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Optimization of Aerosolizable Messenger RNA Lipid Nanoparticles for Pulmonary Delivery
Biomed Sci Letters 2023;29:231-241
Published online December 31, 2023;
© 2023 The Korean Society For Biomedical Laboratory Sciences.

Se-Hee Lee1,* , Jong Sam Lee2,* , Dong-Eun Kim2,** and Keun-Sik Kim1,†,**

1Department of Biomedical Laboratory Science, Konyang University, Daejeon 35365, Korea
2Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea
Correspondence to: Keun-Sik Kim. Department of Biomedical Laboratory Science, Konyang University, Daejeon 35365, Korea.
Tel: +82-42-600-8434, Fax: +82-42-600-8408, e-mail:
*Graduate student, **Professor.
Received November 1, 2023; Revised December 4, 2023; Accepted December 5, 2023.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Messenger RNA (mRNA)-based vaccines and treatments have recently emerged as a promising strategy. Naked mRNA presents various limitations for direct delivery. Therefore, in this paper, Lipid Nanoparticles (LNPs) were utilized for the delivery of mRNA. Lipid nanoparticle (LNP) mRNA systems are highly effective as vaccines, but their efficacy for pulmonary delivery has not yet been fully established. Additionally, research on effective delivery systems and administration methods for vaccines is required to resolve the stability and degradation issues associated with naked mRNA delivery. This study aimed to determine mRNA delivery efficiency via the inhalation of a lipid nanoparticle (LNP) formulation designed specifically for pulmonary delivery. To this purpose, we built a library of seven LNP configurations with different lipid molar and N/P ratios and evaluated their encapsulation efficiency using gel retardation assay. Among the tested LNPs, LNP1, LNP2-2, and LNP3-2 demonstrated high transfection efficiency in vitro based on FACS analyses luciferase assays, and intracellular accumulation tests. The mRNA delivery efficiencies of the selected LNPs after inhalation and intravenous injection were compared and evaluated. LNP2-2 showed the highest mRNA expression in healthy mouse lungs when aerosolized and was found to be non-toxic. These results indicate that LNP2-2 is a promising carrier for lung mRNA delivery via inhalation.
Keywords : Lipid nanoparticle, mRNA, Aerosolization, Pulmonary

Recently, extensive studies have been conducted on the use of messenger RNA (mRNA) for vaccination and therapeutic purposes. Unlike DNA, mRNA has the advantage of not requiring passage through the nuclear membrane and can be translated directly within the cytoplasm. However, owing to its hydrophilic anionic nature, naked mRNA has difficulty passing through cell membranes. Additionally, drawbacks, such as structural instability, rapid degradation by RNase enzymes, and short-term expression, limit its usefulness in vaccination (Liang et al., 2021; Perche et al., 2019). Therefore, an effective delivery system is important for overcoming these challenges and ensuring the stable and efficient delivery of mRNA.

Lipid nanoparticles (LNPs) have emerged as nonviral vectors for the efficient delivery of nucleic acids. They offer several advantages over viral vectors, including lower immunogenicity (Kenjo et al., 2021) and protection of mRNA from degradation by nucleases (Rybakova et al., 2019). Their versatility allows for custom composition, size, and surface molecule design, making them promise for therapeutic applications and vaccine delivery systems targeting various diseases. For example, LNP-based mRNA vaccines for COVID-19, such as Pfizer-BioNTech's BNT162b and Moderna Inc.'s mRNA-1273, have been approved by the FDA. Accordingly, LNPs have shown potential as mRNA vaccine carriers for the influenza virus (Ye et al., 2022; Zhuang et al., 2020), Zika virus (Medina-Mag체es et al., 2021; Richner et al., 2017), and cancer (Rosenblum et al., 2020; Rybakova et al., 2019). Numerous LNP/mRNA therapeutics are currently undergoing preclinical and clinical trials (Gupta et al., 2021; Hou et al., 2021; Vlatkovic, 2021; Wang et al., 2023).

