Elicit: Lipid Nanoparticles in mRNA Vaccine Delivery

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Lipid Nanoparticles in mRNA Vaccine Delivery

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May 5, 2026

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. More on methods

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

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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:

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.

Extract complete lipid nanoparticle composition and design details for SARS-CoV-2 mRNA delivery, including:

Extract detailed characteristics of the nucleoside-modified mRNA for SARS-CoV-2 vaccine antigens, including:

Extract mechanistic details of how LNPs deliver mRNA into target cells, including:

Extract quantitative measures of mRNA delivery effectiveness specifically for SARS-CoV-2 vaccines, including:

Extract physical and structural characteristics that enable mRNA delivery, including:

Extract biodistribution data showing where LNPs deliver mRNA after administration, including:

Extract data on how LNPs contribute to immune activation beyond mRNA delivery, including:

Extract factors affecting LNP-mRNA stability and delivery performance over time, including:

Results

Characteristics of Included Studies

Study

Full text retrieved?

Study focus

LNP formulation

Study design

Suman Alishetty et al., 2021

Yes

Novel C24 ionizable lipid development

C24 lipid (48:13:37:2 ratio)

Mouse immunogenicity and challenge studies

Afshana Quadiri et al., 2024

Yes

Effect of helper lipid composition

LNP4 with DOPE phospholipid

Hamster protection studies comparing 4 LNP formulations

Afshana Quadiri et al., 2025

Yes

DOPE-containing LNP characterization

LNP4 with DOPE

Hamster immunogenicity and protection

Xuexiang Han et al., 2023

Yes

Adjuvant lipidoid substitution

C12-TLRa adjuvant lipidoid (35:16:46.5:2.5)

Mouse immunogenicity with TLR7/8 agonist lipid

J. Szebeni et al., 2023

Yes

Structural analysis of Comirnaty vaccine

BNT162b2 with ALC-0315

Physicochemical characterization using AFM, cryo-TEM

S. Kent et al., 2024

No

Human pharmacokinetics

Moderna SPIKEVAX

Clinical study in 19 vaccinated subjects

M. Alameh et al., 2021

Yes

Adjuvant activity mechanisms

Proprietary Acuitas formulation

Mouse studies with influenza and SARS-CoV-2 mRNA

Natalia Martinez et al., 2025

No

Monoolein as helper lipid

MO-based modified Moderna LNPs

In vivo delivery efficiency studies

Dorottya Laczkó et al., 2020

Yes

Nucleoside-modified mRNA immunogenicity

Standard LNP formulation

Mouse single-dose immunogenicity

Devdoot Majumdar et al., 2023

No

Mechanisms of muscle-based adjuvancy

Moderna sm-102 formulation

Mouse mechanistic studies of immune response

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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.

Component

Example formulations

Molar ratios

Design rationale

Ionizable lipid

C24, ALC-0315, C12-TLRa

35-48%

Enhanced endosomal protonation; TLR7/8 activation

Helper lipid

DSPC, DOPC, DOPE, Monoolein

13-16%

Structural stability; pH-dependent mesophase transitions

Cholesterol

Standard component

37-46.5%

Structural support

PEG-lipid

DMG-PEG2000

2-2.5%

Immune evasion and circulation

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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.

mRNA feature

Specifications

Nucleoside modification

m1ψ (1-methylpseudouridine); Pseudo-U; m1J-5’-triphosphate

Sequence length

3,804 bp; 4,284 nucleotides

Cap structure

Trinucleotide cap1 analog (CleanCap)

Poly-A tail

80-101 adenine residues

Codon optimization

Applied to enhance translation

Encapsulation efficiency

93-95%

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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.

References

Xuexiang Han, M. Alameh, Kamila Butowska, James J Knox, Kendall A. Lundgreen, and 11 more\ (2023).Adjuvant lipidoid-substituted lipid nanoparticles augment the immunogenicity of SARS-CoV-2 mRNA vaccines. Nature Nanotechnology

J. Szebeni, B. Kiss, Tamás Bozó, K. Turjeman, Y. Levi-Kalisman, and 2 more\ (2023).Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA–Lipid Complexes. ACS Nano

Suman Alishetty, Manuel J. Carrasco, M. Alameh, M. Paige, Hooda Said, and 14 more\ (2021).Novel lipid nanoparticle provides potent SARS-CoV-2 mRNA vaccine at low dose with low local reactogenicity, high thermostability and limited systemic biodistribution

S. Kent, Shiyao Li, T. Amarasena, A. Reynaldi, W. Lee, and 9 more\ (2024).Blood Distribution of SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine in Humans. medRxiv

Afshana Quadiri, S. Prakash, Latifa Zayou, N. Dhanushkodi, Amruth Chilukuri, and 4 more\ (2024).Spike mRNA Vaccine Encapsulated in a Lipid Nanoparticle Composed of Phospholipid 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine Induced Potent B- and T-cell Responses Associated with Protection against COVID-19 in Hamsters. bioRxiv

Afshana Quadiri, S. Prakash, Latifa Zayou, N. Dhanushkodi, Amruth Chilukuri, and 5 more\ (2025).A Spike-Based mRNA Vaccine Encapsulated in Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine Containing Lipid Nanoparticles Induced Potent B- and T-Cell Responses Associated with Protection Against SARS-CoV-2 Infection and COVID-19-like Symptoms in Hamsters. Vaccines

M. Alameh, I. Tombácz, Emily Bettini, Katlyn Lederer, Sonia Ndeupen, and 29 more\ (2021).Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses. Immunity

Dorottya Laczkó, Michael J. Hogan, Sushila A. Toulmin, Philip D. Hicks, Katlyn Lederer, and 37 more\ (2020).A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice. Immunity

Devdoot Majumdar, W. Dowell, Sylvester Languon, Zachary D. Miller, Jake Dearborn\ (2023).Understanding Mechanisms of Adjuvancy in Muscle by mRNA/Lipid Nanoparticles. Journal of Immunology

Natalia Martinez, Haitao Yu, Joshua Iscaro, Hao Wang, Brendan P. Dyett, and 5 more\ (2025).Structure–Function Correlation of Clinically Inspired Lipid Nanoparticles for Lung and Spleen Targeted mRNA Delivery. Advanced Healthcare Materials

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Novel lipid nanoparticle provides potent SARS-CoV-2 mRNA vaccine at low dose with low local reactogenicity, high thermostability and limited systemic biodistribution

Suman Alishetty, Manuel J. Carrasco, M. Alameh, M. Paige, Hooda Said, L. Wright, Aarthi Narayanan, F. Alem, Keziah Hernandez, P. Gillevet, O. Soliman, Philip D. Hicks, Tomaz B. Manzoni, Paul Bates, A. Stephens-Shields, T. Cleveland, A. Grishaev, D. Weissman, M. Buschmann

