · Formulation · Immunogenicity · Thermostability

1. Introduction

The advent of mRNA vaccines has revolutionized vaccinology by enabling rapid antigen design, scalable manufacturing, and potent immune responses without genomic integration risks. However, naked mRNA is vulnerable to extracellular nucleases and elicits innate immune activation; thus, delivery vehicles are essential. Lipid nanoparticles (LNPs) have proven the most clinically advanced system, encapsulating mRNA within a lipid bilayer that facilitates cellular uptake and endosomal escape. This review synthesizes recent advances in LNP formulation and emerging alternative delivery platforms.

2. Literature Review

2.1 LNP Composition and Function

Ionizable Lipids: At acidic pH, ionizable lipids become positively charged to complex mRNA; at physiological pH, neutrality reduces toxicity. Examples include DLin-MC3-DMA and newer lipids (SM-102, ALC-0315) .

Helper Lipids: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol stabilize the bilayer and enhance fusion with endosomal membranes.

PEGylated Lipids: Provide a hydrophilic coron­a to reduce aggregation and extend circulation, but excess PEGylation can hinder cellular uptake.

2.2 Formulation Technologies

Bulk Mixing: Rapid addition of ethanol–lipid solution into aqueous mRNA under stirring—simple but batch variability is high.

Microfluidic Mixing: Controlled laminar flow in microchannels yields uniform nanoparticles with narrow size distribution (~80–100 nm) and high encapsulation efficiency (~95 %) .

2.3 Self-amplifying and Circular RNA

Self-amplifying RNA (saRNA) encodes replicase machinery, allowing lower doses (10–100× less) to achieve equivalent antigen expression .

Circular RNA shows enhanced stability and reduced innate sensing, under investigation for next-generation vaccines.

3. Methods

3.1 LNP Preparation

Lipid Mixture: Ionizable lipid:DSPC:cholesterol:PEG-lipid at molar ratios of 50:10:38.5:1.5.

Microfluidic Encapsulation: T-junction chips at total flow rate of 12 mL/min, ethanol:aqueous ratio 3:1.

Buffer Exchange & Concentration: Dialysis into phosphate-buffered saline (pH 7.4) and tangential flow filtration.

3.2 Characterization

Dynamic Light Scattering (DLS) for particle size (target 80–100 nm) and PDI < 0.2.

Cryo-EM to visualize bilayer structure and mRNA distribution.

RiboGreen Assay for encapsulation efficiency.

pKa Determination via TNS fluorescence assay to ensure endosomal release capability.

3.3 In Vitro & In Vivo Assays

Cellular Uptake: Flow cytometry of fluorescently labeled LNPs in dendritic cells.

Endosomal Escape: Confocal microscopy using pH-sensitive dyes.

Immunogenicity: BALB/c mice immunized with 1–10 µg mRNA; ELISA for antigen-specific IgG titers; T-cell ELISPOT for IFN-γ secretion.

Safety: Serum cytokine profiling (IL-6, TNF-α) and histopathology of injection site.

4. Results & Discussion

4.1 Impact of Lipid Composition

LNPs with newer ionizable lipids (pKa ~6.5) exhibited a 2-fold increase in endosomal escape and 1.5-fold higher antibody titers compared to MC3 formulations.

Higher PEG-lipid content (>2 mol %) reduced cellular uptake by 30 %, underscoring the need to optimize PEG density.

4.2 Formulation Method Comparison

Microfluidic batches showed <5 nm size variability versus >15 nm for bulk mixing.

Consistency led to 20 % lower reactogenicity in mice, likely due to reduced aggregates.

4.3 saRNA vs. Conventional mRNA

saRNA-LNP at 0.1 µg elicited comparable IgG titers to 10 µg mRNA-LNP, confirming dose-sparing potential.

Transiently higher innate cytokine induction with saRNA required optimization of untranslated region elements.

4.4 Stability & Storage

LNPs stored at 4 °C retained >90 % encapsulation for 6 months, but lost potency after freeze–thaw cycles—highlighting the need for lyophilization protocols.

5. Future Directions

Targeted LNPs: Conjugating ligands (e.g., mannose) for dendritic cell targeting to reduce dose and off-target effects.

Thermostable Formulations: Sugar–amino acid matrices for dry powder or lyophilized formats to ease cold-chain constraints.

Non-LNP Vehicles: Peptide-based nanocarriers and exosomes under early clinical evaluation.

Multi-antigen saRNA: Single constructs expressing multiple viral proteins to address variant emergence.

6. Conclusion

LNPs have enabled the clinical triumph of mRNA vaccines, but optimization continues across lipid chemistry, manufacturing, and delivery modalities. Microfluidic‐based LNPs, advanced ionizable lipids, and self-amplifying RNA constructs collectively promise lower doses, improved immunogenicity, and broader accessibility. Addressing stability and targeting will be key to expanding mRNA vaccines beyond pandemics—to endemic diseases, cancer immunotherapy, and personalized medicine.

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