A Lipid Nanocomplex for Intranasal Delivery of Nucleic Acid Molecule

Abstract

The present invention relates to a lipid complex for the nasal delivery of nucleic acid molecules comprising ionized lipids, structural lipids, cholesterol, and lysoPC as active ingredients. The lipid complex of the present invention can be usefully utilized for the local and intensive delivery of therapeutic nucleic acid molecules specifically to nasal epithelial cells by efficiently being compatible with the microenvironment unique to nasal tissue, easily penetrating the mucus layer barrier, and effectively escaping endosomes. Furthermore, the lipid complex of the present invention can be applied as a combination administration platform that induces a synergistic therapeutic effect by two pharmacological components by loading additional drugs into internal compartments of the lipid complex, as well as improving dispersibility by loading the target nucleic acid molecule externally.

Description

Technology Field

[0001] The present invention relates to a lipid nanocomplex for specifically delivering a nucleic acid molecule encoding a pharmacological protein to nasal tissue. Background Technology

[0003] Gene therapies are receiving significant attention as next-generation treatments due to their various advantages over existing protein therapies, including safety, efficacy, productivity, and cost-effectiveness. In particular, as gene therapies utilizing lipid nanoparticles (LNPs)—such as Patisiran, an intravenous siRNA therapeutic for polyneuropathy, and Comirnaty, an mRNA-based intramuscular COVID-19 vaccine—have demonstrated excellent clinical efficacy and safety, interest in gene delivery technologies targeting organs and tissues other than the liver and muscles is growing. Despite the various benefits of mRNA therapies, numerous barriers still exist that impair their function as pharmacological components under physiological conditions; therefore, delivery vehicles play a crucial role in safely transporting mRNA into the cytoplasm of target cells.

[0004] Meanwhile, while nasal drug delivery offers the advantages of being non-invasive, easy to administer by non-specialists, and allowing for localized administration, existing lipid nanoparticle (LNP) formulations exhibit significantly lower gene delivery efficiency compared to intravenous or intramuscular injections due to the specific microenvironment of nasal tissues. Gene delivery technology to nasal tissues and nasal epithelial cells holds great potential for application not only to infectious diseases but also to various conditions requiring gene therapy, such as sinusitis and rhinitis; to realize this potential, the development of gene delivery vehicles specialized for nasal delivery is necessary.

[0005] Currently, clinically used gene therapies utilize LNPs composed of ionizable lipids, structural lipids, cholesterol, and PEG-lipids to encapsulate and protect genes within a carrier and deliver them to target cells. However, when these types of particles are used for nasal administration, they not only struggle to overcome the mucus barrier within the nasal cavity, but the PEG layer surrounding the nanoparticles also interferes with cell interaction, making effective gene delivery to nasal cells difficult. Accordingly, the inventors aimed to develop a gene delivery vehicle capable of effective gene delivery to nasal cells by considering the nasal microenvironment and cell barriers.

[0007] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention. Prior art literature

[0009] Patent Document 1. U.S. Patent Publication No. 2022 / 0347100 A1 The problem to be solved

[0010] The inventors have made diligent research efforts to develop an optimized lipid nanoparticle-based gene delivery system capable of efficiently delivering nucleic acid molecules encoding therapeutic proteins to the nasal tissue of a subject. As a result, the inventors discovered that when a lipid complex comprising ionizable lipid, structural lipid, cholesterol, and lysoPC (lysophosphatidylcholine) in a specific molar ratio is used as a carrier, compatibility with the nasal tissue-specific microenvironment and penetration into the mucus layer are effectively achieved, thereby enabling significantly higher expression of the target protein specifically in the nasal tissue compared to the conventional nucleic acid delivery system, LNP (lipid nanoparticle), thus completing the present invention.

[0011] Accordingly, the objective of the present invention is to provide a lipid complex for nasal delivery of nucleic acid molecules comprising ionized lipids, structural lipids, cholesterol, and lysoPC as active ingredients; and a method for inducing the expression of a target protein in nasal tissue using the same.

