Lipid Nanoparticles in mRNA Vaccine Delivery

How do lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccine antigens?

Lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccines by encapsulating the mRNA through electrostatic and hydrogen bonding interactions, facilitating cellular uptake via endocytosis, and releasing the mRNA into the cytoplasm when ionizable lipids protonate in acidic endosomes and disrupt the endosomal membrane to enable translation.

Abstract

Lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccines through a coordinated physicochemical and biological process. LNPs composed of ionizable lipids, helper lipids, cholesterol, and PEG-lipids form 50-85 nm particles that encapsulate mRNA via electrostatic interactions and hydrogen bonding. Following intramuscular administration, LNPs are taken up by muscle-resident immune cells through endocytosis, with >10% of myeloid and lymphoid cells successfully transduced. The critical delivery step occurs when ionizable lipids protonate in acidic endosomes (pH ~5-6), triggering ion pairing with endosomal phospholipids that disrupts membranes and releases mRNA into the cytoplasm. Nucleoside modifications such as m1ψ reduce innate immune recognition of the mRNA cargo, while optimized formulations achieve 93-95% encapsulation efficiency and mRNA detectability for 14-28 days post-vaccination in humans.

Beyond mRNA delivery, LNPs function as intrinsic adjuvants by triggering rapid neutrophil and dendritic cell recruitment within 24 hours and inducing IL-6 production critical for germinal center responses. This dual functionality produces potent immunogenicity, with optimized formulations achieving 10-fold higher neutralizing titers than standard LNPs and protective immunity at doses as low as 0.25 µg. Rational lipid design enables independent control of delivery efficiency, tissue targeting, immune activation, and stability: the C24 formulation reduces off-target liver expression 6-fold while maintaining potency, adjuvant lipidoids provide TLR7/8 activation, and specific formulations remain stable at 4°C for at least 19 days. These mechanisms collectively explain the exceptional clinical efficacy of LNP-mRNA SARS-CoV-2 vaccines.

Methods

We analyzed 10 sources from an initial pool of 200, using 6 screening criteria. Each paper was reviewed for 8 key aspects that mattered most to the research question.

Records from Elicit search

• n = 200
• Papers screened using: Lipid Nanoparticle Delivery System, Nucleoside-Modified mRNA, SARS-CoV-2 Antigen Target, Delivery Mechanism Data, Study Type, Delivery System Scope

n = 200
Papers screened out
n = 190
Papers included for extraction
n = 10

Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “How do lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccine antigens?”

The search returned 200 total results from Elicit. We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

• Lipid Nanoparticle Delivery System: Does this study investigate lipid nanoparticles (LNPs) as delivery vehicles for mRNA?
• Nucleoside-Modified mRNA: Does this study involve nucleoside-modified mRNA (such as pseudouridine-modified or other nucleoside modifications) rather than unmodified mRNA?
• SARS-CoV-2 Antigen Target: Does the mRNA in this study encode SARS-CoV-2 antigens (such as spike protein, nucleocapsid, or other viral proteins) rather than non-SARS-CoV-2 antigens?
• Delivery Mechanism Data: Does this study report on delivery mechanisms, cellular uptake, biodistribution, or pharmacokinetics of LNP-mRNA systems?
• Study Type: Is this a preclinical study (in vitro, ex vivo, animal models) or clinical study rather than an opinion piece, editorial, commentary, or conference abstract without peer review?
• Delivery System Scope: Does this study focus on lipid-based delivery systems rather than non-lipid delivery systems (such as polymeric nanoparticles, viral vectors, or protein subunit vaccines)?