2021·

9 citations

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LNP Composition

- Ionizable lipid type and concentration: C24, a novel multiprotic ionizable lipid, with a mole ratio of 48. - Helper lipids and ratios: DSPC (13), cholesterol (37). - PEG-lipid type and percentage: DMG-PEG2000, 2%. - Novel or modified lipid components: C24 has a trivalent head group and degradable primary esters. - Design rationale for each component: Enhanced endosomal protonation and controlled charge. - Particle size and polydispersity: 80 nm diameter. - Formulation buffer and pH conditions: PBS, pH 7.3-7.4.

mRNA Specifications

- Target antigen: Diproline-stabilized membrane-bound spike protein (S2P) immunogen from SARS-CoV-2 - Specific nucleoside modifications: Not mentioned - mRNA sequence length and structure: Not mentioned - Cap structure and poly-A tail details: Not mentioned - Codon optimization strategies: Not mentioned - mRNA-to-lipid ratios in formulation: Not mentioned

Cellular Uptake Mechanism

- Cell types: Not explicitly mentioned, but likely includes muscle cells and antigen-presenting cells (APCs) at the injection site. - Uptake pathways: Endocytosis implied by the context of endosomal protonation and release. - Endosomal escape mechanisms: Protonation of ionizable lipids in the endosome, followed by ion pairing with endosomal phospholipids to open the endosomal membrane. - Role of ionizable lipids: pH-dependent release through protonation in the endosome. - Timeline from uptake to protein expression: Not explicitly detailed. - Subcellular trafficking patterns: Not explicitly detailed. - Membrane interaction studies: Not explicitly detailed.

Delivery Efficiency

- Protein expression levels: C24 LNP shows 2-fold higher luciferase expression at the injection site compared to MC3. - Transfection efficiency percentages: Not explicitly mentioned, but implied by higher protein expression levels. - Duration of protein expression: Not explicitly mentioned. - Dose-response relationships: C24 LNP is effective at a lower dose (0.25 µg) compared to MC3. - Comparison with control formulations: C24 LNP is compared to MC3 LNP, showing improved delivery efficiency. - In vitro vs in vivo delivery efficiency: In vivo data from mouse models is provided. - Target tissue specificity: C24 LNP reduces off-target expression in the liver by 6-fold.

Structural Properties

- LNP morphology: Peripheral bilayer and internal electron dense amorphous core. - mRNA-lipid interaction patterns: Encapsulation efficiency; mRNA inaccessible to ribogreen. - Hydrogen bonding or ionic interactions: Not mentioned. - Membrane organization: Peripheral bilayer. - Mechanical properties: Not mentioned. - Surface charge and zeta potential: Higher pKa for C24 (6.77) than MC3 (6.55); larger increase in surface protonation for C24. - Cryo-TEM or AFM structural observations: Larger size for C24 LNP (80 nm vs. 64 nm for MC3); similar structure with peripheral bilayer and internal electron dense amorphous core.

Biodistribution Pattern

- Primary target tissues: Muscle (though not entirely contained within muscle tissue) - Distribution kinetics and timeline: Rapid increase in surface charge and net charge near neutral pH limits systemic biodistribution - Off-target expression levels: 6-fold reduction in liver off-target expression for C24 compared to MC3 - Route of administration effects: IM administration with deposition in facial plane between muscles - Factors controlling tissue targeting: Rapid increase in surface charge and net charge near neutral pH - Methods used to track biodistribution: IVIS - Clearance mechanisms and timeline: Not detailed in the paper

Inflammatory Effects

- Innate immune responses triggered by LNPs: Systemic adverse reactions and reactogenicity may be due to innate inflammatory responses to lipids or mRNA. - Inflammatory cytokine induction: Not mentioned. - Neutrophil and dendritic cell recruitment: Not mentioned. - Local vs systemic inflammation: C24 LNP shows reduced off-target expression in the liver and lower injection site inflammation compared to MC3. - Adjuvant effects of lipid components: The mechanism that significantly limits systemic biodistribution for the C24 LNP could be related to its rapid increase in surface charge and net charge near neutral pH. - TLR activation pathways: Not mentioned. - Injection site reactogenicity: Reduced for C24 compared to MC3. - Timeline of inflammatory responses: Mixed cell-type lymphoid structures observed 24 hours post-administration.

Stability Factors

- Storage temperature requirements: Stable at 4°C for at least 19 days; frozen storage recommended. - mRNA integrity maintenance: Stable for at least 19 days at 4°C; declines at higher temperatures. - LNP structure preservation: Not explicitly mentioned. - Bioactivity retention timeline: Stable for two weeks at 4°C; declines by 20-40% at room temperature. - Mechanisms of degradation: Phosphodiester transesterification; affected by temperature and LNP environment. - Formulation stability enhancers: Not explicitly mentioned. - Real-time vs accelerated stability data: Real-time data shows stability at 4°C; accelerated data indicates faster degradation at higher temperatures. - Impact on delivery efficiency: Not explicitly mentioned.

Concerns with current mRNA Lipid Nanoparticle (LNP) systems include dose-limiting reactogenicity, adverse events that may be partly due to systemic off target expression of the immunogen, and a very limited understanding of the mechanisms responsible for the frozen storage requirement. We applied a new rational design process to identify a novel multiprotic ionizable lipid, called C24, as the key component of the mRNA LNP delivery system. We show that the resulting C24 LNP has a multistage protonation behavior resulting in greater endosomal protonation and greater translation of an mRNA-encoded luciferase reporter after intramuscular (IM) administration compared to the standard reference MC3 LNP. Off-target expression in liver after IM administration was reduced 6 fold for the C24 LNP compared to MC3. Neutralizing titers in immunogenicity studies delivering a nucleoside-modified mRNA encoding for the diproline stabilized spike protein immunogen were 10 fold higher for the C24 LNP versus MC3, and protection against viral challenge in a SARS-CoV-2 mouse model occurred at a very low 0.25 µg prime/boost dose of the same immunogen in the C24 LNP. Injection site inflammation was notably reduced for C24 compared to MC3. In addition, we found the C24 LNP to be entirely stable in bioactivity and mRNA integrity when stored at 4 ºC for at least 19 days. Storage at higher temperatures reduced both bioactivity and mRNA integrity, but less so for C24 than MC3, and in a manner consistent with the phosphodiester transesterification reaction mechanism of mRNA cleavage. The higher potency, lower injection site inflammation, and higher stability of the C24 LNP present important advancements in the evolution mRNA vaccine delivery.