The efficacy of LNPs varies greatly depending on the route of administration. Using LNPs for pulmonary delivery involves two methods: systemic administration and inhalation. Extensive studies have shown that many LNP preparations are hepatotropic upon systemic delivery because they bind to circulating ApoE proteins, which then transport the particles to the organs (Jayaraman et al., 2012). To solve this problem, inhalation, based on a selective organ-targeting strategy, delivers drugs directly to the lungs. This method directly increases drug accumulation in the lungs as opposed to systemic administration, where the drug accumulates in the liver or spleen. Consequently, drug delivery to the lungs via inhalation can potentially reduce doses and minimize side effects (Garbuzenko et al., 2014; Lee et al., 2018; Leong and Ge, 2022). However, it is important to note that existing LNPs may encounter a variety of environments in terms of blood flow and airway dynamics, and even the best nebulizers can generate shear forces that can affect LNP structure (Lokugamage et al., 2021). Therefore, it is necessary to develop LNPs specifically optimized for inhalation. This study aimed to optimize the composition and N/P ratio of LNPs for the efficient pulmonary delivery of mRNA through inhalation.

This paper presents the development of an LNP library (Table 1) for effective mRNA delivery to the lungs (Fukushige et al., 2020; Idris et al., 2021; Li et al., 2019; Supramaniam et al., 2022; Zhang et al., 2020; Zhuang et al., 2020). In vitro experiments confirmed the encapsulation efficiency through gel retardation, and FACS, luciferase assay, and intracellular accumulation tests demonstrated high transcription efficiency for LNP1, LNP2, and LNP3-2. The mRNA delivery efficiency of the selected LNPs was evaluated after inhalation and intravenous injection. Through these evaluations, it was observed that mRNA, when delivered through inhalation with LNP2-2, exhibited high expression efficiency, indicating the promising nature of LNP2-2 as an mRNA delivery system.

LNP formulations for delivering mRNA to lung

Formulation Lipid molar ratio (%)
DOTAP DOPE DSPE-PEG2000 PA-PEG-mannose Cholesterol
LNP1 50 49 1 0 0
LNP2 50 29 1 0 20
LNP2-1 50 39 1 0 10
LNP2-2 50 19 1 0 30
LNP3 40 40 10 10 0
LNP3-1 50 40 5 5 0
LNP3-2 50 48 0 2 0



DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino(polyethylene-glycol)-2000]), PA-PEG3-Mannose (1,2-dipalmitoyl-sn-glycero-3-phospho ((ethyl-1',2',3'-triazole)triethyleneglycolmannose)) and Rhod PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)) were purchased from Avanti Polar Lipid, Inc. (Alabaster, AL, USA). Enhanced green fluorescent protein (EGFP) mRNA was provided by Dr. Dong-Eun Kim's lab (Konkuk University). CleanCap Fluc mRNA was purchased from TriLink Biotechnologies Inc. (San Diego, CA, USA). Human bronchial epithelial cells (BEAS-2B cells) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Lung adenocarcinoma A549 cells were purchased from the Korea Cell Line Bank (Seoul, South Korea). Liposomal extruders and polycarbonate membranes were purchased from Whatman (Kent, Maidstone, UK).

Cell culture

A549 and BEAS-2B cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco/Invitrogen, Carlsbad, CA, USA) and 1% penicillin and streptomycin (Hyclone, Logan, UT, USA). Cells were cultured at 37꼦 in a 5% CO2 incubator.

Preparation of LNP and LNP/mRNA complex

The library's lipid composition includes key components such as DOTAP, DOPE, and cholesterol, known for their roles in gene delivery. DOTAP, with its amphipathic structure, binds electrostatically to negatively charged nucleic acids. DOPE contributes to LNP stability and aids in endosomal escape under low pH conditions. Cholesterol enhances structural stability, while PEG-linked lipids improve circulation time and mucosal penetration (Albertsen et al., 2022; Qiu et al., 2019). The library was completed by incorporating a mannose moiety and a lipid to target mannose receptors in the lungs (Xu et al., 2010).

All lipids comprising each LNP were dissolved and mixed in chloroform: methanol (2:1 v/v), and the organic solvent was evaporated with nitrogen gas to obtain a lipid film. The residual organic solvent was thoroughly eliminated using a vacuum process lasting for a minimum of 30 min. The lipid film was rehydrated with HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.5) to obtain a concentration of 1 mg /mL. The LNPs were sonicated for 30 min to achieve homogenization. For precise size control, the LNP formulations were extruded through a series of polycarbonate membranes (800, 400, 200, and 100 nm) for 10 cycles for each membrane.

