Charge-assisted stabilization of lipid nanoparticles enables inhaled mRNA delivery for mucosal vaccination

Fabrication of CAS-LNP with negative surface charge

mRNA, which is rich in negative charges due to the phosphate diester on nucleotide monomers, is mainly encapsulated within LNPs through electrostatic attraction with positively charged ionizable lipids. Therefore, if introducing another negatively charged lipid to impart a negative surface charge to LNPs, the group electronegativity and hydrophilicity of this lipid must be carefully considered. The negatively charged lipid could compete with mRNA for binding with ionizable lipid, thus interfering with mRNA encapsulation. In addition, lipids with weak hydrophilic head groups may have difficulty distributing to the LNP surface. Other than phosphate, carboxylic acid is another common negatively charged group in organisms. The pKa of phosphate diester (~ 1.5) is lower than that of carboxylic acid (~ 2–5). Therefore, the positively charged ionizable lipid should have a stronger affinity for binding to the phosphate groups of mRNA than carboxylic acid.

Among different sources of carboxylic acids, we selected amino acids due to their high biocompatibility and facile preparation²⁸. Through the design of amino acid sequences, peptides can effectively enhance hydrophilicity and adjust the charge properties. By linking peptides to long alkyl chain lipids to construct negatively charged peptide-lipid conjugate, it can stably bind to the surface of LNPs. Based on this, we designed the peptide sequence to be aspartic acid, serine, serine, and cysteine (DSSC) because DSSC has one cysteine group for easy conjugation with lipids, one aspartic acid-bearing carboxylic acid side chain to provide a negative charge, and two uncharged, hydrophilic serine to improve the hydrophilicity of peptide-lipid conjugate that drives its presence on the surface of LNP. We then used DSSC to synthesize a negatively charged, amphiphilic oligopeptide-helper lipid conjugate (Fig. 1b). Although PEG-lipid is on the surface of LNP, we did not conjugate DSSC to the PEG-lipid because the amount of PEG has been shown to greatly affect LNP stability during nebulization. To eliminate the effect of PEG on testing our hypothesis of charge-assisted stability of LNP, we chose 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to conjugate oligopeptide because DOPE has been shown to mainly reside on the outer membrane of LNP²⁹.In addition, the amino group of DOPE can be easily modified with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) to yield PDP-DOPE for subsequent conjugation with DSSC. The successful synthesis of PDP-DOPE and DSSC-DOPE was demonstrated using mass spectrometry (MS), ¹H nuclear magnetic resonance (NMR), and ultraviolet-visible spectroscopy (UV-Vis, Supplementary Figs. 1–3).

We adopted the LNP formulation utilized in the mRNA-1273 COVID-19 vaccine, comprising heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM-102) as ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphochline (DSPC) as helper lipid, cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), at a molar ratio of 50:10:38.5:1.5, respectively (SM102-LNP)³⁰. To create CAS-LNP, DSSC-DOPE was incorporated into SM102-LNP by replacing equimolar amounts of DSPC during microfluidic mixing with mRNA encoding firefly luciferase (mFluc). We integrated different amounts of DSSC-DOPE (0.6%, 2.5%, and 10% of all lipids by mole) to modulate the surface charge of CAS-LNP (Fig. 1c). Dynamic light scattering (DLS) measurements showed that CAS-LNPs and SM102-LNP have similar hydrodynamic diameters ranging from 130 to 140 nm with narrow size distributions (Fig. 1d). The mRNA encapsulation efficiencies for both CAS-LNPs and SM102-LNP exceeded 80% (Fig. 1e). Cryogenic transmission electron microscopy (Cryo-TEM) image depicted a spherical morphology of 2.5% CAS-LNP with a solid core (Fig. 1f). These results suggest that the incorporation of DSSC-DOPE did not affect the assembly of LNP and the overall mRNA encapsulation. The pKa of 2.5% CAS-LNP is similar to that of SM102-LNP (Fig. 1g). The 2.5% CAS-LNP maintained its colloidal stability in PBS at 37 °C (Supplementary Fig. 4). Zeta-potential measurements indicated that SM102-LNP possessed a nearly neutral surface charge (− 2.39 mV), consistent with previous findings³¹. The surface potential of CAS-LNPs gradually declined with an increase in the amount of DSSC-DOPE (Fig. 1h), confirming the successful integration of DSSC-DOPE onto the outer membrane of LNP. The zeta-potential data also suggest that the negative charges of DSSC are not entirely shielded by the surface PEG.

Stability and mRNA delivery efficacy of inhaled CAS-LNP

We next evaluated whether CAS-LNP could maintain colloidal stability under high shear force during nebulization. While most LNPs used for intravenous or intramuscular administration are dispersed in phosphate-buffered saline (PBS)³², the high ion concentration in PBS could shield electrostatic repulsions among CAS-LNPs. To mitigate this effect, we dispersed all LNPs in 0.3 × PBS to reduce the solvent’s ionic strength. We employed a clinically used vibrating mesh nebulizer (Aerogen Solo) that utilizes high-frequency oscillation of a porous metal mesh to aerosolize SM102-LNP or CAS-LNP solutions. To evaluate LNP stability during nebulization, we quantified the percentage of intact LNPs by measuring the amount of encapsulated mRNA before and after nebulization. As shown in Fig. 1i, ~ 17% of intact SM102-LNP was detected after nebulization, suggesting significant disintegration and mRNA leakage. In contrast, all CAS-LNPs exhibited improved stability compared to SM102-LNP, suggesting that increasing LNP surface charge is an effective strategy to enhance its colloidal stability. Interestingly, despite CAS-LNP with 10% of DSSC-DOPE (10% CAS-LNP) having the lowest surface potential, its stability was lower than that of 2.5% CAS-LNP and comparable to that of 0.6% CAS-LNP. Previous studies indicated that LNPs formulated with DSPC exhibit higher stability than those with DOPE due to the higher melting point and fully saturated alkyl chain of DSPC³³. These findings suggest that DSPC also contributes to LNP stability during nebulization, but its impact is overshadowed by that of surface charges. Therefore, 2.5% CAS-LNP, which possesses an optimized ratio of DSPC and charged DSSC-DOPE, exhibited the highest stability during nebulization.

