Novel Less Toxic, Lymphoid Tissue‐Targeted Lipid Nanoparticles Containing a Vitamin B5‐Derived Ionizable Lipid for mRNA Vaccine Delivery

Abstract

Following their approval by the Food and Drug Administration, lipid nanoparticles (LNPs) have emerged as promising tools for delivering mRNA vaccines and therapeutics. Ionizable lipids are among the essential components of LNPs, as they play crucial roles in encapsulating mRNA and facilitating its release into the cytosol. In this study, 17 innovative ionizable lipids using vitamin B5 are designed as the core structure, aiming to reduce toxicity, to maintain vaccine efficiency, and to ensure synthetic feasibility. The top‐performing LNP in terms of mRNA vaccine delivery in the mouse model is LNP 5097, which is generated by incorporating ionizable lipid I97. mRNA⊂LNP 5097 demonstrates favorable structural and physicochemical properties, high mRNA transfection efficiency, and long‐term stability. Moreover, mRNA⊂LNP 5097 specifically delivers the mRNA to the spleen and lymph nodes in model mice, induces balanced Th1/Th2 immune responses, and elicits the production of high levels of neutralizing antibodies with low toxicity. The findings here suggest the high utility of LNP 5097, which includes novel vitamin B5‐derived ionizable lipids with reduced toxicity, in mRNA vaccine research for both infectious diseases and cancer.

The top‐performing lipid nanoparticle, incorporating a novel ionizable lipid derived from vitamin B5, demonstrates high mRNA transfection efficiency, low toxicity, favorable stability, targeted delivery to lymphoid tissues, and high immunogenicity, showing promise as a carrier for mRNA vaccines for infectious diseases and cancer.

1. Introduction

Lipid nanoparticles (LNPs) are a versatile and efficient delivery platform for delivering various cargo, including RNA vaccines and therapeutics.^{[ 1} , ^{2 ]} They are primarily composed of ionizable lipids, helper lipids, cholesterols, and polyethylene glycol (PEG)‐ylated lipids, all of which contribute to their encapsulation efficiency, structural integrity, stability, and circulation times.^{[ 2 ]} LNPs have gained significant attention following their successful use in mRNA vaccines approved by the Food and Drug Administration and used during the coronavirus disease 2019 (COVID‐19) pandemic.^{[ 3 ]} Recently, substantial efforts have been made in the development of mRNA vaccines for the treatment and prevention of various diseases.^{[ 4} , ^{5 ]} These mRNA vaccines have shown promising results and are currently undergoing clinical trials;^{[ 2} , ³ , ^{5 ]} the majority of them employ the LNP platform as their delivery system.^{[ 3 ]}

Ionizable lipids have been developed to maximize mRNA delivery efficiency in terms of encapsulation, cellular uptake, and endosomal release with minimal toxicity. Ionizable lipids maintain a neutral LNP surface at physiological pH, reducing the toxicity compared to cationic materials,^{[ 2 ]} and become positively charged in the acidic environment in endosome, facilitating the release of mRNA.^{[ 6} , ^{7 ]} Focusing on the development of bio‐compatible ionizable lipids, efforts have been made to use vitamins as a part of lipids to leverage their low toxicity and intrinsic functions. The most commonly utilized vitamins are the fat‐soluble vitamins A, E, and D. They are not only suitable for use as tails but also act as targeting moiety for the liver (in the case of vitamin A) or as adjuvants to activate an immune response itself after decomposition in the body (in the case of vitamin E).^{[ 8} , ⁹ , ^{10 ]} Additionally, the water‐soluble vitamins B3, C, and H have also been reported to be mostly utilized by conjugating them to the ends of ionizable lipids. For example, LNPs containing a vitamin C‐conjugated ionizable lipid exhibited high in vivo mRNA transfection efficiency and macrophage lysosome‐targeting properties, enabling the killing of multidrug‐resistant bacteria in mouse models.^{[ 11 ]} Unlike the reported vitamin‐conjugated lipids, we designed ionizable lipids with a novel structure by utilizing a vitamin as the central core.

In this study, three ionizable lipid series (I7X, I8X, and I9X) were designed using vitamin B5 as a core structure. Vitamin B5, also known as pantothenic acid, was selected due to its functionality, synthetic feasibility, immunostimulatory effect, and reduced toxicity. It is a naturally occurring, water‐soluble vitamin that plays an important role in metabolism through the tricarboxylic acid cycle as a precursor to coenzyme A.^{[ 12 ]} Vitamin B5 has been reported to have a protective effect against oxidative stress, mitochondrial toxicity, and cardiovascular damage.^{[ 13} , ¹⁴ , ^{15 ]} For this reason, we expected that vitamin B5‐derived ionizable lipids could be used to develop safe LNPs for mRNA vaccine delivery. In addition, recent studies have shown that vitamin B5 exhibits immunostimulatory effects.^{[ 16} , ^{17 ]} For example, the levels of inflammatory cytokines such as tumor necrosis factor α and interleukin (IL) 6 increase in epithelial cells and bone marrow‐derived macrophages treated with vitamin B5.^{[ 16 ]} Vitamin B5 also accelerates the activation of CD4⁺ T cells, enhancing interferonγ and IL‐17 levels. Furthermore, vitamin B5 contains primary and secondary hydroxyl groups, as well as a carboxylic acid group, making it suitable for introducing ionizable heads and lipid tails orthogonally.

We synthesized 17 novel ionizable lipids with structural differences based on the vitamin B5 core. The top‐performing LNP was confirmed through screening of the functional and physicochemical properties of the formulated LNPs, such as their size, pK_a, in vivo mRNA transfection efficiency, and immune response. Interestingly, the top‐performing LNP was found to specifically accumulate in the lymph nodes and spleen, crucial organs of the immune system, without notable toxicity in normal mice. It has been reported that conventional LNPs utilizing the ionizable lipids SM‐102 and ALC‐0315 deliver a significant amount of mRNA to the liver through interactions with apolipoprotein E.^{[ 18 ]} Delivery to non‐hepatic organs that are associated with immunity could enhance the efficacy of mRNA vaccines and result in reduced their side effects. Particularly noteworthy is the minimal toxicity of these LNPs and effective induction of neutralizing antibodies for both infectious diseases and cancer.