Introduction

There are 2 completed and 6 ongoing clinical trials for COVID-19 mRNA vaccines 1 . The BioNTech/Pfizer and Moderna products have received Emergency Use Authorization 2,3 and announced results from phase 3 clinical trials that reported efficacy greater than 94% for reduction of SARS-CoV-2 symptomatic infection after 2 doses of a nucleoside-modified mRNA encoding the spike protein delivered in a lipid nanoparticle (LNP) 4,5 . Recent interim data from a trial by CureVac contained disappointing results with only 47% protection 6 , possibly due to the use of non-nucleoside-modified mRNA, which has higher innate immunogenicity than nucleosidemodified mRNA 7 , and thereby limiting the dose to 12 µg in the CureVac trial versus 30 µg for BioNTech and 100 µg for Moderna. These latter two doses were maximum tolerated doses determined in phase 1 trials where higher doses (250 µg Moderna 8 , 100 µg for BioNTech 9 ) were discontinued due to frequent and severe injection site pain. Data from the clinical trials 10,11 as well as post-approval follow-up data 12,13 indicate the Moderna vaccine has a higher frequency of adverse events (ADEs) and reactogenicity than the BioNTech vaccine which could also be related to its higher dose (100 µg Moderna versus 30 µg BioNTech/Pfizer). Vaccine reactogenicity is thereby dose-related, generating a narrow successful dose range for mRNA vaccines that motivates the identification of more efficient mRNA delivery systems to achieve protection at lower doses.

In addition to injection site pain that is seen in nearly all subjects, systemic adverse reactions are seen in nearly half, and allergy-type reactions can be found in as many as 2% 12 with anaphylactoid reactions in around 1 in 100,000 14 . Cases of myocarditis 15 and thrombocytopenia 16 have also been identified as potentially mRNA vaccine-related. These ADEs and reactogenicity may be due to innate inflammatory responses to the lipids or mRNA, molecular mimicry between viral spike protein and endogenous proteins 15 , and off-target biodistribution and mRNA expression at unintended sites following intramuscular (IM) injection. For example, IM injection of LNPs containing an mRNA-encoded luciferase in mice showed high levels of liver expression at 6 hours (Fig S4 in 17 ). The current emergency-approved vaccines also generated off-target distribution and expression in liver and other organs in rodent preclinical models 18,19 . Our recent work showed a means to minimize off-target expression by using a less negatively charged mRNA-LNP that is more locally retained in the injected muscle and draining lymph nodes versus trafficking systemically as seen by expression in liver 20 . A final challenge for current mRNA vaccines is the need for frozen storage with relatively short stability times for non-frozen conditions, severely limiting global vaccination. Unfortunately, current literature does not provide any data to illuminate the mechanisms involved in loss of mRNA-LNP bioactivity 21,22 during storage although regulatory documentation suggests a loss of mRNA integrity is involved 18,19 . In order to widely implement mRNA-LNP vaccines for infectious diseases in the future, there is an urgent need to increase their potency and reduce dose, as well as to control biodistribution and increase understanding of the mechanisms determining stability during storage.

The design of the ionizable lipid in LNPs is considered the key aspect that determines potency or mRNA delivery efficiency, as well as degradability, toxicity, reactogenicity and the adjuvant properties of the LNP 23 . Early studies using siRNA containing LNPs showed that only 1-2% percent of the siRNA in endosomes is released from the LNP and from endosomes and loads into the RNA-induced silencing complex 24 . The current LNPs in COVID-19 vaccines may increase endosomal release several fold 25,26 but still release only a small fraction of mRNA for cytosolic translation. For this reason, the main design principle guiding improvements to ionizable lipids is to increase endosomal release that occurs due to protonation of the ionizable lipid in the endosome followed by ion pairing with a negatively charged endogenous endosomal phospholipid where this ion pair can open the endosomal membrane to release mRNA into the cytoplasm 27 . The endosomal protonation requirement is well established and is summarized as requiring the pKa of the LNP to be in the 6-7 range 28 . We recently showed that this LNP pKa is different from the ionizable lipid pKa that is in the 8-10 range 20 and this 2-3 point difference as mainly due to proton partitioning between the lipid phase and the aqueous media external to the LNP. This insight has now permitted us in the current study to systematically scan and screen theoretical structures of novel multiprotic head groups of ionizable lipids, since we are able to predict the pKa of the resulting LNP based on the structure of the ionizable lipid.

In addition to endosomal protonation, another principle of ionizable lipid design is the molecular shape hypothesis 29,30 , which states that the lipid tails should have a wider cross-section than the head group, thereby creating a cone-shaped ion pair with an endogenous phospholipid that is not compatible with a lipid bilayer and therefore destabilizes it for endosomal release. The evolution of ionizable lipids displays an increased level of lipid tail branching that augments a cone-shaped morphology. MC3, the standard reference ionizable lipid in the approved Onpattro silencing RNA product from Alnylam has a dilinoleic tail, the Moderna SM-102 ionizable lipid has 3 saturated alkyl branches and the Acuitas ALC 0315 in the BioNTech/Pfizer product has 4 saturated alkyl tails 23 . A third critical component of the ionizable lipid is the linker between the head group and tails that should be degradable in order to permit elimination in the body and limit accumulation 31 .

A favored linker in this respect is a primary ester since it has been shown to degrade quickly in vivo to minimize accumulation and allow repeat administration 31 . This rapid degradability was associated with reduced inflammation at the IM injection site and increased tolerability 26 .

We designed a new ionizable lipid for mRNA-LNP vaccines based on the above criteria. We theoretically screened a broad molecular design space of head groups that were not limited to monoprotic units used in current mRNA LNP vaccines. The incorporation of more than one ionizable nitrogen in the head group can provide an additional dimension to both enhance endosomal protonation and to control charge of the LNP that influences biodistribution. The chosen trivalent head group, 4-methyl-1-piperazinebutanamine, was linked via two degradable primary esters to octyldodecyl tails creating 4 saturated alkyl tails with non-symmetric 8 and 10 carbon lengths. The resulting compound, abbreviated C24, was used along with the standard 3 additional lipids to produce mRNA-LNPs containing a nucleoside-modified mRNA that encoded for either a luciferase reporter or a diproline-stabilized membrane-bound spike protein (S2P) immunogen from SARS-CoV-2 that is equivalent to the immunogen in the current emergency authorized mRNA vaccines. We comprehensively studied the physicochemical properties of the C24 ionizable lipid, the resulting C24 LNP, as well as immunogenicity, protection against lethal SARS-CoV-2 challenge, injection site inflammation and stability of C24 mRNA-LNPs and mRNA integrity during liquid storage. By direct comparison to the standard MC3 LNP, which was used in the first two phase 1 clinical trials of nucleoside-modified mRNA for influenza 32 , the C24 mRNA LNP appears significantly more potent with 10 fold higher neutralizing antibody titers than MC3, protecting against SARS-CoV-2 infection at a low 0.25 µg dose administered twice in mice and is less inflammatory at the injection site while entirely maintaining bioactivity beyond 2 weeks when stored in a liquid format at 4°C.

Trivalent head group of C24 ionizable lipid displays molecular and LNP ionization properties that augment protonation in the endosomal pH range

The initial screening of our ionizable lipid design space included more than 100 head groups, 10 linkers and 50 alkyl tails that combine for over 50,000 potential ionizable lipid candidates. We calculated aqueous phase pKas for ~500 candidates that sampled this design space and selected several head groups for synthesis with acrylate bearing alkyl tails using a synthetic procedure that involved only 2 reactions and one catalyst versus more than 5 reactions and 7 catalysts for the ionizable lipids in the current COVID-19 mRNA vaccines 26,33 . The simplicity of the reaction scheme resulted in much fewer purification steps, much shorter synthetic time, and over 80% yield producing an estimated 10 fold reduction in cost that could faciliate global vaccination campaigns.