We then studied whether the enhanced stability of CAS-LNP during nebulization could improve its mRNA delivery efficacy in mice. To ensure consistent dosing across all groups, equal amounts of SM102-LNP or CAS-LNPs encapsulating mFluc in 0.3 × PBS were nebulized, collected, and administered to mice via oropharyngeal aspiration (Fig. 2a). Mice were imaged by an in vivo imaging system (IVIS) at various time points post-administration. As shown in Fig. 2b and Supplementary Fig. 5, SM102-LNP and CAS-LNPs exhibited similar kinetics of mRNA expression, which peaks at 6 h and gradually declines thereafter. All CAS-LNPs displayed higher luminescence intensity in the lung area compared to SM102-LNP. Quantitative analysis (Fig. 2c) demonstrated that 2.5% CAS-LNP exhibited the highest mRNA expression among all groups. Ex vivo imaging of major organs at 24 h post-administration (Fig. 2d and Supplementary Fig. 6) revealed exclusive mRNA expression in the lung for all LNPs, affirming the efficiency of inhalation as a lung-targeted delivery method with minimal off-target expression. Further quantitative analysis of excised lungs (Fig. 2e) showed a 6.9-fold increase in mRNA expression with 2.5% CAS-LNP compared to SM102-LNP. These findings indicate a direct correlation between the improved colloidal stability of CAS-LNP during nebulization and its enhanced mRNA delivery efficacy.

a Workflow for evaluating mRNA expression of nebulized LNPs in mice. b Representative IVIS images and (c) quantitative analysis of luminescence in mice treated with SM102-LNP or CAS-LNPs in 0.3 × PBS at different time points. d Representative IVIS images of major organs (heart, liver, spleen, lung, and kidney) and (e) quantitative analysis of lungs at 24 h post-administration. Each dose contains 1 µg of mFluc per mouse (n = 5 biologically independent samples). Data are shown as mean ± standard error of the mean (SEM). Statistical significance was analyzed by two-way (c) or one-way (e) ANOVA and Tukey’s multiple comparisons test. f Photograph of custom apparatus for inhaled administration to three mice simultaneously. g Representative IVIS images of major organs and (h) quantitative analysis of lungs treated with SM102-LNP or 2.5% CAS-LNP in 0.3 × PBS at 24 h post-administration (n = 3 biologically independent samples). Data are shown as mean ± SD. Statistical significance was analyzed by unpaired two-tailed Student’s t test. i Percentage of intact LNPs after nebulization in PBS of varying ionic strength (n = 3 technical replicates). Data are shown as mean ± SD. j Representative IVIS images and (k) quantitative analysis of luminescence in mice treated with SM102-LNP or 2.5% CAS-LNP in 1 × or 0.1 × PBS at 6 h post-administration. l Representative IVIS images of major organs and (m) quantitative analysis of lungs at 24 h post-administration (n = 5 biologically independent samples). Data are shown as mean ± SEM. Statistical significance was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. Figure 2a was created in BioRender. Lu, X. (2024) BioRender.com/y08x203. Source data are provided as a Source Data file.

Full size image

To compare the oropharyngeal aspiration method of nebulized solution with inhalation and further validate the enhanced mRNA delivery of CAS-LNP post-inhalation, we connected the nebulizer to a custom apparatus that allowed simultaneous inhalation by three mice (Fig. 2f). Subsequently, nebulized SM102-LNP or 2.5% CAS-LNP containing 100 µg of mFluc was administered. The lungs of mice were excised at 24 h post-inhalation and imaged using IVIS. As shown in Fig. 2g, h and Supplementary Fig. 7, CAS-LNP exhibited a 4.0-fold increase in mRNA expression compared to SM102-LNP, consistent with the results obtained with the oropharyngeal aspiration of nebulized LNP solution (Fig. 2e). These results not only affirm the feasibility of our protocol for evaluating inhaled mRNA delivery of LNP but also reinforce the improved mRNA delivery efficacy of CAS-LNP.

Nevertheless, it is important to rule out the possibility that the increased mRNA delivery efficacy is solely due to changes in LNP formulation, regardless of stability during nebulization. To address this, we administered pre-nebulized 2.5% CAS-LNP or SM102-LNP to mice via intravenous injection. IVIS images (Supplementary Fig. 8) indicated similar biodistribution patterns for both 2.5% CAS-LNP and SM102-LNP, with the liver showing the highest mRNA expression. However, SM102-LNP displayed approximately a 2.3-fold higher mRNA expression compared to 2.5% CAS-LNP, suggesting that the incorporation of DSSC-DOPE negatively affected the mRNA expression of SM102-LNP prior to nebulization. This observation is reasonable considering that CAS-LNP possesses more negative charges, which may exhibit reduced cellular uptake. In vitro cellular uptake studies using dendritic cells indicated that 2.5% CAS-LNP displayed a 2.4-fold lower cellular uptake compared to SM102-LNP (Supplementary Fig. 9). CAS-LNP and SM102-LNP exhibited similar pKa (Fig. 1g), suggesting that DSSC may not affect the endosomal escape of LNP. Indeed, hemolysis study of red blood cell (RBC) indicated that 2.5% CAS-LNP possesses similar membrane disruption capability at endosomal pH with that of SM102-LNP (Supplementary Fig. 10). To better understand the influence of DSSC-DOPE on the cellular uptake, dendritic cells were treated with small-molecule inhibitors of endocytosis, macropinocytosis, and phagocytosis prior to incubation with LNPs. As shown in Supplementary Fig. 11, macropinocytosis was identified as the major pathway for SM102-LNP uptake, consistent with previous reports^34,35. CAS-LNP enters cells through both macropinocytosis and caveolae-mediated endocytosis, which is likely due to the different charges and DSSC modification on the surface.

Taken together, these data demonstrate that the effective inhaled delivery of mRNA by CAS-LNP is dependent on its colloidal stability during nebulization, which compensates for the reduced cellular uptake. These findings emphasize a fundamental difference in LNP design principles for nebulized delivery compared to systemic injections. While the efficacy of LNP following intravenous or intramuscular administrations largely hinges on their cellular uptake and endosomal escape, inhaled LNPs necessitate a delicate balance between their colloidal stability during nebulization and subsequent interactions with cells.