[0012] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0014] According to one aspect of the present invention, the present invention provides a lipocomplex for nasal delivery of nucleic acid molecules comprising ionizable lipid, structural lipid, cholesterol and lysoPC (lysophosphatidylcholine) as active ingredients.

[0015] The inventors have made diligent research efforts to develop an optimized lipid nanoparticle-based gene delivery system capable of specifically delivering nucleic acid molecules encoding therapeutic proteins to nasal tissues. As a result, they discovered that when a lipid complex comprising ionized lipids, structural lipids, cholesterol, and lysoPC in a specific molar ratio is used as a carrier, it is efficiently compatible with the microenvironment of mucinous nasal tissues, can easily penetrate mucin networks, and can effectively escape endosomes, thereby significantly improving the expression efficiency of the target protein.

[0016] In this specification, the term “lipid” is a general term for natural or synthetic biomolecules that are soluble in non-polar solvents. Lipids typically have amphiphilic properties, including hydrophilic and hydrophobic components, and include, for example, phospholipids, fatty acids, fatty alcohols, neutral fats, phosphadides, glycolipids, aliphatic alcohols, waxes, terpenes, and steroids.

[0017] In this specification, the term “ionizable lipid” refers to a lipidoid that is uncharged at neutral pH but positively charged under acidic conditions, and 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 used without limitation as long as it is a lipid capable of being protonated under acidic conditions (e.g., a lipid having a chemical structure of pKa 5 to 7 to be positively charged at 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. More specifically, the ionizable lipid used in the present invention may be Dlin-MC3-DMA.

[0018] In the present invention, the term “structural lipid” refers to a phospholipid that serves as the 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 may be used in the present invention include dipalmitoyl phosphatidylcholine (DPPC), dilauryl phosphatidylcholine (DLPC) (C12:0), dimyristoyl phosphatidylcholine (DMPC) (C14:0), distearoyl phosphatidylcholine (DSPC), dipitanoyl phosphatidylcholine, nonadecanoyl phosphatidylcholine, arachidoyyl phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) (C18:1), dipalmitoleyl phosphatidylcholine (C16:1), linoleyl phosphatidylcholine (C18:2), myristoyl palmitoyl phosphatidylcholine (MPPC), steroidyl myristoyl phosphatidylcholine (SMPC), steroidyl palmitoyl phosphatidylcholine (SPPC), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl palmitooleoyl Phosphatidylcholine (PPoPC), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidyl ethanolamine (DOPE), dimyristoyl phosphatidyl ethanolamine (DMPE), distearoyl phosphatidyl ethanolamine (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) are included, but not limited thereto.

[0019] More specifically, neutral phospholipids may be used as the structural lipids of the present invention.

[0020] In this specification, the term “neutral phospholipid” refers not only to phospholipids in which each atom within the molecule does not carry a charge, but also to phospholipids in which the total net charge of the molecule is zero even if some atoms carry a charge, such as zwitterions. The neutral phospholipids used in the present invention may be one or more selected from the group consisting of, for example, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), phosphatidylcholine, DPPC (dipalmitoyl phosphatidyl choline), DMPC (dimyristoylphosphatidyl choline), DOPC (dioleoyl phosphatidyl choline), and DOPE (dioleyl phosphatidyl ethanolamine), but are not limited thereto.

[0021] Most specifically, DSPC can be used as the structural lipid of the present invention.

[0022] In this specification, the term “liposome” refers to a lipid carrier formed by creating a closed lipid bilayer. Liposomes are biocompatible with lipid bilayers and possess amphiphilicity, allowing them to pass through a hydrophobic membrane while containing internal hydrophilic substances. The diameter of liposomes is generally 20 to 2000 nm, but is not limited thereto and may vary in size depending on the manufacturing method and the length of the nucleotide to be encapsulated and delivered.