2. Results and Discussion

2.1. Design and Synthesis of Vitamin B5‐Derived Ionizable Lipids

To develop LNPs for efficient and safe mRNA vaccine delivery, we intended to develop less toxic ionizable lipids based on the vitamin B5 core. Vitamin B5 is a natural compound with three functional groups suitable for facile chemical modification: primary hydroxyl (R_A), secondary hydroxyl (R_B), and carboxylic acid (R_C) groups. We designed novel ionizable lipids with the vitamin B5 structure as the central core, and the lipids were categorized into three types (I7X, I8X, and I9X) based on the connections of the ionizable heads and lipid tails to these functional groups: (1) I7X: R_A serves as the ionizable head, while the conical lipid tail is positioned exclusively at the R_C; (2) I8X: R_A also serves as the ionizable head, but the lipid tails extend to both the R_B and R_C; and (3) I9X: R_C serves as the ionizable head, with lipid tails placed at both the R_A and R_B (Figure  1 ). All ionizable lipids were successfully synthesized in good yields under mild conditions, typically requiring 4 to 7 steps (Scheme  1 ; Figures S1–S3, Supporting Information). First, five ionizable lipids of the I7X‐type lipids (I71 to I75) were synthesized with the same branched, cone‐shaped tail (R_T1), and 5 types of ionizable heads (R_H): dimethylamino, pyrrolidino, piperidino, 4‐methylpiperazino, and morpholino groups. With the I7X series, we found that the in vivo mRNA transfection efficiency was the highest in LNPs containing I75 with a morpholino head group. Consequently, in subsequent series, the ionizable heads were fixed with either morpholino or the most common dimethylamino groups. For the I8X‐type lipids, two different paring of R_T3 and R_T2 (a linear R_T2L and a branched R_T2B) and two selected heads (R_H) were introduced. Four ionizable lipids (I81 to I84) were synthesized as having homo‐paired tails (if they incorporated both branched R_T2B and R_T3) or hetero‐paired tails (if they incorporated linear R_T2L and branched R_T3) to the R_B and R_C hydroxyl groups of vitamin B5. LNPs containing I8X lipids with homo‐paired tails exhibited superior results, including particle size, monodispersity, and in vivo mRNA transfection efficiency (especially I82), compared to LNPs containing lipids with hetero‐paired tails. Finally, in the I9X‐type lipids, two branched alkyl chains (R_T3 and R_T4), which having different length of linker (C3 and C1, respectively), were utilized as tails. The I9X series were designed a distinct direction from the head to the tails; the ionizable head was attached to carboxylic acid (R_C) rather than alcohol (R_A). For I91 to I94, the hetero‐paired tails, R_T4‐COOH and R_T3‐COOH, were sequentially attached to the primary (R_A) and secondary (R_B) alcohol sites, respectively. In contrast, for I95 to I98, the homo‐paired tails, two R_T3‐COOH units, were simultaneously attached to both the R_A and R_B sites. Then, two types of ionizable heads, R_H‐NH₂ and R_H‐OH, were incorporated separately via amide and ester formation, respectively. With two types of ionizable heads, two bond types, and two pairing of tails, a total of eight ionizable lipids (I91–I98) were synthesized. The structures of all synthesized ionizable lipids were confirmed using ¹H and ¹³C nuclear magnetic resonance spectroscopy and high resolution mass spectroscopy (Figures S4–S20, Supporting Information).

Figure 1.

Design of the vitamin B5‐based ionizable lipids and examples of their structures. Vitamin B5 has a primary alcohol (R_A site), secondary alcohol (R_B site), and carboxylic acid (R_C site). The three ionizable lipid series were designed according to the direction in which the ionizable heads and tails were functionalized at each functional group site: I7X (ionizable head at R_A and tail at R_C), I8X (ionizable head at R_A and two tails at R_B and R_C), and I9X (ionizable head at R_C and two tails at R_A and R_B).

Scheme 1.

Synthetic scheme for vitamin B5‐based ionizable lipids, I71–I75, I81–I84, I91–I94, and I95–I98. The I7X and I8X series were synthesized by incorporating a tail into the carboxylic acid of acetal‐protected vitamin B5 through EDC coupling, followed by acetal deprotection, and then attaching an ionizable head to the primary alcohol through EDC coupling. In the case of the I8X series, an additional tail was attached to the secondary alcohol through EDC coupling. The I9X series was synthesized by incorporating two tails, either identical or different, to Fm‐protected vitamin B5 through EDC coupling and attaching an ionizable head through EDC coupling to the carboxylic acid after Fm deprotection.

2.2. Primary Selection of LNPs Based on the Characterization Data and in vivo mRNA Transfection Efficiency

To select the optimal ionizable lipids, LNPs were formulated and their properties were screened. Using the 17 synthesized ionizable lipids described above (I71–I75, I81–I84, and I91–I98), we next sought to formulate 34 LNPs, each with two types of lipid (Figure  2 ): (1) LNP C0 (ionizable lipid:1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC):cholesterol:DMG‐PEG2000 = 50:10:38.5:1.5), a conventional LNP (Con‐LNP) composition for mRNA delivery,^{[ 19 ]} and (2) LNP 50 (ionizable lipid:6,6′‐trehalose dioleate(TDO):1,2‐Dioleoyl‐sn‐glycero‐3‐phosphoethanolamine(DOPE):n‐butyl lithocholate(L‐Bu):DMG‐PEG2000 = 25:25:10:38.5:1.5), according to our previous product, LNP S050L.^{[ 20} , ²¹ , ²² , ^{23 ]} In the LNP 50 type, 50% of the ionizable lipids were replaced by trehalose glycolipids, which reduced side effects compared to Con‐LNP. LNP S050L and Con‐LNP, containing SM‐102 as the ionizable lipid in the LNP 50 and C0, respectively, were used as the corresponding positive controls.

Figure 2.

Two types of LNP formulations used in this study. LNP 50 type consisted of the ionizable lipid, 6,6′‐trehalose dioleate (TDO), DMG‐PEG2000, 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE), and n‐butyl lithocholate (L‐Bu) at a molar ratio of 25:25:1.5:10:38.5. The LNP C0 type consisted of the ionizable lipid, DMG‐PEG2000, 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC), and cholesterol at a molar ratio of 50:1.5:10:38.5.

The formulated 34 LNPs were assessed their overall functional and physicochemical properties, including size, polydispersity index (PDI), encapsulation efficiency, pK_a, and in vivo mRNA transfection efficiency in mouse models as described below. We visualized all results in a heatmap to identify LNPs that could be suitable for mRNA vaccine delivery (Figure  3A). In the heatmap, each analyzed item is color‐coded to highlight the optimal effective delivery range, marked in red, for the LNPs. Especially in terms of mRNA delivery efficacy, the LNPs depicted in dark red were considered superior to the corresponding control LNPs (LNP S050L and Con‐LNP). Conversely, LNPs with undesirable properties that are not suitable as mRNA carriers are marked in white.

Figure 3.

Identification of structural and physicochemical properties of R/L mRNA⊂LNPs and in vivo transfection efficiency. A) Heatmap representing the characterization of all LNPs based on size, PDI value, pK_a value, and delivery efficacy. The effective range of LNPs as mRNA vaccine carriers is shown in red, with the darker red indicating LNPs that have higher delivery efficacy than the control LNPs containing SM‐102. White indicates LNPs with poor properties that are not suitable for use as an mRNA carrier, while pale red in the middle indicates moderate carrier properties that are not optimal. B–D) Structural and physicochemical properties of control LNPs (Con and S050L) and the best candidate LNPs (5093 and 5097) in terms of B) size and PDI, C) mRNA encapsulation efficiency, and D) pK_a. E) in vivo R/L mRNA transfection efficiency 6 h after intradermal (I.D.) injection of R/L mRNA⊂LNPs (Con, S050L, 5093, and 5097) into the ears of 6‐week‐old ICR mice. F) Schematic illustration of the assay process for in vivo transfection efficiency of R/L mRNA to assess the delivery efficacy of R/L mRNA⊂LNPs. 6 h after intradermal (I.D.) injection of R/L mRNA⊂LNPs into the ears of 6‐week‐old ICR mice, the mice were sacrificed, and their ears were harvested and homogenized in lysis buffer. The bioluminescence signal (relative light units) from the expressed R/L was then measured. The delivery efficiency for each LNP was calculated as a fold change in bioluminescence signal relative to the control LNPs. In all bar graphs, error bars represent mean ± standard deviation. Statistical significance was assessed by Tukey's paired comparison test: ^* p ≤ 0.05, ^** p ≤ 0.01, ^*** p ≤ 0.001.

First, the size and PDI of all LNPs were determined. According to a recent report from Moderna, Inc., the LNPs with a size of ≈ 100 nm effectively produced neutralizing antibodies in a mouse model, and LNPs ranging in size from 60 to 150 nm induced significant immune responses in non‐human primates.^{[ 24 ]} In addition, the PDI values provide information about the uniformity of the formulated LNPs. Generally, a PDI value below 0.2 has been considered to indicate that the formulated LNPs are homogeneous enough.^{[ 25 ]} Overall, the 29 formulated LNPs, excluding LNPs 5083, C095, C083, C074, and C072, were considered suitable carriers for mRNA vaccines in terms of both size and PDI (Figure 3A,B; Figure S21, Supporting Information).