Mechanism of the enhanced delivery efficiency of CAS-LNP

To explore whether the improved stability of inhaled CAS-LNP is due to its surface charge, we first evaluated CAS-LNP stability during nebulization in PBS with varying salt concentrations. The presence of salt can screen electrostatic repulsions among LNPs, potentially compromising the enhanced stability conferred by surface charges. As expected, all CAS-LNPs exhibited nearly identical stability to SM102-LNP in 1 × PBS, indicating the complete loss of charge-assisted stabilization during nebulization (Fig. 2i). However, as we gradually decreased salt concentrations, we observed a progressively more substantial increase in the percentage of intact CAS-LNPs compared to SM102-LNP after nebulization. We then investigate the inhaled delivery efficacy of SM102-LNP and 2.5% CAS-LNP in 0.1 × and 1 × PBS in mice. While SM102-LNP showed low and similar mRNA expression in both solutions, 2.5% CAS-LNP in 0.1 × PBS exhibited robust luminescence signals in the lung (Fig. 2j–m, Supplementary Figs. 12 and 13). Quantitative analysis of mice and excised lungs revealed that 2.5% CAS-LNP in 0.1×PBS achieved approximately 5.5-fold (Fig. 2k) and 20.3-fold (Fig. 2m) higher mRNA expression, respectively, compared to SM102-LNP in 0.1 × PBS. However, mRNA expression significantly reduced when 2.5% CAS-LNP was used in 1 × PBS. Collectively, these findings demonstrate that the colloidal stability of CAS-LNP during nebulization originates from its surface charge. 2.5% CAS-LNP is highly effective for inhaled mRNA delivery when used in a solution with low ionic strength. Therefore, we selected 2.5% CAS-LNP in 0.1 × PBS for further investigation and referred to it as CAS-LNP unless specifically stated otherwise.

To validate the design principles of DSSC that has to carry carboxylic acid groups as the source of negative charge and sufficient hydrophilicity to present on the LNP surface, we tried different negatively charged molecules including 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (bearing a phosphate monoester, Fig. 3a) and heptadecanoic acid (HA) (bearing a carboxylic acid group, Fig. 3b) as alternatives. We replaced DSSC-DOPE in CAS-LNP with an equivalent amount of DOPA or HA to yield DOPA-LNP or HA-LNP, respectively. All LNPs were successfully synthesized with uniform sizes (Supplementary Fig. 14a). However, neither DOPA nor HA improved the stability of LNP during nebulization or nebulized delivery efficiency (Fig. 3d–f and Supplementary Fig. 14c). Specifically, the use of DOPA reduced mRNA encapsulation and exhibited only slightly declined surface potential (− 4.33 mV, Fig. 3c and Supplementary Fig. 14b), suggesting that the phosphate monoester of DOPA may compete with mRNA for binding with ionizable lipids. On the other hand, the incorporation of HA did not affect mRNA encapsulation efficacy (Supplementary Fig. 14b) and the surface potential of LNP (− 2.89 mV, Fig. 3c), suggesting that the hydrophilicity of the carboxylic group of HA was insufficient to allow for its arrangement on the surface of LNPs. Consequently, HA was incorporated into the core of LNPs, failing to impart surface charge to LNPs. Upon modifying HA with DSSC peptide (Fig. 3b and Supplementary Fig. 15), the resulting HA-DSSC conjugate effectively decreased the surface potential of HA-DSSC-LNP (− 9.43 mV, Fig. 3c), significantly enhanced LNP stability during nebulization (Fig. 3d) and mRNA expression after nebulization (Fig. 3e, f and Supplementary Fig. 14c). These results underscore the importance of careful design when selecting negatively charged molecules, which should possess ideal pKa and hydrophilicity.

a Chemical structures of DOPA, HA, and HA-DSSC (b). c Zeta potentials of SM102-LNP, 2.5% DOPA-LNP, 2.5% HA-LNP, and 2.5% HA-DSSC-LNP (n = 3 technical replicates). Data are shown as mean ± SD. d Percentage of intact LNPs after nebulization in 0.1 × PBS (n = 3 technical replicates). Data are shown as mean ± SD. e Representative IVIS images and (f) quantitative analysis of luminescence in mice at 6 h post-administration (n = 3 biologically independent samples). Data are shown as mean ± SEM. Statistical significance was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. g Chemical structures of DSSC, SSSC, DDSC, and DDDC. h Isoelectric points of DSSC, SSSC, DDSC, and DDDC. i Zeta potentials of CAS-LNP, SSSC-LNP, DDSC-LNP, and DDDC-LNP (n = 3 technical replicates). Data are shown as mean ± SD. j Percentage of intact LNPs after nebulization in 0.1 × PBS (n = 3 technical replicates). Data are shown as mean ± SD. k Representative IVIS images of lungs and (l) quantitative analysis at 24 h post-administration (n = 5 biologically independent samples). Data are shown as mean ± SEM. m Chemical structures of D_ESSC-DOPE and D_ESSC_E-DOPE. Either one or both carboxyl groups of DSSC were converted to methyl esters. n Zeta potentials of CAS-LNP, D_ESSC-LNP, and D_ESSC_E-LNP (n = 3 technical replicates). Data are shown as mean ± SD. o Percentage of intact LNPs after nebulization in 0.1 × PBS (n = 3 technical replicates). Data are shown as mean ± SD. p Representative IVIS images and (q) quantitative analysis of luminescence in mice treated with CAS-LNP, D_ESSC-LNP, or D_ESSC_E-LNP at 6 h post-administration (n = 5 biologically independent samples). Data are shown as mean ± SEM. r Representative IVIS images of major organs and (s) quantitative analysis of lungs at 24 h post-administration (n = 5 biologically independent samples). Data are shown as mean ± SEM. The statistical significance of this figure was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Full size image

