Injection site-retained lipid nanoparticles for targeted intramuscular delivery of mRNA RSV prefusion-F vaccine

Abstract

mRNA therapeutics, particularly mRNA vaccines, hold significant promise for a wide range of medical applications. Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicles for mRNA, but issues such as off-target effects and liver accumulation hinder their broader clinical adoption. In this study, we designed and synthesized a library of 26 novel ionizable lipids to screen for better delivery efficiency and tissue specificity. After formulating into LNPs, these ionizable lipids exhibited favorable physicochemical properties. In vitro transfection and cytotoxicity assays revealed that LNPs formulated with YK-201, YK-202, and YK-209 showed superior transfection efficiency and low cytotoxicity. In a mouse model, intramuscular injection of Fluc mRNA-LNPs resulted in sustained and localized protein expression at the injection site. When applied to prepare RSV preF-mRNA vaccines, these novel LNPs elicited robust humoral immune responses and reduced lung damage, outperforming the clinically used SM-102. The safety of the LNP formulations was subsequently demonstrated in a mouse model. Collectively, these findings highlight the potential of these novel ionizable lipids as effective injection site-retained mRNA vaccine delivery vehicles.

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Introduction

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus that primarily affects the lungs and respiratory tract.¹ It can cause severe symptoms, particularly in young children and older adults, leading to complications that result in up to 100 000 deaths annually worldwide.^2,3 The development of an RSV vaccine has been hindered by the failure of the formalin-inactivated RSV vaccine in the 1960s, which led to the observation of vaccine-associated enhanced respiratory disease (VAERD) and resulted in two fatalities.^4,5 The first approved RSV vaccine was introduced in 2023, and as of now, three RSV vaccines have been authorized, including an mRNA-based vaccine, mRESVIA, developed by Moderna.⁶ More recently, the FDA paused ongoing clinical trials of RSV vaccines for children following the detection of more severe lower respiratory tract infections (LRTI) in two participants after receiving Moderna's RSV mRNA vaccine. While the exact cause remains unclear, this event underscores the urgent need to develop RSV vaccines with enhanced safety profiles.

mRNA therapeutics represent a revolutionary approach offering innovative solutions for various clinical applications, including vaccines,^7,8 protein replacement therapies,^9,10 immunotherapy,^11,12 and gene editing.^13,14 Delivery methods are the fundamental technology for mRNA therapies, as RNA molecules are large and negatively charged, which limits their ability to pass through the phospholipid-based cell membrane.¹⁵ Additionally, naked RNA molecules are unstable and rapidly degraded by nucleases in a biological environment.¹⁶ Various methods have been developed for mRNA delivery,¹⁷ among which lipid nanoparticles (LNPs) are the only mRNA delivery vehicles approved by FDA, which have proved their effectiveness and safety in two SARS-CoV-2 mRNA vaccines developed by Pfizer/BioNTech and Moderna.^18,19

Typical LNP formulations consist of four components: ionizable lipids, helper lipids, cholesterol, and polyethylene glycol (PEG)-lipids.²⁰ Among these, ionizable lipids are considered to be the most important.²¹ Ionizable lipids typically contain a tertiary amine head group and several hydrophobic tails.²² The polar head group allows the LNP to remain neutrally charged under physiological conditions, minimizing cellular toxicity, while becoming positively charged upon entering the endosome to facilitate endosomal escape.²³ Due to their critical role, extensive efforts have been made to optimize the structure of ionizable lipids to improve delivery efficiency and enhance clinical outcomes.²⁴

Despite promising progress, LNPs still face several challenges, particularly safety concerns. LNPs are known be absorbed by the liver via the ApoE-mediated pathway,²⁵ leading to off-target effects and reduced therapeutic efficacy.^26,27 In vaccine applications, where intramuscular injection is commonly used, the ideal mechanism of action is to target local lymph nodes to induce humoral and cellular immunity.²⁸ Therefore, off-target effects in other organs should be minimized.²⁹ Previous studies have shown that optimizing ionizable lipids can enable muscle-selective delivery.^21,30,31 However, there are still limited muscle-targeting LNPs available, which constrains their potential for widespread clinical use.