Then, we confirmed the mRNA encapsulation efficiency of all of the LNPs using a gel retardation assay. The results indicated almost no free RNAs observed in the LNP solutions (Figure 3C; Figure S22, Supporting Information), indicating that all LNPs effectively encapsulated mRNAs.

Next, the pK_a values of the LNPs were determined with a 6‐(p‐toluidinyl)naphthalene‐2‐sulfonic acid (TNS) assay.^{[ 26 ]} When LNPs enter the cell through endocytosis, the pH inside the resulting endosome gradually decreases from 7.0 to 5.5.^{[ 6 ]} If the pK_a of the LNPs is between 6 and 7, which is the ideal range for potent delivery, protons gradually attach to them in the mature endosome, making them cationic and facilitating endosomal release.^{[ 6} , ^{7 ]} If the pK_a of the LNPs is greater than 7.5, the LNP may exhibit cationic properties even at physiological pH values, which could lead to toxicity through non‐specific interactions with serum proteins. Conversely, when the pK_a is lower than 5.5, the LNPs may not have enough ionic charges at neutral pH, inducing their aggregation through hydrophobic interactions. In both cases, whether the pKa is higher than 7.5 or lower than 5.5, their delivery efficacy is also reduced. The assay revealed that the positive control LNPs, LNP S050L and Con‐LNP, had pK_a values of 6.51 and 6.73, respectively, falling within the well‐known optimal range. Among our LNPs, 21 developed LNPs had pK_a values that fits within the optimal range, including LNP 5097 (pK_a 6.23), which had the best mRNA vaccine delivery performance, as described below. Another 6 LNPs had moderate pK_a values ranging from 5.5 to 6.0, and the remaining 7 LNPs had pK_a values below 5.5 (Figure 3A,D; Figure S23, Supporting Information). Generally, the pK_a values depended on the structure of the ionizable head (R_H) and not the LNP type. LNPs with ionizable lipids containing a dimethylamino group as the ionizable head exhibited higher pK_a values (5.90 to 7.01) than those with morpholino group as the ionizable head (4.57 to 5.94). Moreover, LNPs with an amide linker between the ionizable head and vitamin B5 core (LNPs constructed with I91, I92, I95, and I96) had higher pK_a values (5.60 to 7.01) than corresponding LNPs with an ester bond between the two (LNPs constructed with I93, I94, I97, and I98, respectively) (5.03 to 6.85). That is, the pK_a of the LNP reflects the pK_a tendency of the ionizable head. Furthermore, LNPs containing lipids with hetero‐paired tails (LNPs constructed with I81, I83, I91, I93, and I94) showed a higher tendency of pK_a values compared to their corresponding LNPs containing lipids with homo‐paired tails (LNPs constructed with I82, I84, I95, I97, and I98, respectively). We speculate that the tail structures of the ionizable lipids may also affect the pK_a value by influencing the compactness of the self‐assembled LNPs. These findings suggest that the structure of the ionizable lipids has a substantial influence on the pK_a of the resulting LNPs, providing information on the structural design of ionizable heads and tails that would be suitable for the development of new ionizable lipids with appropriate pK_a values.

Finally, we assessed the delivery efficacy of each LNP by measuring the in vivo transfection efficiencies of Renila luciferase (R/L) mRNA, as indicated by the bioluminescence by the expressed R/L. LNPs encapsulating R/L mRNA (10 µg) were administered to the ears of 6‐week‐old ICR mice via intradermal (I.D.) injection, and bioluminescence was measured 6 h post‐administration. LNPs 5075, 5093, 5097, 5098, and C098 showed significantly higher delivery efficacy than did the control LNPs containing SM‐102 (Figure 3A,E; Figure S24, Supporting Information). In addition, compared with the LNP C0, the LNP 50 showed similar or higher delivery efficacy for almost all ionizable lipids. The I9X group demonstrated a higher number of candidates with delivery efficacy similar to that of SM‐102 compared to both the I7X and I8X groups, indicating that positioning the ionizable head group at the R_C position may be more favorable than at the R_A position. The I7X group, characterized by a funnel‐shaped structure with the cone‐shaped tail R_T1 positioned farther away from the ionizable head group, generally exhibited lower delivery efficacy. Although the funnel‐type structure is similar to the conical structure, it appears to be less suitable for ionizable lipids. Nevertheless, when the ionizable head was a morpholino group, the LNP 50 types displayed exceptional delivery efficacy, even though they were not selected due to their pK_a values being outside the optimal range of 6 to 7. The I8X group was designed to modify the funnel structure of the I7X group into a cone shape, resembling SM‐102, by adding a tail at the R_B position. Although the overall delivery efficacy was lower, I82 in LNP C0 types exhibited the highest delivery efficacy, even though it was only 57% of that of SM‐102. Among the I9X structures, where the orientation of the ionizable head group and the hydrophobic tail group is reversed compared to the I7X and I8X groups, several LNP 50 types showed delivery efficiencies comparable to SM‐102. LNPs with ester linkages between the vitamin B5 core and the ionizable head group outperformed those with amide linkages, and LNPs with homo‐paired tails generally exhibited higher delivery efficacy than those with hetero‐paired tails. Although the dimethylamino group tended to outperform the morpholino group as the ionizable head, the difference was minimal and insufficient to establish a general trend. These trends were not consistent across all LNP C0 types, where only I98 showed a 1.48‐fold higher delivery efficacy compared to SM‐102. However, due to its pK_a falling outside the optimal range, I98 was excluded from the final selection.

Based on overall results, LNPs 5093 and 5097, which are shown with all red tiles for each evaluated item in heatmap, were considered to be the best candidates for mRNA delivery (Figure 3A). These two LNPs only differ structurally in terms of tail pairing (i.e., homo‐ versus hetero‐pairing); otherwise, both employ dimethylamino heads attached through an ester bond, and the other LNP compositions are identical. Therefore, based on the results of the primary screening, LNPs 5093 and 5097 were primarily selected as the best candidates. Among the suboptimal LNP candidates for vaccine delivery, LNPs 5075, C082, 5098, and C098 also showed good potential with relatively high mRNA delivery efficacy similar to that of the control LNPs. Although these candidates may not have presented with optimal values for all properties investigated, their specific strengths make them valuable contenders for further investigation. It is possible that their performance can be further improved by adjusting the LNP component or the N/P ratio.

2.3. Selection of the Top‐Performing LNP for mRNA Vaccine Delivery Based on Heatmaps and in vivo Immune Responses

To identify the top‐performing LNP, the immune responses in model mice injected with hemagglutinin‐encoding mRNA (HA mRNA)⊂LNPs (5093 and 5097) were compared with those injected with control LNPs. LNPs were administered to 6‐week‐old BALB/c mice by intramuscular (I.M.) injections through prime and booster vaccinations over a 2‐week period (Figure  4A). The levels of both immunoglobulin G1 (IgG1) and immunoglobulin G2a (IgG2a)⊥ significantly increased in all groups administered HA mRNA⊂LNPs after the primary and secondary vaccination (Figure 4B,C). Notably, the IgG1/IgG2a ratio is calculated to be between 1.29 and 1.54, which falls within the range of 0.5 to 2.0, indicating that a well‐balanced immune response was induced (Figure 4D).^{[ 27 ]} Two weeks after booster immunization, neutralizing antibody levels were measured in the serum through a hemagglutination inhibition (HI) assay. The level of neutralizing antibodies was the highest with LNP 5097, followed by Con‐LNP, LNP 5093, and LNP S050L (Figure 4E). Based on these findings, LNP 5097 has the greatest potential for use as an mRNA vaccine carrier due to its favorable characteristics, including the induction of a balanced immune response, production of the highest neutralizing antibody levels, greater mRNA transfection efficiency, and suitable physicochemical properties with a pK_a of 6.23, an optimal particle size (92.0 ± 0.7 nm), and excellent monodispersity (PDI = 0.11).