To further validate the CAS strategy, we designed three peptides—SSSC, DDSC, and DDDC—carrying one, three, and four carboxylic acid groups, respectively (Fig. 3g). The calculated isoelectric points (pIs) of SSSC, DSSC, DDSC, and DDDC were 5.70, 3.57, 3.51, and 3.49, respectively (Fig. 3h). All these peptides have pIs below 5.7, indicating a negative charge in 0.1 × PBS (pH = 7.4). We synthesized SSSC-DOPE, DDSC-DOPE, and DDDC-DOPE, and used them as replacements for DSSC-DOPE to fabricate SSSC-LNP, DDSC-LNP, and DDDC-LNP, respectively (Supplementary Fig. 16). All LNPs were successfully synthesized with uniform sizes and high mRNA encapsulation efficiencies (Supplementary Fig. 17). The surface potential gradually decreased with the increasing number of carboxylic acid groups (Fig. 3i). These LNPs exhibited greatly enhanced stability during nebulization and inhaled mRNA delivery efficiency compared to SM102-LNP (Fig. 3j–l). While SSSC-LNP showed slightly lower stability, likely due to its less negative surface charge (− 7.6 mV), this did not significantly affect overall mRNA delivery post-inhalation. These results suggest a correlation between the surface charge and LNP stability, with higher negative surface charges providing better stability. However, when the surface charge of LNPs reaches a certain threshold, further increases in negative charge do not result in additional improvements in stability or mRNA delivery efficacy. Collectively, ensuring sufficient negative surface charge is critical for the charge-assisted stability of LNP.

To investigate the role of carboxyl groups on DSSC in conferring negative charges to CAS-LNP, we performed esterification on one carboxyl group of aspartic acid or both carboxyl groups of aspartic acid and cysteine to yield D_ESSC-DOPE or D_ESSC_E-DOPE, respectively (Fig. 3m). UV-Vis and MS measurements confirmed the successful synthesis of both compounds (Supplementary Figs. 18 and 19). Subsequently, we replaced DSSC-DOPE in CAS-LNP with an equivalent amount of D_ESSC-DOPE or D_ESSC_E-DOPE to yield D_ESSC-LNP or D_ESSC_E-LNP, respectively. All LNPs were successfully synthesized with uniform sizes and high mRNA encapsulation efficiencies (Supplementary Fig. 20). The surface potential gradually increased with the increasing number of esterified carboxylic acid groups (Fig. 3n). We then evaluated their stability during nebulization and subsequent mRNA delivery efficacy in vivo. D_ESSC-LNP, bearing one carboxyl group, exhibited slightly decreased stability (Fig. 3o) and mRNA expression compared to CAS-LNP (Fig. 3p–s and Supplementary Figs. 21 and 22), indicating that even a single carboxyl group contributes to LNP stability. In contrast, despite the presence of a phosphate group on DOPE, D_ESSC_E-LNP, with both carboxyl groups blocked, exhibited a marked decrease in stability during nebulization and reduced mRNA delivery efficacy. Both D_ESSC-LNP and SSSC-LNP, which contain one phosphate group on DOPE and one carboxyl group on the peptide, exhibited lower surface potential and greatly improved stability compared to D_ESSC_E-LNP. HA-DSSC-LNP, which contains two carboxyl groups but lacks the phosphate group, also demonstrated comparable zeta potential, stability, and inhaled mRNA delivery efficiency with DOPE-DSSC-LNP. Collectively, these data demonstrate that the carboxyl groups on peptides are crucial for providing the negative surface charges of CAS-LNP, thereby enhancing its stability during nebulization and overall delivery efficacy. While the phosphate group on DOPE may contribute some surface charge to CAS-LNP, its overall impact on stability and delivery efficacy is limited.

To determine whether the enhanced stability of CAS-LNP during nebulization stems from the improved stability of individual LNPs or from the electrostatic repulsions among LNPs, we characterized the mechanical properties of CAS-LNP and SM102-LNP using liquid-phase atomic force microscopy (AFM). Our results showed that Young’s modulus and the maximum force required to break individual CAS-LNP and SM102-LNP are nearly identical (Fig. 4a–d), indicating that the inclusion of negatively charged peptide-lipid conjugate (DSSC-DOPE) had no discernible impact on the mechanical stability of individual LNPs. Therefore, instead of improving the mechanical stability of individual LNPs, the addition of DSSC-DOPE enhances the electrostatic repulsions among LNPs, thus preventing LNPs from aggregation during nebulization.

a Representative aqueous AFM amplitude images and (b) force curves of SM102-LNP and CAS-LNP. c Young’s modulus and (d) the maximum force required to break LNP calculated from the force curves of individual LNP (n = 23 technical replicates). Data are shown as mean ± SD. Statistical significance was analyzed by unpaired two-tailed Student’s t test. e Zeta potentials of MC3-LNP, MC3-CAS-LNP, ALC0315-LNP, and ALC0315-CAS-LNP (n = 3 technical replicates). Data are shown as mean ± SD (f) Percentage of intact LNPs after nebulization (n = 3 technical replicates). Data are shown as mean ± SD. The statistical significance was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. g Representative IVIS images of major organs and (h) quantitative analysis of lungs at 24 h post-administration (n = 4 biologically independent samples). Data are shown as mean ± SEM. The statistical significance was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. i IVIS images of tracheas and lungs of dog and pig (j) at 3 h post-administration. Dog and pig were administered with 0.3 mg/kg of mFluc through the Aerogen Solo nebulizer with a custom nose cone. Figure 4i and j were created in BioRender. Lu, X. (2024) BioRender.com/s18b330. Source data are provided as a Source Data file.