The mRNA was diluted in diethyl pyrocarbonate (DEPC) and mixed with an equal volume of LNP solution based on the desired N/P ratio. The N/P ratio represents the molar ratio between the amine amino group of DOTAP and the nucleic acid phosphate group. The specific N/P ratio used in this study was determined by referring to previous research papers (Ahmad et al., 2019; Robinson et al., 2018). After gentle vortexing for 1 min and incubation at room temperature for 30 min, the LNP/mRNA complexes were prepared for further experimentation.

Gel retardation assay

A gel retardation assay was conducted to assess the encapsulation efficiency of the LNP/mRNA complexes at various N/P ratios. The mRNA/LNP complex was loaded onto a 1.2% agarose gel in 1x Tris/borate/EDTA (TBE) buffer. Electrophoresis was performed at 100 V for 20 minutes. The gel was stained in the dark with a diluted solution of SYBR Green II (Invitrogen, Carlsbad, CA, USA) in a 5,000:1 ratio of SYBR Green II to 1x TBE buffer for 30 min. The stained mRNA was visualized under UV illumination.

Transfection assay

A549 and BEAS-2B cells (5 횞 104 cells/well) were seeded in 24-well plates and incubated at 37꼦 in a 5% CO2 incubator for 24 h. Next, the cells were transfected with various N/P ratios of the LNP/mRNA complexes in serum-free RPMI-1640 medium for 3 h. The cells were then washed with PBS and incubated in 10% RPMI-1640 medium at 37꼦 in a 5% CO2 incubator for 21 h.

To assess the transfection efficiency, FACS analyses and luciferase assays were performed. To determine the expression of the transfected eGFP, the transfected cells were harvested and washed twice with cold PBS, followed by analysis using a flow cytometer (Agilent, California, USA). To quantify the expression of Fluc mRNA, transfected cells were treated with 1x CCL buffer to obtain cell lysates. Luciferase activity was measured using a luciferase assay system (Promega, Southampton, UK, cat# E1501) and a luminometer (Titertek-Berthold, Sirius L, Pforzheim, Germany).

Cell cytotoxicity assay

To assess the cytotoxicity of the LNP/mRNA complexes, an MTT assay was performed. A549 and BEAS-2B cells (2 횞 104 cells/well) were seeded in 96-well plates and incubated at 37꼦 in a 5% CO2 incubator for 24 h. The cells were then treated with LNP/mRNA complexes in serum-free RPMI-1640 medium for 24 h. After that, cells were washed with PBS and incubated with serum-free RPMI-1640 containing MTT reagent (5 mg/mL) for 3 h at 37꼦. The media was removed, and the cells were treated with DMSO to dissolve the purple formazan crystals for 15 min at 37꼦. The absorbance of the resulting solution was measured at 570 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).


The LNP/mRNA complex was diluted in PBS to a total volume of 1 mL. The diluted LNP/mRNA solution was loaded into an AeroNeb Pro reservoir (Aerogen, Galway, Ireland). A 15-minute nebulization cycle was initiated to collect the nebulized LNP in a 15 mL conical tube. Nebulization was stopped when aerosol generation ceased completely. Additionally, Rho-DOPE was used to replace DOPE lipids to generate Rho-LNPs. The nebulized Rho-LNPs were then examined for internalization into cells. BEAS-2B cells were treated with sprayed Rho-LNPs for 1, 3, 6, 18, and 24 h and then analyzed by FACS analysis.

