Lipid nanoparticle-based drug delivery system using ionizable peptide, and method for preparing same

Abstract

The present invention relates to a lipid nanoparticle-based drug delivery system using an ionizable peptide and a method for preparing same. The lipid nanoparticle-based drug delivery system according to the present invention can deliver a gene to a target cell in vivo with high efficiency by using an ionizable peptide having excellent encapsulation efficiency when forming a complex with an anionic gene. In addition, the lipid nanoparticle-based drug delivery system can improve the expression level of the gene in the target cell, and thus can be effectively used in related technical fields such as lipid nanoparticle-mediated gene therapy.

Description

Lipid nanoparticle-based drug delivery system using ionizable peptide and method for preparing the same

[0001] The present invention relates to a lipid nanoparticle-based drug delivery system using an ionizable peptide and a method for producing the same.

[0002] In the pharmaceutical industry, the Drug Delivery System (DDS) is designed to efficiently deliver the required amount of drugs while reducing side effects and maximizing efficacy and effectiveness. It is a high-value-added core technology with a high probability of success that can generate economic benefits comparable to new drug development. Its purpose is to improve the quality of patient treatment by making drug administration more efficient.

[0003] The solubilization of poorly soluble drugs, a key technology in drug delivery systems and a key component of drug absorption enhancement, is considered the most rational way to reduce the development costs of new drugs while simultaneously increasing the added value of existing drugs. Especially in countries like Korea, where conditions for new drug development are challenging, the development of improved new drugs through the development of drug solubilization technology offers the potential to generate significant added value at low cost.

[0004] Gene therapy using gene drug delivery systems holds great promise for correcting genetic mutations and treating numerous diseases. Effective gene delivery is a key challenge in successfully and safely implementing this gene therapy, and viral vectors have proven effective for gene delivery. However, the use of viruses as a gene delivery system is limited by several issues, including immunogenicity, tumorigenicity, cytotoxicity, limited DNA size within viral vectors, complex production processes, and limitations in repeated administration due to immune responses. Therefore, non-viral gene carriers, such as cationic liposomes and polymers, which can overcome these issues, are beginning to attract attention as alternatives to viral systems.

[0005] An ideal nonviral gene carrier should condense genetic material into a nanoscale complex that can be absorbed by cells, maintain a stable structure to facilitate delivery into cells, and be stable against degradation by enzymatic reactions in the body until the external genetic material is delivered to the cell nucleus. In particular, most genes are anionic, and cationic materials must be used to effectively load them into nonviral carriers. Attempts have been made to manufacture ideal nonviral gene carriers, such as forming gene complexes with only cationic polymers, proteins, or peptides, or using cationic lipid nanoparticles. For example, it has been reported that nucleic acid-lipid particles comprising nucleic acids, cationic lipids, non-cationic lipids, and conjugated lipids that suppress particle aggregation can be employed as a means of facilitating nucleic acid delivery within cells or the body. In addition, lipid nanoparticles comprising pH-sensitive cationic lipids as constituent lipids have been reported as carriers for the efficient delivery of nucleic acids such as siRNA into target cells.

[0006] Under these technical backgrounds, the inventors of the present invention have made great efforts to develop a drug delivery system capable of efficiently delivering anionic drugs, nucleic acids, etc. to a target organ or cell, and as a result, have prepared a pH-sensitive ionizable peptide with improved stability when forming a complex with nucleic acids, and have confirmed that the lipid nanoparticles encapsulating the complex of the peptide and nucleic acid have excellent stability and remarkable drug delivery ability to the target, thereby completing the present invention.

[0007] [Prior Art Literature]

[0008] [Patent Document]

[0009] (Patent Document 1) U.S. Patent No. 9,404,127 (Title of invention: Non-liposomal systems for nucleic acid delivery, Applicant: PROTIVA BIOTHERAPEUTICS, INC., Registration date: August 2, 2016)

[0010] (Patent Document 2) Republic of Korea Patent Publication No. 10-2020-0018782 (Title of the invention: Lipid membrane structure for intracellular delivery of siRNA, Applicant: Hokkaido Daigaku, Publication date: February 20, 2020)

[0011] (Patent Document 3) Republic of Korea Patent Publication No. 10-2019-0093816 (Title of invention: Lipid nanoparticle mRNA vaccine, Applicant: Curevac Age et al., Publication date: August 26, 2019)

[0012] (Patent Document 4) Republic of Korea Patent No. 10-1308591 (Title of the invention: Anionic drug delivery system containing cationic lipid and method for producing the same; Applicant: Samyang Biopharm Co., Ltd.; Registration date: September 9, 2013)

[0013] The purpose of the present invention is to provide a lipid nanoparticle-based drug delivery system using an ionizable peptide and a method for manufacturing the same.

[0014] The present invention comprises: (a) a complex comprising a nucleic acid, and an ionizable peptide comprising a water-soluble enhancement tag and an amino acid sequence of SEQ ID NO: 4;

[0015] (b) anionic lipid; (c) neutral lipid; (d) cholesterol; and, (e) PEG-modified lipid; and is characterized by a mixing ratio thereof.

[0016] The nucleic acid is characterized in that it comprises at least one selected from the group consisting of plasmid DNA, single-stranded DNA, double-stranded DNA, miRNA, siRNA, ssRNA, shRNA, mRNA, ncRNA, antisense RNA, and LNA.

[0017] The nucleic acid can be encapsulated in the lipid nanoparticles at an encapsulation rate of 70% or more, preferably 70 to 94%, and more preferably 80 to 94%.

