Lipid nanocomplex for inhalation delivery of nucleic acid molecules

Abstract

The present invention relates to: a lipid complex for nucleic acid molecule delivery, comprising an ionizable lipid, a structural lipid, and cholesterol as active ingredients; and a preparation method therefor. The lipid complex of the present invention can be usefully utilized as a drug delivery system for treating various intractable lung diseases such as cystic fibrosis, pulmonary fibrosis, and α1-antitrypsin deficiency by maintaining the structure of nucleic acids even under shear force during spraying and effectively delivering target genes to deep lung cells by being efficiently fused with a lung surfactant membrane. In addition, the lipid complex of the present invention has further improved spray stability by loading target nucleic acid molecules externally, and can be applied as a combined administration platform that induces a synergistic therapeutic effect by means of two pharmacological components by loading an additional drug in the internal compartment of the lipid complex.

Description

Lipid nanocomplexes for inhalational delivery of nucleic acid molecules

[0001] The present invention relates to a lipid nanocomplex for inhalation delivery for specifically delivering a nucleic acid molecule encoding a pharmacological protein to lung tissue.

[0002] mRNA therapeutics have garnered significant attention over the past several years due to their superior safety, efficacy, and cost-effectiveness compared to conventional protein therapeutics. However, despite extensive research to enhance the function of mRNA, numerous barriers still exist that hinder its pharmacological function under physiological conditions. Therefore, carriers play a crucial role in safely delivering mRNA into the cytoplasm of target cells. Lipid nanoparticles (LNPs) are widely used and approved by the U.S. Food and Drug Administration (FDA) due to their excellent delivery efficiency and biocompatibility. LNPs are formed through ionic interactions between mRNA and ionizable cationic lipids, resulting in a structure that carries the mRNA within its internal compartment. Furthermore, conjugating polyethylene glycol (PEG) to the exterior of the particles can prevent particle aggregation and enhance systemic circulation. These types of nanoparticles are specialized for therapeutic protein expression via intravenous or intramuscular vaccination. They have been used as a vaccine platform worldwide, particularly during the COVID-19 pandemic, and are currently being used to deliver mRNA to various target tissues, including the lungs, bone marrow, and brain. In particular, mRNA delivery to the lungs is one of the most promising methods for treating various intractable diseases, including infectious diseases, cystic fibrosis, idiopathic pulmonary fibrosis, and α-antitrypsin deficiency.

[0003] Systemic administration of conventional LNPs primarily induces protein expression in the liver. Recent studies have attempted to induce lung-specific delivery by adding cationic lipids or lipid-like engineering after systemic administration. However, the particles were primarily taken up by endothelial cells, limiting their delivery to the endothelial cells. Furthermore, systemic administration of cationic LNPs has been reported to induce massive coagulation. A promising alternative for pulmonary mRNA delivery is inhalation using a nebulizer, which offers an efficient, noninvasive, and patient-friendly method for direct drug delivery to the lungs. However, shearing forces during nebulization can destabilize the nanoparticle structure, significantly reducing mRNA stability and delivery efficiency. Studies have been conducted to modify the composition of LNPs to increase shear resistance, but these methods do not fundamentally address the structural instability of the particles and the reduction in delivery efficiency after nebulization.

[0004] Another obstacle to delivering LNPs to the lungs lies in the unique specificity of the lung microenvironment, which differs significantly from the blood microenvironment. For example, the protein concentration in the lung microenvironment (0.05-0.08 mg / mL) is significantly lower than that in plasma (60-80 mg / mL). Therefore, LNPs that rely on serum protein-PEG lipid exchange for cellular uptake maintain their PEGylated state in the lung microenvironment, resulting in reduced uptake into lung cells. The residual PEGylation induces pulmonary surfactant (PS) to form multiple layers at the interface between the airway and lung cells, inhibiting their penetration.

[0005] Therefore, there is a growing need for the development of optimized delivery systems to efficiently deliver mRNA encoding pharmacological proteins to the lung microenvironment.

[0006] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0007] [Prior Art Literature]

[0008] U.S. Patent Publication No. 2010 / 0324120 A1

[0009] The present inventors have conducted extensive research efforts to develop an optimized lipid nanoparticle-based gene delivery system capable of selectively delivering nucleic acid molecules encoding therapeutic proteins to the lung tissue of a subject. As a result, when the target nucleic acid molecule is loaded into a lipid complex comprising ionizable lipids, structural lipids, and cholesterol in a specific molar ratio to have a specific range of N / P (Nitrogen / Posphate) ratio, the system exhibits stable particle characteristics equivalent to or superior to those of LNPs (lipid nanoparticles) used in the past for similar purposes, while also overcoming shear forces during spraying and efficiently being compatible with the lung microenvironment, enabling the target protein to be efficiently and specifically expressed in the lungs, thereby completing the present invention.

[0010] Accordingly, the purpose of the present invention is to provide a lipid complex for nucleic acid molecule delivery comprising ionizable lipids, structural lipids and cholesterol as active ingredients, and a method for producing the same.

[0011] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0012] According to one aspect of the present invention, the present invention provides a lipocomplex for nucleic acid molecule delivery comprising an ionizable lipid, a structural lipid, and cholesterol as active ingredients.

[0013] The present inventors have conducted extensive research to develop an optimized lipid nanoparticle-based gene delivery system capable of selectively delivering nucleic acid molecules encoding target proteins to the lung tissue of a subject. As a result, we discovered that loading a target nucleic acid molecule into a lipid nanocomposite comprising a specific molar ratio of ionizable lipids, structural lipids, and cholesterol to achieve a specific N / P ratio significantly improves shear resistance during nebulization and compatibility with the lung microenvironment, making it an excellent platform for inhaled mRNA delivery.

[0014] The term “lipid” as used herein refers to a general term for natural or synthetic biomolecules that dissolve in non-polar solvents. Lipids typically have amphipathic properties, including hydrophilic and hydrophobic components, and include, for example, phospholipids, fatty acids, fatty alcohols, triglycerides, phosphatides, glycolipids, fatty alcohols, waxes, terpenes, and steroids.

[0015] The term “ionizable lipid” as used herein refers to a lipid analogue that is uncharged at neutral pH but positively charged under acidic conditions, and that contains a functional group (e.g., an amine) that can be easily protonated as the pH decreases. The ionizable lipid used in the present invention may be any lipid that can be protonated under acidic conditions (e.g., a lipid having a chemical structure of pKa 5 to 7 so that it can be positively charged under acidic pH), and may be, for example, one or more selected from the group consisting of Dlin-MC3-DMA, SM-102, and ALC-0315, but is not limited thereto.

[0016] In the present invention, the term “structural lipid” means a phospholipid that serves as a structural basis for forming a liposome of a closed lipid bilayer in the lipid complex for nucleic acid molecule delivery of the present invention, and various phospholipids known in the art to be applicable for similar purposes may be used. Exemplary phospholipids that can be used in the present invention include dipalmitoyl phosphatidylcholine (DPPC), dilauryl phosphatidylcholine (DLPC) (C12:0), dimyristoyl phosphatidylcholine (DMPC) (C14:0), distearoyl phosphatidylcholine (DSPC), dipittanoyl phosphatidylcholine, nonadecanoyl phosphatidylcholine, arachidoyl phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) (C18:1), dipalmitoleoyl phosphatidylcholine (C16:1), linoleoyl phosphatidylcholine (C18:2), myristoyl palmitoyl phosphatidylcholine (MPPC), stearoyl myristoyl phosphatidylcholine (SMPC), stearoyl palmitoyl phosphatidylcholine (SPPC), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyl palmitooleoyl These include, but are not limited to, phosphatidylcholine (PPoPC), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidylglycerol (DOPG), palmitoyl oleoyl phosphatidylglycerol (POPG), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidylserine (DSPS), and palmitoyl oleoyl phosphatidylserine (POPS).

[0017] More specifically, a neutral phospholipid can be used as the structural lipid of the present invention.

[0018] As used herein, the term “neutral phospholipid” refers to a phospholipid in which each atom in the molecule has no charge, as well as a phospholipid in which the net charge as a molecule is 0 even if some atoms have charges, such as a zwitterion. The neutral phospholipid used in the present invention may be, for example, one or more selected from the group consisting of phosphatidylcholine, dipalmitoyl phosphatidyl choline (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dimyristoylphosphatidyl choline (DMPC), polyethylene glycolphosphatidyl ethanolamine (PEG-PE), dioleoyl phosphatidyl choline (DOPC), and dioleyl phosphatidyl ethanolamine (DOPE), but is not limited thereto.