Figure 4.

In vivo immune response following injection of HA mRNA⊂LNPs (Con, S050L, 5093, and 5097). A) Schematic illustration of the immunization schedule. Six‐week‐old BALB/c mice were administered prime/booster injections with LNPs (Con‐LNP, LNP S050L, 5093, and 5097) encapsulating HA mRNA (10 µg) or DPBS intramuscularly. Serum was collected 2 weeks after the first and second administrations of HA mRNA⊂LNPs or DPBS. B–D) Serum levels of B) IgG1 and C) IgG2a and the D) IgG1/IgG2a ratio in mice after receiving prime or booster injections with HA mRNA⊂LNPs or DPBS. E) HI titers in the serum of mice after receiving prime/booster injections with HA mRNA⊂LNPs or DPBS. Statistical significance was assessed by Tukey's paired comparison test: ^* p ≤ 0.05, ^** p ≤ 0.01, ^*** p ≤ 0.001.

2.4. Morphology and Long‐Term Stability of LNP 5097

To assess the structural characteristics of the LNPs, the size and morphology of the top‐performing LNP, LNP 5097, and the control LNPs (LNP S050L and Con‐LNP) were further confirmed through cryo‐transmission electron microscopy (cryo‐TEM). The diameters of LNPs observed by cryo‐TEM are consistently ≈ 70 nm (Figure  5A). Specifically, the diameter of LNP 5097 observed by cryo‐TEM is 73.5 nm, which is slightly smaller than its hydrodynamic diameter (92.0 ± 0.7 nm). This observation reflects the hydration of the hydrophilic surface of the LNP in aqueous solution due to PEG moieties. Additionally, the cryo‐TEM images revealed characteristic morphologies for each LNP: Control LNPs and LNP 5097 exhibited a typical spherical shape with an electron‐dense core.

Figure 5.

Morphology and storage stability test for mRNA⊂LNP 5097. A) Cryo‐TEM images of R/L mRNA⊂LNPs (Con, S050L, and 5097). B) Storage stability test for the colloidal stability of R/L mRNA⊂LNPs (Con, S050L, and 5097) in pH 7.4 DPBS solutions over time. C) Storage stability test for in vitro mRNA transfection efficiency (symbol/line) and size (column) of EGFP mRNA⊂LNP 5097 in pH 8.0 Tris buffer solutions containing 10% trehalose and 10% sucrose as the preservatives according to the storage condition. The LNP solutions were divided into four groups and stored under different conditions: blue square, fresh LNP; red square, stored at −80 °C in a deep freezer; black circle, stored at 2 to 8 °C in a refrigerator; gray triangle, freeze‐dried and then stored at 2 to 8 °C in a refrigerator; and white inverted triangle, freeze‐dried and then stored at −20 to −15 °C in a freezer.

The colloidal stability of LNP 5097 was determined by measuring the change in the size of the LNPs after a month. Aliquots of the formulated LNP solutions were stored in a refrigerator at 2 to 8 °C in DPBS (pH 7.4) and the size of the LNPs was measured every week. For all aliquots, no significant changes in their size were observed over time (Figure 5B). That is, the colloidal stability of LNP 5097 at 2 to 8 °C was maintained for a month, suggesting that the LNPs can be stably refrigerated.

Furthermore, the stability of LNP 5097 after lyophilization was investigated under various storage conditions. COVID‐19 mRNA vaccines require frozen storage and distribution, which presents significant challenges. This requirement has limited the distribution of the vaccine, especially in regions where the cold chain is difficult to maintain. To overcome this limitation, there have been explorations into lyophilization technology, aiming to simplify storage and distribution by improving the stability of the LNP formulation at elevated temperature.^{[ 28} , ^{29 ]} The lyophilized LNPs were also recently evaluated via molecular dynamic simulations.^{[ 30 ]} This study revealed that the cryopreservatives are distributed on the surface of LNPs and interact with the lipid membrane via hydrogen bonding in an anhydrous state. It was thus hypothesized that the cryopreservatives protect the LNP structure by surrounding it during lyophilization. Therefore, in this study, enhanced green fluorescent protein‐encoding (EGFP) mRNA⊂LNP 5097 formulations in cryopreservation buffer (Tris buffer supplemented with 10% sucrose and 10% trehalose, pH 8.0) were prepared and their storage stabilities were monitored. The formulations were stored under various conditions: frozen at −80 °C, refrigerated at 2 to 8 °C, and either refrigerated (2 to 8 °C) or frozen (−20 to −15 °C) after lyophilization. After four weeks, all LNP samples were assessed for their in vitro EGFP mRNA transfection efficiency and particle size (Figure 5C; Figure S25, Supporting Information). The stability trend is similar to that observed with commercially available mRNA vaccines for frozen and refrigerated LNPs. The LNPs stored at −80 °C for 4 weeks maintained their size and mRNA transfection efficiency. For refrigerated storage, the LNPs in the presence of cryopreservatives showed an increase in particle size, and macroscopic aggregation was observed from the second week. Additionally, the mRNA transfection efficiency decreased beginning in the second week. Notably, after storage via lyophilization, the size of the LNPs and mRNA transfection efficiency were maintained across both freezing and refrigeration temperatures for the entire four‐week period.

In conclusion, mRNA⊂LNP 5097 is stable for a minimum duration of one month under refrigeration (2 to 8 °C) in the absence of cryopreservatives, and for period of one week when cryopreservatives are incorporated. Furthermore, the lyophilized mRNA⊂LNP 5097 remains stable for at least one month when stored either in a refrigerator (2 to 8 °C) or a freezer (−20 to −15 °C) in the presence of cryopreservatives. The enhanced stability of lyophilized LNP formulations at refrigeration temperature could potentially alleviate the storage and distribution challenges associated with current mRNA vaccines.

2.5. Lymphoid Tissue‐Targeted Delivery of mRNA⊂LNP 5097

The in vivo and ex vivo biodistributions of Firefly luciferase (F/L) mRNA⊂LNP 5097 were assessed by bioluminescence imaging in a mouse model, administered to mice via an I.M. or intravenous (I.V.) injection. After a 6‐h incubation period, a bioluminescence signal in mice that received an I.M. injection was observed at the injection site and the closest inguinal lymph node (InLN) (Figure  6A; Figure S26A, Supporting Information). In mice that received an I.V. injection, the bioluminescence signal was specifically detected in the spleen (Figure 6B; Figure S26B, Supporting Information). All bioluminescence signals persisted for 24 h post‐injection, indicating sustained expression of luciferase from the F/L mRNA. Con‐LNP, containing SM‐102, has been reported to widely distribute the mRNA to the liver.^{[ 18 ]} Conversely, LNP 5097 was not detected in the liver but was found in the spleen (via I.V.) and lymph node (via I.M.). Consistent with our previous report,^{[ 20 ]} S050L (LNP 50 with SM‐102) appeared to target the spleen after I.V. administration and lymph nodes after I.M. administration, despite variations in ionizable lipids. Although the exact mechanism remains unclear, we hypothesize that the trehalose glycolipid may act as a ligand for macrophage inducible C‐type lectin, which are expressed on immune cells such as macrophages, dendritic cells, and neutrophils.^{[ 31 ]} This interaction could enhance the affinity of LNPs for immune cell surfaces, potentially explaining their preferential distribution to lymphoid tissues. Additionally, the use of L‐Bu in LNP 50, instead of cholesterol, could influence their non‐hepatic distribution. A notable distinction between SM‐102 and I97 is observed in our studies: S050L retained half the liver signal compared to the spleen 6 h post‐I.V. administration,^{[ 20 ]} whereas LNP 5097 showed almost no signal in the liver, indicating superior spleen‐directing by I97 (Figure 6B). This reduced hepatic distribution supports the idea that structural changes in ionizable lipids can enhance targeting to non‐hepatic organs. Recent studies confirm that lipid nanoparticles can target the spleen based solely on their chemical structure and composition of lipids, without the need for specific ligands.^{[ 32} , ^{33 ]} I97 serves as a clear example of this phenomenon, reinforcing the concept that structural modifications in ionizable lipids can direct tissue‐specific distribution, particularly to the spleen, without the need for active targeting ligands or negatively charged auxiliary lipids.^{[ 34} , ^{35 ]} The observed expression of F/L in the lymphoid tissue— crucial in immune system regulation—suggests that LNP 5097 could be utilized for mRNA‐based vaccination via both I.M. and I.V. injection routes.