Full size image

CAS is a universal strategy

To evaluate the universality of the CAS strategy, we applied the CAS strategy to other clinical LNP formulations utilized in the patisiran for the treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis (MC3-LNP) and the BNT162b2 COVID-19 vaccine (ALC0315-LNP), respectively. MC3-CAS-LNP and ALC0315-CAS-LNP were fabricated by incorporating 2.5% DSSC-DOPE to replace equimolar amounts of DSPC (Supplementary Fig. 23a). Zeta-potential measurements (Fig. 4e) indicated that MC3-LNP and ALC0315-LNP also possessed nearly neutral surface charges (− 3.99 mV and − 3.92 mV, respectively), and the surface potentials of MC3-CAS-LNP and ALC0315-CAS-LNP decreased (− 11.33 mV and − 9.14 mV). We then evaluated their stability during nebulization and subsequent mRNA delivery efficacy in vivo. While MC3-LNP and ALC0315-LNP are unsuitable for nebulized delivery, MC3-CAS-LNP and ALC0315-CAS-LNP exhibited significantly enhanced stability during nebulization (Fig. 4f) and mRNA expression compared to their original formulations (Fig. 4g, h and Supplementary Fig. 23b). These data clearly demonstrate that the CAS strategy can be easily applied on other LNP formulation, making it an ideal strategy to convert LNPs that are originally unsuitable for nebulized delivery into an inhalable formulation.

Furthermore, we have compared the mRNA delivery efficacy of CAS-LNP with recently reported inhaled formulations (Supplementary. Fig. 23a). Our results showed that CAS-LNP shows significantly higher mRNA delivery efficacy compared to T1-5²⁵, NLD1⁹ and LNP2-2³⁶ (Supplementary. Fig. 23b, c). It is worth noting that those inhaled formulations all utilize the positively charged DOTAP lipid (28% in T1-5, 5% in NLD1, and 50% in LNP2-2). DOTAP carries a quaternary amine that has a stronger binding affinity to mRNA compared to the tertiary amine of ionizable lipids. Therefore, DOTAP mainly resides inside of LNP. For example, SORT LNPs using 10% or 40% DOTAP by mole showed surface potential of ~ 1.34 and 7.57 mV³⁷. Lung SORT LNP with 50% DOTAP showed a surface potential of − 0.89 mV³⁸. These results suggest that adding DOTAP did not introduce a surface charge to LNP. DOTAP may improve pulmonary delivery of LNP through another mechanism that is still not fully elucidated.

Inhaled CAS-LNP delivers mRNA to the lungs of large animals

We next studied whether CAS-LNP could efficiently deliver mRNA in larger animals, which possess more comparable physiology of respiratory system to humans than mice^39,40. We first compared the mRNA expression efficiency in Bama miniature pigs after inhalation of SM102-LNP and CAS-LNP. The pigs received 0.3 mg/kg of mFluc in LNPs via nebulization. Three hours post-administration, the trachea and lungs were isolated after intraperitoneal injection of luciferin for subsequent bioluminescence imaging. As depicted in Fig. 4i, the expression of mFluc in the CAS-LNP-treated group is higher than that in the SM102-LNP-treated group. Furthermore, we validated that CAS-LNP can also successfully transfect the lung in Beagle dogs (Fig. 4j). These results demonstrated that CAS-LNP could efficiently deliver mRNA through inhalation across different species, including mouse, dogs, and pigs, suggesting its great potential for further clinical translation.

Inhaled CAS-LNP penetrates the mucus layer and transfects immune cells

After nebulization, CAS-LNPs need to traverse the mucus layer to reach lung cells for mRNA expression⁴¹. Given the preserved structural integrity of CAS-LNP during nebulization, we hypothesized that the PEG-coating of intact CAS-LNP could facilitate its trafficking across the mucus barrier⁴². To investigate this, we encapsulated Cy5-labeled mRNA encoding enhanced green fluorescent protein (Cy5-mEGFP) in CAS-LNP or SM102-LNP. These LNPs were nebulized, collected, and administered to mice through oropharyngeal aspiration. The whole lungs were isolated at 30 min post-administration and imaged by IVIS and three-dimensional fluorescent imaging. As shown in Fig. 5a, b, and Supplementary Fig. 24, CAS-LNP treated mouse exhibited greatly stronger Cy5 and EGFP fluorescence in the parenchyma than SM102-LNP. We then separated the tracheas and lungs and dissected them into thin slices for confocal laser scanning microscopy. As shown in Fig. 5c–f and Supplementary Figs. 25 and 26, both Cy5 and EGFP signals in lung parenchyma were exclusively found in the CAS-LNP treated group. This suggests effective penetration of the mucus layer by CAS-LNP, leading to EGFP expression in pulmonary cells. In contrast, strong Cy5 signals from the trachea and minimal Cy5 and EGFP signals from parenchyma were observed in the SM102-LNP treated group. Such observation is likely due to the leakage of Cy5-mEGFP from SM102-LNP during nebulization. Leaked mRNA can barely penetrate the mucus layer and is prone to degradation by enzymes or clearance from the airway via mucociliary clearance⁴³.

Three-dimensional fluorescent imaging of lungs in mice treated with inhaled SM102-LNP (a) or CAS-LNP (b) encapsulating Cy5-mEGFP at 30 min post-administration. Green and red signals represent EGFP and Cy5, respectively. Representative confocal microscopy images of excised tracheas (c) and lungs (e). Quantitative analysis of Cy5 fluorescence intensity in trachea (d) (n = 8 technical replicates) and pulmonary parenchyma (f) (n = 10 technical replicates). Data are shown as mean ± SD. Statistical significance was analyzed by unpaired two-tailed Student’s t test. g Treatment scheme for evaluating the mRNA expression in different cell types of inhaled CAS-LNP or intravenously injected IV-LNP. Each mouse received 5 µg of mCre per dose. h Schematic illustrating that the expression of Cre recombinase deletes the stop cassette and activates the expression of tdTomato in C57BL/6-Rosa26-CAG-LSL-tdTomato mice. i Representative flow cytometry measurements and quantitative analysis (j) of immune cells (CD45), endothelial cells (CD31), and epithelial cells (CD326) expressing tdTomato in the lungs after different treatments (n = 3 biologically independent samples). Data are shown as mean ± SEM. k The percentage of tdTomato + dendritic cell, alveolar macrophage, interstitial macrophage, neutrophil, B cell, T cell, and NK cell among immune cells of CAS-LNP treated lungs (n = 4 biologically independent samples). Figure 5c and e were created in BioRender. Lu, X. (2024) BioRender.com/m16g625. Figure 5g was created in BioRender. Lu, X. (2024) BioRender.com/j51w648. Figure 5h was created in BioRender. Lu, X. (2024) BioRender.com/m96i222. Source data are provided as a Source Data file.