[0019] Most specifically, DSPC can be used as the structural lipid of the present invention.

[0020] As used herein, the term "liposome" refers to a lipid carrier formed by forming a closed lipid bilayer. Liposomes are biocompatible and amphiphilic due to their lipid bilayer membrane, allowing them to pass through the hydrophobic membrane while still containing hydrophilic substances. Liposomes typically range in diameter from 20 to 2000 nm, but this is not limited thereto. Liposomes can vary in size depending on the manufacturing method and the length of the nucleotide to be encapsulated and delivered.

[0021] In this specification, the term “encapsulation” refers to a process of encapsulating a delivery substance in a closed membrane structure or covalently or non-covalently binding it to the surface of a membrane to efficiently introduce it into a living body.

[0022] According to a specific embodiment of the present invention, the ionizable lipid is included in a molar ratio of 50-90% of the total lipid, more specifically 55-85%, even more specifically 65-75%, even more specifically 67-73%, even more specifically 69-71%, and most specifically about 70%.

[0023] According to a specific embodiment of the present invention, the structural lipid is included in a molar ratio of 5-15% of the total lipid, more specifically 7-13%, even more specifically 9-11%, and most specifically about 10%.

[0024] According to a specific embodiment of the present invention, the cholesterol is contained in the total lipid at a molar ratio of 5-35%, more specifically 10-30%, even more specifically 17-23%, even more specifically 19-21%, and most specifically about 20%.

[0025] According to a specific embodiment of the present invention, the ionizable lipid, structural lipid, and cholesterol are contained in the lipid complex of the present invention in a molar ratio of 6-8:0.5-1.5:1.5-2.5. More specifically, they are contained in a molar ratio of 6.5-7.5:0.7-1.2:1.7-2.3, and most specifically, they are contained in a molar ratio of 7:1:2.

[0026] According to a specific embodiment of the present invention, the molar ratio (N / P) of the amine group in the ionizable lipid to the phosphate group in the nucleic acid molecule to be delivered in the lipid complex for nucleic acid molecule delivery of the present invention is 5 to 25, more specifically 9 to 21, even more specifically 11 to 19, even more specifically 13 to 17, and most specifically about 15.

[0027] According to a specific embodiment of the present invention, the lipid complex does not contain PEG (polyethylene glycol). The inventors of the present invention have discovered that when PEG is bound to a lipid structure for nucleic acid molecule delivery, such as conventional LNPs, the PEG does not dissociate in the lung microenvironment, where serum concentration is low and pulmonary surfactant (PS) is formed at the air-alveolar fluid boundary, and remains on the outside of the particle, thereby hindering the cellular uptake of the nucleic acid. Therefore, the lipid complex of the present invention can efficiently be compatible with the lung microenvironment while maintaining excellent particle properties even without containing PEG.

[0028] According to a specific embodiment of the present invention, the lipid complex has an average particle diameter of 50 nm to 200 nm, more specifically, an average particle diameter of 50 nm to 150 nm, and most specifically, an average particle diameter of 50 nm to 100 nm.

[0029] According to a specific embodiment of the present invention, a nucleic acid molecule to be delivered using the lipid complex of the present invention is attached to the outer surface of the lipid complex.

[0030] According to the present invention, the lipid complex of the present invention not only improves spray stability by loading a target nucleic acid molecule to the outside, but also induces a synergistic therapeutic effect by co-delivery of two pharmacological ingredients by loading an additional drug such as a hydrophilic low-differentiation compound into the internal compartment of the lipid complex.

[0031] In this specification, the term “nucleic acid molecule” has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified. When the lipid complex of the present invention delivers RNA, it may be mRNA (messenger RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), aRNA (antisense RNA), miRNA (micro RNA), siRNA (small interfering RNA), or piRNA (piwi interacing RNA).

[0032] According to a specific embodiment of the present invention, the nucleic acid molecule to be delivered using the lipid complex of the present invention is an mRNA molecule.

[0033] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base portion is modified.

[0034] According to a specific embodiment of the present invention, the nucleic acid molecule used in the present invention is an mRNA molecule. When mRNA is used as the nucleic acid molecule of the present invention, various modifications may be applied, such as changing the length of the poly(A) tail or substituting some adenine bases; modifying the 5'cap; applying one or more modified nucleosides, in order to improve the expression (translation) efficiency of the target pharmacological protein. Modified nucleosides that may be applied include, but are not limited to, N1-methylpseudouridine, pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, and 5-methoxyuridine, and any modified nucleoside known in the art to be capable of reducing the immunogenicity of the mRNA molecule may be applied.

[0035] According to a specific embodiment of the present invention, the mRNA molecule used in the present invention has a 5'-UTR and a 3'-UTR bound to each of its two ends, and more specifically, a 5'-cap bound to the 5'-UTR. As used herein, the term "UTR (untranslated region)" refers to an untranslated region bound to each end of a coding sequence encoding a target protein in mRNA, and includes a 5'-UTR located upstream of the coding sequence and a 3'-UTR located downstream. The term "5'-cap" refers to a component of mRNA that is linked to the 5'-UTR and binds to eIF4E (eukaryote translation initiation factor 4E), thereby binding the 40S ribosomal subunit to the mRNA and initiating protein synthesis from the 5' start site of the mRNA, as well as protecting the mRNA from nucleases.

[0036] The term "express" as used herein means artificially introducing a gene using a gene vector to cause a subject to express an exogenous gene or to increase the natural expression level of an endogenous gene, thereby making the gene replicable within the subject's cells as an extrachromosomal element or through chromosomal integration. Therefore, the term "expression" has the same meaning as "transformation," "transfection," or "transduction."

[0037] According to a specific embodiment of the present invention, the lipid complex is a complex for inhalation delivery for lung-specific nucleic acid molecule delivery.

[0038] As used herein, the term "inhalation delivery" refers to a non-invasive drug delivery method that involves aerosolizing a lipid complex loaded with a pharmacological agent (e.g., an mRNA molecule encoding a therapeutic protein) and applying it to the respiratory tract. Inhalation delivery can be performed using a nebulizer.

[0039] According to another aspect of the present invention, the present invention provides a method for preparing a lipocomplex for nucleic acid molecule delivery, comprising the following steps:

[0040] A step of forming a lipid film by drying a mixed solution containing ionizable lipid, structural lipid, and cholesterol;

[0041] A step of rehydrating the above lipid film with a C1-C3 alcohol solvent; and

[0042] A step of mixing and stirring the rehydrated lipid and the nucleic acid molecule to be delivered.

[0043] The composition and ionizability of the lipid used in the lipid complex for nucleic acid molecule delivery of the present invention and the N / P ratio of the nucleic acid molecule have already been described above, so description thereof is omitted to avoid excessive duplication.

[0044] According to a specific embodiment of the present invention, the C1 to C3 alcohol solvent is an ethanol solvent.

[0045] According to a specific embodiment of the present invention, the method of the present invention further comprises the step of dialyzing the rehydrated lipid film using a dialysis membrane having a cutoff value of 5-15 kDa.

[0046] According to a specific embodiment of the present invention, the rehydrated lipid and the nucleic acid molecule to be delivered are stirred in equal volumes.

[0047] The features and advantages of the present invention are summarized as follows:

[0048] (a) The present invention provides a lipid complex for nucleic acid molecule delivery comprising ionizable lipids, structural lipids and cholesterol as active ingredients, and a method for producing the same.

[0049] (b) The lipid complex of the present invention maintains the structure of nucleic acids even under shear force when sprayed, efficiently fuses with the lung surfactant membrane, and effectively delivers the target gene to deep lung cells, so that it can be usefully used as a drug delivery system for treating various intractable lung diseases such as cystic fibrosis, pulmonary fibrosis, and α-antitrypsin deficiency.

[0050] (c) The lipid complex of the present invention can be applied as a combination administration platform that not only further improves spray stability by loading the target nucleic acid molecule to the outside, but also induces a synergistic therapeutic effect by two pharmacological ingredients by loading an additional drug into the internal compartment of the lipid complex.