In this work, we designed and synthesized a library of 26 novel ionizable lipids to screen for compounds with better delivery efficacy and tissue selectivity. When formulated into lipid nanoparticles with other components, these novel ionizable lipids exhibited favorable particle size and polydispersity index. In vitro transfection and cytotoxicity assay revealed that LNPs formulated with YK-201, YK-202, and YK-209 achieved 1.4- to 2.6-fold higher protein expression compared to YK-009, which was previously developed for the delivery of the COVID-19 Omicron vaccine.³² In a mouse model, intramuscular injection of Fluc-mRNA LNPs resulted in sustained protein expression for up to 48 hours. Organ tropism analysis confirmed the targeted retention of protein expression at the injection site for all three LNP formulations. These formulations were then applied for the delivery of an RSV preF-mRNA vaccine, where the RSV preF-mRNA-LNPs induced robust humoral immune responses and reduced lung damage in the mouse model, outperforming the clinically used SM-102. Additionally, the three LNP formulations demonstrated favorable safety profiles in vivo. Overall, these novel ionizable lipids represent promising candidates for injection site-retained mRNA delivery and hold significant potential for clinical applications, including RSV mRNA vaccines.

Results

Design and synthesis of novel ionizable lipids

Previous studies have shown that efficient mRNA delivery using ionizable lipids requires specific structural features, including one or more tertiary amines in the head group³³ and hydroxyl groups that promote closer lipid–lipid interactions and minimize hydration layer formation.³⁴ These characteristics have recently been identified as crucial for effective intramuscular mRNA delivery.²¹ Building on these findings, we designed and synthesized a series of novel ionizable lipids (Fig. 1a). These new lipids incorporate at least one additional tertiary amine and, in some cases, a hydroxyl group in the head group (Fig. 1b). The resulting library consists of 26 structurally distinct ionizable lipids, formed by combining 8 distinct head groups and 10 lipid tails (Fig. 1b, c and Fig. S1, ESI†). Detailed synthetic routes for these compounds are provided in the ESI.†

Fig. 1 Design and synthesis of novel ionizable lipids. (a) Representative chemical structure of the ionizable lipids used in this study. (b) Head group building blocks used in the construction of the ionizable lipid library. (c) Lipid tail building blocks used in the construction of the ionizable lipid library.

Physicochemical characterization and in vitro screening of LNP formulations

To formulate the LNPs, DSPC and DMG-PEG2000 were selected as the helper lipid and polymer lipid, respectively, based on their high delivery efficiency demonstrated in previous studies when combined with YK-009.³² LNPs were then formulated by encapsulating firefly luciferase (Fluc) mRNA using microfluidic mixing (Fig. 2a). The lipid molar ratio was set as follows: ionizable lipid : DSPC : cholesterol : DMG-PEG2000 = 45 : 10 : 43.5 : 1.5, which was determined through screening of four candidate ratios using YK-201 as the model ionizable lipid (Fig. S2a, ESI†). The resulting LNPs were characterized via dynamic light scattering (DLS), which revealed a particle size of approximately 70–100 nm and a monodisperse distribution with a polydispersity index (PDI) of 0.05–0.13 (Fig. 2b).

Fig. 2 Preparation and in vitro characterization of LNPs. (a) Overview of LNP formulation and preparation process. (b) Particle size and polydispersity index (PDI) of different LNP formulations. (c) Relative luminescence intensity in HEK293T cells 24 h after incubation with LNPs encapsulating Fluc mRNA (n = 5). Data were normalized to the luminescence intensity of YK-009. (d) Cell viability of HEK293T cells post-LNP treatment, assessed via a CCK-8 assay (n = 5). (e) Chemical structures of YK-201, YK-202, and YK-209, which were used for subsequent evaluation. Data are presented as mean ± SEM.

The formulated LNPs were evaluated for transfection efficiency in an HEK293T cellular model, with luminescence intensity normalized to YK-009. Seven formulations showed higher luminescence signals than YK-009, with YK-201 exhibiting the highest signal, showing a 2.6-fold increase compared to YK-009 (Fig. 2c). YK-202 and YK-209 followed, with increases of 1.8- and 1.4-fold, respectively (Fig. 2c). Notably, ionizable lipids lacking hydroxyl groups in their head groups, such as YK-215 to YK-226, displayed significantly lower protein expression, consistent with previous studies²¹ (Fig. 2c). Cytotoxicity of these formulations was also assessed, and YK-201, YK-202, and YK-209 demonstrated comparable cytotoxicity to YK-009 (Fig. 2d). To optimize further for in vivo use, we screened the molar ratio of ionizable lipids for YK-202 and YK-209, finding that the 45 : 10 : 43.5 : 1.5 ratio provided the best efficiency for both formulations, consistent with YK-201 (Fig. S2b, ESI†). Additionally, the transfection efficiency of YK-201, YK-202, and YK-209 was confirmed by outperforming SM-102, a clinically validated benchmark (Fig. S3, ESI†). Based on these results, we proceeded to explore the use of YK-201, YK-202, and YK-209 for in vivo applications (Fig. 2e).