Animal Experiments

Eight-week-old, male BALB/c mice were purchased from Koatech (Pyeongtaek, South Korea). The mice were maintained in accordance with the regulations for the care and use of laboratory animals of the Animal Ethics Committee at Konkuk University (No. KU22098). All the animal studies were conducted under protocols approved by the Committee on Use and Care of Animals at Konkuk University, South Korea. In the in vivo experiments, Clean Cap Fluc mRNA was used. The LNP/mRNA complexes were administered to the mice via intravenous (i.v.) injection or inhalation at a dosage of 0.5 mg/kg (Qiu et al., 2022; Tam et al., 2022; Zhuang et al., 2020). The mice were immobilized in a mouse holder and inhalation exposure was performed by connecting a nosecone-shaped funnel designed to expose only the nose to the AeroNeb Pro nebulizer. The mice were sacrificed 6 h after administration. Bronchoalveolar lavage (BAL) fluid was collected by washing the lungs with 1 ml of cold PBS and centrifuged at 3,000 rpm for 5 min at 4꼦. Liver samples were homogenized using a tissue grinder and the resulting tissue homogenate was centrifuged at 12,000 rpm for 4 min at 4꼦.

Enzyme-linked immunosorbent assay

BAL fluid collected from the mice was analyzed to measure the secretion levels of IL-6 and IL-1棺. Human IL-6 DuoSet Enzyme-linked immunosorbent assay (ELISA) and human IL-1棺 DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA) were used for the analysis, following the manufacturer's instructions. The absorbance of the samples was measured at 450 nm using a microplate reader.

Statistical Analysis

The experimental results are presented as means 짹 standard deviations (SDs). Statistical analyses were conducted using two-tailed Student's t-tests. The significance of the data is indicated by nsP > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.


mRNA encapsulation efficiency of LNPs at different N/P ratios

Gel retardation analysis was performed to determine the N/P ratio at which the mRNA was encapsulated within each LNP preparation (Fig. 1). The results showed the successful encapsulation of mRNA under all conditions when the N/P ratio was above 3:1, except for LNP3 and LNP3-1. LNP3 and LNP3-1 exhibited distinct mRNA bands at all N/P ratios, indicating a lack of mRNA encapsulation. Consequently, the LNP3 and LNP3-1 cells were excluded from the experimental group.

Fig. 1. Gel retardation assay for mRNA encapsulation efficiency of LNPs at various N/P ratios.
The encapsulation efficiency of various LNP formulations was determined using gel retardation assay. For each LNP, encapsulation was performed using 100 ng of mRNA at various N/P ratios for 30 min at room temperature. This was followed by gel lag on a 1.2% agarose gel at 100 V for 20 min. The mRNA bands were then stained with SYBR Green II diluted 1:10,000 in TBE buffer and measured using UV illumination.

Optimization of N/P ratio for in vitro transfection of LNP/mRNA complexes

Based on the experimental results shown in Fig. 1, LNPs were prepared with various N/P ratios ranging from 3:1 to 12:1 to determine the optimal N/P ratio for mRNA delivery among LNP types other than LNP3 and LNP3-1. The transfection efficiency of EGFP mRNA into A549 and BEAS-2B cells was evaluated. EGFP was used as a reporter protein to monitor mRNA transfection, and the FACS analysis showed that EGFP expression increased with increasing N/P ratios (Fig. 2A and 2B).

Fig. 2. Optimization of N/P ratio for in vitro transfection by FACS.
GFP mRNA was encapsulated in each LNP and transfected into A549 and BEAS-2B cells to determine the optimal N/P ratio. Encapsulation was performed at room temperature for 30 min at each N/P ratio, followed by transfection into A549 and BEAS-2B cells. After 3 h of transfection, cells were incubated at 37꼦 with 5% CO2 for 21 h. Post-trypsinization, cells were collected, and GFP expression efficiency was assessed using a Flow cytometer. (A) Percentage of GFP expression efficiency measured by FACS analysis. (n = 3; mean 짹 SD, ***P < 0.001 as compared with control; Student's t-test, two-tailed). (B) Confirmation of the shift in GFP expression efficiency measured by FACS analysis, shown in the histogram.

Additionally, for quantitative analysis of transfection efficiency, luciferase-expressing mRNA was used to analyze the expression efficiency in A549 and BEAS-2B cells treated with LNPs at N/P ratios of 3:1 to 12:1 (Fig. 3A and 3B). Most LNPs showed maximal luciferase activity at a 6:1 N/P ratio in both cell lines, whereas LNP2, LNP2-1 showed maximal luciferase activity at an N/P ratio of 6:1 and 9:1.