More than 30 candidate ionizable lipids were initially synthesized and characterized physicochemically and for certain biological performance indicators resulting in the selection of C24 (Fig 1a ) for further investigation in the current study. C24 bears a trivalent 4-methyl-1piperazinebutanamine head group with theoretical aqueous phase pKas ranging from 4 to 8, two of which were close in predicted values (7.7 and 7.8). We synthesized a water soluble analogue (C24-WSA) and measured the pKa of each nitrogen using an established 1 H NMR method 20 confirming theoretical pKas to within 0. was assessed using the TNS dye-binding assay that measures surface charge and with zeta potential by electrophoretic mobility that measures net charge of the LNP, as we described recently 20 . The TNS dye-binding assay revealed a higher pKa for C24 (6.77) than MC3 (6.55) and a larger increase in surface protonation when pH drops from 7. Taken together, the above analyses relate molecular protonation events on monoprotic versus multiprotic head groups to their protonation in the condensed LNP environment and demonstrates an approximately 2 fold increase in protonation of the C24 LNP versus MC3 in the endosomal pH range.

Size characterization showed C24 LNPs to be slightly larger than MC3 (80 nm versus 64 nm diameter in Fig 2a ) and to have a slightly lower fraction of mRNA that is inaccessible to ribogreen dye-binding (68 % versus 79 % in Fig 2b ). The fraction of mRNA that is inaccessible to ribogreen is often called encapsulation efficiency, however it is now known that the accessible portion is not a free fraction of mRNA since it may not migrate on a gel-based assay 35 . We used our recently published molecular volume model 20 of the LNP to estimate the number of copies of the Firefly Luciferase (FLuc) encoding mRNA in each LNP finding 4.5 copies in the MC3 LNP and 6 for the C24 LNP (Fig 2c ), on average, due to its larger size. CyroTEM analyses revealed a larger size for the C24 LNP consistent with DLS measurements and both LNPs had a similar structure showing a peripheral bilayer 36 and an internal electron dense amorphous core (Fig 2d, e ).

MC3

In vivo expression of the mRNA FLuc encoding reporter after intramuscular (IM) administration of the LNPs in mice at a relatively high dose of 5µg mRNA showed 2 fold higher expression at the injection site for C24 at 4hrs and 24hrs (Figs 3a and 3b ). Systemic biodistribution and offtarget expression in liver was high for MC3 (Figs 3). In contrast, C24 at this high dose virtually eliminated systemic biodistribution with a 6 fold reduction of off-target expression in liver compared to MC3 (Fig 3C ), an important finding since systemic reactogenicity and adverse events associated with mRNA-LNP vaccines may be linked to systemic biodistribution and off-target expression in sites other than the injection site and draining lymph nodes. The high off-target expression seen here for MC3 is consistent with previous findings for MC3 17 and regulatory documentation 18,19 suggest it also occurs in rodent models for the current emergency authorized COVID-19 mRNA vaccines. The mechanism that significantly limits systemic biodistribution for the C24 LNP could be related to its rapid increase in surface charge and net charge near neutral pH (Fig 1d and 1e ), since we found previously that a less negatively charged LNP was more locally contained upon IM adminsitration 20 . IM administration of Fluc-encoding mRNA-LNPs at a lower 0.5µg dose that is more representative of vaccination doses in mice showed C24 to express 4 fold higher than MC3 at the injection site (Fig 3d and 3e ). Off target expression could not be detected by IVIS at this 10 fold lower dose due to low signal to noise at the liver site. Daily imaging of mice showed an initial burst of expression lasting 48 hours with a gradual decline to baseline over

5days (Fig 3f).

Immunogenicity towards mRNA-encoded SARS-CoV-2 spike protein shows higher binding titers and 10 fold higher pseudoneutralization titers for C24 LNP versus MC3 LNP against the original Wuhan strain and against two prominent variants LNPs were assembled with nucleoside-modified mRNA encoding for the diproline-stabilized membrane-bound spike protein immunogen (S2P) that is in the current emergency-authorized COVID-19 mRNA vaccines. We performed immunogenicity studies in Balb/c mice by IM administration of two immunizations with dose ranging from 0.1 µg to 1 µg with 3 weeks between prime and boost and serum analyses for binding antibodies to the receptor-binding domain of the spike protein, as well as neutralization assays to a SARS-CoV-2 pseudovirus. Optical density of the ELISA binding assay at transitional dilutions showed binding antibodies were significantly higher for the C24 LNP versus MC3 at all doses (Fig 4a and 4b ). Neutralization assays to a SARS-CoV-2 pseudovirus showed C24 LNPs with significant ~10 fold increases of neutralization titers versus MC3 at all doses after the boost (Fig 4C ). A very similar assay done in Balb/c mice with an identical mRNA-encoded immunogen (other mRNA structures differed) in the SM-102 LNP of Moderna revealed titers similar to those of MC3 (1,000 at 1 µg) 37 suggesting that the C24 LNP may also be more potent than the SM-102 LNP. The 1 µg dose delivered in the C24 LNP was also capable of inducing neutralization after a single dose where MC3 did not produce neutralization (Prime in Fig 4c ). Finally, we found that serum from animals vaccinated with the highest 1 µg dose were capable of neutralizing two variants of SARS-CoV-2 and that titers of C24 were higher than those of MC3 for the tested variants (Fig 4d ).

Lethal challenge in the K18-hACE2 mouse reveals complete protection for the C24

LNP at a low, 0.25 µg, prime/boost dose of nucleoside-modified S2P mRNA and complete elimination of lung infection at 0.5 µg dose Protection against lethal infection with SARS-CoV-2 after vaccination with mRNA-LNPs containing the S2P immunogen was investigated in the K18-hACE2 transgenic mouse bearing the human ACE2 receptor. C24 and MC3 LNPs containing the S2P immunogen were administered twice at mRNA dose levels ranging from 0.1 to 1 µg, each with a group of 5 animals, and then challenged with a lethal intranasal dose of the Italian strain (Isolate Italy-INMI1) of SARS-CoV-2. Two of the five animals in each group were sacrificed on day 5 to assess viral titers in the lung and the remaining 3 followed until euthanasia criteria were met. We found C24 mRNA LNPs completely protected mice at 0.25 µg with one of 3 animals at 0.1 µg dose also surviving versus MC3 where protection occurred at 0.5µg dose (Fig 5a ). Neutralization titers in a plaque assay showed C24 LNPs achieved titers at 0.25 µg that were equivalent to those of MC3 at 1 µg dose after both the prime and the boost (Fig 5b and c ). Lung viral titers examined 5 days after infection found C24 mRNA LNPs entirely blocked lung infection at 0.5µg dose and that MC3 did not block infection entirely even at the highest 1 µg dose. Taken together, these results indicate that C24 LNPs achieve protection against infection at a dose~4X lower than MC3. The 0.25 µg protective dose applied twice in our study is 60X lower than the single effective dose (15 µg) found in an LNP 38 containing a self-replicating mRNA for the spike protein and 10X lower than another single dose found with a different LNP 39 also containing a self-replicating mRNA. Although these latter studies with self-replicating mRNA are single dose, the required dose is much greater and at a level that would be expected to generate unacceptable reactogenicity in humans. The study that is most similar to ours is prime/boost nucleoside modified approach using the S2P immunogen in the SM-102 LNP of Moderna 37 where doses as high as 1 µg of the S2P immunogen in the SM-102 LNP were not capable of blocking lung infection, although they did reduce lung infection compared to PBS controls. The potency of the SM-102 LNP for blocking lung infection in mice therefore appears similar to that of MC3 in our study supporting with the similar potency we found for SM-102 and MC3 neutralization titers against a SARS-CoV-2 pseudovirus (both at ~1,000 Fig 4c ). Taken together these results suggest the C24 LNP system exceeds the potency of MC3 and of SM-102 LNP in mice models. Often, translation to non-human primates and humans is not predictable from mouse models 40 so that results in larger animal models and clinical studies are required to further assess C24 mRNA LNP vaccines.