[0051] Figure 1 is a diagram showing the development process of lipid-based nanoparticles for inhalation delivery of mRNA of the present invention. Figure 1a is a schematic diagram showing the major obstacles during inhalation delivery. First, the particles become unstable due to the shear force generated during aerosolization in a vibrating mesh. Second, the PS layer present at the air-alveolar fluid interface inhibits the penetration of the particles into the alveolar fluid, and the low serum protein level in the alveolar fluid interferes with the cellular uptake of the PEGylated particles. Figure 1b is a diagram showing the results of analyzing the A549 infection efficiency of existing LNPs under various conditions, including the presence of serum, nebulization, and PS encapsulation. The data are expressed as the mean ± standard deviation (n = 4). Nluc mRNA dose: 1 μg / mL. The luminescence signal was normalized as the fold change compared to the untreated group. Figure 1c is a diagram showing the structural features of the ionizable lipid complex (iLPX) of the present invention.

[0052] Figure 2 is a diagram showing the optimization process of iLPX for inhalation delivery through a multi-step screening. Figure 2a is a schematic diagram showing the multi-step screening process for iLPX optimization. Figure 2b is a diagram showing the molar ratio of lipid components for 13 core liposome candidates (C1-C13). Figure 2c is a diagram showing the in vitro infection efficiency of iLPX candidates before and after nebulization. A549 cells were injected with iLPX candidates containing Nluc mRNA, and the luminescence intensity was measured using a luciferase assay 24 hours later (dosage: 0.5 μg / mL). Figure 2d is a diagram showing the in vitro infection efficiency of nebulized iLPX candidates encapsulated with animal-derived surfactant (5 mg / mL). UT: Untreated Figures 2e-g show the comparison of quantified luminescence signals in the lungs 24 hours after inhalation of iLPX candidates with various lipid compositions (e), tails of structural lipids (f), and N / P ratios (g) in C57BL / 6 mice (dose: 10 μg / head). Figure 2h shows the comparison of infection efficiencies between conventional LNPs and IH-iLPX in the mouse lungs. The luminescence signals were compared 24 hours after inhalation of particles loaded with Nluc mRNA in C57BL / 6 mice (dose: 10 μg / head). Figure 2i shows the quantified luminescence signals and the comparison results between the groups in Figure 2h, and each data above is expressed as the mean ± standard deviation (n = 3).

[0053] Figure 3 is a diagram showing the physicochemical properties of IH-iLPX during spraying. Figures 3a-c compare the physical properties of LNP, iLPX, and cLPX before and after spraying, measuring the size (Figure 3a), PDI (Figure 3b), and encapsulation efficiency (Figure 3c). Figure 3d compares the infection efficiencies of LNP, iLPX, and cLPX before and after spraying. A549 cells were treated with particles loaded with GFP mRNA (dosage: 0.5 μg / mL), and GFP expression was compared by flow cytometry 24 hours later. Figure 3e compares the infection efficiencies mediated by LNP and IH-iLPX after spraying and RNase treatment. Depending on the group (2 μg / mL), nanoparticles loaded with Nluc mRNA (dosage: 0.5 μg / mL) were sprayed and / or treated with RNase, and then delivered to A549 cells, and the luminescence signal was measured 24 hours after treatment. Figure 2f is a picture visualizing the morphological changes of LNP and IH-iLPX after spraying using Cryo-TEM. Figure 2g is a schematic diagram comparing the morphological changes and mRNA protection effects of LNP and IH-iLPX after spraying.

[0054] Figure 4 shows the infectious capacity of IH-iLPX in the lung microenvironment. Figures 4a and 4b show the results of analyzing protein expression after delivery of GFP mRNA-loaded nanoparticles in serum-free medium using fluorescence imaging (Figure 4a) and single-cell analysis (Figure 4b). A549 cells were treated with LNPs, IH-iLPX, and cLPX (at a dose of 0.5 μg / mL) in serum-free RPMI medium, and GFP signals were analyzed 24 hours later. Figures 4c and 4d show the results of evaluating the effect of PS encapsulation on mRNA delivery using fluorescence imaging (Figure 4c) and single-cell analysis (Figure 4d). A549 cells were treated with surfactant-encapsulated LNPs, IH-iLPX, and cLPX (at a dose of 0.5 μg / mL), and GFP signals were analyzed 24 hours later. Figure 4e shows the results comparing the A549 infection efficiency of nanoparticles in simulated lung fluid (dosage: 0.5 μg / mL). Figures 4f and 4g show the results comparing protein expression mediated by LNP and IH-iLPX in mouse lungs. LNPs loaded with Nluc mRNA (dosage: 4 μg / head) and IH-iLPX were intratracheally injected into mice, and the luminescence signal in the mouse lungs was imaged (Figure 4f) and its luminescence intensity was quantified (Figure 4g).

[0055] Figure 5 is a diagram showing the tissue and cellular distribution of inhaled IH-iLPX in the lungs. Figure 5a shows the results of analyzing the luminescence signals in various organs at various time points for 72 hours after inhalation of IH-iLPX loaded with Nluc mRNA. Figure 5b shows representative images of the luminescence signals in major organs 24 hours after inhalation of IH-iLPX. Figure 5c shows the results of analyzing the distribution of Nluc expression in lung lobes, showing a schematic diagram of the lung lobes and trachea (left) and an image of the expression distribution by lung lobe 24 hours after inhalation of IH-iLPX loaded with 20 μg of Nluc mRNA (right). Figure 5d shows the results of quantifying the expression signals by lung lobe in Figure 5c. Figure 5e shows the pulmonary distribution of delivered mRNA after inhalation of IH-iLPX. Figure 5f shows the percentage of Cy5-positive cells in the lungs of IH-iLPX or LNP-inhaled mice, as quantified by flow cytometry.

[0056] Figure 6 shows the results of evaluating the biocompatibility of inhaled IH-iLPX in the pulmonary and systemic environments. Figure 6a shows the results of evaluating the pulmonary toxicity of IH-iLPX by measuring the levels of inflammatory cytokines (IL-1β, IL-6, TNF-α) in BAL fluid. BAL fluid was collected 24 hours after treatment with IH-iLPX (inhalation, dose: 100 μg / head) or LPS (intranasal injection, 3 mg / kg), and the cytokine levels were quantified by ELISA. Figure 6b shows the total white blood cell counts in BAL fluid for the untreated, IH-iLPX, and LPS groups. Figure 6c shows lung tissue images stained with H&E for histopathological evaluation. Figure 6d shows the results of analyzing the systemic toxicity of inhaled IH-iLPX through blood chemistry tests. Serum was extracted 24 hours after iLPX inhalation (dose: 100 μg / head) and analyzed for AST, ALT, and total protein levels.

[0057] Figure 7 is a schematic diagram of the manufacturing process for ionizable lipid complexes (iLPX). Core liposomes are formed by emulsifying an ethanol phase containing ionizable lipids, structural lipids, and cholesterol with a deionized water solution. The ethanol is then removed from the solution through dialysis to complete the formation of the core liposome. The core liposome then complexes with mRNA in deionized water through charge interactions to form iLPX.

[0058] Figure 8 shows the results of analyzing the effect of the ion concentration of the ignition solvent on the spray stability, and the X-axis is the ion concentration of the sodium acetate (NaAc) buffer.

[0059] Figure 9 shows the results of analyzing the effect of nebulization on the biochemical properties of iLPX. The size and PDI (Figure 9a), encapsulation efficiency (Figure 9b), and zeta potential (Figure 9c) of 13 iLPX candidates with various liposome compositions were compared before and after nebulization.

[0060] Figure 10 is a diagram showing the optimization process of structural lipids based on physicochemical properties and transfection efficiency, showing particle size and PDI (Figure 10a), encapsulation efficiency (Figure 10b), zeta potential before and after spraying (Figure 10c), A549 transfection efficiency before and after spraying (Figure 10d), and A549 transfection efficiency after PS encapsulation of sprayed iLPX (24 h culture, serum-free medium, dose: 0.5 μg / mL) as the tail length of phosphocholine changes from 14:0 to 22:0.

[0061] Figure 11 is a diagram showing the process of optimizing the N / P ratio according to physicochemical properties and infection efficiency, and shows the size and PDI according to each N / P ratio (Figure 11a), encapsulation efficiency (Figure 11b), zeta potential before and after spraying (Figure 11c), A549 infection efficiency before and after spraying (Figure 11d), and A549 infection efficiency after PS encapsulation of sprayed iLPX (24-hour culture, serum-free medium, dose: 0.5 μg / mL) (Figure 11e), respectively.

[0062] Figure 12 shows the results of confocal microscopy imaging after treating A549 cells with sprayed or non-sprayed LNPs, IH-iLPX, and cLPX to evaluate the effect of nanoparticle atomization on protein expression efficiency (blue: nuclei, green: GFP, dose: 1 μg / mL).