Fig. 3. Optimization of N/P ratio for in vitro transfection by luciferase assay.
(A and B) A549 and BEAS-2B cells were transfected with Fluc mRNA encapsulated in each LNP to determine the optimal N/P ratio. Encapsulation was carried out at room temperature for 30 min at each N/P ratio. Following transfection, cells were incubated for 3 h and then stabilized at 37꼦 with 5% CO2 for an additional 21 h. After this incubation period, cell lysates were obtained by treating with 1X CCL buffer at room temperature for 2 h, collected in microtubes, and centrifuged at 12,000 rpm for 1 min to obtain the supernatant. Subsequently, the supernatant was measured with luciferase assay reagent by luminometer (n = 3; mean 짹 SD, nsP >0.05, *P < 0.05, **P < 0.01, and ***P < 0.001; Student's t-test, two-tailed).

Finally, The MTT assay revealed that the toxicity increased slightly with increasing cations at higher N/P ratios. However, no significant cytotoxicity was observed in any of the LNPs (Fig. 4). Therefore, considering both transfection efficiency and cytotoxicity, the optimal N/P ratio was 6:1, and LNP2-2 was likely to be the most efficient mRNA delivery system.

Fig. 4. Cytotoxicity assessment of LNP/mRNA complexes.
(A and B) The cytotoxicity of LNP/mRNA complexes was verified by encapsulating LNPs with mRNA at various N/P ratios, followed by a 24 h incubation at 37꼦 with 5% CO2. A positive control was transfected using lipofectamine 3000. Subsequently, MTT reagent was diluted to 10% in serum-free media and incubated at 37꼦 with 5% CO2 for 2 h 30 min. The resulting formazan particles were dissolved by treatment with DMSO for 30 min. The optical density (OD) value was measured using a microplate reader at 570 nm.

In vitro transfection efficiency of nebulized LNP/mRNA complexes

To assess whether LNP/mRNA retains transfection capability after aerosolization through a nebulizer, after nebulizing the LNP/mRNA complexes, the transfection efficiency was assessed by a luciferase assay in A549 and BEAS-2B cells (Fig. 5). The results showed decreased luciferase expression in all the LNPs after nebulization. LNP2-2, which exhibited the highest luciferase activity in BEAS-2B cells before nebulizing, maintained the highest activity after spraying. LNP1, which showed low expression before nebulization, exhibited the second-highest luciferase activity after nebulization. LNP3-2 showed a relatively high expression in both cell lines before and after nebulization. LNP2 and LNP2-1 showed no significant increase in luciferase activity after nebulization. Based on these results, it was determined that LNP2 and LNP2-1 were not suitable for inhalation testing.

Fig. 5. Intracellular expression of luciferase by LNP formulations before and after nebulization.
All formulations of LNPs were prepared with 250 ng of mRNA and an N/P ratio of 6:1. They were then nebulized using nebulizer, treated in A549 and BEAS-2B cells, and incubated at 37꼦 with 5% CO2 for 3 h. Subsequently, the media was changed to 10%. After an additional 21 h of incubation, cell lysate was obtained by treating with 1X CCL buffer. Following centrifugation at 12,000 rpm for 1 min, the supernatant was collected, treated with luciferase assay reagent, and measured with a luminometer (n = 3; mean 짹 SD, **P < 0.01, ***P < 0.001; Student's t-test, double-tailed).

Intracellular accumulation of sprayed Rho-LNPs in BEAS-2B cells

To determine the period showing the highest accumulation efficiency, DOPE was replaced with rhodamine fluorescence-labeled Rho-DOPE lipids to generate Rho-LNPs to investigate the intracellular accumulation time of the three LNPs (LNP1, LNP2-2, and LNP3-2). The results showed that the accumulation of LNP1 increased for up to 24 h. In contrast, the accumulation of LNP2-2 and LNP3-2 peaked 3 h after Rho-LNP treatment and then gradually decreased (Fig. 6). Based on these results, we designed an in vivo experiment to collect samples 6 h after LNP/mRNA administration with consistently high efficiency across all LNPs.