Injection site inflammation for the C24 LNP is milder than for the MC3 LNP

Injection site inflammation for MC3 mRNA LNPs was visually evident 24 hrs post IM administration by macroscopic swelling and a high level of stiffness of the injected leg. Injection site inflammation of C24 mRNA LNPs injected at a 5 µg dose was macroscopically lower than the swelling of MC3 LNP injected sites. We therefore fixed and processed the injected legs for standard histological analyses (Paraffin and H&E staining). We found that the injected 50 µL depot of mRNA LNPs was not inside the muscle tissue but was rather deposited mainly in the facial plane between the medial and lateral gastrocnemius (IS in Fig 6a, d, g ) and was difficult to distinguish from adipose tissue at these sites. This non-intramuscular site for the LNP depot was due to the standard 3mm injection depth passing through the medial gastrocnemius that is ~2 mm thick. The volume of the medial gastrocnemius is only ~150 µL 41 so that the standard 50 µL LNP volume would not likely be contained inside the muscle even if the injection depth were reduced.

In comparing C24 histology to MC3 we observed greater levels of inflammation for MC3, for example in the synovium which was multicellular and thickened for MC3 versus a normal appearance for C24 (Fig 6 e, f versus b, c ). In addition to infiltration of leukocytes and vasculature at the injected site, we observed the presence of mixed cell-type lymphoid structures (Fig 6 a, g, i ) at 24 hrs post administration for both C24 and MC3 LNPs that were absent in control uninjected legs. These rapidly forming (in 24 hrs) lymphoid structures may play an important role in the immune response in mice and are being further characterized.

Storage of LNPs at 4°C reveal C24 LNPs are stable for at least 19 days while storage at higher temperatures induces loss of bioactivity and mRNA cleavage that is consistent with the phosphodiester transesterification reaction mechanism of mRNA cleavage

mRNA LNPs generally require frozen storage and can be stored at refrigerated temperatures for up to 30 days according to instructions for use of the emergency authorized LNPs. Recent reviews 21,22 have highlighted a nearly total lack of information on mechanisms contributing to instability during storage of mRNA LNPs although European regulatory documentation 18,19 states that instability is temperature-dependent and involves a loss of mRNA integrity as well as changes in LNP size and generation of impurities. We therefore stored C24 and MC3 FLuc mRNA LNPs for 2 weeks at one of 2 temperatures, 4°C or room temperature (RT≈22°C) in PBS and tested bioactivity by Luciferase expression in vitro. We found that bioactivity was stable (to within the high variability of this bioactivity assay performed on cells seeded on different days) for both C24 and MC3 LNPs for 2 weeks when stored at 4°C but declined by 20-40% when stored at room temperature (Fig 7a ). We did not find any change in LNP size or accessibility to Ribogreen for either storage temperature over 2 weeks (Fig 7b, c ). We calculated the loss of mRNA integrity of the 2,061 nucleotide long FLuc over time using a model derived from data characterizing the temperature-and pH-dependence of the base-catalyzed phosphodiester transesterification and cleavage of mRNA backbone (Eq e from 42 ). The model predicted half-lives of 2,300 days at 4°C and 125 days at 22°C (RT) and 11 days at 37°C, all at pH 7.4 (Fig 7d ). mRNA backbone cleavage is thereby expected to be exquisitely sensitive to temperature and these trends are qualitatively consistent with previous findings for free mRNA (Fig E2 from 43 and Fig 2 from 44 ). We then stored additional mRNA LNPs in PBS at 4°C and at 22°C (RT) as well as at 37°C to accelerate degradation and extracted mRNA in chloroform/methanol to analyze mRNA integrity on a microfluidic electrophoresis device. We found mRNA integrity to be maintained for at least 19 days when stored at 4°C while the higher temperatures could produce mRNA cleavage (Fig 7e ). mRNA integrity was estimated by the area under the curve (AUC) corresponding to the FLuc mRNA peak normalized to the day 0 value of C24 and MC3 LNPs taken together. The C24 LNP appeared to maintain higher levels of mRNA integrity than the MC3 LNP at the higher temperatures although sample numbers were too low to permit statistical analyses (Fig 7f ).

Increased mRNA cleavage at higher temperatures is consistent with the model of based-mediated phosphodiester transesterification reaction mechanism of mRNA cleavage 42 (Fig 7d ) but may exhibit a dependence on the LNP environment and on the specific structure of the ionizable lipid in the LNP. The LNP environment may for example accelerate mRNA degradation by the proton partitioning phenomena we found to be responsible for the difference in pKa of the ionizable lipid in aqueous media versus in the LNP. Namely, the higher proton solvation energy in the lipid environment will exclude protons and raise the pH in the LNP compared to the external aqueous phase and could thereby accelerate base-mediated mRNA cleavage as predicted by the pHdependence of the model of base-catalyzed phosphodiester transesterification and cleavage of the mRNA backbone 42 .

Conclusions

Through a rational design process that includes a theoretical ionization assessment of ionizable lipid candidates, followed by structural considerations that include high levels of branching alkyl tails and quickly degradable primary esters, we identified and synthesized a new ionizable lipid, C24, with a multiprotic trivalent head group. The ionization behavior of the C24 LNP was consistent with molecular ionization characteristics reflecting the multiple protonation stages of the multivalent head group, resulting in an overall doubling of protonation in the endosomal pH range compared to the MC3 reference mRNA LNP. Intramuscular administration in mouse models showed that mRNA translation was 4 fold higher for C24 versus MC3 and this translated to 10 fold higher neutralizing titers in immunogenicity studies and a significantly greater protective capacity in a SARS-CoV-2 challenge model with protection at a low 0.25 µg prime/boost dose in mice. Additional important observations for C24 were a 6 fold lower level of systemic biodistribution compared to MC3 and lower levels of inflammation at the injected site indicating the potential for lower reactogenicity and a wider therapeutic window if these results translate to larger animal models and to human studies. Finally, we found the C24 LNP to maintain bioactivity and mRNA integrity for at least 19 days. When stored at higher temperatures our data suggests base-mediated mRNA cleavage is likely responsible for loss of mRNA integrity and bioactivity in LNPs. The important improvements in potency, reactogenicity and storage properties of the C24 LNP motivate further preclinical studies for eventual use in human mRNA vaccine studies.