[0023] In this specification, the term “encapsulation” refers to the process of capturing a delivery substance by surrounding it with a closed membrane structure or by covalently or non-covalently binding it to the surface of a membrane in order to efficiently incorporate it into the body.

[0024] According to a specific embodiment of the present invention, the lysoPC is selected from the group consisting of 12:0 lysoPC, 14:0 lysoPC, 18:0 lysoPC, 20:0 lysoPC, and 22:0 lysoPC, more specifically selected from the group consisting of 14:0 lysoPC, 16:0 lysoPC, and 18:0 lysoPC, and most specifically 16:0 lysoPC.

[0025] The inventors have discovered that when lysoPC is added as a component of a core liposome, it lowers the energy barrier during the fusion process between ionized lipids and the endosome membrane, thereby allowing nucleic acid molecules to effectively escape the endosome, which enables efficient transfection into nasal cells.

[0026] According to a specific embodiment of the present invention, the ionized lipid is included in a molar ratio of 40 to 60% of the total lipid, and more specifically, in a molar ratio of 50 to 60%.

[0027] According to another specific embodiment of the present invention, the ionized lipid is included in a molar ratio of 45-55% of the total lipid, more specifically in a molar ratio of 47-53%, and most specifically in a molar ratio of about 50%.

[0028] 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%.

[0029] According to a specific embodiment of the present invention, the cholesterol is included in a molar ratio of 20-40% of the total lipids, more specifically 25-35%, even more specifically 27-33%, even more specifically 29-31%, and most specifically about 20%.

[0030] According to a specific embodiment of the present invention, the lysoPC is included in a molar ratio of 5-15% of the total lipids, more specifically 7-13%, even more specifically 9-11%, and most specifically about 10%.

[0031] According to a specific embodiment of the present invention, ionized lipids, structural lipids, cholesterol, and lysoPC are included in the lipid complex of the present invention in a molar ratio of 4-6 : 0.5-1.5 : 2-4 : 0.5-1.5. More specifically, they are included in a molar ratio of 4.5-5.5 : 0.7-1.3 : 2.5-3.5 : 1.7-2.3, and most specifically, in a molar ratio of 5 : 1 : 3 : 1.

[0032] According to a specific embodiment of the present invention, the lipid complex of the present invention does not contain PEG (polyethylene glycol). The inventors have identified that when PEG is bound to a lipid structure for delivering nucleic acid molecules, such as conventional LNPs, the PEG remains outside the particle without dissociating in the nasal microenvironment where serum protein concentrations are very low, thereby blocking interactions with nasal epithelial cells and hindering the intracellular absorption of nucleic acids. Accordingly, the lipid complex of the present invention can efficiently deliver target nucleic acids into the nasal microenvironment while maintaining high dispersibility through the electrostatic repulsion of ionized lipids, even without containing PEG.

[0033] According to a specific embodiment of the present invention, the lipid complex of the present invention has an average particle size of 50 nm to 200 nm, more specifically, an average particle size of 50 nm to 150 nm, and most specifically, an average particle size of 50 nm to 100 nm.

[0034] As described above, the lipid complex of the present invention can effectively deliver mRNA to nasal cells by adding lysoPC as a component of the core liposome to increase the curvature of the lipid bilayer and reduce the average particle size to less than 100 nm, thereby facilitating penetration into the nasal mucus layer composed of a dense mucin network having pores of 100-200 nm.

[0035] According to a specific embodiment of the present invention, the nucleic acid molecule of the present invention is attached to the outer surface of the lipid complex.

[0036] According to the present invention, the lipid complex of the present invention not only improves dispersibility by loading a target nucleic acid molecule externally, but also induces a synergistic therapeutic effect through the combined delivery of two pharmacological components by loading an additional drug, such as a hydrophilic low-molecular-weight compound, into an internal compartment of the lipid complex.

[0037] 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 building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites 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 (pi interfering RNA).

[0038] According to a specific embodiment of the present invention, the nucleic acid molecule to be delivered through the lipid complex of the present invention is an mRNA molecule.