Figure 6.

Ex vivo biodistribution of F/L mRNA⊂LNP 5097. A,B) Bioluminescence images of various organs from two C57BL/6 mice who received an A) intramuscular (I.M). or B) intravenous (I.V.) injection of F/L mRNA⊂LNP 5097. InLN = inguinal lymph node, L = left, R = right.

2.6. Application of OVA mRNA⊂LNP 5097 for Cancer Vaccination

The potential of LNP 5097 as an mRNA vaccine carrier was assessed in a mouse model using mRNA encoding the ovalbumin (OVA) antigen for OVA‐expressing cancer cells (E.G7‐OVA). Mice immunized with OVA mRNA can elicit a specific, potent immune response against E.G7‐OVA, resulting in the elimination of growing tumor from these cells. Mice received primary and secondary subcutaneous (S.C.) injections of OVA‐mRNA(10 µg)⊂ LNP 5097 at four‐day intervals; four days after the second injection, E.G7‐OVA cells were implanted subcutaneously (Figure  7A). Changes in body weight and tumor volume were observed every 2 to 3 days after cancer inoculation. The difference in mouse weight tended to increase by 1 to 2 g per week (Figure 7B). In the negative control group (DPBS), the tumors exhibited exponential growth in volume (Figure 7C,D). In contrast, in both the OVA‐mRNA⊂LNP 5097 and OVA‐mRNA⊂LNP S050L groups, no tumors had developed, even after 30 days (Figure 7C,D). In addition, it was confirmed that tumor growth was inhibited even at lower doses (2 and 5 µg) of OVA‐mRNAs (Figure S27, Supporting Information). At a 5 µg dose, tumor growth was suppressed for 37 days in both the LNP S050L and LNP 5097 groups, while tumors in 1 or 2 mice in the 2 µg dose group began to slowly increase ≈ 30 days after tumor inoculation in both cases (Figure S27B, Supporting Information). Interestingly, in the LNP 5097 group with a 2 µg dose, the tumor in one mouse started to grow ≈30 days after the cancer inoculation, but with continuous observation, the tumor volume subsequently decreased within a week, while the tumors in two mice were growing in S050L group (Figure S27C, Supporting Information). This suggests that I97 exhibits superior properties compared to SM‐102 as an ionizable lipid component in LNPs for mRNA‐based cancer vaccine delivery, and that even if the tumor initially grows, the humoral immunity induced by OVA mRNA⊂LNP 5097 can suppress further tumor growth. Whereas, in the case of EGFP mRNA⊂LNP 5097, which did not induce OVA antigen, tumors grew similarly to those in the DPBS group (Figure S27B,D, Supporting Information). Overall, these findings highlight the potential of LNP 5097 containing I97 as a promising carrier for mRNA‐based cancer vaccines, with the capability to outperform LNP S050L containing SM‐102 in terms of antigen expression and neutralizing antibody production.

Figure 7.

Cancer vaccination study with OVA mRNA⊂LNPs. A) Schematic illustration of the injection schedule. Seven‐week‐old C57BL/6 mice were administered prime/booster injections with LNP 5097 encapsulating OVA mRNA (10 µg), LNP S050L encapsulating OVA mRNA (10 µg) or DPBS through subcutaneous (S.C.) injection, followed by inoculation with E.G7‐OVA cancer cells (1 × 10⁶ cells). B,C) Changes in B) body weight and C) tumor volume in the LNP 5097, LNP S050L, and DPBS groups according to the time after cancer cell inoculation. D) Representative photographs of mice in the LNP 5097, LNP S050L, and DPBS groups 30 days after cancer cell inoculation.

2.7. Assessment of the in vitro and in vivo Toxicity of LNP 5097

Before assessing the toxicity of LNP 5097, we conducted in vitro studies on the pharmacokinetics and stability of I97, including cytocrome P450 (CYP) inhibition assay, patch clamp assay for human ether‐a‐go‐go‐related gene (hERG) channels, the Ames test, and a microsomal stability assay (Figure S28; Tables S1–S4, Supporting Information). The results indicated that I97 itself could be considered almost nontoxic and exhibits high microsomal stability, making it a suitable candidate for consideration in constructing LNPs as carriers for mRNA vaccines. To assess the in vitro cytotoxicity of LNP 5097 compared to the control LNPs (Con‐LNP and LNP S050L), HepG2 cells and fibroblasts were incubated with the LNPs for 24 h. The results showed almost no changes in cell viability regardless of the concentration of any of the LNPs (Figure S29, Supporting Information). These findings indicate that LNP 5097 exhibited minimal toxicity to these cell lines within the tested concentration range (0.1 to 2.0 mm total lipid concentration).

The absence of a significant change in body weight upon injection of LNP 5097 (Figure 7B) suggested the safety of this LNP, and additional tests were conducted to confirm its in vivo safety. We monitored organ weights, hematologic parameters, and clinical chemical values in the blood of C57BL/6 mice receiving I.M. injections of EGFP mRNA⊂LNP 5097 or DPBS. At 2‐week intervals, EGFP mRNA⊂LNP 5097 (50 µg of mRNA per mouse, which is the maximum mass that does not induce LNP aggregation at 4 °C overnight) was injected twice into the mice (Figure  8A). After 2 weeks of the second injection, the mice were euthanized, and their organs and blood were collected. First, no significant differences were observed between the two groups in the weights, shapes or colors of the heart, liver, or spleen (Figure 8B–E). Conversely, the InLN near the injection site weighed 1.3 times more in the LNP 5097 group than in the DPBS group. Lymph node swelling is a typical response to many vaccines, typically occurring on the same side as the injection site.^{[ 36 ]} Such swelling generally diminishes over time, and the lymph node eventually returns to its original state.

Figure 8.

In vivo safety assessment of LNP 5097. A) Schematic illustration of the injection schedule. Seven‐week‐old C57BL/6 mice were administered prime/booster intramuscular (I.M.) injections of LNP 5097 encapsulating EGFP mRNA (50 µg) or DPBS. B‐E) Weights of the B) heart, C) liver, D) spleen, and E) inguinal lymph node (InLN) in the LNP 5097 and DPBS groups. F) Clinical chemistry values for liver‐related alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma glutamyl transferase (GGT), heart‐related lactate dehydrogenase (LDH), kidney‐related blood urea nitrogen (BUN) and creatinine (Crea), and pancreas‐related lipase (Lip) in the serum of the mice in the LNP 5097 and DPBS groups. Statistical significance was assessed by Tukey's paired comparison test: ^* p ≤ 0.05.