[0018] The above water-soluble tag may be selected from the group consisting of an S-tag, a b'a' domain of protein disulfide isomerase (PDIb'a'), maltose binding protein (MBP), hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), N-utilization substance protein A (NusA), small ubiquitin related modifier (SUMO), and protein disulfide isomerase (PDI).

[0019] The above complex may contain nucleic acid:peptide in a weight ratio of 1:5 to 1:15. Preferably, it is contained in a weight ratio of 1:5 to 1:10.

[0020] The lipids of the above (b) to (e) may include 30 to 50 mol % of anionic lipid, 30 to 50 mol % of neutral lipid, 10 to 30 mol % of cholesterol, and 0.5 to 5 mol % of PEG-modified lipid.

[0021] The above anionic lipids are,

[0022] DSPG (1,2-distearoyl-sn-glycero-3-phosphatidylglycerol),

[0023] DPPG (dipalmitoylphosphatidylglycerol),

[0024] DOPG (dioleoylphosphatidylglycerol),

[0025] SOPG (1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol),

[0026] Soy PG (L-α-phosphatidylglycerol (Soy)),

[0027] Egg PG (L-α-phosphatidylglycerol (Egg, Chicken)),

[0028] DLPG (1,2-dilauroyl-sn-glycero-3-phosphatidylglycerol),

[0029] Cardiolipin (1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol),

[0030] DMPG (dimyristoylphosphatidylglycerol),

[0031] POPG (palmitoyloleoylphosphatidylglycerol),

[0032] DOPS (dioleoylphosphatidylserine),

[0033] DTPA (diethylenetriamine pentaacetic acid),

[0034] DPTGA (1,4-dipalmitoyl-tartarate-2,3-diglutaric acid),

[0035] DSTSA (1,4-disteroyl-tartarate-2,3-disuccinic acid),

[0036] CHHDA (2-carboxyheptadecanoyl heptadecylamide),

[0037] DMPS (dimyristoylphosphatidylserine),

[0038] DPPS (dipalmitoylphosphatidylserine),

[0039] POPS (palmitoyloleoylphosphatidylserine),

[0040] DMPA (dimyristoylphosphatidic acid),

[0041] DPPA (dipalmitoylphosphatidic acid),

[0042] DOPA (dioleoylphosphatidic acid),

[0043] POPA (palmitoyl-oleoylphosphatidic acid),

[0044] CetylP(Cetyl phosphate) and

[0045] It is characterized by being a lipid comprising at least one selected from the group consisting of CHEMS (cholesterol hemisuccinate) or a salt thereof.

[0046] 상기 중성지질은 HSPC(hydrogenated soy phosphatidylcholine),

[0047] DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine),

[0048] DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine),

[0049] POPE(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine),

[0050] DSPE(1,2-distearoyl-sn-glycero-3-phosphoethanolamine),

[0051] DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine),

[0052] POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine),

[0053] DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine),

[0054] SM(N-palmitoyl-D-erythro-sphingosylphosphorylcholine),

[0055] DLPE(1,2-dilauroyl-sn-glycero-3-phosphoethanolamine),

[0056] DPPE(1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine),

[0057] 포스파티딜 콜린(phosphatidyl choline),

[0058] 디미리스톨글리세롤(dimyristoylglycerol),

[0059] succinoyl-diacylglycerol,

[0060] phosphatidyl ethanolamine,

[0061] tetraether lipid,

[0062] sphingolipid,

[0063] diacryl glycerol and

[0064] It may include one or more selected from the group consisting of glycerides or a salt thereof.

[0065] The above PEG-modified lipid is characterized by polyethylene glycol (PEG) being bound to anionic lipid or neutral lipid.

[0066] In another aspect, the present invention provides a method for producing a nucleic acid-peptide complex, comprising the steps of: (a) mixing a nucleic acid solution and a water-soluble enhancing tag and an ionizable peptide comprising an amino acid sequence of SEQ ID NO: 4;

[0067] (b) introducing a lipid solution containing an organic solvent, anionic lipid, neutral lipid, cholesterol, and PEG-modified lipid into a reactor, and removing the organic solvent to form a lipid film; and

[0068] (c) a step of adding and dispersing the complex prepared in step (a) to the lipid film formed in step (b) to form a lipid nanoparticle dispersion; a method for preparing a lipid nanoparticle-based drug delivery system is provided.

[0069] Additionally, after maintaining pH 3.8 to 4.2 during the drug delivery vehicle manufacturing step, the pH can be adjusted to pH 7.0 to pH 7.4 for drug delivery when completing the lipid nanoparticle manufacturing.

[0070] As another method, in the present invention, instead of freezing and thawing the lipid, a method of preparing a dispersion using a droplet-based microfluidic system in the above-mentioned (c) can be applied to prepare a lipid nanoparticle-based drug delivery system.

[0071] The present invention is described in detail below.

[0072] The 'ionizable peptide' of the present invention is a 'peptide whose cohesion with nucleic acid and peptide changes depending on acidity', and is characterized by a strong cohesion with nucleic acid in an acidic state and a weak cohesion with nucleic acid in a neutral state.

[0073] These properties effectively induce complex formation with mRNA under acidic conditions and enable high-efficiency mRNA encapsulation into negatively charged LNPs. The ionizable nature of the peptide contributes to increased transfection efficiency of mRNA loaded into LNPs.

[0074] LNPs encapsulating mRNA deliver the mRNA into the cytoplasm through endocytosis. Upon endocytosis, LNPs become trapped within endosomes. A key factor in transfection efficiency is determining how much mRNA escapes from the endosome before fusion with lysosomes. Ionizable peptides have the property of absorbing protons in the slightly acidic endosomal environment, becoming cationized. This increases osmotic pressure and structural instability of the endosome, contributing to mRNA escape from the endosome.