[0039] When mRNA is used as the nucleic acid molecule of the present invention, various modifications may be applied to improve the expression (translation) efficiency of the target pharmacological protein, such as, for example, a change in the poly(A) tail length or a substitution of some adenine bases; a modification of the 5' cap; or the application of one or more modified nucleosides. The modified nucleosides that may be applied include, for example, N1-methylpseudouridine, pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, and 5-methoxyuridine, but are not limited thereto, and any modified nucleoside known in the art to reduce the immunogenicity of the mRNA molecule may be applied.

[0040] 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 attached to each of its ends, and more specifically, the 5'-UTR has a 5'-cap attached to it. In this specification, the term “UTR (untranslated region)” refers to an untranslated region attached to both ends of a coding sequence encoding a target protein within the 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 the mRNA that is connected to the 5'-UTR and binds to elF4E (eukaryote translation initiation factor 4E), thereby binding the 40S ribosomal subunit to the mRNA to initiate protein synthesis from the 5' start region of the mRNA, as well as protecting the mRNA from nucleases.

[0041] In this specification, the term “to express” means that a gene becomes replicable within a subject’s cell as an extrachromosomal factor or through the completion of chromosomal integration by artificially introducing it using a gene carrier to cause the subject to express an exogenous gene or to increase the natural expression level of an endogenous gene. Accordingly, the term “expression” has the same meaning as “transformation,” “transfection,” or “transduction.”

[0042] According to another aspect of the present invention, the present invention provides a method for expressing a target protein in the nasal cavity of a subject, comprising the following steps:

[0043] - A step of encapsulating a nucleic acid molecule encoding a target protein in the lipid complex of the present invention as described above; and

[0044] - A step of administering the lipid complex containing the above nucleic acid molecules into the nasal cavity of the subject.

[0045] Since the lipid complex used in the present invention and the nucleic acid molecules captured therein have already been described above, their description is omitted to avoid excessive duplication. Effects of the invention

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

[0048] (a) The present invention provides a lipid complex for nasal delivery of nucleic acid molecules comprising ionized lipids, structural lipids, cholesterol, and lysoPC as active ingredients.

[0049] (b) The lipid complex of the present invention can be effectively used to deliver therapeutic nucleic acid molecules locally and intensively to nasal epithelial cells specifically, by efficiently being compatible with the microenvironment unique to nasal tissue, easily penetrating the mucus layer barrier, and effectively escaping endosomes.

[0050] (c) The lipid complex of the present invention can be applied as a combination administration platform that improves dispersibility by loading a target nucleic acid molecule externally, and induces a synergistic therapeutic effect by two pharmacological components by loading an additional drug into an internal compartment of the lipid complex. Brief explanation of the drawing