[0063] Figure 13 shows the results of agarose gel electrophoresis of mRNA harvested from nanoparticles subjected to various spraying conditions and RNase treatment (2 μg / mL) to evaluate the effect of nanoparticle atomization on the integrity of loaded mRNA. Red arrows indicate a comparison of mRNA integrity between LNP and IH-iLPX after atomization and RNase treatment.

[0064] Figure 14 shows the results of evaluating intracellular mRNA delivery in a simulated lung microenvironment. Figures 14a and 14b show the results of analyzing mRNA delivery after delivery of Cy5-tagged nanoparticles in serum-free media using confocal imaging and flow cytometry. Cy5 signals were analyzed 24 hours after treating A549 cells in serum-free RPMI media with LNPs, IH-iLPX, and cLPX (at a dose of 0.5 μg / mL). Figures 14c and 14d show the results of evaluating the effect of PS encapsulation on mRNA delivery using fluorescence imaging (Figure 14c) and single-cell analysis (Figure 14d). Cy5 signals were analyzed 24 hours after treating A549 cells with in vitro surfactant-encapsulated LNPs, IH-iLPX, and cLPX (0.5 μg / mL). Yellow: Cy5, blue: nucleus.

[0065] Figure 15 is a diagram showing the results of intravenous and intramuscular delivery of nanoparticles to mice. Figures 15a and 15b show the results comparing the protein expression levels of LNP and IH-iLPX after intravenous injection, showing the in vivo luminescence signal (Figure 15b) and the quantified luminescence signal in the liver 24 hours after intravenous injection of IH-iLPX and LNP (occipital) (dose: 3 μg / head) (Figure 15b), respectively. Figures 15c and 15d show the results comparing the protein expression levels of LNP and IH-iLPX after intramuscular injection, showing the in vivo luminescence signal (Figure 15c) and the quantified luminescence signal in the thigh 24 hours after intramuscular injection of IH-iLPX and LNP (dose: 3 μg / head) (Figure 15d), respectively.

[0066] Figure 16 shows the pulmonary distribution of delivered mRNA after LNP inhalation, with fluorescence images of untreated C57BL / 6 mouse lung tissue (Figure 16a) and LNP-treated C57BL / 6 mouse lung tissue (Figure 16b), respectively. Dose: 100 μg / head. Blue: nuclei, yellow: Cy5.

[0067] Figure 17 is a diagram showing the gating strategy for single-cell analysis. After inhaling Cy5-tagged mRNA, mouse lung cells were analyzed by flow cytometry to identify immune cells (CD45 + ), epithelial cells (CD45 - EpCAM + CD31 - ) and endothelial cells (CD45 - EpCAM - CD31 + ) were distinguished.

[0068] Figure 18 shows the results of analyzing the protein expression levels in the lungs after inhalation of IH-iLPX at various Nluc mRNA doses. Lungs were harvested 24 hours after inhalation delivery of IH-iLPX at mRNA doses of 10 μg / head, 20 μg / head, and 30 μg / head. The results are expressed as representative images of the dose-dependent luminescence signal (Figure 18a) and quantification of the dose-dependent luminescence (Figure 18b) using a linear regression model and an R-squared value of 0.863 (n=3).

[0069] Figure 19 is a diagram showing the simultaneous delivery of mRNA and hydrophilic substances into cells using iLPX. It is a fluorescence image of A549 cells treated with IH-iLPX co-loaded with calcein (dosage: 10 mM) and mCherry mRNA (dosage: 0.5 μg / mL) 24 hours later. Green: calcein, red: mCherry, blue: nucleus.

[0070] Forty-eight core liposomes were prepared by varying the molar ratios of the ionizable lipid Dlin-MC3-DMA (MC3), the structural lipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol. Thirteen core liposome formulations were selected based on size and PDI. The selected liposomes were then complexed with mRNA at a weight ratio (mRNA:liposome) of 1:40 to formulate iLPXs, and the characteristics of the 13 iLPXs (C#, #: 1-13) were analyzed before and after spraying. As a result, no significant changes were observed in any group after spraying. However, the infection efficiency differed depending on the formulation, and the protein expression of C13 was the highest after spraying. This may be because the higher the content of ionizable lipid, the higher the encapsulation efficiency, resulting in differences in the ability to protect mRNA during the spraying process. In addition, the group with a higher cholesterol content tended to have a higher infection efficiency. Eight groups were selected based on infection efficiency after spraying, and infection efficiency in serum-free medium after PS encapsulation was evaluated to simulate the lung microenvironment. Animal-derived PS lipid membranes were rehydrated with the sprayed particle solution, incubated at 37°C for 15 minutes, and then treated with A549 cells. Group C13 exhibited the highest infection efficiency.

[0071] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0072] Example

[0073] [Experimental Method]

[0074] Experimental materials

[0075] Dlin-MC3-DMA (MC, HY-112251) was purchased from MedChemExpress, and 1,2-dimyristoyl-sm-glycero-3-phosphocholine (14:0 PC(DMPC), 850345P), dipalmitoyl-sn-glycero-3-phosphocholine (16:0 PC(DPPC), 850355P), 1,2-distearoyl-sn-glycero-3-phosphocholine (18:0 PC(DSPC), 850365P), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (20:0 PC, 850368P), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (22:0 PC, 850371P), cholesterol (700000P), 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000, 880151P) and 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP, 890890P) were purchased from Avanti Polar Lipids. Nanoluc encoding plasmid vector and superfolder GFP (sfGFP) encoding plasmid vector were kindly provided by the Department of Chemical Engineering and Applied Chemistry, Chungnam National University. Nano-Glo luciferase assay system (N1110) for Nluc analysis and VivoGlo luciferin (P1041) for in vivo Fluorescence analysis were purchased from Promega. Newfactan was purchased from Yuhan Corporation (Seoul, Korea). SnakeSkin dialysis tubing (10 kDa MWCO, 16 mm) (88243) and Hoechst 33342 solution (#62249) were purchased from Thermo Fisher. Amicon centrifuge tubes (10 kDa MWCO, UFC9010) were purchased from Merk / milipore. Chloroform (366927) and methanol (34860) were purchased from Sigma. Dulbecco's phosphate-buffered saline (DPBS, LB001) was purchased from Welgene.For single-cell analysis, PE-Cyanine 7-conjugated anti-mouse CD31 (#102417), Brilliant Violet 605-conjugated anti-mouse CD326 (#118227), and Zombie Aqua fixable viability stain (#423102) were purchased from Biolegend, and APC-eFlour 780-conjugated anti-mouse CD45 (#47-0451-82) and propidium iodide (#424902) were purchased from Invitrogen.

[0076] In vitro transcription of mRNA

[0077] The plasmid vector encoding the target mRNA was linearized with appropriate restriction enzymes, and the linearized plasmid DNA was purified using a PCR purification kit (QIAGEN). Using the purified template DNA, mRNA was synthesized for 2 hours using the HiScribe T7 High Yield RNA Synthesis Kit (NEB). To synthesize mRNA for protein expression, UTP was completely replaced with N1-methylpseudo-UTP (Jena Bioscience). To synthesize Cy5-tagged mRNA, 25% of the UTP was replaced with Cy5-UTP (APExBIO). The synthesized mRNA was purified using the Monarch RNA Cleanup Kit (NEB). The purified in vitro-transcribed mRNA was then capped for 1 hour using Vaccinia capping enzyme (NEB) and mRNA Cap 2'-O-Methyltransferase (NEB). The capped mRNA was then tailed using E. coli poly(A) polymerase (NEB) for 30 minutes. The resulting mRNA was purified using the Monarch RNA Cleanup Kit. All procedures were performed according to the manufacturer's instructions.

[0078] Nanoparticle production

[0079] Nanoparticles were prepared by ethanol emulsion method. To prepare iLPX core liposomes, Dlin-MC3-DMA, structural lipid (phosphocholine), and cholesterol were mixed at various molar ratios and the solvent was evaporated in a desiccator. The lipid film was rehydrated to 1.5 mM in ethanol and mixed with aqueous solvent (ethanol:aqueous = 1:2, v / v) by vortexing at 37°C. The particles were incubated at room temperature for 10 min and then dialyzed overnight against deionized water (500 times more volume, pH = 5.2–5.5) using 10 kDa snakeskin dialysis tubing. The prepared core liposomes (200 μg / mL) were mixed dropwise with an equal volume of mRNA and vortexed. iLPX was then concentrated to the desired concentration using a 10 kDa Amicon. cLPX was prepared using the same method as iLPX with a composition of DOTAP:DSPC:Chol = 70:10:20 (molar ratio). For conventional LNPs, the FDA-approved Onpattro formulation (Dlin-MC3-DMA: DSPC: Cholesterol: DMG-PEG200 = 50:10:38.5:1.5) was used. The lipid in the ethanol phase and the mRNA in the aqueous phase (50 mM pH 4 citrate buffer) were rapidly mixed at an N / P ratio of 5 and a volume ratio of 1:3. The mixed solution was incubated at room temperature for 10 min and then dialyzed overnight against DPBS (pH 7.4) using a 10 kDa snakeskin dialysis tubing.