[0075] In the present invention, the nucleic acid is a therapeutic agent for intracellular or intravital delivery, and can form a complex through electrostatic interaction with a cationic ionizable peptide, and may be, for example, at least one selected from the group consisting of plasmid DNA, single-stranded DNA, double-stranded DNA, miRNA, siRNA, ssRNA, shRNA, mRNA, ncRNA, antisense RNA, and LNA, but is not limited thereto.

[0076] In the present invention, "miRNA (microRNA)" refers to a single-stranded RNA molecule consisting of 21 to 23 nucleotides in length that regulates gene expression. miRNA is an oligonucleotide that is not expressed within cells and has a short stem-loop structure. miRNA has complete or partial homology with one or more messenger RNAs (mRNAs) and suppresses target gene expression through complementary binding with said mRNAs.

[0077] In the present invention, the term "siRNA" refers to a double-stranded RNA (duplex RNA) capable of inducing RNA interference (RNAi) by cleavage of a specific mRNA, or a single-stranded RNA having a double-stranded form within a single-stranded RNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary to it. siRNA is provided as an efficient gene knockdown method or as a method of gene therapy because it can suppress the expression of a target gene. The bond between the duplexes is formed through hydrogen bonds between nucleotides, and not all nucleotides within the duplex need to be completely complementary.

[0078] The length of the siRNA may be about 15 to 60 nucleotides, specifically about 15 to 50, about 15 to 40, about 15 to 30, about 15 to 25, about 16 to 25, about 19 to 25, about 20 to 25, or about 20 to 23 nucleotides. The siRNA length refers to the number of nucleotides on one side of the double-stranded RNA, i.e., the number of base pairs, and in the case of single-stranded RNA, refers to the length of the double strand within the single-stranded RNA. In addition, the siRNA may be composed of nucleotides introduced with various functional groups for purposes such as increasing blood stability or weakening the immune response.

[0079] In the present invention, the term "ssDNA" means a single-stranded oligonucleotide that selectively binds to a specific target DNA and induces an antigenic effect.

[0080] In the present invention, the term "shRNA" refers to a single-stranded RNA consisting of 50 to 70 nucleotides, which forms a stem-loop structure in vivo. A long RNA of 19 to 29 nucleotides complementarily pairs with a loop region of 5 to 10 nucleotides on either side to form a double-stranded stem.

[0081] In the present invention, the term "mRNA" means synthetic mRNA (in vitro transcribed mRNA) capable of expressing a gene.

[0082] In the present invention, the term "ncRNA" refers to RNA that functions as itself without being translated into protein. NCRNA includes the commonly known tRNA and rRNA, as well as snoRNA, miRNA, and lncRNA, and is involved in transcription, splicing, and translation throughout the cell.

[0083] In the present invention, the term "antisense RNA" refers to RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, which binds to the complementary sequence within the mRNA and inhibits the translation of the mRNA into protein. An antisense RNA sequence refers to an RNA sequence that is complementary to the mRNA of the genes and can bind to the mRNA. This can inhibit essential activities for the translation, cytoplasmic translocation, maturation, or any other overall biological function of the gene mRNA. The length of the antisense RNA may be 6 to 100 bases, specifically 8 to 60 bases, and more specifically 10 to 40 bases. The antisense RNA may be synthesized in vitro by a conventional method and administered in vivo, or the antisense RNA may be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase. One example of how to synthesize antisense RNA in vivo involves using a vector with the multiple cloning site (MCS) origin in the opposite direction to allow antisense RNA to be transcribed. Preferably, the antisense RNA sequence includes a translation stop codon within its sequence, preventing it from being translated into a peptide sequence.

[0084] In the present invention, the term "LNA (Locked nucleic acids)" refers to a nucleic acid analog containing a 2'-O, 4'-C methylene bridge. LNA nucleosides contain common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rule. However, due to the molecule's "locking" caused by the methylene bridge, LNA cannot form an ideal shape in Watson-Crick bonding. When LNA is included in a DNA or RNA oligonucleotide, LNA can pair more quickly with a complementary nucleotide chain, thereby increasing the stability of the double helix.

[0085] In the present invention, the ionizable peptide may be composed of a water-solubility enhancing tag and an amino acid sequence of a human PA R cluster (-SRRRRRRS-) of SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 4 may be composed of a repeating bond structure after the water-solubility enhancing tag, but is preferably composed of one water-solubility enhancing tag and one amino acid sequence of SEQ ID NO: 4.

[0086] Accordingly, as an example of the ionizable peptide, a peptide consisting of the amino acid sequence of sequence number 2 can be mentioned.

[0087] The above solubility enhancing tag (S-tag) refers to a labeling substance (tag) that can improve the aqueous stability of a complex when the ionizable peptide of the present invention is combined with an anionic nucleic acid to form a complex, and any tag that can express the ionizable peptide in a water-soluble form can be used without limitation. For example, the water-soluble enhancement tag may be selected from the group consisting of an S-tag, the b'a' domain of protein disulfide isomerase (PDIb'a'), maltose binding protein (MBP), hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), N-utilization substance protein A (NusA), small ubiquitin related modifier (SUMO), and protein disulfide isomerase (PDI). The above water-soluble enhancement tag may be each bound to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the human PA R cluster of the present invention, and may be bound one or more times in succession.

[0088] In the present invention, the water-solubility enhancing tag bound to sequence number 4, i.e., the water-solubility enhancing tag forming part of the amino acid sequence of sequence number 2, may be an S-tag (KETAAAKFERQHMDS), which may be bound to the N-terminus of the human-derived arginine cluster (human PA R cluster; -SRRRRRRS-) to form the ionizable peptide of the present invention.