Synthesis of ionizable lipids and ionizable lipid analogues

Solvents were purchased from Sigma Aldrich, Combi blocks, Oakwood chemicals, Alfa Aesar, VWR and Thermofisher. Anhydrous methylene chloride (DCM), anhydrous tetrahydrofuran (THF) and anhydrous DMF were purchased from Sigma Aldrich. Reactions were monitored using Opt. KMnO4 glass-based Silica Plates, F254 thin layer chromatography plates (TLC) and column purification was done using silica gel chromatography (TLG-R10014BK-323) purchased from Silicycle. NMR spectra were recorded on a Bruker 400 MHz Spectrometer using CDCl3, D2O (Sigma Aldrich), as d-solvents and internal standards (δ 7.26 for 1 H NMR and δ 77.00 for 13 acrylate 46 (4) (3.13 mmol, 0.40 g), were added to an oven dried 5ml biotage microwave vial and solvent free conditions were maintained 46 . The sealed microwave vial was stirred for 2-3 days at 90°C. Reaction progress was monitored by TLC (chloroform/methanol 9:1 v/v, can be visualized with iodine stain, and Phospomolybdic Acid stain), until complete consumption of 1. The crude product was purified on a silica gel column eluted with chloroform containing 0-5% methanol.

Column fractions were analyzed by thin layer chromatography (TLC) and fractions containing pure product (Rf = 0.34) were concentrated, to obtain product as yellow oil ( 5). (0.30 g, 53% yield).

DCM, rt, 8h HCl

DLin-MC3-DMA water soluble analogue (8 above) was synthesized as per the previous literature reports 47,48 . A mixture of 4-(dimethylamino)butanoic acid hydrochloride salt (6) (59.7 mmol, 1.0 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC•HCl) (7.16 mmol, 0.63 g), 4-(dimethylamino)pyridine (DMAP) (1.49 mmol, 0.18 g), triethylamine (42.0 mmol, 4.25 g, 5.83 mL), and CH2Cl2 (100 mL) were placed in a 250-mL two-neck round bottom flask. The reaction was stirred for 20 minutes at room temperature and neopentyl alcohol (7) (7.16 mmol, 0.78 mL) was added dropwise and the reaction stirred overnight. Reaction progress was monitored by TLC (chloroform/methanol 9:1 v/v, can be visualized with iodine stain) until complete consumption of 6 and the solvent CH2Cl2 was removed by rotary evaporation. The mixture was dissolved in 100 mL of CH2Cl2 and washed with 150 mL of water and 150 mL of saturated NaHCO3 solution. The organic phase was dried over magnesium sulphate and evaporated. The crude product was purified on a silica gel column eluted with chloroform containing 0-1% methanol. Column fractions were analyzed by thin layer chromatography (TLC) and fractions containing pure product (Rf = 0.4) were concentrated, to obtain product as yellow oil (8) 1 .

NMR measurement of pKa of water-soluble ionizable lipid analogues

The pH-dependence of proton NMR chemical shifts was used to measure the pKa's of the ionizable lipid water-soluble analogues (WSA) following published methods 5,6 . Chemical shifts of piperazine, imidazole, 2-chloroacetic acid and acetic acid were used as internal pH indicators.

Solutions for MC3-WSA were prepared with 100 mM KCl, 2 mM piperazine, 2 mM imidazole, and 5 mM water soluble ionizable lipid analogue in 95% H2O-5% D2O. Solutions for the terminal(N1) and aza amines(N2) at the β-position of carboxylic esters of C24-WSA were prepared with 100 mM KCl, 2 mM piperazine, 2 mM imidazole, 0.2 mM DSS, and 5 mM water soluble ionizable lipid analogue in 95% H2O-5% D2O. Solutions for the internal amine (N3) of C24-WSA were prepared with 100 mM KCl, 2 mM chloroacetic acid, 2 mM acetic acid, 0.2 mM DSS, and 5 mM water soluble ionizable lipid analogue in 95% H2O-5% D2O. Solutions were split into two equal volumes and one titrated to a lower pH using 0.1 M HCl and the other to an upper pH using 0.1 M NaOH where the lower and upper pH bracketed the desired range of pH (Supplementary Figure 2 ). Intermediate pH values were obtained by mixing different proportions of these two solutions. NMR measurements were performed on a Bruker 400 MHz spectrometer where 1 H spectra were acquired at each of ~24 pH values ranging from the lower to upper pH.

Peaks for MC3 were calibrated to D2O (400 MHz, D2O, δ = 4.79 ppm as standard) and the peaks for C24 were calibrated to DSS (400 MHz, DSS, δ = 0.00 ppm as standard). Chemical shifts from piperazine, imidazole, 2-chloroacetic acid, and acetic acid were then used to calculate the pH of each solution according to published methods 5,6 and the chemical shifts of protons adjacent to each nitrogen in the ionizable lipid water sluble analogue headgroup were fit to the Henderson-Hasselbalch equation to provide the pKa of each Nitrogen. Spectra are in Supplementary Figure 2 .

Theoretical Calculation of pKa

Experimentally determined pKa values from NMR, Zeta Potential and TNS assays were compared against theoretically calculated values using Advanced Chemistry Development, Inc. (ACD/Labs) software. The ACD/pKa database algorithmically estimates pKa values of whole molecules in an aqueous environment based on their constituent fragments using two approaches. The Classic algorithm employs a database of Hammett-type equations parameterized to cover most ionizable functional groups, each characterized by several equations involving variations of sigma constants.

The Galas algorithm estimates pKa microconstants for all possible ionization centers in a hypothetical uncharged state based on the surroundings of the reaction center and neighboring ionization centers to produce microconstants from which pKa macroconstants are obtained.

Classic algorithm calculations were used in this study.

Preparation of mRNA Lipid Nanoparticles

mRNA-loaded LNPs were formulated by preparing lipids in ethanol using % mole ratios for ionizable-lipid/DSPC/Cholesterol/DMG-PEG2000 of 50/10/38.5/1.5 for MC3 and 48/13/37/2 for C24 and preparing mRNA in aqueous buffers. These two solutions were mixed in a Spark NanoAssmblr™ (Precision NanoSystems) at volumes and concentrations to achieve a molar ratio of ionizable lipid to phosphate on the mRNA backbone of 4 and ejected into PBS pH 7.4. The MC3 LNP was prepared using standard procedures described previously 49 while C24 LNP assembly optimized some parameters for this specific formulation. The resulting mixtures were diluted 1:1 into PBS pH 7.4 and dialyzed against PBS to reach pH 7.3-7.4 after 6 buffer exchanges over 6 hours using a Slide-A-Lyzer MINI Dialysis Device (MWCO, 10 kDa).