Injection site-retained mRNA expression of YK-201, YK-202, and YK-209

The in vivo delivery efficiency of these novel LNP formulations was evaluated in a BALB/c mouse model. LNPs encapsulating Fluc mRNA were intramuscularly injected into the lateral thigh of mice (Fig. 3a). At various time points, a fluorescence imaging substrate was administered intraperitoneally. Whole-body imaging revealed that bioluminescence mainly occurred at the injection site for YK-201, YK-202, and YK-209 LNP formulations throughout the experiment, while SM-102-treated mice exhibited noticeable protein expression in the liver area (Fig. 3b). Time-dependent analysis of the injection site bioluminescence showed persistent Fluc expression from 6 to 48 hours, with YK-201 and YK-202 exhibiting slightly higher expression levels than YK-209 and SM-102 (Fig. 3c).

Fig. 3 YK-201, YK-202, and YK-209 exhibit sustained mRNA expression at the injection site. (a) Schematic of study design. BALB/c mice were injected intramuscularly (i.m.) with Fluc-mRNA-LNPs, and luminescence intensity at the liver and injection site was measured. (b) and (c) Representative IVIS images (b) and quantification of injection-site bioluminescence (c) at various time points post-injection of different Fluc-mRNA LNPs (0.25 mg kg⁻¹). (d) and (e) IVIS images (d) and quantification of luminescence intensity (e) in the whole body, injection site, and liver 6 h post i.m. injection of Fluc-mRNA-LNPs (0.25 mg kg⁻¹).

Next, we assessed the tissue tropism of these LNP formulations by quantifying the signal of liver area and injection site area. All three novel LNPs predominantly delivered mRNA to the injection site (Fig. 3d and e), with less than 10% of the mRNA expression detected in the liver compared to the injection site. In contrast, the clinically used SM-102 delivered a significant amount of mRNA to the liver, with only 27% of the protein expression found at the injection site (Fig. 3d and e). These findings indicate that the novel LNP formulations are highly efficient in vivo, with mRNA expression predominantly retained at the injection site. Additionally, the protein expression in inguinal lymph nodes were also measured and we found that while SM-102 LNPs produced a measurable luminescence signal in the lymph nodes, none of the three novel formulations (YK-201, YK-202, and YK-209) elicited any detectable signal (Fig. S4, ESI†). These findings are in line with previous reports on muscle-targeting LNPs, which similarly observed minimal lymph node expression.³⁰

RSV preF-mRNA-LNP vaccines activate strong immune responses

The promising site-specific mRNA expression of these novel LNP formulations prompted us to explore their potential application in RSV vaccine development. For this purpose, the RSV prefusion glycoprotein (preF) was chosen as the antigen. The preF protein is the primary antigenic target for RSV vaccine development due to its role in mediating viral entry and its genetic conservation.^35,36 It has been selected for all three FDA-approved RSV vaccines and was successfully used in the development of the first RSV mRNA vaccine, making it an ideal candidate for further RSV vaccine development.⁶

To this end, we prepared RSF preF-mRNA and combined it with SM-102, YK-201, YK-202, YK-209, and other components to form LNPs. BALB/c mice were immunized intramuscularly with LNP encapsulated mRNA encoding the RSV preF protein at Weeks 0 and 3, and blood samples were collected at Week 4 to measure antibody titers (Fig. 4a). After that, the mice were intranasally challenged with RSV A2 on day 43 after the second immunization, and lung samples were collected on day 47 to evaluate viral titers and pathology (Fig. 4a). We found that vaccination with SM-102, YK-201, YK-202, and YK-209 successfully induced anti-RSV preF antibodies in the sera (Fig. 4b). However, YK-202 and YK-209 elicited antibody levels that were 1.2-fold and 1.5-fold higher than SM-102, respectively (Fig. 4b). The 50% neutralization titer (NT50) was determined using the half-maximal inhibitory concentration of plasma samples from serial dilutions. We tested two different RSV strains, A2 and B18537, and found that NT50 values for SM-102, YK-201, and YK-202 were similar (Fig. 4c and d). In contrast, YK-209 demonstrated a 2.0-fold higher NT50 for A2 and a 2.7-fold higher NT50 for B18537 (Fig. 4c and d), indicating superior protective potential.

Fig. 4 Immunogenicity of different preF-mRNA-LNPs in BALB/c mice. (a) Schematic of vaccination regimen. Mice received intramuscular injections at weeks 0 and 3. Serum was collected at week 4, followed by RSV virus challenge on day 43. Mice were euthanized on day 47 for tissue harvesting. (b) RSV preF-specific total lgG titers in the serum were measured by ELISA on day 47 (n = 5). Control refers to DPBS-treatment. (c) and (d) Neutralizing antibody titers (NT50) in serum against RSV/A2 (c) and RSV/B18537 (d) on day 47 (n = 5). (e) Viral titers in the lung on day 47. (f) Body weight change of mice throughout the vaccination (n = 5). (g) Pathology scores for different LNP formulations (n = 5). Statistics were assessed by one-way ANOVA with Kruskal–Wallis multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significant difference. Data represents the mean ± SEM.