[0089] In the case of the ionizable peptide containing the above S-tag, when bound to a nucleic acid to form a complex, the aqueous stability of the complex can be improved, and when encapsulated within a lipid nanoparticle, the stability of the lipid nanoparticle can be enhanced. This allows for the stable delivery of nucleic acids into target cells and contributes to high gene expression levels.

[0090] In the present invention, the lipid in the PEG-modified lipid can be used without limitation as long as it is a lipid that can bind to PEG (polyethylene glycol), and for example, any neutral lipid or anionic lipid can be used.

[0091] The above PEG-modified lipid refers to a form in which lipid and PEG are conjugated, and refers to a lipid having a polyethylene glycol (PEG) polymer, which is a hydrophilic polymer, bound to one end. The PEG-modified lipid contributes to the stability of nanoparticles in serum within lipid nanoparticles and acts as a stabilizer lipid that prevents aggregation between lipid nanoparticles. In addition, the PEG-modified lipid protects nucleic acids from degrading enzymes during in vivo delivery of nucleic acids, thereby enhancing the stability of nucleic acids in the body, and can increase the half-life of drugs encapsulated within the lipid nanoparticles.

[0092] In the above PEG-modified lipid, PEG may be directly conjugated to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for linking PEG to the lipid may be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties. The non-ester-containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, as well as combinations thereof (e.g., linkers containing both a carbamate linker moiety and an amido linker moiety). The ester-containing linker moieties include, but are not limited to, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0093] PEG in the above PEG-modified lipid is a hydrophilic polymer that has the ability to inhibit the adsorption of plasma proteins, thereby increasing the circulation time of lipid nanoparticles in the body and preventing aggregation between nanoparticles. Furthermore, PEG-modified lipids exhibit stealth properties in vivo, preventing the degradation of nanoparticles by immune cells.

[0094] The above PEG may be a functionalized PEG in which a functional group is bonded to the side that is not bonded to the lipid. In this case, the usable functional group may be at least one selected from the group consisting of a succinyl group, a carboxylic acid, a maleimide, an amine group, biotin, a cyanuric group, and a folate.

[0095] In the present invention, cholesterol provides morphological stability to the lipid filling within the lipid nanoparticles, and is dispersed in the core and surface of the nanoparticles to enhance the stability of the nanoparticles.

[0096] In the present invention, the average particle size of the lipid nanoparticles may be 10 to 2,000 nm. If the size of the lipid nanoparticles is less than 10 nm, the complex may be difficult to encapsulate in the lipid nanoparticles, and stability may be reduced when injected into the body, which is undesirable. Furthermore, if the size exceeds 2,000 nm, stability of the composition containing the lipid nanoparticles may be reduced when injected into the body, which is undesirable.

[0097] In addition, the present invention comprises the steps of (a) preparing a peptide-nucleic acid complex by mixing a nucleic acid solution and an ionizable peptide comprising a water-soluble enhancing tag and an amino acid sequence of SEQ ID NO: 4;

[0098] (b) introducing a lipid solution containing an organic solvent, anionic lipid, neutral lipid, cholesterol, and PEG-modified lipid into a reactor, and removing the organic solvent to form a lipid film; and

[0099] (c) a step of adding and dispersing the complex prepared in step (a) to the lipid film formed in step (b) to form a lipid nanoparticle dispersion; a method for preparing a lipid nanoparticle-based drug delivery system is provided.

[0100] In the method for manufacturing a lipid nanoparticle-based drug delivery system of the present invention, step (b) is a step of introducing a lipid solution containing an organic solvent, anionic lipid, neutral lipid, cholesterol, and PEG-modified lipid into a reactor, and removing the organic solvent to form a lipid film.

[0101] As the above anionic lipids, neutral lipids, cholesterol and PEG-modified lipids are as described above, their description is omitted here.

[0102] The step of removing the organic solvent may include maintaining the interior of the reactor in a vacuum state and evaporating the organic solvent in the lipid solution introduced into the reactor. At this time, the temperature within the reactor may be set to a high temperature to evaporate only the organic solvent. By evaporating and removing the organic solvent in this way, the remaining lipid components in the lipid solution, excluding the organic solvent, form a lipid film on the inner wall and bottom of the reactor.

[0103] The type of the organic solvent is not particularly limited, and any organic solvent that can dissolve the lipid used in the production of the lipid nanoparticle-based drug delivery system of the present invention can be used. The organic solvent may preferably be a non-polar or low-polar organic solvent, and may be selected from the group consisting of benzene, butanol, butyl acetate, carbon tetrachloride, chloroform, cyclohexane, dichloroethane, dichloromethane, diethyl ether, diisopropyl ether, ethyl acetate, heptane, hexane, isooctane, methyl ethyl ketone (MEK), methyl t-butyl ether (MTBE), pentane, toluene, trichloroethylene, xylene, and mixed solvents thereof.

[0104] In the method for manufacturing a lipid nanoparticle-based drug delivery system of the present invention, step (c) is a step of adding and dispersing the complex manufactured in step (a) into the lipid film formed in step (b) to form a lipid nanoparticle dispersion.

[0105] In the above step (c), a freezing / thawing process of freezing the dispersion and thawing the frozen dispersion may be further included. Specifically, the process of freezing the dispersion by exposing it to liquid nitrogen for 5 minutes and then exposing it to warm water again to thaw it may be repeated. At this time, the freezing / thawing process may be repeated 1 to 12 times, and by repeating the steps of freezing and thawing the lipid film composition in this way, a lipid nanoparticle dispersion having a more uniform size can be formed, and the drug encapsulation efficiency of the lipid nanoparticles can be increased. However, if the number of times exceeds 12, the encapsulation efficiency of the lipid nanoparticles may actually decrease, so the number of times is preferably 12 or less.