[0080] Analysis of particle characteristics before and after nebulization

[0081] The size and zeta potential of the nanoparticles were measured at room temperature using a dynamic light scattering (DLS) instrument (Nano-ZS90, Malvern). Encapsulation efficiency was measured using a low-range standard curve with the Quant-it RiboGreen RNA assay kit (Thermo Fisher) according to the manufacturer's instructions. The nanoparticles were nebulized using an Aeroneb Lab Nebulizer unit (Kent Scientific) according to the manufacturer's instructions to produce aerosols with a size of 2.5–4.0 μm. After nebulization, the aerosol was rapidly liquefied in a 15 mL conical tube on ice, and the resulting nanoparticles were characterized as before nebulization.

[0082] Extracorporeal lung surfactant capture

[0083] Bovine-derived PS (Newfactan) was dissolved in a 2:1 mixture of chloroform and methanol, placed in a vial, and dried until the solvent was completely removed to form a lipid membrane. The preheated nanoparticle solution was added to the vial and rapidly mixed to rehydrate the lipid membrane, resulting in a final solution with a surfactant concentration of 5 mg / mL. The mixture was incubated at 37°C for 30 minutes, and the produced nanoparticles were subjected to an in vitro infection assay in serum-free medium.

[0084] Cryogenic transmission electron microscopy

[0085] 3 μL (~5 mg / mL) of nanoparticle solution was applied to glow-discharged Quantifoil R1.2 / 1.3 Cu 300 grids and flash-frozen in liquid ethane using a Vitrobot mark IV (Thermo Fisher) with the chamber set to 100% humidity, 8°C, and a blotting time of 3–4 s. Cryo-TEM images were acquired using a Glacios microscope (Thermo Fisher) operating at an accelerating voltage of 200 kV and a 70 μm C2 aperture. A Falcon IV direct electron detector was used to acquire images of the samples with a 100 μm objective aperture.

[0086] In vitro transfection studies

[0087] A549 cells were cultured in complete RPMI 1640 medium (Hyclone) supplemented with 10% heat-inactivated FBS and 1% penicillin / streptomycin in a 5% CO2 humidified incubator at 37°C. For in vitro Nluc mRNA transfection assays, cells were seeded at 1.8 × 10 4 Cells were seeded in white 96-well plates at a density of 10 cells / well and cultured overnight. To compare the transfection efficiency in serum-containing and serum-free environments, cells were appropriately treated with particles loaded with 1 μg / mL Nluc mRNA. For serum-containing environments, the particles were diluted in complete RPMI 1640 medium containing serum and then treated with cells. For serum-free environments, the particles were diluted in RPMI medium without FBS and then treated with cells. After 6 h, the cells were washed with DPBS, and the medium was changed to fresh complete RPMI. After 24 h of treatment, the medium was replaced with a 1:1 (v / v) mixture of Nluc substrate solution and Nluc buffer. The luminescence signal was quantified using an in vivo imaging system (IVIS, PerkinElmer) after 10 min of incubation at room temperature. For single-cell analysis and fluorescence imaging, 1.0 × 10 cells were seeded at 1 × 10 5 Cells were seeded in clear 6-well and 24-well plates at a density of 10 cells / mL. Particles loaded with sfGFP mRNA were treated in the same manner as those loaded with Nluc mRNA. GFP signals were analyzed 24 h later by flow cytometry or confocal microscopy.

[0088] RNase susceptibility analysis

[0089] Particle samples loaded with 20 μg / mL of mRNA were mixed with 2 μg / mL of RNase A (NEB) and incubated at 37°C for 30 minutes, followed by the addition of 4 units / mL of protein kinase K (NEB) and incubation for an additional 10 minutes. Samples were either directly treated with A549 cells for in vitroluciferase assay or mRNA was isolated for integrity measurements.

[0090] gel electrophoresis

[0091] mRNA was purified from nanoparticles using a PCR purification kit (QIAGEN), and the concentration was quantified using a NanoDrop One (Thermo Fisher). A 1% agarose gel containing formaldehyde and MOPS buffer was prepared, and the RNA sample was heated at 70°C for 10 minutes and then cooled on ice for 3 minutes. The sample was loaded onto the gel and electrophoresis was performed at 5–6 V / cm until the ladder migrated sufficiently.

[0092] animal

[0093] All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Advanced Institute of Science and Technology (KAIST). To determine the in vivo protein expression, mRNA delivery, and biocompatibility of iLPX, 7-week-old female C57BL / 6N mice were purchased from Coretech. To determine the distribution of protein expression in the lung, 7-week-old female B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze / J mice were purchased from Jackson Laboratories. All mice were acclimated for at least 1 week before use in the experiments.

[0094] Administration of nanoparticles to mice via inhalation

[0095] For inhalation administration of nanoparticles, mice were placed in nose-only restraints and exposed to aerosol using an Aeroneb Lab nebulizer unit and the InExpose system (SCIREQ). Nanoparticles containing 50–100 μg / mL mRNA were nebulized and allowed to inhale at an airflow rate of 1 L / min until the desired mRNA dose was reached. Mice were anesthetized 3 or 24 hours after inhalation of Cy5-tagged mRNA or Nluc mRNA, respectively, and perfused transcardially with 0.9% NaCl. After sacrifice, the lungs and other major organs (liver, spleen, heart, and kidney) were removed and analyzed.

[0096] In vivo luciferase assay

[0097] Lung tissue from mice that had inhaled nanoparticles loaded with Nluc mRNA was collected and washed with PBS. The lungs were then cut into lobes and immersed in Nano-Glo substrate (Promega) diluted 50-fold in DPBS. After 10 minutes of incubation, the lung lobes were placed on black plates, and the luminescence signal was quantified using IVIS.

[0098] Flow cytometry

[0099] To perform single-cell analysis of in vitro cell lines, cells were prepared in 24-well cell culture plates. Cells were dissociated by treating with 500 μL trypsin-EDTA (Gibco) for 5 minutes, then 200 μL RPMI medium was added, the cells were collected, and centrifuged at 350 × g for 5 minutes at 4°C. The supernatant was discarded, and the cells were washed twice in PBS with 1% FBS. The cells were then resuspended in 500 μL 1% FBS and treated with 1 μg / mL propidium iodide (Invitrogen) to confirm viability. After 15 minutes, the cells were analyzed using a BD LSRFortessa X-20 cell analyzer (BD Bioscience).

[0100] To perform single-cell analysis on mouse lung cells, the collected lungs were minced in 100 μL of dissociation solution (1x RPMI + 2 mg / mL collagenase D (Sigma) + 1.5 mg / mL collagenase / dispase (Sigma) + DNase I (ThermoFisher Scientific)). 900 μL of dissociation solution was then added, and the mixture was incubated at 37°C for 1 hour with gentle agitation. After filtering through a 70 μm cell strainer and washing three times with 5% FBS solution, an appropriate number of cells were aliquoted and incubated with Zombie Aqua fixable viability dye (Biolegend) for 15 minutes at room temperature. Anti-mouse CD16 / 32 antibody (Fc blocker, Biolegend) was then added and incubated at 4°C for 5 minutes, followed by anti-EPCAM, anti-CD31, and anti-CD45 antibodies, which were then added and incubated at 4°C for 30 minutes. Stained single cells were analyzed using an LSRFortessa X-20 cell analyzer.

[0101] confocal microscopy

[0102] To visualize GFP expression or Cy5-tagged mRNA delivery in A549 cells, cells were cultured in 6-well plates. Twenty-four hours after nanoparticle administration, cells were fixed with 10% neutral buffered formalin (Sigma) for 15 minutes at room temperature. Cells were then washed three times with PBS and incubated with a 1000-fold dilution of Hoechst 33342 (Thermo Fisher) solution in PBS for 5 minutes. After another three washes with PBS, the wells were filled with 2 mL of PBS and imaged using a confocal microscope (Nikon).

