Novel polymer compounds and nucleic acid delivery compositions containing the same

Abstract

To provide high-performance polymer delivery vehicles, and compositions for nucleic acid delivery comprising the same.SOLUTION: Provided are novel polymer compounds and compositions for nucleic acid delivery comprising the same, where the polymer compounds have high binding affinity to nucleic acids and effectively protect the nucleic acids from nucleases, thereby having effects of improving the nucleic acid delivery effect and stability.SELECTED DRAWING: Figure 23

Description

[Technical Field]

[0001] The present invention relates to amphiphilic polymer compounds and nucleic acid delivery compositions containing the same. [Background technology]

[0002] mRNA vaccines, which are attracting attention as next-generation vaccines, activate immunity by artificially synthesizing and injecting mRNA containing the genetic information of a virus, causing cells to directly produce spike proteins. Because mRNA vaccines utilize the genes of the virus, rapid development is possible as long as the genetic structure of the virus is known, and they have the advantage of being able to be produced quickly without the need to culture antigens or antibodies from outside. mRNA vaccines have been used all over the world to respond to the COVID-19 pandemic, and currently, various research and development projects are being conducted using them to overcome not only infectious diseases but also various diseases such as cancer and rare diseases.

[0003] On the other hand, since mRNA can be easily damaged by degrading enzymes in the human body, nano-deliverers are attracting attention as a technology for safely delivering mRNA to cells. Currently, the most widely used nano-deliverers are lipid nanoparticles (LNPs), and the COVID-19 vaccine recently developed by Pfizer and Moderna also uses LNPs as deliverers. However, in the case of lipid nanoparticles, the lipids easily become rancid, making storage and distribution at room temperature difficult, and side effects such as hypersensitivity reactions due to the toxicity of the LNPs themselves have been reported.

[0004] To overcome this, research into synthetic polymer nanodeliverers is being actively pursued. Synthetic polymer nanodeliverers are manufactured by repeatedly bonding small units, allowing for free design of structure, size, ionization properties, and stimulus-responsive drug release. They can also be used in future gene-editing gene therapy agents. Furthermore, by introducing other components into polymer nanodeliverers, it is possible to simultaneously achieve more advantages while maintaining the properties of polymers, and the configuration can be adjusted as multipolymer systems and polymer-based hybrid systems, such as polymer-lipid and polymer-inorganic hybrid delivery nanoplatforms.

[0005] Polymer nanoparticles loaded with mRNA can have various structures, including polyflexes, polymer micelles, polymersomes, and core-shell polymer-lipid hybrids. Such polymer nanoparticle delivery bodies exhibit low toxicity, biodegradability, and excellent biocompatibility, thus reducing toxicity and immune responses. They can provide fundamental data for the development of next-generation vaccines and drugs where mRNA delivery via LNPs is difficult. Furthermore, polymer-based mRNA delivery body technology can be used not only for mRNA vaccines but also for the development of other RNA drugs, offering high potential for the treatment of various diseases.

[0006] Cationic dendrimers, as an example of polymer nanoparticle delivery bodies, have been used in non-clinical trials against various viruses such as Ebola, H1N1 influenza, and Zika. However, due to the structural characteristics of the highly branched molecules themselves, they are difficult to degrade in vivo and exhibit toxicity, necessitating supplementation. Polymer nanoparticles have been reported in numerous publications, either used alone or as lipid-polymer complexes with lipids. However, compared to research findings, accurate physical property analysis and structural elucidation are difficult, creating a somewhat high barrier to clinical application, and commercialization has not yet occurred. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention was created to solve the above problems, and an object thereof is to provide a polymer delivery agent having high performance and a nucleic acid delivery composition containing the same.