[0106] In the present invention, the term "encapsulation" means encapsulating a delivery substance to surround it and efficiently introduce it into a living body.

[0107] Additionally, the encapsulation rate of nucleic acids encapsulated in lipid nanoparticles can be confirmed according to the following equation.

[0108] Encapsulation rate (%) = (Weight of nucleic acid loaded on lipid nanoparticles / Weight of nucleic acid introduced into lipid nanoparticles) x 100

[0109] The present invention relates to a lipid nanoparticle-based drug delivery system using an ionizable peptide and a method for producing the same. The lipid nanoparticle-based drug delivery system of the present invention uses a pH-sensitive ionizable peptide having an excellent encapsulation rate when forming a complex with a gene having an anionic property, thereby enabling high-efficiency delivery of a gene to a target cell in the body and also enhancing the level of gene expression in the target cell, and therefore can be usefully used in related technical fields such as lipid nanoparticle-mediated gene therapy.

[0110] Figure 1 is a schematic diagram showing the process of preparing a nucleic acid complex using an ionizable peptide at pH 4.0 and then finally storing the lipid nanoparticles at pH 7.2.

[0111] Figure 2 is a photograph showing the results of electrophoresis of a composition in which Fluc mRNA (Fly luciferase) and No. 2 peptide are mixed at each pH and weight ratio to explore the mixing ratio conditions of nucleic acid and ionizable peptide complexes.

[0112] Figure 3 shows the mRNA delivery efficiency after subcutaneously injecting the lipid nanoparticles of Manufacturing Example 1, Fluc mRNA, into experimental animals. The upper photo shows the protein expression of the gene due to mRNA delivery at 24 and 48 hours, and the lower graph shows the results in numerical form.

[0113] Figure 4 is a photograph showing the results of electrophoresis of a composition in which No. 3 peptide or No. 2 peptide was mixed with Fluc mRNA to confirm the conditions for preparing a complex according to the human PA R cluster structure of an ionizable peptide.

[0114] Figure 5 is a photograph showing the results of electrophoresis of a composition in which Fluc mRNA was mixed with No. 3 peptide or No. 1 peptide, respectively, to confirm the conditions for preparing a complex depending on the presence or absence of an S-tag of an ionizable peptide.

[0115] Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the contents introduced herein are provided to ensure thoroughness and completeness, and to sufficiently convey the spirit of the present invention to those skilled in the art.

[0116] <Example 1. Synthesis of an ionizable peptide>

[0117] The following sequences 1 to 3 were manufactured by GenScript, a peptide biosynthesis organization. They were synthesized according to the method of Patent No. 10-2549868. These sequences were synthesized based on the human PA R cluster peptide sequence of SEQ ID NO: 4 (NCBI, #NP_002752.1) and the S-Tag peptide sequence of SEQ ID NO: 5.

[0118] SEQ ID NO. Peptide sequence1SRRRRRRSSRRRRRRS(human PA R cluster)-(human PA R cluster)2KETAAAKFERQHMDSSRRRRRRRS(S-tag)-(human PA R cluster)3KETAAAKFERQHMDSSRRRRRRSSRRRRRRRS(S-tag)-(human PA R cluster)-(human PA R cluster)4SRRRRRRS(human PA R cluster, #NP_002752.1)5KETAAAKFERQHMDS(S-tag)

[0119] <Example 2. Confirmation of the appropriate mixing ratio of mRNA and ionizable peptide>

[0120] As disclosed in Fig. 1, in order to manufacture the lipid nanoparticle of the present invention, an ionizable peptide and mRNA are mixed under acidic conditions (e.g., pH 4.0) to obtain a complex, and the complex and each lipid are mixed under neutral conditions (e.g., pH 7.2) to manufacture a complete lipid nanoparticle. Fluc mRNA (MG IVT Fluc mRNA) was produced in-house, and was synthesized manually through in vitro transcription (IVT) using Fluc plasmid DNA as a template and T7 polymerase (Cat #: 2540A, TaKaRa), and then purified using phenol: chloroform: isoamyl alcohol, and the purity and concentration were confirmed using Nanodrop (BIOTEK Synergy H1) and agarose gel, and small amounts were dispensed and stored in a deep freezer for use.

[0121] Accordingly, in this experiment, the optimal mixing conditions of mRNA and ionizable peptide were first confirmed, and when the mRNA content was fixed at 1 part by weight (0.25 μg) under each of the pH 4.0 condition, the pH 4.0 to pH 7.2 conversion condition, and the pH 7.2 condition, the content of the ionizable peptide (SEQ ID NO: 2) was varied from 0.5 parts by weight (0.125 μg) to 10 parts by weight (2.5 μg), and these were mixed, respectively, and the formation of a complex was confirmed by agarose gel electrophoresis analysis. At this time, if the complex is not formed, mRNA appears on the gel, showing a band or drag phenomenon. The experimental results are shown in Fig. 2.

[0122] As confirmed in Figure 2, when mRNA and ionizable peptide are mixed at a weight ratio of 1:0.5 under pH 4.0 conditions, no complex is formed, and thus the dragging phenomenon of mRNA is observed (fourth lane). (Only mRNA in Figure 2: MG IVT Fluc mRNA, only hLMWP2: No. 2 ionizable peptide)

[0123] In comparison, when mixed at a weight ratio of 1:1, a complex is formed and mRNA is not observed in the gel (first arrow). Mixing mRNA and peptide at weight ratios of 1:5 and 1:10 confirms that the complex is well formed.