[0103] To visualize delivery of nanoparticles to deep lung tissue after inhalation, excised lungs were incubated overnight in 10% neutral buffered formalin at 4°C. The lungs were then placed in optimal cutting temperature (OCT) compound (Leica) and frozen in a deep freezer. Frozen lung samples were sectioned into 10-μm slices using a Cryostat cryocut microtome (Leica CM1850). Lung tissue sections were mounted on Histobond adhesive microscope slides (Marienfeld) and washed three times with phosphate-buffered saline (PBS). The sections were then stained with a 5000-fold diluted Hoechst 33342 solution for 5 minutes, followed by a final wash with PBS. Fluoromount aqueous mounting medium (Sigma) was then applied, and a cover glass was placed over the samples. Finally, the samples were imaged using a confocal microscope.

[0104] Toxicity studies

[0105] To evaluate the toxicity of IH-iLPX, IH-iLPX loaded with 100 μg / head of mRNA was administered intranasally, and 3 mg / kg of LPS was used as a positive control. After 24 h, mice were anesthetized, and blood was collected from the orbital vein using heparinized capillary tubes (DWK Life Science). The collected blood was centrifuged twice at 2000× speed at 4°C, and the supernatant was collected for blood chemistry tests.

[0106] To obtain BAL fluid, a catheter (BD Angiocath Plus) was inserted approximately 0.5 cm into the trachea using a 23G needle inserted into a plastic polyethylene tube, and the needle was then removed. Then, 1 mL of 4°C PBS was slowly infused through the tube. After waiting for approximately 3 seconds, the BAL fluid was carefully collected. The BAL fluid was centrifuged at 400x for 10 minutes at 4°C to separate the supernatant and the cell pellet. The cell pellet was resuspended in PBS, and the cell number in the BAL fluid was counted using a Luna automated cell counter (Logosbio). IL-1, IL-6, and TNF-α in the BAL fluid supernatant were analyzed using an ELISA kit (Invitrogen) according to the manufacturer's instructions.

[0107] After BAL fluid extraction, transcardiac perfusion was performed, and the lungs were removed. The removed lungs were imaged using an optical microscope (Nikon), stained with H&E, and subjected to histopathological analysis.

[0108] Statistical analysis

[0109] Data are expressed as mean ± standard deviation (SD), and statistical significance was determined using independent-samples t-test, one-way ANOVA, and two-way ANOVA with Tukey's multiple comparison test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). All statistical analyses were performed using GraphPad (Prism 8.4) software.

[0110] [Experimental Results]

[0111] Design of an inhalable nanoparticle platform for mRNA delivery to the lungs

[0112] To effectively deliver nanoparticles to deep lung cells, a nebulizer must atomize the particles to a size of 5 μm or less. When using a vibrating mesh nebulizer, the solution is aerosolized by a piezoelectric actuator that converts electrical energy into mechanical vibrations. The rapid vibration of the particle solution into an aerosol generates shear forces that disrupt the particle structure. Therefore, a carrier for inhalation delivery of mRNA must be able to withstand these shear forces.

[0113] Meanwhile, after inhalation, nebulized aerosol interacts with pulmonary surfactant (PS) at the air-alveolar fluid interface deep in the lungs. PS is a complex of lipids and proteins that forms multiple layers at the air-lung interface, posing a major obstacle to inhaled therapeutics. Particles that pass through surfactant and reach lung cells are encapsulated by PS, making interaction with PS a crucial factor. PS, produced by type II alveolar epithelial cells, circulates and crosses the lung barrier via a "squeeze-out" mechanism during respiratory movement. Another characteristic of the lung microenvironment is that type II alveolar epithelial cells differentiate the composition of blood and alveolar fluid, with bronchoalveolar lavage (BAL) fluid having a significantly lower protein concentration than blood. Therefore, for mRNA to be effectively delivered to the lungs via inhalation, nanocarriers must overcome shear forces during nebulization and be compatible with the lung microenvironment (Figure 1a).

[0114] To evaluate whether lipid nanoparticles (LNPs), approved by the US FDA for delivering siRNA to hepatocytes, are suitable for inhaled delivery, LNPs containing nanoluciferase (Nluc) mRNA were delivered to A549 cells, a type II alveolar adenocarcinoma cell line, and infected under various conditions, including nebulization, the presence or absence of serum in the medium, and surfactant encapsulation. Quantification of bioluminescence levels revealed that protein expression levels were reduced 5.88-fold after nebulization, 40-fold in the absence of serum, and 1.54-fold after surfactant incubation (Fig. 1b). The decrease in expression due to nebulization is primarily due to the structural limitations of LNPs, which have a partially separated lipid layer, making them unable to withstand the shear forces generated during nebulization. The decrease in expression under serum-free and surfactant conditions is likely due to the PEG surrounding the exterior of the LNPs. In a systemic environment, the exchange of serum protein apo-E and PEG is known to be a major driving force behind particle uptake into cells in the liver. However, in the absence of serum proteins, PEG cannot dissociate from the particles. Consequently, PEG remaining on the particle exterior interferes with surfactant interaction and cellular uptake, suggesting that PEG inclusion acts as an inhibitor in the lung microenvironment.

[0115] To address these issues with LNPs, we developed a novel particle platform, the ionizable liposome-mRNA lipocomplex (iLPX). iLPX is formed by complexing a core liposome with mRNA. The core liposome of iLPX was prepared by emulsifying ionizable lipids, structural lipids, and cholesterol (Figure 7). This structure enhances spray stability due to strong hydrophobic interactions between the lipid tails, and is designed to allow mRNA to be attached to the exterior of the liposome to maintain its internal structure (Figure 1c). Furthermore, a PEG-free lipid composition was used to maximize mRNA loading outside the liposome, promote PS-based penetration, and achieve high intracellular delivery under low serum conditions. The core liposome preparation process was optimized based on spray stability. Because ionic concentration can affect the aggregation and kinetic stability of nanoparticles in solution, particle size changes during spraying were investigated as a function of ionic concentration during complexation of the core liposome with mRNA. Size measurements revealed that the absence of ions between the mRNA and liposomes contributed to the spray stability (Fig. 8). Because smaller nanoparticles may decrease their clearance by macrophages, deionized water was used as a solvent during the manufacturing process of iLPX. In summary, iLPX was designed to exhibit excellent resistance to external physical forces, maintaining its properties during spraying and enabling easy transport into lung cells.

[0116] Optimization of iLPX for inhalation through multi-stage screening

[0117] A sequential multi-step screening procedure was performed to select iLPX optimized for inhalation (IH-iLPX) (Fig. 9). A particle library was constructed by varying lipid composition, molar ratio of ionizable lipid to nucleotide (N / P ratio), and tail length of the structural lipids. Multi-step screening was performed for each variable, and then fixed for subsequent optimization. In the first screening step, particles were selected based on their physicochemical properties (hydrodynamic size, polydispersity index (PDI), and mRNA capture efficiency). The second screening step focused on the infection efficiency of nebulized iLPX in A549 cells. In the third screening step, infection efficiency was assessed in serum-free medium after in vitro PS encapsulation, which simulates the lung environment encountered by inhaled particles. Finally, protein expression was measured after inhalation in mice, completing the multi-step screening.

[0118] First, we optimized the lipid composition of the core liposome. Forty-eight core liposomes were prepared by varying the molar ratios of the ionizable lipid Dlin-MC3-DMA (MC3), the structural lipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol. Thirteen core liposome formulations were selected based on size and PDI (Table 1) (Fig. 2a). The selected liposomes were then complexed with mRNA at a weight ratio of 1:40 (mRNA:liposome) to formulate iLPXs. The characteristics of the 13 iLPXs (C#, #: 1-13) were analyzed before and after spraying. As a result, no significant changes were observed in any group after spraying (Fig. 9). However, the infection efficiency varied depending on the formulation, with the protein expression of C13 being the highest after spraying (Fig. 2b). This may be because the encapsulation efficiency was higher with a higher content of ionizable lipids, resulting in differences in the ability to protect mRNA during the nebulization process (Fig. 9b). Furthermore, the infection efficiency tended to be higher in the group with a higher cholesterol content. Eight groups were selected based on the infection efficiency after nebulization, and the infection efficiency in serum-free medium after PS encapsulation was evaluated to simulate the lung microenvironment. The animal-derived PS lipid membrane was rehydrated using the nebulized particle solution, incubated at 37°C for 15 minutes, and then treated on A549 cells. Group C13 showed the highest infection efficiency (Fig. 2c). Among groups with the same ionizable lipid content, the particle with the highest cholesterol ratio showed the highest efficiency, so one candidate was selected for each ionizable lipid content (50%, 60%, and 70%) and the infection efficiency in the lungs after inhalation was evaluated. Significantly higher signals were observed in the C13 group (Figs. 2d and 2e), which is consistent with the in vitro optimization results in the lung bioluminescence images (Figs. 2b and 2c).