[0052] Figure 1 is a schematic diagram illustrating the mechanism by which the intranasal lipid complex (IN-iLPX) of the present invention delivers mRNA to nasal cells. Figure 2 shows the results of measuring the zeta potential of iLPX according to the type of ionized lipid in iLPX (Figure 2a), the degree of mRNA expression in lung tissue (Figure 2b), the degree of mRNA expression in the nasal cavity (Figure 2c), and in vivo fluorescence images visualizing expression in the lungs and nasal cavity of mice (Figure 2d), respectively. Figure 3 shows the results of comparing the nasal mRNA expression efficiency according to the ratio of ionized lipids in iLPX. Figure 4 is a figure showing the results of evaluating the expression efficiency in HEK293T cells by conjugating 18 lysoPC iLPX candidate substances (#1-#18) of the present invention with mRNA, and the changes in particle size (Fig. 4a), PDI value (Fig. 4b), mRNA capture efficiency (Fig. 4c), and mRNA expression efficiency (Fig. 4d) as the lysoPC ratio is increased while the ratio of structural lipids is fixed. Figure 5 shows the results of comparing mRNA expression efficiency according to the type and ratio of lysoPC in lysoPC iLPX, including the expression efficiency (Figure 5a) when 18:0, 16:0, and 14:0 lysoPCs were included in ratios of 0-20%, respectively, and the particle size (Figure 5b), PDI value (Figure 5c), capture efficiency (Figure 5d), and luciferase expression amount (Figure 5e) according to the molar fraction of 16:0 lysoPC, which was selected as the most effective lysoPC, in IN-iLPX. Figure 6 is a figure showing the mRNA expression levels (Figure 6a) and changes in mRNA development patterns over time (Figure 6b) after nasal injection of the IN-iLPX of the present invention and conventional LNP, respectively. Figure 7 shows lysotracker-stained cell images (Figure 7a) showing the colocalization of Cy5 fluorescent conjugated mRNA with acidic cell organelles to compare the endosome escape ability of iLPX and IN-iLPX, and the results of quantifying the % colocalization of lysotracker and mRNA (Figure 7b), respectively. Figure 8 shows the results of comparing the endosome escape ability of iLPX, IN-iLPX, and conventional LNP, respectively, and the results of RBC hemolysis assays in acidic (Figure 8a) and neutral (Figure 8b) environments. Figure 9 is a figure showing the results of analyzing tissue confocal images after nasal injection of LNP loaded with Cre mRNA and IN-iLPX to confirm the intranasal expression pattern of mRNA delivered by IN-iLPX of the present invention and conventional LNP, respectively. Figure 10 is a figure showing the results of analyzing the types of cells expressing mRNA delivered into the nasal cavity using the IN-iLPX of the present invention by FACS. Figure 11 is a figure showing the results of comparing the intranasal mRNA expression ability of IN-iLPX formulations and LNP formulations with various ionized lipids introduced. Specific details for implementing the invention

[0053] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0055] Examples

[0056] Overview of IN-iLPX (intranasal mRNA delivery-optimized iLPX) Core Technology

[0057] For mRNA to be delivered to nasal cells and for effective protein translation to occur, it must not only effectively penetrate the mucus layer but also be efficiently introduced into the cells within the nasal tissue fluid environment, and effectively escape the endosome after introduction into the cell. The present invention aims to form a mRNA-lipid nanocomplex (iLPX) by forming a core liposome composed of ionizable lipids, structural lipids, and cholesterol using an ethanol injection method, followed by the formation of a complex with mRNA. iLPX maintains its nanoparticle form by being dispersed in DW (pH 5–6) using electrostatic repulsion through ionizable lipids, without the use of PEG (polyethylene glycol). Furthermore, due to the ionization ability of iLPX, it possesses the characteristic of changing its surface charge according to the nasal microenvironment after injection into the nasal cavity. Therefore, after being injected in a cationic state, it remains in the negatively charged mucus layer through charge interactions, and as acidity decreases, its surface charge gradually decreases, making it possible to pass through the mucus layer. Furthermore, unlike LNPs widely used for conventional mRNA delivery, it does not contain PEG, allowing it to interact efficiently with cells in the nasal tissue fluid environment. Since the nasal microenvironment has very low serum protein concentrations, PEG that has not desorbed from the nanoparticle surface can interfere with cell interaction; thus, a PEG-free composition is a key factor in mRNA delivery to nasal cells. Additionally, to further enhance the mRNA delivery efficiency of iLPX to nasal cells, lysoPC was added as a component of the core liposome to develop IN-iLPX (intranasal mRNA delivery-optimized iLPX), which is more effective for nasal delivery.Since the mucous layer of the nasal cavity consists of a dense mucin protein network with pore sizes of 100–200 nm, it is important to reduce the physical size of the carrier to effectively deliver mRNA to nasal cells. Accordingly, the inventors fabricated IN-iLPX using lysoPC lipids to create smaller core liposomes by increasing the curvature of the lipid bilayer. Additionally, lysoPC functions to lower the energy barrier during the fusion process between the ionized lipid and the endosome membrane, thereby contributing to the effective escape of mRNA from the endosome. Consequently, due to its small physical size, IN-iLPX can easily penetrate the dense mucin network and effectively escape the endosome, enabling a high level of transfection of nasal cells (Fig. 1).