[0124] However, among these complex samples, the 1:1 weight ratio sample was transferred from pH 4.0 to pH 7.2 and gel run revealed that the complex was disassembled and a weak mRNA dragging phenomenon was observed (second arrow). The mRNA and peptide mixtures at weight ratios of 1:5 and 1:10 maintained the complex well even under these conditions.

[0125] This is due to the reversible ionization properties of the peptide, suggesting that the complex may be disassembled before the production of lipid nanoparticles when the mixing ratio of mRNA and peptide is 1:1 under neutral conditions for the production of lipid nanoparticles.

[0126] Therefore, it can be confirmed through this pH 4.0 to pH 7.2 variation experiment that the complex must be at least 5 times more abundant than mRNA, i.e. nucleic acid, to remain stable until lipid nanoparticles are formed.

[0127] This was verified by manufacturing the complex again in the same pH 7.0 condition and performing gel running, and confirming that when the mixing ratio of mRNA and peptide was 1:1, the mRNA dragging phenomenon appeared (third arrow), and when the mixing ratio of mRNA and peptide was 1:5 or higher, the mRNA dragging phenomenon did not appear.

[0128] <Example 3. Preparation, characterization, and encapsulation rate testing of lipid nanoparticles>

[0129] Based on the results of Example 2, lipid nanoparticles were prepared under the conditions shown in Table 2 below. The lipid nanoparticles were prepared using a thin film hydration method.

[0130] For example, in the case of lipid nanoparticles of Manufacturing Example 1, 1.5 mg of total lipids (DOPE (neutral lipid): Cholesterol: DPPG (anionic lipid): DSPE-PEG) in a ratio of 38.5:40:20:1.5 (mol %)) were dissolved in 1 mL of chloroform, and the solvent was evaporated to form a lipid film. Thereafter, the formed lipid film was hydrated with 1 mL of a complex (Acetate buffer, pH 4) formed by mixing 60 μg of Fluc mRNA and 600 μg of the peptide of SEQ ID NO: 2.

[0131] Next, lipid nanoparticles were prepared by replacing the LNP solution (dialysis, PBS, pH 7.2) through a freeze-thaw procedure and then filtering through a 0.2 μm syringe filter.

[0132] The stability of the synthesized lipid nanoparticle samples was evaluated by visual inspection and dynamic light scattering (DLS) analysis, which allows measurement of particles with a size of 0.3–10 μm, immediately after synthesis, after 3 h (stored at 4 °C), and after 24 h (stored at 4 °C).

[0133] Manufacturing Example 1 Peptide Lipid composition of mRNA sequence number 2Fluc mRNAKETAAAKFERQHMDSSRRRRRRS(S-tag)-(human PA R cluster)DOPE:Cholesterol:DPPG:DSPE-PEG(38.5:40:20:1.5 (mol %))Content 60 ㎍600 ㎍1.5 mg

[0134] Lipid nanoparticles, neutral lipids, cholesterol, anionic lipids, PEG-modified lipids, mol % mixing ratio, manufacturing example 1, DOPECholesterolDPPGDSPE-PEG38.5:40:20:1.5, manufacturing example 2, DSPCCholesterolDOPADSPE-PEG38.5:40:20:1.5, manufacturing example 3, DPPCCholesterolDPPSDSPE-PEG38.5:40:20:1.5, manufacturing example 4, DSPCCholesterolPOPSDOPC-PEG38.5:40:20:1.5, manufacturing example 5, HSPCCholesterolDOPSDPPE-PEG38.5:40:20:1.5, manufacturing example 6, DOPECholesterolDPPGDSPE-PEG35:35:27:3, manufacturing example 7, DOPECholesterolDPPGDSPE-PEG40:43:15:2, manufacturing example 8DOPECholesterolDPPGDSPE-PEG39:35:25:1

[0135] The final average particle size of the lipid nanoparticles manufactured in this way was measured and analyzed by DLS (dynamic light scattering), and this was expressed as follows.

[0136] Lipid nanoparticle Size (nm) PDI Manufacturing example 11240.18 Manufacturing example 21320.14 Manufacturing example 31410.12 Manufacturing example 41140.12 Manufacturing example 51370.19 Manufacturing example 61250.12 Manufacturing example 71280.13 Manufacturing example 81150.14

[0137] In addition, the encapsulation rate of nucleic acid (mRNA) in the complex with lipid nanoparticles was confirmed through the Ribogreen assay, and the following high encapsulation rate was confirmed.

[0138] Lipid nanoparticle mRNA encapsulation rate (%) Manufacturing example 194 Manufacturing example 290 Manufacturing example 391 Manufacturing example 490 Manufacturing example 594 Manufacturing example 692 Manufacturing example 791 Manufacturing example 890

[0139] <Example 4. Confirmation of target delivery efficiency of lipid nanoparticles>

[0140] The Fluc mRNA encapsulated in the lipid nanoparticles of Manufacturing Example 1 is a genetic material encoding the fly luciferase protein. When the coding protein is expressed, it oxidizes the additionally injected substrate luciferin to cause it to emit light. The delivery efficiency of Fluc mRNA can be evaluated through the luminescence intensity of luciferin, and the delivery efficiency of the mRNA of the lipid nanoparticles was confirmed through experiments on a 6-week-old male BalB / c mouse animal model.

[0141] To this end, 4.8 μg of mRNA per mouse group was injected intramuscularly (IM) into each group of mice. 24 and 48 hours later, 100 μl of D-luciferin solution (Perkin Elmer) was injected intraperitoneally, and luminescence signals within 5–10 minutes were confirmed using optical imaging equipment (IVIS, PerkinElmer). The luminescence value was calculated as Total Flux (photons / s) after designating a Region of Interest (ROI).