Fig. 6. Determination of accumulation time of nebulized Rho-LNP/mRNAs in BEAS-2B cells.
To determine the accumulation time of LNPs within cells, Rho-LNPs were synthesized by replacing DOPE with Rho-DOPE. The resulting Rho-LNP was nebulized using a nebulizer and subsequently applied to BEAS-2B cells. After incubation for various durations, the cells were stabilized with 10% medium, subjected to trypsin treatment, harvested, and the accumulated rhodamine within the cells was quantified using a flow cytometer.

In vivo evaluation of LNP/mRNA delivery and toxicity

LNP/mRNA candidates were subjected to in vivo experiments following both i.v. injection and inhalation to assess mRNA delivery efficiency. LNP/mRNA was administered to mice via i.v. injection and inhalation using a nebulizer at a 0.5 mg/kg dose per mouse to validate the results obtained from the in vitro experiments in animal models. The mice were euthanized 6 h, and tissue samples were prepared for analysis (Scheme 1).

Fig. 8. In vivo Experiments with Selected LNPs.
Based on the in vitro experiment results, LNP1, LNP2-2, and LNP3-2 were chosen for further evaluation in animal experiments. CleanCap Fluc mRNA was prepared at a concentration of 0.5 mg/kg in mice and encapsulated with each LNP at an N/P ratio of 6:1. The formulations were then administered via intravenous injection or nebulization. After 6 h, the mice were euthanized, and bronchoalveolar lavage (BAL) fluid was collected using 1 mL of PBS. The lungs and liver were harvested and immediately frozen at -80꼦. Subsequently, tissue samples and BAL fluid were subjected to Luciferase assay to quantify mRNA expression and ELISA to assess toxicity.

First, we assessed the mRNA transport to the lungs and liver via luciferase assay (Fig. 7A). Overall, minimal luciferase activity was observed in the liver tissue. In the lung tissue, the group administered LNP2-2 showed highest luciferase activity in the inhalation group and LNP1 and LNP3-2 groups showed a slight increase in luciferase activity after inhalation. This indicates efficient delivery via inhalation to the lungs. These results indicate that LNP/mRNA delivery to the lungs was most effective in the LNP2-2 group via inhalation.

Fig. 7. Luciferase and cytokine expression in vivo after LNP/mRNA administration.
Mice were administered with LNP/mRNAs via i.v. injection and inhalation. After 6 h, the mice were euthanized, and the lungs, livers and BAL fluid were promptly harvested, then immediately frozen at -80꼦. (A) Tissue lysates were prepared by homogenizing the lungs and liver in 1X CCL buffer. The resulting supernatant was collected through centrifugation, and a luciferase assay was conducted by luminometer. (B) Expression levels of IL-1棺 and IL-6 were assessed using the ELISA method in cells from the BAL fluid. The procedure was conducted as instructed in the manual, and measurements were taken using a microplate reader at 450 nm. (n = 3; mean 짹 SD, *P < 0.05, ***P < 0.001 as compared with control; Student's t-test, two-tailed).

Additionally, analysis of proinflammatory cytokines in the experimental group showed no significant increase compared to that in the control group (Fig. 7B), indicating that the LNPs used in this study did not induce a notable inflammatory response.


Recent research has focused on mRNA-based vaccination and treatment. These mRNAs have the advantage of being directly translated in the cytoplasm, resulting in a high transfection efficiency. However, their inherent instability has prompted the use of various carriers, such as polymers, peptides, and LNPs, to develop more robust delivery systems for effective mRNA delivery to cells (Pardi et al., 2020). In this study, LNPs were used as a delivery system to transport mRNA to the lungs, and inhalation was selected as the direct route of administration of the mRNA/LNP complex to the lungs. Clinical trials of the inhalation delivery of MRT5005 (NCT03375047), developed as a treatment for cystic fibrosis, were conducted in previous studies (Hou et al., 2021; Leong and Ge, 2022). This approach holds real promise for increasing the efficacy of mRNA therapies in combating various diseases.