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

• LNP Composition:
  Extract complete lipid nanoparticle composition and design details for SARS-CoV-2 mRNA delivery, including:
  • Ionizable lipid type and concentration
  • Helper lipids (DSPC, cholesterol, etc.) and ratios
  • PEG-lipid type and percentage
  • Novel or modified lipid components
  • Design rationale for each component
  • Particle size and polydispersity
  • Formulation buffer and pH conditions
• mRNA Specifications:
  Extract detailed characteristics of the nucleoside-modified mRNA for SARS-CoV-2 vaccine antigens, including:
  • Target antigen (spike protein variants, epitopes, etc.)
  • Specific nucleoside modifications (pseudouridine, 5-methylcytidine, etc.)
  • mRNA sequence length and structure
  • Cap structure and poly-A tail details
  • Codon optimization strategies
  • mRNA-to-lipid ratios in formulation
• Cellular Uptake Mechanism:
  Extract mechanistic details of how LNPs deliver mRNA into target cells, including:
  • Cell types that take up LNPs (muscle cells, APCs, etc.)
  • Uptake pathways (endocytosis, fusion, etc.)
  • Endosomal escape mechanisms
  • Role of ionizable lipids in pH-dependent release
  • Timeline from uptake to protein expression
  • Subcellular trafficking patterns
  • Membrane interaction studies
• Delivery Efficiency:
  Extract quantitative measures of mRNA delivery effectiveness specifically for SARS-CoV-2 vaccines, including:
  • Protein expression levels (luciferase, spike protein)
  • Transfection efficiency percentages
  • Duration of protein expression
  • Dose-response relationships
  • Comparison with control formulations
  • In vitro vs in vivo delivery efficiency
  • Target tissue specificity
• Structural Properties:
  Extract physical and structural characteristics that enable mRNA delivery, including:
  • LNP morphology (core-shell, solid core, etc.)
  • mRNA-lipid interaction patterns
  • Hydrogen bonding or ionic interactions
  • Membrane organization (bilayer, monolayer)
  • Mechanical properties (elasticity, compliance)
  • Surface charge and zeta potential
  • Cryo-TEM or AFM structural observations
• Biodistribution Pattern:
  Extract biodistribution data showing where LNPs deliver mRNA after administration, including:
  • Primary target tissues (muscle, liver, spleen, etc.)
  • Distribution kinetics and timeline
  • Off-target expression levels
  • Route of administration effects
  • Factors controlling tissue targeting
  • Methods used to track biodistribution
  • Clearance mechanisms and timeline
• Inflammatory Effects:
  Extract data on how LNPs contribute to immune activation beyond mRNA delivery, including:
  • Innate immune responses triggered by LNPs
  • Inflammatory cytokine induction
  • Neutrophil and dendritic cell recruitment
  • Local vs systemic inflammation
  • Adjuvant effects of lipid components
  • TLR activation pathways
  • Injection site reactogenicity
  • Timeline of inflammatory responses
• Stability Factors:
  Extract factors affecting LNP-mRNA stability and delivery performance over time, including:
  • Storage temperature requirements
  • mRNA integrity maintenance
  • LNP structure preservation
  • Bioactivity retention timeline
  • Mechanisms of degradation
  • Formulation stability enhancers
  • Real-time vs accelerated stability data
  • Impact on delivery efficiency

Results

Characteristics of Included Studies

All 10 studies investigated lipid nanoparticle delivery systems for nucleoside-modified mRNA encoding SARS-CoV-2 spike protein antigens. Seven studies had full text available, while three were abstract-only. The studies employed diverse approaches including novel lipid development, structural characterization, immunogenicity testing in animal models, and clinical pharmacokinetic analysis in humans.

LNP Composition and Formulation Design

LNP formulations for SARS-CoV-2 mRNA delivery consist of four core lipid components: ionizable lipids, helper lipids, cholesterol, and PEG-lipids. The ionizable lipid component is critical for mRNA complexing and pH-dependent release.

The C24 LNP formulation demonstrated a specific composition of 48:13:37:2 for ionizable lipid/DSPC/cholesterol/DMG-PEG2000, formulated in PBS at pH 7.3-7.4. The adjuvant lipidoid formulation used a 35:16:46.5:2.5 ratio and achieved a particle size of approximately 52 nm with low polydispersity (PDI 0.127). Monoolein incorporation as a structural helper lipid induced pH-dependent mesophase transitions promoting inverse hexagonal structures to enhance mRNA release.

LNP morphology varied across formulations. Comirnaty exhibited a granular solid core enclosed by mono- and bilipid layers, with mechanical properties showing soft, compliant structures with stiffness of approximately 9 pN/nm. The particle size ranged from 52 nm to 80-85 nm with polydispersity indices of 0.02-0.06. Surface charge was generally neutral in PBS, though Comirnaty showed a less negative zeta potential of 8.6 ± 5.3. The C24 formulation displayed higher pKa (6.77) compared to MC3 (6.55).

mRNA Cargo Characteristics

The nucleoside-modified mRNA cargo encoded the SARS-CoV-2 spike protein with specific structural modifications. The target antigens included full-length spike protein with deleted furin cleavage site, diproline-stabilized spike protein (S2P), and prefusion-stabilized spike with two proline mutations.

The nucleoside modifications served to reduce immune recognition and enhance stability. The mRNA sequences were codon-optimized for efficient translation in human cells, with coding sequences identical to FDA-approved vaccines mRNA-1273 and BNT162b2 in some formulations.

Cellular Uptake and Delivery Mechanisms

LNPs deliver mRNA through a multi-step process involving cellular uptake, endosomal escape, and cytoplasmic release. The ionizable lipids play a central role in pH-dependent delivery.