[0142] Looking at Figure 3, in the group of mice injected subcutaneously with only Fluc mRNA (mRNA only), 10 3 The low luminescence at the photons / s level made it appear as if no mRNA response was observed when viewed with the naked eye.

[0143] In contrast, in the group of mice subcutaneously injected with lipid nanoparticles of Manufacturing Example 1 (LNP-mRNA), 10 6 A high transmission efficiency of photons / s was observed, which also left a very strong luminescence image in the photograph.

[0144] Therefore, it can be confirmed that the lipid nanoparticles of Manufacturing Example 1 are very useful in delivering Fluc mRNA to the target well and maintaining its expression.

[0145] <Example 5. Testing conditions for manufacturing nucleic acid complexes according to the structure of ionizable peptides>

[0146] Next, additional experiments were conducted to determine which position in the structure of the ionizable peptide has a more important influence on the formation of the nucleic acid complex, or which amino acid sequence is essential for the construction of this peptide.

[0147] Accordingly, as performed in Example 2, a complex was obtained by mixing Fluc mRNA and peptides of various structures, and then the complex was run on a gel to determine whether the mRNA dragging phenomenon occurred and the complex was not formed well, or whether the dragging phenomenon disappeared and the complex was formed well.

[0148] For this purpose, the No. 2 peptide synthesized in the present invention has a structure in which one human PA R cluster is bound to an S-tag, and the No. 3 peptide has a structure in which two human PA R clusters are connected in series to an S-tag.

[0149] The upper photo of Fig. 4 shows the result of electrophoresis (gel running) of a complex of Fluc mRNA and No. 3 peptide, and the lower photo of Fig. 4 shows the result of electrophoresis of a complex of Fluc mRNA and No. 2 peptide. It can be confirmed that the complex in which mRNA and No. 3 peptide are mixed at a weight ratio of 1:1 is not disintegrated even when the pH becomes 7.2.

[0150] Meanwhile, although the complex was disassembled at pH 7.2, it appears that the complex was formed even at pH 4.0 when mRNA and No. 3 peptide were mixed at a ratio of 1:0.5 (no dragging phenomenon is observed in the fourth lane). This suggests that in the ionizable peptide structure, the electrostatic interaction with mRNA becomes stronger as the human PA R cluster increases. This means that a strong complex can be formed even with a small amount of peptide.

[0151] The results of the lower gel image of Fig. 4 are the same as those of Example 2 because No. 2 peptide was used, and still show that No. 2 peptide at least 5 times the weight of mRNA is required.

[0152] Meanwhile, as shown in the lower gel image of Fig. 5, the same experiment was repeated using No. 1 peptide without an S-tag and with only the human PA R cluster repeated twice in series. It was confirmed that the complex in which mRNA and No. 1 peptide were mixed at a weight ratio of 1:1 maintained the complex state well at both pH 4.0 and pH 7.2.

[0153] However, when mRNA and No. 1 peptide were mixed at a weight ratio of 1:0.5, complex formation did not occur well at either pH 4.0 or 7.2. In comparison, as shown in the upper gel image of Fig. 5, No. 3 peptide still formed a complex at a mixing ratio of 1:0.5 at pH 4.0.

[0154] This suggests that the pH adaptability of the human PA R cluster sequence varies depending on the presence or absence of the S-tag, and that the S-tag may be an important factor in the ionizable nature of the peptide itself.

[0155] Accordingly, we decided to more clearly confirm the influence of the presence or absence of the S-tag and the repetitive sequence of the human PA R cluster on the production of lipid nanoparticles through the following encapsulation rate experiment.

[0156] <Example 6. Confirmation of the encapsulation rate of lipid nanoparticles according to the characteristics of ionizable peptides>

[0157] Lipid nanoparticles were manufactured under the conditions of Manufacturing Example 1 using the complexes of various mixing conditions manufactured in Example 5.

[0158] The lipids used at this time were DOPE (neutral lipid): Cholesterol: DPPG (anionic lipid): DSPE-PEG in a ratio of 38.5:40:20:1.5 (mol %). Based on 60 ㎍ of Fluc mRNA, the peptides were mixed according to the mixing ratio in Table 6 below, and the encapsulation rate was also shown. In addition, this experiment was repeated, and the results for two representative samples are shown below.

[0159] Lipid nanoparticle peptide sequence mRNA:peptide mixing ratio mRNA encapsulation rate (%) Comparative manufacturing example 1 No. 11:1 Aggregation Comparative manufacturing example 2 No. 11:1 Aggregation Comparative manufacturing example 3 No. 11:5 Aggregation Comparative manufacturing example 4 No. 11:5 Aggregation Comparative manufacturing example 5 No. 11:10 Aggregation Comparative manufacturing example 6 No. 11:10 Aggregation Comparative manufacturing example 7 No. 21:19 Comparative manufacturing example 8 No. 21:15 Manufacturing example 9 No. 21:580 Manufacturing example 10 No. 21:594 Manufacturing example 11 No. 21:1090 Manufacturing example 12 No. 21:1088 Manufacturing example 13 No. 21:1592 Manufacturing example 14No.21:1591Comparative manufacturing example 15No.31:1AggregationComparative manufacturing example 16No.31:1AggregationComparative manufacturing example 17No.31:5AggregationComparative manufacturing example 18No.31:5AggregationComparative manufacturing example 19No.31:10AggregationComparative manufacturing example 20No.31:10Aggregation

[0160] Looking at the results in Table 6, the mRNA (complex) encapsulation rate within the lipid nanoparticles was confirmed to be in a state where aggregation occurred with the naked eye due to strong charge interaction with mRNA when manufacturing lipid nanoparticles containing No. 1 peptide consisting of only two human PA R clusters, and the particle size was non-uniform to the extent that PDI measurement was difficult. In addition, a similar aggregation phenomenon occurred in lipid nanoparticles containing No. 3 peptide in which an S-tag and two human PA R clusters were linked.