Means for Solving the Problems

[0008] The present invention relates to the following formula (1) JPEG0007842172000001.jpg51170[wherein, R 1 is an alkyl group of C1 to C 10 ; -(CH2)3-O-CH3; or -(CH2) X -[(CH2)2-O] y -CH3 (where X is 0 or 1, y is an integer of 1 to 45, provided that when y is 1, X is 1, and when y is 2 or more, X is 0); and m and n are each independently an integer of 1 to 100, provided that m + n ≧ 10, a is an integer of 0 to 2, R 2 and R 3 are each independently one of the following formula (2) JPEG0007842172000002.jpg24170{wherein, R 4 is hydrogen; an amino group; a hydroxy group; an alkoxy group of C1 to C 20 ; an aliphatic ring group of C3 to C 20 ; an aryl group of C6 to C 20 ; a heteroaryl group of C1 to C 20 ; a heterocyclic group of C1 to C 20 ; -NH-C1 to C 20 alkenyl group -NH2; -NH-C1 to C 20 hydroxyalkyl group; -N(C1 to C 20 alkyl group)(C1 to C 20 alkyl group); -N(C1 to C 20 alkyl group)(C1 to C 20 alkenyl group -NH2); -(OH)(C1 to C 20 alkyl group); and -(C1 to C 20 hydroxyalkyl group)(C1 to C 20Selected from the group consisting of hydroxyalkyl groups; The compound is represented by} where z is an integer between 0 and 45.

[0009] The aforementioned R 1 This can be expressed by any one of the following equations (1-1) to (1-3): JPEG0007842172000003.jpg47170

[0010] The aforementioned R 2 or R 3 This can be expressed by any one of the following equations (2-1) to (2-14): JPEG0007842172000004.jpg178170

[0011] In another embodiment, the present invention provides a nucleic acid delivery composition comprising the compound.

[0012] The nucleic acid may be selected from the group consisting of DNA, RNA, PNA, interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), antisense oligonucleotides, and mixtures thereof.

[0013] Furthermore, it may further contain a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt.

[0014] Furthermore, in another embodiment, the present invention provides nanoparticles formed by the binding of the compound and nucleic acid.

[0015] The ratio of protonable nitrogen to nucleic acid phosphate (N / P ratio) of the above compound is 1 to 30.

[0016] In another embodiment, the present invention provides a vaccine composition comprising the compound.

[0017] Furthermore, in another embodiment, the present invention provides a method for producing the compound represented by formula (1), comprising: 1) a step of synthesizing a β-amino acid N-thiocarboxyanhydride (β-NTAs) monomer; 2) a step of synthesizing a polymer precursor by ring-opening polymerization of the β-NTAs monomer using butylamine as an initiator; and 3) a step of modifying the side chain of the polymer precursor by an aminolysis reaction.

[0018] In another embodiment, the present invention provides a method for delivering nucleic acids into cells using the compound.

[0019] The intracellular location mentioned above is either the cytoplasm or the nucleus. [Effects of the Invention]

[0020] The amphiphilic polymer compound according to the present invention can be easily produced by a simple reaction, exhibits high binding ability to therapeutic nucleic acids such as RNA drugs, effectively protects nucleic acids from nucleolytic enzymes, and can improve the stability and delivery capacity in the blood as a nucleic acid delivery body. [Brief explanation of the drawing]