Uptake pathways and cell types:

Muscle-resident myeloid and lymphoid cells were directly transduced, with >10% of these cells successfully receiving mRNA. Lung macrophages, epithelial cells, and cancer cells also took up LNPs. Intramuscular administration of empty LNPs elicited strong neutrophil and dendritic responses within 24 hours, indicating rapid immune cell engagement. Skeletal muscle played a role in the early immune response.

Endosomal escape mechanism:

The cellular uptake occurred via endocytosis. Following endocytosis, ionizable lipids became protonated in the acidic endosomal environment. This protonation triggered ion pairing with endosomal phospholipids to open the endosomal membrane. The C24 formulation demonstrated greater endosomal protonation due to its multistage protonation behavior. Neutralization of LNP charge by anionic lipids inside cells disrupted nanoparticle structure, releasing mRNA.

The adjuvant lipidoid formulation enhanced endosomal escape through interaction with TLR7/8 receptors, with structural characterization showing LNPs initially trapped in endo/lysosomes before escaping into the cytoplasm. Monoolein incorporation promoted pH-dependent mesophase transitions that facilitated enhanced mRNA release.

Structural interactions:

The Comirnaty formulation revealed unique mRNA-lipid interaction patterns. Atomic force microscopy demonstrated that molecular strands corresponding to mRNA could be pulled from nanoparticles with stepwise rupture of mRNA-lipid bonds. The neutral intra-LNP core suggested hydrogen bonding rather than ionic interactions as the primary stabilization mechanism. The ionizable lipid ALC-0315 contained free =O and −OH groups capable of hydrogen bonding with nitrogenous bases in mRNA.

Delivery Efficiency and Protein Expression

Quantitative delivery efficiency varied across formulations, with several showing superior performance compared to standard formulations.

Protein expression levels:

The C24 LNP showed 2-fold higher luciferase expression at injection sites compared to MC3 at both high and low doses. Neutralizing antibody titers were 10-fold higher for C24 versus MC3. The adjuvant lipidoid formulation (C12-113/TLRa) outperformed standard C12-113 in mRNA transfection efficiency both in vitro and in vivo, with protein expression lasting over 14 days at injection sites. Monoolein-based LNPs achieved superior mRNA transfection efficiency compared to original Moderna LNPs across diverse cell types.

Dose-response relationships:

The C24 LNP was effective at very low doses, with protection against SARS-CoV-2 occurring at 0.25 µg prime/boost. The C12-113/TLRa formulation consistently outperformed control formulations at any mRNA dose tested.

Tissue specificity:

Enhanced mRNA transfection occurred at both injection sites and inguinal lymph nodes for the adjuvant formulation. The C24 LNP reduced off-target liver expression 6-fold compared to MC3. Monoolein-based LNPs achieved targeted pulmonary delivery by intranasal administration and spleen delivery by intravenous administration.

Biodistribution Patterns

Following intramuscular administration, LNPs demonstrated specific biodistribution kinetics with varying degrees of systemic exposure.

Distribution timeline in humans:

In a clinical study of 19 subjects receiving Moderna SPIKEVAX, both mRNA and ionizable lipid peaked in blood 1-2 days post-vaccination, with median peak levels of 0.19 ng/mL for mRNA and 3.22 ng/mL for ionizable lipid. The mRNA remained detectable for 14-28 days post-vaccination in most subjects. The decay kinetics of intact mRNA and ionizable lipid were identical, suggesting intact lipid nanoparticles recirculated in blood.

Tissue targeting:

The primary target tissues included skeletal muscle at the injection site and draining lymph nodes. Animal studies showed mRNA expression observed within 6-24 hours at injection sites and lymph nodes, with sustained expression over 14 days. Minimal accumulation occurred in major organs including liver, heart, spleen, lung, and kidney.

Factors controlling distribution:

Rapid increase in surface charge near neutral pH limited systemic biodistribution for C24 formulations. The magnitude of mRNA and ionizable lipid detected in blood correlated with boost in PEG antibodies. The ability of monocytes to phagocytose lipid nanoparticles showed an inverse relationship with PEG antibody rise.

Immune Activation and Adjuvant Properties

Beyond mRNA delivery, LNPs demonstrated intrinsic adjuvant activity contributing to robust immune responses.

Innate immune activation:

LNP formulations triggered strong innate immune responses through multiple mechanisms. The ionizable lipid component induced IL-6 cytokine production, critical for T follicular helper cell differentiation. Empty LNPs alone elicited strong neutrophil and dendritic responses within 24 hours of intramuscular administration. Enhanced dendritic cell maturation occurred with specific formulations, particularly LNP4.