Subsequently, serum biochemical and hematological parameters were compared between the two groups. The values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma‐glutamyltransferase (GGT) fell within the normal range in both groups, indicating that LNP 5097 does not cause hepatotoxicity (Figure 8F). Although AST levels in both the DPBS and LNP 5097 groups remained within the normal range, the LNP 5097 group exhibited AST levels ≈2.7‐fold lower than those in the DPBS group. Since both values are within the normal range, this difference cannot be considered a meaningful change in liver function. Interestingly, similar reductions in AST have been observed in studies on the hepatoprotective effects of vitamin B5.^{[ 37 ]} Specifically, these studies have shown that vitamin B5 administration in normal rats led to approximately twofold decrease in AST levels compared to untreated controls. Given the findings that vitamin B5 significantly reduces AST levels in models of hepatotoxicity, suggesting a protective effect on the liver, it is plausible that I97, a vitamin B5‐derived ionizable lipid, may also provide hepatoprotective effects. However, further studies are needed to confirm this potential benefit and demonstrate functional improvements. The serum levels of lactate dehydrogenase (LDH, reflecting heart function), blood urea nitrogen and creatinine (BUN and Crea, reflecting kidney function), and lipase (Lip, reflecting pancreatic function) were similar to or lower than those in the DPBS group, suggesting the minimal impact of LNP 5097 on the heart, kidney, and pancreas (Figure 8F). Additionally, the hematological parameters from a complete blood count showed similar levels between for the LNP 5097 and DPBS groups, indicating that LNP 5097 is not hemotoxic as well (Table S5, Supporting Information). These serum biochemical and hematological results suggest that LNP 5097 is a safe carrier for delivering mRNA vaccines even at the highest LNP concentrations, exerting little effect on the body while stimulating an immune response.

3. Conclusion

We generated novel ionizable lipids derived from vitamin B5 with various ionizable heads, tails, linkages, and directionalities. Comprehensive assessments of all LNPs containing the novel ionizable lipids were carried out, focusing on their structural and physicochemical characteristics, as well as their in vivo mRNA transfection efficiency. The use of an ester bond to connect the ionizable lipid to the vitamin B5 core yielded an LNP with greater mRNA transfection efficiency than LNPs constructed with lipids employing an amide bond. Furthermore, the presence of a dimethylamino group as the ionizable head resulted in a more suitable pK_a for LNPs than the presence of a morpholino group. Detailed structural differences, such as linker length in the overall lipid structure, also influenced the properties of the LNPs and their mRNA transfection efficiency. The top‐performing LNP containing a novel vitamin B5‐derived ionizable lipid exhibited favorable attributes, including an appropriate pK_a and size, great monodispersity, excellent storage and lyophilization stability, the ability to induce a balanced Th1/Th2 immune response and in vivo safety. In particular, regarding the delivery of mRNA vaccines, the novel top‐performing LNP 5097 demonstrated better antigen expression efficiency from the delivered mRNA, the formation of more neutralizing antibodies, and the accurate delivery of mRNA to immune‐related organs compared to conventional LNPs containing SM‐102. Currently, in our laboratory, the structure of this vitamin B5‐derived ionizable lipid is being further optimized, as is the lipid composition of the LNPs. The optimized LNPs will be utilized for mRNA vaccine delivery research for the prevention and treatment of target diseases. It is anticipated that this delivery system could be used to safely deliver vaccines for the next generation of infectious diseases that may arise.

4. Experimental Section

Materials

All chemical reagents used for the synthesis of ionizable lipids were purchased from Sigma‒Aldrich (Burlington, MA 01803, United States), Tokyo Chemical Industry (Tokyo, Japan), Thermo Fisher Scientific (Waltham, Massachusetts, United States), and Combi‐Blocks (San Diego, CA, United States) and used without further purification.

SM‐102 was purchased from Hanmi Fine Chemical Co., Ltd. (Gyeonggi‐do, Republic of Korea). Cholesterol, trehalose, and sucrose were purchased from Sigma‒Aldrich. Trehalose‐6,6′‐dioleate (TDO) and n‐butyl lithocholate (L‐Bu) were synthesized following the previous report.^{[ 20 ]} 1,2‐Dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE), 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC), and 1,2‐dimyristoyl‐rac‐glycero‐3‐methoxypolyethylene glycol‐2000 (DMG‐PEG2000) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL 35007, United States).

Two types of RNA platforms were used: an internal ribosome entry site (IRES)‐based cap‐independent RNA platform for R/L mRNA and a cap‐dependent RNA platform for HA mRNA with capping.^{[ 20 ]} mRNA was generated from DNA templates through in vitro transcription using the EZTM T7 High‐Yield in vitro Transcription Kit (Enzynomics, Daejeon, Korea). The specific protocol is described in the Supplementary Information.

5‐Methoxyuridine‐modified CleanCap Enhanced Green Fluorescent Protein (EGFP) mRNA, 5‐methoxyuridine‐modified CleanCap F/L mRNA, and 5‐methoxyuridine‐modified CleanCap OVA mRNA were purchased from TriLink BioTechnologies (San Diego, CA, United States).

Dulbecco's phosphate‐buffered saline (DPBS) and high‐glucose Dulbecco's modified essential medium (DMEM) were purchased from WELGENE (Gyeongsangbuk‐do, Republic of Korea). A Cell Counting Kit‐8 (CCK‐8) was purchased from Dojindo Laboratories (Kumamoto, Japan). HepG2 cells, fibroblasts, HEK293 cells were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea).

Mice

Female ICR mice aged 6 weeks were obtained from Daehan Biolink and used to assess in vivo R/L mRNA delivery efficacy. BALB/c mice were used for immunization experiments. Mice were housed under specific pathogen‐free conditions with a 12/12 h light/dark cycle. These animal experimental procedures conducted on animals in this study followed the guidelines of and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (approval No. CUK‐IACUC‐2022‐024).

C57BL/6 female mice, aged 6–8 weeks, were obtained from JA BIO for the biodistribution, cancer vaccination, and in vivo safety experiments. These experiments involving animals were conducted in accordance with the applicable guidelines of the IACUC at the Korea Institute of Science and Technology (KIST) (approval Nos. KIST‐IACUC‐2023‐009‐3 and KIST‐IACUC‐2024‐003).

Synthesis of the Vitamin B5‐Derived Ionizable Lipids

The ionizable lipids (I71–I75, I81–I84, and I91–I98) were synthesized under mild conditions. Detailed synthetic methods are included in the Supplementary Information.

Formulation of the mRNA⊂LNPs

All lipid components were dissolved in a 1:1 mixture of chloroform and methanol at the concentration of 10 µg µL⁻¹. The lipid mixtures were prepared by mixing at the following specific molar ratios to formulate two types of LNPs: LNP 50 type (25:25:10:38.5:10, ionizable lipids:TDO:DOPE:L‐Bu:DMG‐PEG2000) and LNP C0 type (50:10:38.5:1.5, ionizable lipids:DSPC:cholesterol:DMG‐PEG2000). The lipid mixtures were concentrated under reduced pressure and redissolved in ethanol. The mRNA was dissolved in a pH 4.0 buffer (sodium citrate, 50 mm). The charge ratios (N/P) of lipids and mRNAs were 3 and 6 for the LNP 50 and LNP C0 types, respectively. LNPs were formulated using NanoAssemblr Spark (for less than 50 µg of mRNA, a mixing volume ratio of 1:2 for organic lipid and aqueous mRNA solutions) or NanoAssemblr Ignite (for more than 50 µg of mRNA, a total flow rate of 10 mL min⁻¹, a mixing volume ratio of 1:3 for organic lipid and aqueous mRNA solutions). The mRNA‐encapsulated LNPs were washed twice with DPBS (1X) and concentrated to 0.25 µg µL⁻¹ based on the amount of RNA obtained using an Amicon Ultra‐15 Centrifugal Filter with a LABOGENE 624R centrifuge (848 × g, 10 min, 4 °C).