[0059] Optimization process of ionized lipids for nasal delivery of iLPX

[0060] Primarily, we aimed to select ionized lipids suitable for nasal delivery by analyzing the differences in surface charge according to the type of ionized lipid in iLPX and the resulting protein expression patterns after nasal injection. The nasal mucus layer is slightly acidic with a pH of approximately 5.5–6.5, which is similar to the typical pKa values ​​(6–7) of ionized lipids used for nucleic acid delivery. Therefore, depending on the type of ionized lipid, the surface charge of iLPX in the mucus layer varies (Fig. 2a), and the interaction pattern with the negatively charged mucus layer can differ significantly. For example, if the surface charge is too low, the nanoparticles cannot interact with the mucus layer and migrate to the lungs (Fig. 2b), while if the surface charge is too high, they become trapped in the mucus layer and cannot be delivered to nasal epithelial cells. Accordingly, the inventors discovered the ionized lipid DLin-MC3-DMA; MC3, which has low protein expression in the lungs and can effectively deliver mRNA to nasal cells, by screening the optimal iLPX using various ionized lipids with different pKa values ​​(Figs. 2c and 2d). The core liposome of the iLPX used in the screening was composed of ionized lipid : DSPC : cholesterol in a molar ratio of 70:10:20.

[0062] Next, the composition ratio of ionized lipids was optimized to maximize the mRNA nasal delivery capability of iLPX. The luciferase expression intensity in the mouse nasal cavity was analyzed for each group while fixing the DSPC inclusion ratio at 10% and adjusting the inclusion ratio of ionized lipids (MC3) within iLPX to 40 to 70%. As a result, it was confirmed that nasal delivery was most suitable when the ratio of ionized lipids to total lipids was 50 to 60% (Fig. 3).

[0064] Optimization of IN-iLPX mRNA Nasal Delivery

[0065] The inventors additionally screened core liposome compositions containing 18:0 lysoPC for selected ionized lipids (MC3, 50-60%). The range of the primary screening based on physical properties included all intervals in 5% intervals for DSPC 0-10%, lysoPC 0-20%, and cholesterol 20% or more (Table 1). Eighteen compositions (#1-#18) with a particle size not exceeding 80 nm and a PDI of 0.3 or less passed the primary screening and were selected as candidates for iLPX production.

[0066] Primary screening results of material property-based lysoPC core liposomes MC3 / DSPC / Chol / lysoPC Size (nm) PDI 50 / 10 / 40 / 0 (#1) 66.4 0.190 50 / 0 / 45 / 5 118.6 0.375 50 / 0 / 40 / 10 84.4 0.180 50 / 0 / 35 / 15 (#2) 75.9 0.178 50 / 0 / 30 / 20 (#3) 70.3 0.177 50 / 5 / 40 / 5 (#4) 73.1 0.171 50 / 5 / 35 / 10 (#5) 70.1 0.182 50 / 5 / 30 / 15 (#6) 67.6 0.184 50 / 5 / 25 / 20 (#7) 61.7 0.196 50 / 10 / 35 / 5 (#8) 63.3 0.183 50 / 10 / 30 / 10 (#9) 66.4 0.213 50 / 10 / 25 / 15 (#10) 60.6 0.241 50 / 10 / 20 / 20 (#11) 64.6 0.235 60 / 10 / 30 / 0 (#12) 72.6 0.187 60 / 0 / 35 / 5 103.5 0.227 60 / 0 / 30 / 10 85.0 0.141 60 / 0 / 25 / 15 (#13) 79.1 0.169 60 / 0 / 20 / 20 86.1 0.182 60 / 5 / 30 / 5 (#14) 77.0 0.161 60 / 5 / 25 / 10 (#15) 78.3 0.134 60 / 5 / 20 / 15 (#16) 74.4 0.185 60 / 10 / 25 / 5 (#17) 71.4 0.157 60 / 10 / 20 / 10 (#18) 71.6 0.142