Throughout the vaccination process, no significant body weight loss was observed in any of the groups (Fig. 4e). Viral titers were only detectable in the control group, indicating effective viral clearance in the vaccinated groups (Fig. 4f). Next, lung pathology scores were assessed based on perivascular, peribronchial, alveolar, and interstitial infiltrations. Mice vaccinated with SM-102, YK-201, YK-202, and YK-209 displayed significantly lower pathology scores than controls, suggesting robust protection against RSV infection (Fig. 4g). At the same time, SM-102-treated mice exhibited slightly higher pathology scores compared to other LNPs, suggesting potential lung injury associated with the formulation, independent of RSV infection (Fig. 4g). Taken together, these results demonstrate that YK-201, YK-202, and YK-209 LNP formulations are optimal for delivering the RSV preF-mRNA vaccine, which confers robust viral protection.

RSV preF-mRNA-LNPs demonstrated benign safety profile in a mouse model

To assess the potential toxicity of preF-mRNA-LNPs, BALB/c mice were intramuscularly (i.m.) injected with 0.5 mg kg⁻¹ RSV preF-mRNA-LNPs. Hematological analysis showed minimal impact on alanine transaminase (ALT) and aspartate transaminase (AST) levels (Fig. 5a). In contrast, SM-102 significantly upregulated ALT levels (Fig. 5a), indicating minimal liver toxicity associated with these novel ionizable lipids, potentially due to the absence of liver off-target effects. Additionally, there were no significant changes in serum urea (URE) or creatinine (CRE) levels, suggesting the absence of kidney toxicity (Fig. 5a).

Fig. 5 Safety evaluation of RSV preF-mRNA-LNP constructs in vivo. (a) Serum levels of aspartate transaminase (AST), alanine transaminase (ALT), urea (URE), and creatinine (CRE) in BALB/c mice vaccinated with preF-mRNA-LNPs formulated with SM-102, YK-201, YK-202, and YK-209, measured on day 12 after three vaccinations on day 0, 3, and 7 (n = 3). Blank refers to DPBS-treatment. (b) Serum cytokines 6 h after the first vaccination (n = 3). Statistics were assessed by one-way ANOVA with Tukey's multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns means no significant difference. Data represents the mean ± SEM.

Since the release of pro-inflammatory cytokines is a known response to ionizable lipids, we measured the levels of key cytokines, including IL-1β, TNF-α, IL-6, and IFN-γ, following preF-mRNA-LNP injection. SM-102 induced upregulation of all four cytokines, indicating strong immunogenicity (Fig. 5b). In contrast, YK-201, YK-202, and YK-209 did not significantly affect IL-1β or TNF-α levels, though all three induced a significant upregulation of IL-6, with levels much lower than those observed with SM-102 (Fig. 5b). Additionally, YK-202 and YK-209 were found to slightly upregulate IFN-γ levels (Fig. 5b). These results suggest that, while exhibiting some immunogenicity, these novel ionizable lipids may offer a safer profile and show promise for further clinical development.

Conclusions

In conclusion, we designed and synthesized a library of 26 novel ionizable lipids and assessed their efficacy and tissue selectivity for mRNA delivery. We found that the resulting LNP formulations exhibited favorable physicochemical properties. After in vitro screening, three formulations (YK-201, YK-202, and YK-209) demonstrated 1.4- to 2.6-fold higher protein expression and comparable cytotoxicity to YK-009. In a mouse model, these LNPs exhibited sustained and localized protein expression at the injection site. When formulated with RSV preF-mRNA to build mRNA vaccines, they elicited robust humoral immune responses and minimal lung damage, outperforming the clinically used SM-102. Additionally, these formulations exhibited a strong safety profile. Therefore, our data indicated that these site-retained LNP formulations are promising clinical candidates for the development of RSV and other mRNA vaccines.

Author contributions

X. C., H. Z. and D. L. conceived this study, designed the methodology, conducted the experiments. H. Z. and D. L. drafted this manuscript. J. W. and G. S. validated the results and reviewed and edited this manuscript. All authors have read and agreed to the published version of the manuscript.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

G. S., X. C. and H. Z. are inventors on a pending patent related to this work filed by Beijing Youcare Kechuang Pharmaceutical Technology Co. Ltd.

Acknowledgements

We thank Dr Kai Dong, Dr Jinyu Zhang and Huanyu Wang for helpful advice and discussion.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00117j

‡ These authors contributed equally.

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