In this study, we performed an LNP library analysis for nucleic acid delivery to the lungs, re-established LNP compositions, and developed seven new LNP compositions and ratios. First, a gel retardation assay was performed to verify the encapsulation efficiency of the LNP/mRNA. The results confirmed that most LNPs were encapsulated at a 3:1 N/P ratio or higher. However, LNP3 and LNP3-1 were not encapsulated because of their high PEG molar ratios. Previous studies have suggested that increasing the PEG content may reduce encapsulation efficiency by making vesicles smaller and less capable of encapsulating mRNA (Miao et al., 2023). Additionally, FACS analyses and luciferase assays confirmed the optimal N/P ratio for mRNA delivery. It was confirmed that the higher the N/P ratio, the better the delivery efficiency up to a ratio of 9:1. However, as the positive charge on the LNP surface increased, cell damage and toxicity increased slightly. Based on these results, the optimal LNP/mRNA N/P ratio was determined to be 6:1.

Next, in the experimental results on the delivery efficiency of LNP/mRNA before and after the cardia, we confirmed that the delivery efficiency of LNP/mRNA decreased somewhat after spraying. A similar decrease in protein expression after nebulization was observed in another study, which most likely occurred because of the shear forces experienced while passing through the nebulizer (Zhang et al., 2020). Among the tested LNPs, LNP2-2 showed the highest expression rate after spraying. LNP1 and LNP3 also showed relatively high expression levels. Therefore, LNP1, LNP2-2, and LNP3-2 were selected for in vivo experiments. Additionally, the intracellular accumulation time of Rho-LNPs was assessed. The accumulation of LNP1 continued to increase until 24 h, whereas LNP2-2 and LNP3-2 reached their maximum accumulation at 3 h after Rho-LNP treatment and then gradually decreased. Based on these results and the data from other studies (Deng et al., 2022; Miao et al., 2023; Qiu et al., 2022; Zhang et al., 2020; Zhuang et al., 2020), we designed an in vivo experiment to investigate the expression of LNPs/mRNAs within 6 h after administration.

In animal experiments, intravenous administration of LNP/mRNA did not significantly increase luciferase activity in the BAL fluid cells and liver. However, a significant increase in luciferase activity in the BAL fluid cells was observed in the inhalation group. This indicates that when LNP/mRNA is delivered via inhalation, it does not circulate in the bloodstream but targets the lungs directly, suggesting that mRNA expression patterns vary depending on the method of administration. LNP 2-2 showed the highest efficiency. This is believed to be affected by the LNP components. LNP2, LNP2-1, and LNP2-2 are characterized by the highest molar ratio of cholesterol among the tested groups with the same 1% molar ratio as the high-molecular-weight PEG lipids with long carbon tails. These results indicate the importance of the presence or absence of an appropriate amount of cholesterol in improving the encapsulation efficiency of LNPs (Miao et al., 2023).

Finally, we found no significant increase in the inflammatory cytokines IL-6 and IL-1棺 in the BAL fluid in response to inhalation or i.v. injection, confirming that there was no toxicity. In addition, the cytokine expression rate in our group was significantly lower than that in the LPS-administered positive control group mentioned in other studies. These observations confirmed that the toxicity of LNPs was negligible (Jiang et al., 2015; Shen et al., 2015). Based on the results presented in this study, we suggest that among the seven types of LNPs generated, LNP 2-2 has the potential to efficiently deliver mRNA to the lungs via inhalation.

In conclusion, LNPs were used to deliver mRNA stably and efficiently to the lungs via inhalation, and various LNP compositions and ratios were analyzed to determine the optimal formulation for nucleic acid delivery to the lungs. In vivo experiments showed a significant increase in mRNA expression in the inhalation group, and LNP 2-2 proved to be a highly efficient transporter. Furthermore, the absence of identified toxic candidates among the LNPs employed in this investigation substantiates the superiority of LNP 2-2 as the optimal candidate for mRNA delivery through inhalation. This observation suggests its potential utility in the future for treating or preventing diseases associated with the lung system.


This research was funded by grants from the National Research Foundation of Korea (NRF-2021R1F1A1062932). Scheme1 adapted from "Rat Timeline", by (2023). Retrieved from


Lipid nanoparticle, LNP; 1,2-dioleoyl-3-trimethylammonium-propane, DOTAP; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene-glycol)-2000], DSPE-PEG2000; 1,2-dipalmitoyl-sn-glycero-3-phospho ((ethyl-1',2',3'-triazole)triethyleneglycol-mannose), PA-PEG3-Mannose; (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl), Rhod PE


The authors have no conflict of interest to declare.

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