[0068] For compositions #1 - #18, lysoPC iLPX complexed with mRNA was synthesized and In vitro Secondary screening based on protein expression levels was performed using a luciferase assay in an environment (HEK293T cells). When the structural lipid ratio was constant, it was confirmed that as the lysoPC content increased, the size decreased and mRNA capture efficiency also tended to decline (Figs. 4a and 4c). Additionally, it was confirmed that mRNA expression was enhanced compared to the reference iLPX (#1, #12) when lysoPC was included at a DSPC ratio of 10% (Fig. 4d). All candidate groups were confirmed to exhibit a stable PDI within 0.3 (Fig. 4b). Overall, a range was selected to include 50% MC3 and 10% DSPC with 10% or less lysoPC (#8, #9) to achieve i) a high effect of size reduction; ii) without significantly reducing mRNA capture efficiency; and iii) a high effect of enhancing mRNA expression (Fig. 4).

[0070] To analyze and select the effects of lysoPC types and ratios in greater depth, lysoPC iLPXs were constructed containing 18:0, 16:0, and 14:0 lysoPCs at ratios ranging from 0% to 20%. The constructed lysoPC iLPXs In vitro As a result of comparing protein expression levels via luciferase assay in the environment (HEK293T cells), it was confirmed that the 16:0 lysoPC was the most effective lysoPC, and the ~10% molar ratio group In Vibo It was selected as a candidate group for evaluation (Fig. 5a). The particle size reduction effect according to the LysoPC inclusion ratio (Fig. 5b), stable PDI values ​​(Fig. 5c), and high level of mRNA capture efficiency were also confirmed in the 16:0 lysoPC iLPX group, the final candidate group for nasal delivery optimization (Fig. 5d). In Vibo To evaluate the intranasal mRNA expression capability, lysoPC iLPX loaded with luciferase mRNA was intranasally injected into mice, and the luciferase expression signal in the nasal cavity was analyzed to determine the IN-iLPX optimized for nasal mRNA delivery (Fig. 5e). The finally selected IN-iLPX has a molar ratio of DLin-MC3-DMA: DSPC: Cholesterol: 16:0 lysoPC = 50: 10: 30: 10.

[0071] When comparing the luciferase expression signals after intranasal injection of IN-iLPX and the existing LNP (Onpattro formulation, MC3:DSPC:Choleterol:DMG-PEG2k = 50:10:38.5:1.5) into mice, a signal approximately 62 times higher was observed in IN-iLPX (Fig. 6a), and when examining the expression pattern over time, it was confirmed that the expression intensity reached its maximum at 24h (Fig. 6b).

[0073] Analysis of IN-iLPX's Endosomal Escape Ability

[0074] To evaluate the endosome escape ability of IN-iLPX due to the addition of lysoPC, the degree of colocalization of Cy5-fluorescence-conjugated mRNA with intracellular acidic organelles stained with lysotracker was analyzed. As a result, a higher degree of mRNA cytoplasmic delivery was confirmed in IN-iLPX compared to iLPX (Figs. 7a and 7b).

[0075] In addition, through an RBC hemolysis assay under acidic conditions, it was confirmed that the membrane rupture ability of the iLPX form was higher than that of LNP, and the highest degree of hemolysis was observed in IN-iLPX (Fig. 8a). Furthermore, biocompatibility was confirmed, as it did not indiscriminately cause RBC hemolysis in a pH 7.4 environment (Fig. 8b). Through the above results, it was confirmed that iLPX has a higher endosome escape efficacy compared to LNP, and that this can be further increased by the addition of lysoPC.