[0161] This confirms that the presence or absence of the S-tag has no effect at all on the aggregation of peptides in which two human PA R clusters are linked.

[0162] Ultimately, even though complex formation was successful, the No. 3 peptide was ultimately found to be unsuitable as an ionizable peptide due to the adverse effect of aggregation during lipid nanoparticle manufacturing. Therefore, the No. 2 peptide of the present invention is proven to be the most superior ionizable peptide for increasing mRNA encapsulation rates.

[0163] Accordingly, the lipid nanoparticle carrier of the present invention comprises a complex in which a nucleic acid and an ionizable peptide having an amino acid sequence of SEQ ID NO: 2 are mixed, and includes various anionic lipids, neutral lipids, cholesterol, and PEG-modified lipids.

[0164] Although the present invention has been described through specific matters and limited examples as described above, these are provided only to help with the overall understanding of the present invention, and the present invention is not limited to the above examples, and those skilled in the art to which the present invention pertains can make various modifications and variations based on this description.

[0165] Therefore, the spirit of the present invention should not be limited to the described embodiments, and all things that are equivalent or equivalent to the claims described below as well as the claims are considered to fall within the scope of the spirit of the present invention.

Claims

1. (a) A complex comprising a nucleic acid, and an ionizable peptide comprising a water-soluble tag and an amino acid sequence of SEQ ID NO: 4; A lipid nanoparticle-based drug delivery system comprising: (b) anionic lipid; (c) neutral lipid; (d) cholesterol; and (e) PEG-modified lipid.

2. In paragraph 1, A lipid nanoparticle-based drug delivery system, characterized in that the nucleic acid comprises at least one selected from the group consisting of plasmid DNA, single-stranded DNA, double-stranded DNA, miRNA, siRNA, ssRNA, shRNA, mRNA, ncRNA, antisense RNA, and LNA.

3. In paragraph 1, A lipid nanoparticle-based drug delivery system characterized in that the nucleic acid is encapsulated in the lipid nanoparticles at an encapsulation rate of 70% or more.

4. In paragraph 1, A lipid nanoparticle-based drug delivery system characterized in that the water-soluble tag is a tag selected from the group consisting of an S-tag, a b'a' domain of protein disulfide isomerase (PDIb'a'), maltose binding protein (MBP), hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), N-utilization substance protein A (NusA), small ubiquitin related modifier (SUMO), and protein disulfide isomerase (PDI).

5. In paragraph 1, The above anionic lipids are, DSPG (1,2-distearoyl-sn-glycero-3-phosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), DOPG (dioleoylphosphatidylglycerol), SOPG (1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol), Soy PG (L-α-phosphatidylglycerol (Soy)), Egg PG (L-α-phosphatidylglycerol (Egg, Chicken)), DLPG (1,2-dilauroyl-sn-glycero-3-phosphatidylglycerol), Cardiolipin (1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol), DMPG (dimyristoylphosphatidylglycerol), POPG (palmitoyloleoylphosphatidylglycerol), DOPS (dioleoylphosphatidylserine), DTPA (diethylenetriamine pentaacetic acid), DPTGA (1,4-dipalmitoyl-tartarate-2,3-diglutaric acid), DSTSA (1,4-disteroyl-tartarate-2,3-disuccinic acid), CHHDA (2-carboxyheptadecanoyl heptadecylamide), DMPS (dimyristoylphosphatidylserine), DPPS (dipalmitoylphosphatidylserine), POPS (palmitoyloleoylphosphatidylserine), DMPA (dimyristoylphosphatidic acid), DPPA (dipalmitoylphosphatidic acid), DOPA (dioleoylphosphatidic acid), POPA (palmitoyl-oleoylphosphatidic acid), CetylP(Cetyl phosphate) and A lipid nanoparticle-based drug delivery system characterized in that it is a lipid comprising at least one selected from the group consisting of CHEMS (cholesterol hemisuccinate) or a salt thereof.

6. In paragraph 1, The above neutral lipid is HSPC (hydrogenated soy phosphatidylcholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), SM (N-palmitoyl-D-erythro-sphingosylphosphorylcholine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine), phosphatidyl choline, Dimyristoylglycerol, succinoyl-diacylglycerol, phosphatidyl ethanolamine, tetraether lipid, sphingolipid, diacryl glycerol and A lipid nanoparticle-based drug delivery system characterized in that the lipid comprises at least one selected from the group consisting of glycerides or a salt thereof.

7. In paragraph 1, The above PEG-modified lipid is a lipid nanoparticle-based drug delivery system characterized in that polyethylene glycol (PEG) is bound to an anionic lipid or neutral lipid. 8.(a) A step of preparing a nucleic acid-peptide complex by mixing a nucleic acid solution and an ionizable peptide comprising a water-soluble enhancing tag and an amino acid sequence of SEQ ID NO: 4; (b) introducing a lipid solution containing an organic solvent, anionic lipid, neutral lipid, cholesterol, and PEG-modified lipid into a reactor, and removing the organic solvent to form a lipid film; and (c) A method for producing a lipid nanoparticle-based drug delivery system, characterized by comprising the step of adding and dispersing the complex prepared in step (a) to the lipid film formed in step (b) to form a lipid nanoparticle dispersion.

9. In paragraph 8, A method for producing a lipid nanoparticle-based drug delivery system, characterized in that, instead of freezing and thawing the lipid in step (c), a method for producing a dispersion using the droplet-based microfluidic system is applied.

Images