Assay of mRNA Inaccessibility to Ribogreen

Tris(10 mM, pH=7.5)/EDTA(1 mM) (TE) and Triton/TE (2% v/v Triton in TE Buffer) were added in duplicates to a black microplate. Total mRNA in the LNP was diluted to ~4 ng/µL in TE and added to each TE and TE/Triton well in a 1:1 volume ratio. Two standard curves were included in the Ribogreen Assay, one containing mRNA and TE, and the other containing mRNA and Triton/TE. Each standard curve was used to calculate the mRNA accessibility to ribogreen in each respective buffer. This approach using two standard curves is required, in comparison to a single standard curve in Triton/TE, which can overestimate inaccessibility by 5-10%, since Ribogreen

TNS Assay

LNP pKa was determined using the TNS assay. The TNS reagent was prepared as a 300 μM stock solution in DMSO. Following Sabnis et al 2018 25 , LNPs were diluted to 24 μM ionizable lipid, TNS to 6.

to provide the pKa, n and low pH and high pH zeta potential limits Ψ and Ψ , respectively. The extended model is used here, since the LNP pKa is ionization-state-dependent in a way similar to a polyelectrolyte 34,51 . TNS data did not require the extended model since TNS dye binding only detects LNP surface charge. The isoelectric pI was the pH found by interpolating zeta potential to zero.

Cryoelectron miscroscopy

Grids for electron microscopy were plunge-frozen using a Vitrobot Mark IV system. The chamber was set to 25 °C and 100% humidity. LNPs (3 µL) were applied to grids (ultrathin carbon film on lacey carbon support, Ted Pella #01824G), incubated for 1 minute, and blotted twice for 3 s each time at a blot force of 25 before plunging into liquid ethane. Grids were imaged on a Talos Arctica system (Thermo Fisher Scientific) at 200 kV with a Falcon 3EC detector, using EPU for data collection. The nominal magnification was 45,000x, with a calibrated pixel size of 0.223 nm.

Images were collected in integrating mode using 5 s exposures, with a total dose of 60 e/Å2, and 66 fractions which were motion-corrected using Motioncor2 52 .

In vivo live animal imaging for luciferase expression following intramuscular administration of C24 and MC3 LNPs containing FLuc mRNA

The investigators adhered to the ''Guide for the Care and Use of Laboratory Animals'' by the in 50µl injected into the medial gastrocnemius muscle The two injections were spaced 3 weeks apart and blood was collected through the retro-orbital route one day prior to the first injection (Pre-bleed), prior to the second injection (Prime) and 2 weeks after the second injection (Boost).

Serum was separated from blood following an incubation period of 30 minutes at room temperature, and samples were centrifuged at 10,000 g for 5 minutes in a non-refrigerated Eppendorf 5424 centrifuge (Eppendorf, Enfield, CT, USA). Separated serum was stored at -20°C until used. Total antibody titers were determined using an Endpoint Enzyme Linked ImmunoSorbent Assay (ELISA). Briefly, High Bind Stripwell™ Corning 96 Well Clear

Polystyrene Microplates were coated overnight with 1μg/ml purified SARS-CoV-2 RBD (cat# Z03501), washed once with wash buffer (0.05% Tween-20 in PBS), and blocked for two hours at room temperature using 2% w/v BSA in PBS. Plates were then washed three times, and mouse sera was added at 1:27,000 (prime sera) and 1:54,000 (boost sera)and in the blocking solution and incubated for 2 hours at room temperature, washed three times before incubation with (HRP)conjugated anti-mouse secondary antibody in blocking buffer (1:10 000) for 1.5 hours. After incubation, plates were washed three times before the addition of 100 µl per well of KPL TMB substrate for 8 minutes. The reaction was stopped using 2N sulfuric acid, and the absorbance measured at 450nm using a SpectraMax™ 190 microplate reader. To determine neutralization potential, a VSVΔG-RFP pseudotyped virus (50-200 focus forming units/well) was incubated with 2-fold serially diluted serum samples and incubated for 1 h at 37°C prior to addition of the virusantibody mixture to VeroE6 TMPRSS2 cells. 20 hours post infection, the cells were washed and fixed with 4% paraformaldehyde before visualization on an S6 FluoroSpot Analyzer (CTL, Shaker Heights OH). Individual infected foci were enumerated and the values compared to control wells without antibody. The Focus Reduction Neutralization Titer 50% (FRNT50) was measured as the greatest serum dilution at which focus count was reduced by at least 50% relative to control cells that were infected with pseudotype virus in the absence of mouse serum. FRNT50 titers for each sample were measured in two technical replicates performed on separate days. were challenged 2 weeks after the second immunization with SARS-CoV-2 by intranasal administration at dose of 5X10 5 pfu and followed until death or euthanasia criteria were met.

Protection against SARS-CoV

Weight and temperature were recorded daily. Two of the 5 mice in each group were sacrificed on day 5 post-challenge to assess lung viral titers by plaque assay. Lung tissues were homogenized and spun down. The supernatant recovered for assessment of viral load by plaque assay. The remaining three mice were monitored daily for signs of morbidity and mortality. Weight and temperature reading were also recorded daily for the surviving mice until the end of the study.

For PRNT50 assays, mouse sera were diluted 1:10 in DMEM (supplemented with 5% FBE, 1%

L-glutamate and 20 U/mL penicillin, and 20 μg/mL streptomycin). Serial two-fold dilutions were then prepared from the 1:10 dilution and mixed with 100 pfu of SARS-CoV-2 virus and incubated at 37˚C for 1h. The sera/virus mixture was then overlayed onto a confluent layer of Vero cells in a 12-well plate format and incubated for 1h at 37˚C incubator with 5% CO2. The inoculated wells were then overlaid with a 1:1 mixture of Eagle's Minimum Essential Medium (without phenol red, supplemented with 5% FBE, nonessential amino acids, 1 mM sodium pyruvate (VWR, 45000-710, Dixon, CA, USA), 2 mM L-glutamine, 20 U/mL penicillin, and 20 μg/mL streptomycin) and 0.6% agarose (ThermoFisher, 16500100) and incubated for 48h at 37˚C with 5% CO2. Cells were then fixed with 10% formaldehyde (FisherSci, F79p-4) for 1h. Media was removed, cells were washed with diH2O and stained with 1% crystal violet (FisherSci, C581-25) and 20% ethanol solution (FisherSci, BP2818-4). Plaques were counted and plotted as pfu/dilution.