[0077] Analysis of target cells in IN-iLPX

[0078] To confirm the expression pattern of IN-iLPX in nasal tissues, tissue confocal images were analyzed after intranasal injection of LNP loaded with Cre mRNA and IN-iLPX into Ai14 mice. The results confirmed that the protein was effectively expressed in the nasal respiratory epithelium compared to LNP (Fig. 9a). It was confirmed that mRNA delivery within the nasal tissues mainly occurred in the levels I and II regions where the nasal respiratory epithelium is located and in the lower part of level III, compared to the olfactory epithelium (upper part of level III) (Figs. 9a and 9b).

[0080] To analyze the mRNA expression cell types in nasal tissue in depth, single-cell analysis was performed using a flow cytometer after injecting IN-iLPX loaded with Cre mRNA into Ai14 mice, and it was confirmed that protein expression occurred specifically in nasal epithelial cells (Fig. 10).

[0082] Confirmation of universality for ionized lipids in IN-iLPX

[0083] To determine whether excellent mRNA delivery ability into nasal tissues is maintained even when lipids other than MC3 are used as ionized lipids in the IN-iLPX formulation, the inventors prepared IN-iLPX formulations by replacing MC3 with SM102 and ALC0315, which are representative lipids used as drug delivery vehicles in clinical practice, and compared the mRNA delivery efficiency into the nasal cavity with each LNP formulation. As a result, it was confirmed that when the remaining composition of IN-iLPX (structural lipids, cholesterol, and lysoPC) is the same, it exhibits significantly superior mRNA nasal delivery ability compared to LNP regardless of the type of ionized lipid (Fig. 11).

[0085] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A lipocomplex for nasal delivery of nucleic acid molecules comprising ionizable lipid, structural lipid, cholesterol, and lysoPC (lysophosphatidylcholine) as active ingredients. Claim 2 A lipid complex according to claim 1, characterized in that the ionized lipid is one or more selected from the group consisting of Dlin-MC3-DMA, SM-102, and ALC-0315. Claim 3 A lipid complex according to claim 1, characterized in that the structural lipid is one or more selected from the group consisting of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), phosphatidylcholine, DPPC (dipalmitoyl phosphatidyl choline), DMPC (dimyristoylphosphatidyl choline), DOPC (dioleoyl phosphatidyl choline), and DOPE (dioleyl phosphatidyl ethanolamine). Claim 4 A lipid complex according to claim 1, characterized in that the lysoPC is a 16:0 lysoPC. Claim 5 A lipid complex according to claim 1, characterized in that the ionized lipid is included in a molar ratio of 40 to 60 percent of the total lipid. Claim 6 A lipid complex according to claim 1, characterized in that the structural lipid is included in a molar ratio of 5 to 15% of the total lipid. Claim 7 A lipid complex according to claim 1, characterized in that the cholesterol is included in a molar ratio of 20 to 40 percent of the total lipids. Claim 8 A lipid complex according to claim 1, characterized in that the lysoPC is included in a molar ratio of 5 to 15% of the total lipids. Claim 9 A lipid complex according to claim 1, characterized in that the ionized lipid, structural lipid, cholesterol, and lysoPC have a molar ratio of 4-6 : 0.5-1.5 : 2-4 : 0.5-1.

5. Claim 10 A lipid complex according to claim 1, characterized in that the lipid complex does not contain PEG (polyethylene glycol). Claim 11 A lipid complex according to claim 1, characterized in that the lipid complex has an average particle size of 50 nm to 200 nm. Claim 12 A lipid complex according to claim 1, characterized in that the nucleic acid molecule is attached to the outer surface of the lipid complex. Claim 13 A lipid complex according to claim 1, characterized in that the nucleic acid molecule is an mRNA molecule. Claim 14 A method for expressing a target protein in the nasal cavity of a subject, comprising the following steps: encapsulating a nucleic acid molecule encoding the target protein in a lipid complex of any one of claims 1 to 13; and administering the lipid complex in which the nucleic acid molecule is encapsulated into the nasal cavity of a subject. Claim 15 A method according to claim 14, characterized in that the nucleic acid molecule is an mRNA molecule.

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