Size and PDI of the mRNA⊂LNPs

The size and PDI of all the formulated LNPs were measured on a Zetasizer Ultra dynamic light scattering instrument. To measure the size and PDI, LNPs were diluted with DPBS (1X) to the concentration of 2.5 ng µL⁻¹ based on the amount of RNA.

Assays for Determining the pK_a of mRNA⊂LNPs

A 6‐(p‐toluidino)‐2‐naphthalene sulfonic acid sodium salt (TNS) solution (300 µm) and LNP solutions (1 mm) were prepared in DIW and DPBS (1×), respectively. A total of 18 buffers (20 mm sodium phosphate monobasic, 20 mm ammonium acetate, 25 mm sodium citrate, and 150 mm sodium chloride) were also prepared at pH 0.5 intervals from pH 2.5 to pH 11.0. After mixing the TNS solution with various pH buffers at a volume ratio of 1:48, 98 µL of the solution was dispensed into a black 96‐well plate. Two microliters of each LNP solution was added to buffer solutions containing TNS. The fluorescence intensity at 431 nm was recorded on a BioTek Cytation 5 cell imaging multimode reader at 25 °C using a λ_ex of 322 nm. TNS fluorescence intensities were normalized by the following equation: $100\times \frac{(\mathrm{F}.\mathrm{I}.-\mathrm{F}.\mathrm{I}{.}_{min})}{(\mathrm{F}.\mathrm{I}{.}_{Max}-\mathrm{F}.\mathrm{I}{.}_{min})}(\%)$, where F.I. = the measured fluorescence intensity of LNP solutions at each pH, F.I. _Max = the maximum fluorescence intensity of the measured values, and F.I. _min = the minimum fluorescence intensity of the measured values. For each LNP, the change in the normalized TNS fluorescence intensity according to pH was represented by the sigmoidal curve equation obtained by the KaleidaGraph program. The pK_a value was the pH corresponding to 50% of the difference between the F.I. _Max and F.I. _min on the curve.

mRNA Encapsulation Efficiency of the LNPs

The mRNA encapsulation efficiency of the LNPs was evaluated through gel retardation assays. The process involved preparing a 1% agarose gel by dissolving agarose in MOPS buffer in a microwave, cooling it to ≈50–60 °C, and then adding formaldehyde and Midori green dye. The solution was poured into a gel tray with the comb, and the gel was allowed to solidify for 40 min. To prepare mRNA standard solutions, the mRNA stock solution (50 µg mL⁻¹) was serially diluted with RNase‐free water. The formulated LNP solutions were then prepared. After the addition of loading dye (consisting of 36% glycerol and 1.2 × blue juice) to all the samples, the samples were loaded into wells with 1% agarose gel. The sample‐loaded gel was then immersed in 1X MOPS running buffer within the Mupid‐2plus electrophoresis system. The equipment ran for 7 min at full voltage (≈100 V). The fluorescence image of the gel was recorded using the GelDoc Go Imaging System in gel green mode.

In Vivo mRNA Transfection Efficiency

To compare LNP‐specific R/L expression, all LNPs encapsulating R/L mRNA (5 µg) were intradermally injected into the ears of the mice, female ICR mice aged 6 weeks were obtained from Daehan Biolink, using a 30G insulin syringe (BD, NJ, United States). The mice were anesthetized with 5% isoflurane prior to injection. After 6 h, the mice were sacrificed, and their ears were harvested. The harvested ears were placed in 300 µL of Renilla lysis buffer and finely homogenized using a homogenizer. After a brief 1‐min spin‐down, the R/L activity was measured using the R/L Assay system and the GloMax instrument, strictly following the manufacturer's instructions.

Analysis of the Immune Response Elicited by HA mRNA⊂LNP 5097

To start the experiment, HA mRNA (10 µg) formulated with LNPs (Con‐LNP, LNPs S050L, 5093, and 5095) was intramuscularly injected into the upper thigh muscle of 6‐week‐old BALB/c mice 2 weeks apart. Then serum samples were collected from the injected mice 2 weeks after the prime and booster injections following the protocol. To obtain the samples, blood was collected from the facial vein using an 18‐G needle or from the abdominal vena cava using a 1 mL syringe. Afterward, the samples were microcentrifuged at 6026 × g and 22413 × g for 15 min. Finally, the resulting serum was stored at −80 °C until use.

To measure antigen‐specific immunoglobulin G1 and IgG2a in mouse serum, enzyme‐linked immunosorbent assays (ELISAs) were used. First, the membranes of 96‐well plates (SPL, United States) were coated with 50 ng well⁻¹ of recombinant influenza A H1N1 (A/Puerto Rico/8/1934) hemagglutinin protein (11684‐V08H, Sino Biological, China) overnight at 4 °C. Then, the wells were blocked with 200 µL of blocking buffer (PBS containing 1% bovine serum albumin) for 1 h at room temperature. Diluted serum samples were added to the plates and incubated them for 1 h at room temperature. The wells were washed 3 times with 200 µL of PBS‐T (PBS containing 0.05% Tween 20). Anti‐mouse IgG1‐ and IgG2a‐conjugated antibodies (Invitrogen, Carlsbad, CA, United States, and Novus, Centennial, CO, United States) were diluted 1/1000–1/10000 in PBS and incubated for 1 h at room temperature. After 3 washes with PBS‐T, 50 µL of tetramethylbenzidine substrate (N301, Thermo Scientific, United States) was added for 15 min, and then, a 2 N H₂SO₄ aqueous solution was added to stop the reaction. The optical density (O.D.) values were measured at 450 nm by a GloMax Explorer microplate reader (Promega).

The hemagglutination inhibition assay was conducted following the guidelines outlined in the “Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza”.^{[ 38 ]} The mouse serum samples obtained were treated with receptor‐destroying enzyme (Denka Seiken, Tokyo, Japan) and then incubated overnight at 37 °C. Subsequently, the samples were inactivated at 56 °C for 30 min to prevent adsorption to chicken red blood cells (RBCs) and the induction of nonspecific reactions. The samples were serially diluted twofold with 25 µL of PBS in V‐bottom 96‐well microtiter plates. Afterward, they were incubated with a standardized virus suspension (4 HA U/25 µL) for 30 min at room temperature. Next, 50 µL of 0.5% chicken RBCs was added, and the mixture was incubated for 1 h at room temperature. The initial dilution was 1/10, so the lower limit of detectable antibody titers was 1:10. Titers below 1:10 were assigned a value of 1:5 for calculation. Antibody titers, expressed as the reciprocal of the highest serum dilution that showed complete inhibition of agglutination in duplicate experiments, are reported as geometric mean titers (GMTs).

Assessment of the Morphology of mRNA⊂LNP 5097

Cryo‐TEM images of the R/L mRNA⊂LNPs (Con‐LNP, LNP S050L, and LNP 5097; 0.5 mg mL⁻¹ based on the amount of RNA) were obtained on a FEI Tecnai G2 F20 instrument from the Advanced Analysis Center at the Korea Institute of Science and Technology (KIST, Seoul Headquarters, Republic of Korea).

Assessment of the Storage Stability of mRNA⊂LNP 5097

Con‐LNP, LNP S050L, and LNP 5097 were formulated with R/L mRNA (0.1 mg mL⁻¹ based the amount of RNA). The R/L mRNA⊂LNPs in pH 7.4 DPBS solution were aliquoted and stored at 4 °C. The size of the LNPs was measured using a Zetasizer Ultra at intervals of a week for 1 month.