Full size image

Recent studies have shown successful lung-targeted mRNA delivery through intravenous injection using diverse LNP formulations^44,45. However, it is important to note that the specific lung cell subtype targeted by intravenously administered LNPs may differ substantially from those of inhaled LNPs, as the former reaches the lungs through the bloodstream, while the latter enters through the airway. To identify the specific lung cell subtype transfected by intravenously injected LNP (IV-LNP) and inhaled CAS-LNP, we synthesized a lung-targeting IV-LNP following a previously published formulation⁴⁴. The lung-targeted mRNA expression of IV-LNP was validated (Supplementary Fig. 27). Subsequently, we encapsulated mRNA encoding Cre recombinase (mCre) into CAS-LNP or IV-LNP and administered the same doses of mCre to C57BL/6-Rosa26-CAG-LSL-tdTomato mice via inhalation or intravenous injection, respectively (Fig. 5g). These mice possess a loxP-flanked stop cassette controlling the expression of the fluorescent tdTomato protein, which is activated only when Cre recombinase is present (Fig. 5h). Thus, cells with mCre expression produce tdTomato fluorescence.

After administering three doses of LNPs, we analyzed lung cells with mCre expression using flow cytometry, with specific markers for immune (CD45), endothelial (CD31), and epithelial (CD326) cells (Supplementary Fig. 28). As shown in Fig. 5i, j, IV-LNP exhibited exclusive mRNA expression in endothelial cells, with minimal expression in immune and epithelial cells. In contrast, inhaled CAS-LNP showed the highest mRNA expression in immune cells, intermediate expression in epithelial cells, and minimal expression in endothelial cells. We further analyzed the subpopulations of CAS-LNP transfected immune cells (Supplementary Figs. 29 and 30). As shown in Fig. 5k and Supplementary Fig. 31, over 60% of tdTomato + immune cells are dendritic cells (DCs), the primary antigen-presenting cells, making CAS-LNP an ideal candidate for delivering mRNA vaccines. Inhaled CAS-LNP also showed mRNA expression in neutrophils (~ 5.3%), alveolar macrophages (~ 2.6%), B cells (~ 2.3%), T cells (~ 1.6%), and slight mRNA expression in interstitial macrophages, and natural killer (NK) cells. These results showed the distinct transfection profiles of IV-LNP and inhaled CAS-LNP, highlighting the importance of considering the administration route when utilizing LNPs for mRNA delivery to the lung. The choice of LNP formulation should be carefully tailored to the specific lung cell types targeted. IV-LNP has implications where precise delivery to endothelial cells is desired, whereas inhaled CAS-LNP holds great potential for the development of vaccines or other mRNA-based therapies aimed at modulating immune responses.

Inhaled CAS-LNP induced strong systemic and mucosal immune responses

To assess the potential of CAS-LNP as an inhaled vaccine for COVID-19, we synthesized mRNA encoding the spike protein of the SARS-CoV-2 Omicron variant (mCOVID) and encapsulated it into CAS-LNP or SM102-LNP. These LNPs were then nebulized, collected, and administered through oropharyngeal aspiration to female C57BL6 mice on days 0, 14, and 28, with each dose containing 5 µg of mCOVID per mouse (Fig. 6a). Mice receiving inhaled PBS were used as the controls. Subsequently, we isolated serum, bronchoalveolar lavage fluid (BALF), and lung tissues to analyze the systemic and mucosal immune responses one week after the third immunization. We first evaluated the antigen-specific total IgG antibodies in serum by enzyme-linked immunosorbent assay (ELISA). CAS-LNP induced higher total IgG responses compared to SM102-LNP (Fig. 6b and Supplementary Fig. 32). We then analyzed the neutralizing ability of serum using an Omicron pseudovirus-based assay. Notably, mice vaccinated with CAS-LNP exhibited significantly enhanced neutralizing ability compared to those receiving SM102-LNP and PBS treatments (Fig. 6c), indicating the effectiveness of CAS-LNP in inducing a systemic humoral immune response.

a Vaccination regimen in mice. CAS-LNP or SM102-LNP containing mCOVID were nebulized and administered to mice on days 0, 14, and 28. Each dose contains 5 µg of mCOVID per mouse. b ELISA analysis of Omicron spike protein-specific IgG in serum and IgA in BALF (d) from mice treated with PBS, SM102-LNP, or CAS-LNP. The virus-neutralizing ability of serum (c) and BALF (e) was measured using an Omicron pseudovirus assay in ACE2-expressing HEK-293T cells (n = 5 biologically independent samples). Data are shown as mean ± SD. f Optical images and (g) quantitative analysis of IFN-γ-spot-forming cells via ELISpot assay. Lung cells of mice were plated and stimulated with an Omicron peptide pool (n = 5 biologically independent samples). Data are shown as mean ± SD. Number of CD4 + (h) and CD8 + (k) T cells in the lung of mice (n = 5 biologically independent samples). Representative flow cytometry plots and quantitative analysis of CD4 + (i, j) (n = 5 biologically independent samples) and CD8 + (l, m) (n = 5 biologically independent samples for PBS and SM102-LNP treated groups, n = 4 biologically independent samples in CAS-LNP treated group) T_RM cells among cells in the lung of mice. Flow cytometry analysis showing the number of CD8 + T cells (n) (n = 5 biologically independent samples) and CD8 + T_RM cells (o, p) (n = 5 biologically independent samples for PBS and SM102-LNP treated groups, n = 4 biologically independent samples for CAS-LNP treated group) in BALF. Data are shown as mean ± SD. Statistical significance was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. Figure 6a was created in BioRender. Lu, X. (2024) BioRender.com/c71w817. Figure 6f was created in BioRender. Lu, X. (2024) BioRender.com/a39t997. Source data are provided as a Source Data file.