Cytokine profiles:

LNPs induced proinflammatory cytokines including IL-6, GM-CSF, IL-1β, IL-5, IP-10, KC, LIF, Lix, and MIP-2. IL-6 production peaked at 4 hours post-injection and remained elevated for at least 24 hours. Increased secretion of IL-6, IL-21, and IFN-γ indicated robust inflammatory responses. The adjuvant lipidoid formulation increased production of TNF-α, IL-12p70, and IL-1β.

TLR activation:

The C12-TLRa adjuvant lipidoid conferred Toll-like receptor 7/8-agonistic activity to LNPs. This TLR7/8 activation enhanced dendritic cell maturation. MyD88-based signaling enhanced mRNA-LNP vaccine potency but was not required for LNP adjuvanticity. The adjuvant activity relied on the ionizable lipid component and IL-6 induction but not on MyD88- or MAVS-dependent sensing.

Reactogenicity:

Injection site inflammation was notably reduced for C24 compared to MC3. The inflammatory responses were primarily localized to injection sites and draining lymph nodes with minimal systemic inflammation for the adjuvant formulation.

Stability and Storage Considerations

LNP-mRNA stability depended critically on storage temperature and formulation design.

Temperature-dependent stability:

The C24 LNP was entirely stable in bioactivity and mRNA integrity when stored at 4°C for at least 19 days. Bioactivity remained stable for two weeks at 4°C but declined by 20-40% at room temperature. Storage at higher temperatures reduced both bioactivity and mRNA integrity, though less so for C24 than MC3. Standard formulations required frozen storage, typically at -80°C, though some could be stored at refrigerated temperatures for up to 30 days.

Mechanisms of degradation:

mRNA degradation occurred through phosphodiester transesterification reactions, which were temperature-dependent. The mechanism involved base-mediated phosphodiester transesterification. mRNA half-lives were significantly longer at 4°C compared to higher temperatures. The LNP environment affected mRNA degradation due to proton partitioning phenomena.

Formulation stability:

The adjuvant lipidoid formulation showed minimal changes in physicochemical parameters after one month of storage at 4°C. High encapsulation efficiency (>93%) contributed to stability. Nucleoside modifications and LNP formulation enhanced translational capacity and biological stability. The hydrogen bonding between mRNA and lipids in some formulations was weaker than ionic interactions, potentially affecting long-term stability.

Synthesis

The mechanisms by which lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccines involve an integrated series of physicochemical and biological processes. The delivery cascade initiates with formulation design, where ionizable lipids complex with negatively charged mRNA through electrostatic and hydrogen bonding interactions. Following intramuscular administration, LNPs interact with muscle cells and immune cells through endocytic uptake, with >10% of muscle-resident myeloid and lymphoid cells successfully transduced.

The critical endosomal escape step relies on pH-dependent protonation of ionizable lipids in acidic endosomes, triggering membrane disruption through ion pairing with endosomal phospholipids. Different ionizable lipids achieve this through distinct mechanisms: the C24 formulation employs multistage protonation, while adjuvant lipidoids enhance escape through TLR7/8 receptor engagement. Upon cytoplasmic release, the nucleoside-modified mRNA is translated by cellular ribosomes to produce spike protein antigen.

Beyond simple mRNA delivery, LNPs function as adjuvants by triggering innate immune responses. Empty LNPs alone elicit neutrophil and dendritic cell recruitment within 24 hours, while the ionizable lipid component induces IL-6 production critical for germinal center formation. This dual functionality—mRNA delivery plus immune activation—underlies the exceptional efficacy of LNP-mRNA SARS-CoV-2 vaccines, with some formulations achieving protection at doses as low as 0.25 µg and neutralizing titers 10-fold higher than conventional formulations.

Human pharmacokinetic data reveals that intact LNPs recirculate in blood for weeks post-vaccination, with mRNA detectable for 14-28 days, supporting sustained antigen expression. The formation of PEG antibodies correlates with the magnitude of circulating mRNA/lipid and is inversely related to monocyte phagocytic clearance, suggesting immune-mediated clearance pathways influence delivery kinetics.

Structural innovations continue to optimize this delivery platform. Replacing phospholipids with monoolein induces pH-dependent mesophase transitions promoting inverse hexagonal structures that enhance mRNA release. The C24 formulation reduces off-target liver expression 6-fold while maintaining potency, and adjuvant lipidoids provide TLR7/8 activation to boost cellular immunity. These advances demonstrate how rational lipid design can independently modulate delivery efficiency, tissue targeting, immune activation, and stability—the four key parameters determining LNP-mRNA vaccine performance.