LNP 5097 was formulated with EGFP mRNA, and the buffer was changed from DPBS to 20% sugar‐containing Tris buffer (pH 8.0; 10% trehalose and 10% sucrose). The EGFP mRNA⊂LNP 5097 solution was aliquoted into 30 µL portions and stored in a deep freezer at −80 °C or in a refrigerator at 2 to 8 °C. Portions of LNPs frozen at −80 °C were lyophilized overnight at −80 °C and 5 mTorr using a freeze dryer. The freeze‐dried LNPs were stored under refrigeration (2 to 8 °C) or frozen (−20 to −15 °C) conditions. For a period of 4 weeks, LNPs were prepared at one‐week intervals and stored according to the specified storage conditions. After the extended storage period, the sizes of all the LNPs under various storage conditions, as well as the size of freshly formulated LNP 5097, were measured using a Zetasizer Ultra (Malvern Panalytical. UK).

HEK293 cells were seeded at a density of 8 × 10⁴ cells mL⁻¹ (1 mL per well) in 24‐well plates and incubated overnight at 37 °C. All EGFP mRNA⊂LNP 5097 solutions (0.5 µg RNA per well) stored under various conditions were added to the cells. The growth media containing EGFP mRNA or DPBS was used as the negative control. The cells were harvested 24 h after treatment. in vitro EGFP expression efficiencies were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Inc., United States)

Biodistribution of F/L mRNA⊂LNP 5097

C57BL/6 mice (female, 7 weeks old, 16–18 g, n = 3 for each in vivo and ex vivo imaging) were injected with LNP 5097 encapsulating F/L mRNA (10 µg) intramuscularly into the right thigh or intravenously into the lateral tail vein. For in vivo imaging, a D‐luciferin solution (3.75 mg/100 µL per mouse) was injected intraperitoneally into the mice at 6 and 24 h after the injection of F/L mRNA⊂LNP 5097. Bioluminescence images of the dorsal, lateral, and ventral sides of the mice were obtained using an InVivo Smart‐LF Fluorescent and Bioluminescent Imaging and Analysis System (Vieworks, Republic of Korea). Ex vivo luminescence images were obtained at 6 h after the injection of F/L mRNA⊂LNP 5097. After injecting the D‐luciferin solution, the mice were euthanized, and the following organs were excised: the InLN, brain, heart, lung, liver, spleen, and kidney, as well as the leg thigh muscle. Luminescence images of the organs were obtained using an InVivo Smart‐LF Fluorescent and Bioluminescent Imaging and Analysis System (Vieworks, Republic of Korea).

Application of OVA mRNA⊂LNP 5097 for Cancer Vaccination

C57BL/6 mice (female, 7 weeks old, 16–18 g, n = 4) were injected subcutaneously with LNP 5097 encapsulating OVA mRNA (10 µg) twice at an interval of 4 days. Four days after the second dose, the right flanks of the mice were subcutaneously inoculated with E.G7‐OVA cancer cells (1 × 10⁶ cells). The mice that were injected twice with DPBS and LNP S050L encapsulating OVA mRNA (10 µg) were also inoculated in the same manner as the negative and positive control groups. Changes in the body weight and tumor volume of the mice in the three groups were observed at intervals of 2 to 3 days. The tumor volume was calculated using the following formula: tumor volume (mm³) = 0.5 × length × width². At the humane endpoint, the mice bearing tumors of more than 1500 mm³ were euthanized, and the tumor tissues were harvested and weighed.

Pharmacokinetics and Stability Assessment of I97

The Ames test, hERG channel patch clamping, a CYP inhibition test, and a microsomal stability test were performed at the New Drug Development Center at the Daegu‐Gyeongbuk Medical Innovation Foundation (KMEDIhub, Deagu, Republic of Korea). The detailed experimental methods are described in the Supplementary Information.

Assessment of LNP 5097 Cytotoxicity

Cell viability was assessed with a CCK‐8 assay. LNPs were formulated without mRNA in DPBS, and the resulting LNP 5097 solution was diluted to 2, 1.5, 1, 0.5, and 0.1 mm in DMEM containing 10% DPBS. HepG2 cells and fibroblasts (1 × 10⁴ cells well⁻¹) were seeded into 96‐well cell culture plates and incubated overnight at 37 °C in a CO₂ incubator. The cells were treated with the LNP solutions for 24 h, after which the solution was removed. Subsequently, a 10% CCK‐8 solution in DMEM was added to the cells. After incubation for 2 h in a CO₂ incubator, the O.D. at 450 nm was recorded using a Cytation 5 cell imaging multimode reader (Bio Tek Instruments, United States) at 25 °C. Cells treated with only DMEM were used as a control group.

In Vivo mRNA⊂LNP 5097 Toxicity Assay

C57BL/6 mice (female, 7 weeks old, 16–18 g, n = 7) were injected subcutaneously with LNP 5097 encapsulating GFP mRNA (50 µg) or DPBS twice at an interval of 2 weeks. Two weeks after the second dose, the mice were euthanized, and blood (≈400 µL) was collected. Two hundred microliters of the collected blood were placed in an ethylenediaminetetraacetic acid (EDTA) tube and mixed well. The remaining blood was incubated at room temperature for 2 h and then centrifuged at 3000 rpm for 10 min. The serum was obtained by collecting the supernatant of the centrifuged blood. Blood and serum samples were refrigerated and delivered to KP&T within 24 h. Hematology (CBC), a liver panel (ALT, AST, ALP, GGT, Alb), a cardiac panel (LDH), a renal panel (BUN and Crea), and a pancreas panel (lip) were performed by KP&T (Cheongju, Republic of Korea).

Statistical Analysis

All experimental data presented in bar graphs were expressed as the mean ± standard deviation (SD). Statistical significance between two groups was determined using Tukey's paired comparison test in Origin 2022 (OriginLab Inc., Northampton, MA, United States). Differences were considered statistically significant at ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001.

Conflict of Interest

A patent family application for the Republic of Korea (No. 10‐2023‐0136909), PCT (No. PCT/KR2023/016630), and the US (No. 18/536271) has been filed by the Korea Institute of Science and Technology and The Catholic University of Korea with E.K.B., G.K., M.F., S.P.K., S.Y., J.H.N., and S.B. listed as inventors; all patents in the family cover novel lipid compound and lipid nanoparticle composition comprising the same. These patents have been transferred to SML Biopharm Co., Ltd. Korea. J.H.N serves as the CEO of SML Biopharm Co., Ltd.

Author Contributions

S.Y., M.F., S.‐H.B., K.Y. and H.‐J. P. contributed equally to the work in this paper. M.F., G.K., and E.‐K.B. designed and synthesized the vitamin B5‐based ionizable lipids. S.Y., K.Y., and E.‐K.B. formulated the LNPs with mRNAs, analyzed the structural and physicochemical properties and biodistribution of the mRNA⊂LNPs, and evaluated the in vitro and in vivo toxicity of I97 and LNP 5097 and the applicability of LNP 5097 to cancer vaccination. S.‐H.B., H.‐J.P., and J.‐H.N. evaluated the in vivo mRNA transfection efficiency and immune responses of mRNA⊂LNPs, including the preparation of the assessed mRNAs. S.P.K., I.K.H., and H.J.K. formulated the LNPs with mRNAs and analyzed their structural and physicochemical properties and storage stability. S.Y. and E.‐K.B. drafted the manuscript. E.‐K.B., G.K., and J.‐H.N. organized the study.

Supporting information

Supporting Information

Yoo S., Faisal M., Bae S.‐H., Youn K., Park H.‐J., Kwon S. P., Hwang I. K., Lee J., Kim H. J., Nam J.‐H., Keum G., Bang E.‐K., Novel Less Toxic, Lymphoid Tissue‐Targeted Lipid Nanoparticles Containing a Vitamin B5‐Derived Ionizable Lipid for mRNA Vaccine Delivery. Adv. Healthcare Mater. 2025, 14, 2403366. 10.1002/adhm.202403366

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.