Full size image

COVID-19 viruses enter the bloodstream via the respiratory tract. Mucosal IgA plays a pivotal role as the initial defense against viruses attempting entry through the respiratory mucosa⁴⁶. To investigate the mucosal immune response, we assessed antigen-specific IgA levels in BALF using ELISA. SM102-LNP exhibited IgA levels comparable to the PBS group (Fig. 6d), indicating its limited ability to trigger an IgA response. In contrast, CAS-LNP induced significantly higher IgA levels, resulting in the potent virus-neutralizing ability of BALF, as measured by the Omicron pseudovirus-based assay (Fig. 6e). These results suggest that CAS-LNP stimulated potent humoral immune responses within the airway, which could confer superior protection against respiratory viruses. Furthermore, we analyzed the cellular immune response in the lung by restimulating lung cells with the peptide pool of Omicron. The interferon-gamma (IFN-γ)-producing cells were analyzed using the IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay. Remarkably, CAS-LNP treatment showed the highest number of IFN-γ-producing cells, which was 15.5-fold and 46.4-fold greater than those observed in the SM102-LNP and PBS-treated mice, respectively (Fig. 6f, g). The robust induction of IFN-γ-producing cells by CAS-LNP indicates the activation of a potent, antigen-specific cellular immune response in the lung, which can efficiently target and eliminate infected cells, thus enhancing pathogen clearance and limiting infection severity.

Tissue-resident memory T (T_RM) cells are crucial for protection against various respiratory pathogens and have been proposed as the benchmark of mucosal immune responses⁴⁷. Recent studies indicate that T_RM cells in mucosal tissue offer long-term protection against mucosal pathogens, including SARS-CoV-2, where T_RM cells are induced in the lungs of both severe and mild COVID-19 patients and persist for up to 10 months⁴⁸. We analyzed the population of T cells and T_RM in the lungs and BALFs by flow cytometry (Supplementary Figs. 33–37). SM102-LNP and CAS-LNP treatments exhibited similar numbers of CD4 + and CD8 + T cells in the lung, which are both higher than those of PBS treatment (Fig. 6h, k), suggesting that LNPs elicit proinflammatory responses following inhalation. The development of T_RM requires exposure of T cells to antigens first. Thus, the enhanced mRNA expression of CAS-LNP could contribute to elevated T_RM levels compared to SM102-LNP. Indeed, CAS-LNP treatment led to a significant increase in both CD4 + T_RM (CD4 + CD44 + CD69 + CD11a +, Fig. 6i, j) and CD8 + T_RM cells (CD8 + CD44 + CD69 + CD103 + Fig. 6l, m) in the lung compared to SM102-LNP and PBS treatments. In addition, CAS-LNP increased the population of CD8 + T cells and CD8 + T_RM cells within BALF (Fig. 6n–p).

Taken together, these findings highlight the potency of CAS-LNP in triggering robust systemic and mucosal immune responses. As an inhaled COVID-19 vaccine candidate, CAS-LNP demonstrates the ability to stimulate multiple arms of the immune system to provide comprehensive protection against the virus. The superior stability of CAS-LNP during nebulization plays a crucial role in its efficacy, as it results in significantly enhanced mRNA delivery and expression of the antigen in the respiratory tract compared to SM102-LNP. These findings highlight the importance of lipid nanoparticle formulations in modulating mucosal immune responses and provide valuable insights for the design of future immunization strategies aimed at strengthening mucosal immunity for respiratory protection.

CAS-LNP effectively delivers mRNA cancer vaccines

CAS-LNP could serve as a platform for delivering diverse mRNA-based vaccines and therapeutics to the lungs. To demonstrate its versatility, we conducted a proof-of-concept experiment using CAS-LNP as a prophylactic cancer vaccine to inhibit lung metastasis. We encapsulated an ovalbumin-encoded mRNA (mOVA) into CAS-LNP and SM102-LNP and administered them to mice through inhalation at days 0, 7, and 14 (Fig. 7a). The lungs of mice were isolated on day 13 for the analysis of antigen-specific cytotoxic T cells (Supplementary Fig. 38). CAS-LNP increased the percentage of OVA-specific IFN-γ + CD8 + T cells by approximately 8.7-fold and 21.4-fold compared to SM102-LNP and PBS treatments, respectively (Fig. 7b, c). ELISpot analysis of antigen-specific, IFN-γ-producing cells (Fig. 7d and Supplementary Fig. 39) in the spleen showed the same trend, indicating that CAS-LNP triggered systemic anti-tumor immune response. The enhanced local and systemic anti-tumor immune responses imply good efficacy in inhibiting lung metastasis. To evaluate the prophylactic efficacy of CAS-LNP, we injected 2 × 10⁵ B16F10-OVA (a mouse melanoma cell line expressing OVA) cells into mice through intravenous injection one day after the third immunization. On day 22, after tumor inoculation, the lungs were collected to evaluate tumor metastasis. Notably, CAS-LNP treatment significantly reduced the number of metastatic foci (Fig. 7e, f) and the relative area of tumors in the lungs (Fig. 7g, h and Supplementary Fig. 40) compared to the SM102-LNP and PBS-treated groups, demonstrating its efficacy as a prophylactic cancer vaccine.

a Schematic of treatment regimen on a metastatic B16F10-OVA tumor model. CAS-LNP or SM102-LNP containing mOVA were nebulized and administered to mice on days 0, 7, and 14. Each dose contains 5 µg of mOVA per mouse. b Representative flow cytometry plots and (c) quantitative analysis of IFN-γ + CD8 + T cells among lung cells on day 13 post-prime (n = 3 biologically independent samples). Data are shown as mean ± SEM. d Optical images and quantitative analysis of IFN-γ-spot-forming cells among splenocytes on day 13 post-prime via ELISpot assay. Splenocytes of mice were stimulated with SIINFEKL peptide (n = 3 biologically independent samples). Data are shown as mean ± SEM. e Photographs of lungs bearing metastatic tumors from treated mice. Scale bar is 1 cm. f Number of metastatic foci on the lung of treated mice (n = 5 biologically independent samples). Data are shown as mean ± SEM. g Representative images of H&E-stained lung sections following treatments. h Quantitative analysis of metastatic tumor area among the overall lung area (n = 3 biologically independent samples). Data are shown as mean ± SEM. i Schematic of treatment regimen on a metastatic B16F10 tumor model. CAS-LNP or SM102-LNP containing mGP70 were nebulized and administered to mice on days 2, 7, and 12. Each dose contains 5 µg of mGP70 per mouse. j Photographs of lungs bearing metastatic tumors from treated mice. Scale bar is 1 cm. k Number of metastatic foci on the lung and (l) Survival analysis of treated mice (n = 5 biologically independent samples). Data are shown as mean ± SEM. m Schematic showing the polarization of tumor-associated macrophages (TAMs). n Representative flow cytometry plots and quantitative analysis of M1 and M2 macrophages (n = 5 biologically independent samples). Data are shown as mean ± SEM. Statistical significance was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. Figure 7a was created in BioRender. Lu, X. (2024) BioRender.com/n34x441. Figure 7i was created in BioRender. Lu, X. (2024) BioRender.com/h65g080. Figure 7m was created in BioRender. Lu, X. (2024) BioRender.com/m806034. Source data are provided as a Source Data file.