[0119]

[0120] Next, we aimed to optimize the tail length of the structural lipids constituting the core liposome. Five types of core liposomes were prepared using phosphocholines with tail lengths ranging from 14:0 to 22:0, depending on the lipid composition used in C13. There was no significant change in particle characteristics after spraying across all groups (Figures 10a-10c). Notably, the group with longer lipid tails exhibited relatively higher encapsulation efficiency, suggesting a correlation between membrane stability and mRNA loading. Conversely, the group with shorter lipid tails exhibited higher protein expression (Figure 10d). This is because shorter lipid tails increase liposome membrane fluidity, which may promote membrane destabilization during endosomal escape. However, in the PS-encapsulated environment, the 18:0 group exhibited the highest expression rate (Figures 2f and 11e). Because PS and mRNA can compete with core liposomes for protein encapsulation, the presence of PS may significantly enhance both encapsulation efficiency and core stability. Therefore, C13 (C13-DSPC), which is composed of DSPC with 18:0 lipid tails, was finally selected as the optimal core liposome.

[0121] Finally, the N / P ratio for complexing the liposome core and mRNA was optimized. Various iLPXs were prepared by complexing C13-DSPC liposomes and mRNA at various N / P ratios from 1 to 21, and the physicochemical properties of the particles before and after spraying were evaluated. As a result, the zeta potential was maintained constant at +25-30 mV in the N / P ratio group of 3-21 (Fig. 11c), and the size and mRNA encapsulation were relatively stable in the N / P ratio groups of 11-21 (size < 200 nm, encapsulation efficiency > 80%) (Figs. 11a and 11b). In vitro analysis of the N / P ratio groups of 11-21 showed no significant difference in protein expression in the presence of PS (Figs. 11d and 11e). However, in vivo analysis showed that protein expression was slightly higher in the N / P ratio group of 15 than in the other groups (Fig. 2g), so 15 was selected as the optimal N / P ratio. As a result, iLPX with a lipid composition of 70:10:20 (ionizable lipid:structural lipid:cholesterol), an N / P ratio of 15, and DSPC as the structural lipid was selected as the iLPX (IH-iLPX) optimized for inhalation. IH-iLPX exhibited similar values ​​to conventional LNPs except for the zeta potential, with a hydrodynamic size of 86.45 nm, a polydispersity index of 0.062, a zeta potential of +25.49 mV, and an encapsulation efficiency of 92.6% (Table 1). Surprisingly, inhaled IH-iLPX showed 26.3-fold higher protein expression in the lungs than inhaled LNPs (Figs. 2h and 2i), indicating that a carrier with significantly superior inhalation delivery efficiency of mRNA was derived through a multi-step screening targeting nebulization stability and lung microenvironment compatibility.

[0122]

[0123] Physicochemical properties of IH-iLPX during spraying

[0124] To confirm that the excellent stability of IH-iLPX during spraying was due to the lipid bilayer of the core liposome and to verify that ionizable lipids are useful for mRNA delivery, cationic liposome-based particles (cLPX) were prepared as a control. Comparison of the physicochemical properties of each group before and after spraying revealed that LNPs did not maintain physicochemical properties such as size, PDI, and encapsulation efficiency, whereas IH-iLPX and cLPX maintained these properties (Fig. 3a-c). These results indicate that the lipoplex structure with mRNA loaded on the outside of the liposome is more stable during spraying than the LNP structure with mRNA loaded inside.

[0125] To determine the effect of nebulization on protein expression, A549 cells were treated with non-nebulized / nebulized particles and particles loaded with GFP mRNA in medium containing 10% fetal bovine serum (FBS). After 24 h of incubation, GFP signals were evaluated by flow cytometry and confocal imaging. In the non-nebulized state, IH-iLPX showed a 0.7-fold higher expression level than LNP (Fig. 3d). In contrast, after nebulization, it showed a 2.9-fold higher expression level than LNP, which may be due to the instability of the LNP structure during nebulization. Despite maintaining its properties during nebulization, cLPX showed a low infection efficiency. This may be due to excessive electrostatic interactions with serum proteins in the medium.

[0126] To further analyze the effect of nebulization on the structure and function of the particles, the combined effect of nebulization and RNase was analyzed through protein expression levels and mRNA integrity. The results of the free mRNA group showed that both nebulization and RNase treatment resulted in mRNA degradation (Fig. 13). IH-iLPX maintained both mRNA integrity and infection efficiency during nebulization, whereas LNP maintained integrity but had a reduced infection efficiency (Fig. 3e and Fig. 13). Furthermore, after post-nebulization treatment with RNase, the infection efficiency of IH-iLPX decreased only 2.13-fold, whereas that of LNP decreased by up to 62.5-fold, indicating that the mRNA integrity of IH-iLPX was superior to that of LNP under these conditions. Furthermore, the morphological changes of LNP and IH-iLPX after nebulization were visualized by cryo-TEM. IH-iLPX maintained its morphology and no membrane destabilization was observed, whereas LNP exhibited complete membrane destruction (Fig. 3f). These results demonstrate that IH-iLPX has superior nebulization stability compared to conventional LNPs. This is because mRNA loaded inside LNPs is exposed and dissociated by the shear force of the nebulizer, which reduces protein expression and increases vulnerability to RNase, whereas mRNA loaded into IH-iLPX maintains high integrity due to the stable core liposome induced by strong hydrophobic interactions between lipid tails, and in particular, the persistent charge interaction between the negatively charged phosphate backbone and the positively charged ionizable lipid may maintain protein expression levels and exert a protective effect against RNase (Fig. 3g).

[0127] Transfection ability of IH-iLPX in the lung microenvironment

[0128] To verify the suitability of IH-iLPX for inhaled mRNA delivery in the lung microenvironment, A549 cells in serum-free medium were treated with LNPs, IH-iLPX, and cLPX loaded with GFP-mRNA, and GFP signals were examined by confocal imaging and flow cytometry 24 h later (Figs. 4a and 4b). Fluorescence images showed that the IH-iLPX group had higher overall GFP expression than the other groups. This was also confirmed by single-cell analysis using flow cytometry, where 93.5% of cells in the IH-iLPX group expressed GFP, compared to 30.8% in the LNP group and 35.8% in the cLPX group. Similar results were obtained when confocal imaging and analysis of Cy5-positive single-cell populations after delivery of Cy5-tagged mRNA (Figs. 14a and 14b). The superior efficacy of IH-iLPX compared to LNPs in serum-free medium suggests that PEGylation may interfere with intracellular delivery. Furthermore, these results provide strong evidence that PEG-free IH-iLPX is more effective than PEGylated particles in the lung microenvironment, where serum concentrations are low.

[0129] Next, to mimic the air-alveolar fluid interface (ALF), as performed in the optimization step, each particle was encapsulated with pulmonary surfactant and treated with A549 cells. As a result, the IH-iLPX group showed an overall higher GFP signal than the other groups, with 93.8% of cells expressing GFP (vs. 23.1% in LNP and 0.2% in cLPX) (Figures 4c and 4d). In the case of cLPX, the GFP signal was reduced 125-fold by PS incubation. Analysis of the Cy5 signal after delivery of Cy5-tagged mRNA showed a similar pattern to GFP expression (Figures 14c and 14d). As observed in serum-containing medium, cLPX can aggregate in the PS environment due to excessive interaction with the negatively charged PS. Unlike permanently cationic lipids, ionizable cationic lipids are neutral at physiological pH, so they can be delivered to cells without aggregation after encapsulation in PS.

[0130] Next, to compare the particle-to-particle GFP expression efficiency in simulated lung fluid, a simulated lung fluid mimicking the surrounding environment of lung epithelium was used (Table 3).