For plaque assays, the Vero cells (WT Veros, ATCC) were plated in 12-well plates at a density of 2X10 5 cells per well and incubated overnight. Supernatants from tissue homogenates were serially diluted to 10 -6 and overlaid on cells for 1h (37˚C, 5% CO2O). Cells were then overlaid with Eagle's Minimum Essential Medium (without phenol red, supplemented with 5% FBE, nonessential amino acids, 1 mM sodium pyruvate (VWR, 45000-710, Dixon, CA, USA), 2 mM L-glutamine, 20 U/mL penicillin, and 20 μg/mL streptomycin) with 0.6% agarose (ThermoFisher, 16500100) and incubated for 48h. Cells were then fixed with 10% formaldehyde (FisherSci, F79p-4) for 1h.

Medium was removed, cells were washed with diH2O and stained with 1% crystal violet (FisheSci, C581-25) and 20% ethanol solution (FisherSci, BP2818-4). Plaques were manually counted and datasets represent plaque forming units per milliliter (PFU/mL).

Storage/Stability Studies of mRNA LNPs

C24 and MC3 LNPs containing FLuc mRNA were stored in PBS at 4°C or at room temperature (RT≈22°C). They were assayed on days 0, 2, 4, 7, 14 of storage for size by DLS as described above, mRNA encapsulation as described above and for bioactivity via transfection into HEK293 cells and assessment of luciferase expression. For bioactivity, HEK293 cells were seeded in white 96-well plates at a density of 12x10 3 cells per well in 100 µL EMEM medium (10% FBS) the day before transfection and incubated at 37°C 5% CO2. FLuc mRNA-loaded LNPs were diluted so that 8µL contained 25 ng mRNA FLuc and HEK293 cells were transfected at this dose using triplicates 24 hours after seeding. After a further 24 hours for transfection, 100 µL of One-Glo substrate was added directly to the wells to detect luciferase expression based on luminescence in Cytation 5 luminometer plate reader. Additional C24 and MC3 LNPs containing FLuc mRNA were stored for 19 days in PBS at 4°C, RT and 37°C. FLuc mRNA was extracted from LNPs using chloroform/methanol and analyzed for mRNA integrity via microfluidic electrophoresis on a 2100 Bioanalyser (Agilent) following manufacturer's instructions. mRNA concentration was quantified by Nanodrop, then diluted to 1 ng/ul to load 1ng mRNA of each sample in the Bioanalyser. The % mRNA integrity was calculated by integrating the area under the curve (AUC) of the main mRNA FLuc peak and normalizing to the average day 0 value for C24 and MC3 LNPs taken together.

Statistics and Reproducibility

The Linear Least Squares Multivariate Model in the JMP Pro 15.1.0 software was used to perform comparisons between groups in Figures 1 and 3 . For In Vivo IVIS imaging data the 4 and 20/24 hour time points were considered repeated measures. Log10 transformations were applied to FRNT50 and viral titers. Linear regression models were used to compare OD and transformed FRNT50 and viral titers by LNP adjusted for dose in Figures 4a-4c , 5d, and 5h. Unadjusted linear regression with LNP as a predictor was used to compare neutralization of pseudovirus in Figure 4d . The proportion of mice dead after lethal challenge of SARS-CoV-2 was compared by LNP using Fishers Exact Test, excluding PBS (Figure 5a ). Linear mixed effects models with dose, dilution, and LNP as fixed effects and a random intercept for mouse evaluated the effect of LNP on the percent of virus detected (Figures 5bcfg). b) The ribogreen assay showed slightly lower levels of mRNA inaccessible to ribogreen for C24 versus MC3. We performed this assay using 2 standard curves, with and without triton. When only one standard curve with triton is used (such as in most publications to date) the mRNA inaccessible to ribogreen increases by ~8% shown in the yellow hatched portions. This assay is often interpreted as % encapsulation efficiency versus % mRNA inaccessible to ribogreen shown here. The latter is more accurate since it has been shown that dye accessibility of mRNA does not indicate that the mRNA is free and unencapsulated 35 . It may however reflect a different packing and internal structure of the LNP. c) mRNA copy number was calculated using a previously published molecular volume model 20 ELISA optical density at the transitional dilution of 54,000 for S2P binding antibody assays for higher doses 0.5 µg and 1.0 µg of mRNA-encoded S2P immunogen for serum collected 2 weeks after the boost. A Linear Regression Model analyses including LNP and dose as predictors showed that C24 binding antibody OD were significantly higher than MC3 (p=0.01). Mean +/-SEM. c) C24 LNPs generated a detectable FRNT50 titer against VSV∆G-RFP SARS-CoV-2 pseudovirus 3 weeks after a single Prime injection of a 1µg dose while MC3 LNPs did not (left Prime panel).

Two weeks after the Boost (right Boost panel), C24 LNPs at 0.25 to 1µg dose revealed FRNT50 titers that were ~10 fold higher than MC3 LNPs at the same doses. A Linear Regression Model analysis of log-transformed FRNT50 titers including dose and LNP as predictors showed that C24 FRNT50 titers were significantly higher than MC3 for the Prime (p=0.00002) and for the Boost (p=0.011). Mean +/-SEM. d) Neutralization of pseudovirus variants representing the variants D614G and ZA suggested FRNT50 titers were higher for C24 than the MC3 LNP, although statistical analyses did not find significant differences. Mean +/-SEM.

Data availability

The source data for the graphs and charts in the figures is available as Supplementary data Files.

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

Disclaimer

Certain commercial equipment, instruments, materials, suppliers, or software are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology (NIST), nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. half-lives at pH 7.4 of 2,300 days at 4°C, 125 days at RT and 11 days at 37°C. e) mRNA was extracted from LNPs using chloroform/methanol just after being produced on day 0 as well as after 19 days of storage in PBS at 4°C, RT and 37°C and analyzed by microfluidic electrophoresis. No detectable degradation was found after 19 days at 4°C (compared to day 0) while storage at higher temperatures (RT and 37°C) produced increasing amounts of mRNA cleavage that was qualitatively consistent with higher degradation at higher temperature predicted by the model in (d). The least degraded example of the 3 LNPs measured for each condition is shown in (e). The peak below 20s is a standard while the peak near 40s above the main mRNA Fluc peak is the product of an untemplated extension that is not amenable to elimination using the cellulose method used to purify the mRNA 45 . mRNA integrity was estimated by the area under the curve (AUC) corresponding to the FLuc mRNA peak and was normalized to the average day 0 value for C24 and MC3 LNPs taken together. f) mRNA integrity was stable for at least 19 days at 4°C and declined at higher temperatures but less so for C24 than for MC3. N=3 or for some N=2 due to technical irregularities in the trace of some samples that precluded quantification.

Supplementary Information

Supplementary Figure 1 . 1 H NMR, C13 and DEPT NMR spectra for synthesized compounds.

Acknowledgements

Acknowledgements M.D.B. acknowledges financial support from the Commonwealth Research Commercialization Fund (CRCF) Center for Innovative Technology (CIT) Award ER17-002-LS.M.P. acknowledges supported by resources provided by the Office of Research Computing at George Mason University (URL: https://orc.gmu.edu) and by grants from the National Science Foundation (Awards Number 1625039 and 2018631).

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