Full size image

To assess the efficacy of CAS-LNP as a therapeutic cancer vaccine, we designed an mRNA encoding the envelope glycoprotein 70 (GP70), a native B16F10 tumor antigen⁴⁹. After intravenously injecting C57BL/6 mice with 2 × 10⁵ B16F10 cells, we encapsulated mRNA encoding GP70 (mGP70) into CAS-LNP or SM102-LNP and administered these LNPs to mice through inhalation on days 2, 7, and 12 post-tumor inoculation (Fig. 7i). As shown in Fig. 7j–l, CAS-LNP treatments significantly reduced the number of metastatic foci in the lung and prolonged animal survival compared to SM102-LNP or PBS treatments. We further analyzed the phenotype of macrophages within the lung (Supplementary Fig. 41) since the M1/M2 ratio is a commonly reported prognostic indicator of cancer vaccines^50,51. M1 macrophages possess pro-inflammatory and tumor-inhibiting properties, while M2 macrophages exhibit immunosuppressive and tumor-promoting characteristics (Fig. 7m). As shown in Fig. 7n, CAS-LNP treatment greatly improved the population of M1 macrophages, resulting in an elevated M1/M2 ratio compared to SM102-LNP or PBS-treatments. These results demonstrated that inhaled CAS-LNP stimulated proinflammatory macrophages and effectively inhibited tumor metastasis as a therapeutic vaccine. Although preclinical models like B16F10 may not fully represent metastatic lung cancer, our study demonstrates the potential of an inhaled mRNA vaccine for treatment. With mRNA’s ability to encode diverse tumor antigens, CAS-LNP shows promise for targeting various lung cancers. Future research should investigate its efficacy and safety in clinically relevant models.

Safety profiles of CAS-LNP

CAS-LNP is prepared by integrating a small quantity of DSSC-DOPE conjugates into the clinically approved LNP formulation. DOPE has been widely used in liposomal formulations for biomedical applications and exhibited good safety profiles⁵². DSSC is an oligopeptide made of natural amino acids. Therefore, we did not anticipate the apparent toxicity of CAS-LNP. Throughout the treatment period of all in vivo studies, we did not observe any weight loss (Supplementary Fig. 42) or behavior changes in the animals treated with CAS-LNP. Hematoxylin and eosin (H&E) staining of histological sections of major organs (heart, liver, spleen, lung, and kidney) showed no obvious change in morphology (Supplementary Figs. 40 and 43). We also evaluated the blood routine, serum biochemical indicators, cytokines, and C-reactive protein at 48 h after administration of CAS-LNPs at three dosages. As shown in Supplementary Fig. 44, CAS-LNP at three different dosages (2.5, 5, and 7.5 µg of mRNA) did not induce noticeable changes in all tests compared to PBS treatment. The CAS-LNP is dissolved in 0.1 × PBS for inhalation. We measured the osmotic pressure (OS) of CAS-LNP in 0.1 × PBS at two different mRNA concentrations. The results showed that CAS-LNP in 0.1 × PBS has comparable OS compared with saline, which is greatly higher than that of 0.1 × PBS (Supplementary Fig. 45). Inhaled drugs are generally tolerant to variations in osmotic pressure, allowing safe use of inhaled solutions such as distilled water⁵³ and hypertonic saline⁵⁴ in clinical settings. Collectively, these data demonstrated the good safety profile of CAS-LNP.

Collectively, we presented a strategy to enhance the colloidal stability of LNP during nebulization – a critical step for the development of inhaled mRNA vaccines or therapies. Through the integration of negatively charged DSSC-DOPE, the carboxyl groups on the LNP surface induced electrostatic repulsions among LNPs, thus preventing their aggregation and disintegration during nebulization. The molecular design of DSSC-DOPE is critical to endow LNP with a negative surface charge without affecting mRNA encapsulation. The proposed charge-assisted stabilization is inherently different from previous strategies, which use cationic lipids in the core of LNP, excessive PEG on the LNP surface, or excipients in the solution. We demonstrated that CAS is a general strategy that can be easily applied to different LNP formulations to improve their mRNA delivery efficiencies after inhalation. Furthermore, the flexibility of the peptide head group opens broad chemical spaces for optimization of the DSSC-DOPE conjugates to further improve inhaled mRNA delivery efficiency or achieve targeted transfection in different cell types.

Inhaled CAS-LNP exhibits efficient mucus penetration, mRNA expression, and dendritic cell targeting within the lung. Moreover, inhaled CAS-LNP induces robust systemic and mucosal immune responses, positioning CAS-LNP as a promising platform for delivering mucosal mRNA vaccines to control infection, replication, and spread of respiratory pathogens. We further demonstrated the applicability of CAS-LNP as both prophylactic and therapeutic cancer vaccines, indicating its versatility in delivering diverse mRNA vaccines or therapeutics. These findings exemplify the design principles behind inhaled LNPs, which require harmony between colloidal stability during nebulization and subsequent interactions with cells. These results pave the way for a broad spectrum of inhaled mRNA vaccines or treatments targeting a wide range of pulmonary diseases.

• SEO Powered Content & PR Distribution. Get Amplified Today.
• PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
• PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
• PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
• PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
• Source: https://www.nature.com/articles/s41467-024-53914-x