[0131]

[0132] As a result, consistent with the results in serum-free medium, IH-iLPX showed the highest GFP-positive cell population (82.4%) compared to LNP and cLPX (LNP 31.2%, cLPX 10.8%) (Fig. 4e). Furthermore, to verify the efficacy of IH-iLPX compared to conventional LNP in mouse lungs, LNP and IH-iLPX were directly injected into the trachea to exclude the nebulization effect, and IH-iLPX induced a significantly higher luminescence signal (Fig. 4f and 4g). Considering that LNP mediated higher protein expression than IH-iLPX in both intravenous and intramuscular delivery (Fig. 15), these results demonstrate that IH-iLPX is specialized for mRNA delivery to the lung microenvironment. Overall, IH-iLPX showed higher intracellular delivery in the lung microenvironment with low serum concentrations, which is thought to be due to its excellent penetration into lung cells due to its PEG-free composition and favorable interaction of ionizable lipids with PS.

[0133] Tissue and cellular distribution of inhaled IH-iLPX in the lungs

[0134] Major organs, including the lungs, liver, heart, spleen, and kidney, were collected, and luminescence signals were measured at various time points over 72 hours. The organ biodistribution and expression kinetics after inhalation of IH-iLPX loaded with Nluc-mRNA were analyzed. As a result, luminescence signals were observed only in the lungs, and protein expression reached its peak 24 hours after inhalation (Figures 5a and 5b). Next, to confirm the protein expression distribution in the lungs, IH-iLPX loaded with Nluc-mRNA was administered via inhalation, and the lungs were collected 24 hours later. Each lung lobe and trachea were isolated, and the luciferase expression efficiency in each region was quantified. The results showed that expression was evenly distributed across all lung lobes and deep into the lungs (Figures 5c and 5d). In contrast, luciferase was not detected in the trachea, indicating that spraying the IH-iLPX solution generates small aerosol particles (less than 5 μm), which can preferentially accumulate deep in the lungs.

[0135] To further investigate the distribution in deep lung tissue, a high dose (100 μg) of Cy5-tagged mRNA was loaded into IH-iLPX or LNP and administered to mice via inhalation. After 3 h, lungs were harvested and analyzed using confocal microscopy and flow cytometry. Compared with the LNP group (Fig. 16), the IH-iLPX group showed widespread Cy5 signal distribution in deep lung tissue, observed in both alveolar spaces and airways (Fig. 5e). Single-cell analysis revealed that Cy5-positive lung epithelial cells were significantly higher in the IH-iLPX group than in the LNP group (Fig. 5f), indicating that IH-iLPX is superior to LNP in mRNA delivery to lung epithelial cells. The gating strategy for lung cells was adopted from the method described above (Fig. 17).

[0136] In summary, we demonstrated that IH-iLPX is a promising platform for lung-specific protein expression via inhaled delivery because it effectively delivers mRNA to the alveolar space and airways. Alveoli and airways play a crucial role in various lung diseases, including respiratory viral infections, idiopathic pulmonary fibrosis, surfactant protein B deficiency, cystic fibrosis, and asthma. Since many lung disease therapeutics target epithelial cells, IH-iLPX has the potential to mediate therapeutic effects in these diseases. Analysis of the transfection efficiency as a function of mRNA dose revealed a linear dose-dependent increase in luminescent signal (R 2 =0.8639, p=0.0003) (Fig. 18b). In addition, since IH-iLPX has an internal compartment, it also has the possibility of simultaneous delivery of hydrophilic drugs. This possibility was evaluated by loading the hydrophilic small molecule calcein (MW: 622.5 Da) inside the core liposome and mCherry mRNA on the outside, and as a result, calcein delivery and mCherry expression were confirmed simultaneously in A549 cells (Fig. 19). Based on these results, it is expected that the platform of the present invention can be used to promote synergistic therapeutic effects through simultaneous delivery of mRNA and small molecule chemical drugs.

[0137] Biocompatibility of IH-iLPX inhaled in pulmonary and systemic environments

[0138] The structure of liposomes is similar to that of cell membranes in living organisms, and because their components are derived from natural substances, they have high biocompatibility compared to synthetic materials such as polymers. In addition, ionizable lipids have a neutral charge at physiological pH, so they can solve the toxicity problem of existing cationic lipids. To verify the tolerability of IH-iLPX in vivo, the concentrations of inflammatory cytokines (IL-1, IL-6, and TNF-α) were measured, and lung histopathological analysis and clinical blood tests were performed. IH-iLPX at 100 μg / head mRNA was administered to mice by inhalation, and the untreated group served as the negative control, and the LPS-inhaled group served as the positive control. BAL fluid, blood, and lung samples were collected 24 hours after inhalation. The cytokine concentrations in the BAL fluid of mice administered IH-iLPX were similar to those in the untreated group, but the LPS group had significantly higher concentrations of IL-6 and TNF-α (Fig. 6a). The number of white blood cells in the BAL fluid was similar in the IH-iLPX and untreated groups, but the LPS group had a significantly higher cell count (Fig. 6b). Next, hematoxylin & eosin (H&E) staining of lung tissues showed no difference between the untreated and IH-iLPX groups (Fig. 6c). In contrast, the LPS group showed severe lung damage, including thickened alveolar walls and interstitial infiltration. In addition, clinical blood test results showed that aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were similar in all groups, and total protein in the IH-iLPX group did not increase compared to the untreated group (Fig. 6d). This indicates that inhaled IH-iLPX does not induce severe liver toxicity or systemic inflammation. In summary, mice treated with IH-iLPX showed similar pathological characteristics to untreated mice in the pulmonary and systemic environments, demonstrating the high biosafety of the nanoparticles.

[0139] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0140] The lipid complex of the present invention maintains the structure of nucleic acids even under shear force during spraying, efficiently fuses with the lung surfactant membrane, and effectively delivers the target gene to deep lung cells, thereby being useful as a drug delivery system for treating various intractable lung diseases such as cystic fibrosis, pulmonary fibrosis, and α-antitrypsin deficiency.

Claims

1. A lipid complex (lipocomplex) for nucleic acid molecule delivery containing ionizable lipid, structural lipid, and cholesterol as active ingredients.

2. A lipid complex according to claim 1, characterized in that the ionizable lipid is at least one selected from the group consisting of Dlin-MC3-DMA, SM-102, and ALC-0315.

3. A lipid complex according to claim 1, wherein the structural lipid is at least one selected from the group consisting of phosphatidylcholine, DPPC (dipalmitoyl phosphatidyl choline), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DMPC (dimyristoylphosphatidyl choline), PEG-PE (polyethylene glycolphosphatidyl ethanolamine), DOPC (dioleoyl phosphatidyl choline), and DOPE (dioleyl phosphatidyl ethanolamine).

4. A lipid complex according to claim 1, characterized in that the ionizable lipid is included in a molar ratio of 50 to 90% of the total lipid.

5. A lipid complex characterized in that the structural lipid is included in a molar ratio of 5 to 15% of the total lipid in the first paragraph.

6. A lipid complex characterized in that, in the first paragraph, the cholesterol is included in a molar ratio of 5-35% of the total lipid.

7. A lipid complex according to claim 1, characterized in that the ionizable lipid, structural lipid, and cholesterol have a molar ratio of 6-8:0.5-1.5:1.5-2.

5.

8. A lipid complex according to claim 1, characterized in that the molar ratio (N / P) of the amine group in the ionizable lipid to the phosphate group in the nucleic acid molecule to be delivered in the lipid complex for nucleic acid molecule delivery is 5 to 25.

9. A lipid complex according to claim 1, characterized in that the lipid complex does not contain PEG (polyethylene glycol).

10. A lipid complex according to claim 1, characterized in that the lipid complex has an average particle diameter of 50 nm to 200 nm.

11. A lipid complex, characterized in that the nucleic acid molecule to be delivered in the first paragraph is attached to the outer surface of the lipid complex.

12. A lipid complex according to claim 1, characterized in that the nucleic acid molecule to be delivered is an mRNA molecule.

13. A lipid complex according to claim 1, characterized in that the lipid complex is a complex for inhalation delivery for lung-specific nucleic acid molecule delivery.

14. A step of forming a lipid film by drying a mixed solution containing ionizable lipid, structural lipid, and cholesterol; A step of rehydrating the above lipid film with a C1-C3 alcohol solvent; and A method for producing a lipid complex (lipocomplex) for nucleic acid molecule delivery, comprising the step of mixing and stirring the rehydrated lipid and the nucleic acid molecule to be delivered.

15. A method according to claim 14, wherein the mixed solution comprises ionizable lipids, structural lipids, and cholesterol in a molar ratio of 6-8:0.5-1.5:1.5-2.

5.

16. A method according to claim 14, characterized in that the molar ratio (N / P) of the amine group in the ionizable lipid to the phosphate group in the nucleic acid molecule to be transferred in the lipid complex is 5 to 25.

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