[0021] [Figure 1] This is the 1H NMR spectrum of a benzyl-β3-LCHG monomer according to one embodiment of the present invention. [Figure 2] This is the 1H NMR spectrum of a benzyl-β3-LCHA monomer according to one embodiment of the present invention. [Figure 3] This is the 1H NMR spectrum of a monomer (A=2) according to one embodiment of the present invention. [Figure 4] (A) SEC chart and (B) 1H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt) of a butyl-poly(benzyl-β3-LCHG) precursor polymer according to one embodiment of the present invention. [Figure 5](A) SEC chart and (B) 1H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt) of a PEG1-poly(benzyl-β3-LCHG) precursor polymer according to one embodiment of the present invention. [Figure 6] (A) SEC chart and (B) 1H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt) of a PEG4-poly(benzyl-β3-LCHG) precursor polymer according to one embodiment of the present invention. [Figure 7] (A) SEC chart and (B) 1H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt) of a PEG12-poly(benzyl-β3-LCHG) precursor polymer according to one embodiment of the present invention. [Figure 8] (A) SEC chart and (B) 1H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt) of a PEG24-poly(benzyl-β3-LCHG) precursor polymer according to one embodiment of the present invention. [Figure 9] (A) SEC chart and (B) 1H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt) of a butyl-poly(benzyl-β3-LCHA) precursor polymer according to one embodiment of the present invention. [Figure 10] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a butyl-PGly(DET / CHE) converter according to one embodiment of the present invention. [Figure 11] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a PEG1-PGly(DET / CHE) converter according to one embodiment of the present invention. [Figure 12] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a PEG4-PGly(DET / CHE) converter according to one embodiment of the present invention. [Figure 13] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a PEG12-PGly(DET / CHE) converter according to one embodiment of the present invention. [Figure 14]This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a PEG24-PGly(DET / CHE) converter according to one embodiment of the present invention. [Figure 15] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a butyl-PGly(DET / ADO) converter according to one embodiment of the present invention. [Figure 16] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a PEG1-PGly(DET / ADO) converter according to one embodiment of the present invention. [Figure 17] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a PEG4-PGly(DET / ADO) converter according to one embodiment of the present invention. [Figure 18] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a butyl-PGly(AEP / ADO) converter according to one embodiment of the present invention. [Figure 19] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a butyl-PGly(HED / ADO) converter according to one embodiment of the present invention. [Figure 20] This is the 1H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt) of a butyl-PGly(MDE / ADO) converter according to one embodiment of the present invention. [Figure 21] This figure shows the measurement results of the size and surface charge of high molecular weight nanoparticles of poly(benzyl-b3-LCHG) converter and mRNA according to one embodiment of the present invention. [Figure 22] TEM images (A-C) and particle size distribution histograms (E-G) of polymer nanoparticles are shown. (A,E) Butyl-PGly (DET / CHE), (B,F) PEG4-PGly (DET / CHE), and (C,G) PEG24-PGly (DET / CHE). [Figure 23] This shows the results of measuring the luciferase mRNA expression level of high molecular weight nanoparticles in C2C12 cells according to one embodiment of the present invention, with (A) the state with FBS added and (B) the state without FBS added. [Figure 24] Figure A shows the intracellular uptake efficiency of polymer nanoparticles in C2C12 cells, and Figure B shows the stability analysis results of the polymer nanoparticles. [Figure 25] This paper presents the results of an analysis of cell viability in C2C12 cells against PEGn-PGly(DET / CHE) polymer nanoparticles. [Figure 26] Figure A shows a schematic diagram of the fluorescence expression design for HEK293-loXP-GFP-RFP cells, and Figure B shows the results of analyzing the fluorescence expression levels in cells using ImageJ software after treatment with high molecular weight nanoparticles. [Figure 27] The images shown are CLSM images observed 48 hours after transfection of HEK293-loXP-GFP-RFP cells with various samples containing Cre mRNA. [Best Mode for Carrying Out the Invention]

[0022] The present invention will be described in more detail below. In this specification, the term "amphiphilic" means the property of having both a water-soluble region and a hydrophobic region within a single molecular structure.

[0023] In the present invention, the term "alkyl" or "alkyl group" means an aliphatic hydrocarbon radical, and refers to a saturated aliphatic functional group radical, including linear alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. For example, C1-C6 alkyl groups are aliphatic hydrocarbons having 1 to 6 carbon atoms, and include methyl, ethyl, propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and the like.

[0024] As used in this invention, the terms "alkenyl group" or "alkynyl group" refer to a group consisting of at least two carbon atoms with at least one carbon-carbon double bond, or at least two carbon atoms with at least one carbon-carbon triple bond, and include, but are not limited to, linear or branched chain groups.

[0025] In this invention, the terms "alkoxyl group" or "alkoxy group" mean, unless otherwise defined, a radical in which a hydrogen atom of a hydroxyl group is replaced with an alkyl group. For example, C1-C6 alkoxys include methoxy, ethoxy, propoxy, n-butoxy, n-pentyloxy, isopropoxy, sec-butoxy, tert-butoxy, neopentyloxy, isopentyloxy, and the like.

[0026] As used in this invention, the terms "heterocyclic ring" or "heterocyclic group" include, unless otherwise specified, compounds comprising one or more heteroatoms or heteroatomic groups such as SO2, comprising at least one monocyclic and polycyclic ring, and including heteroaliphatic rings and heteroaromatic rings. They may be formed by the bonding of adjacent functional groups.

[0027] In this invention, the terms "aryl group" or "arylene group" refer to monocyclic or polycyclic aromatic compounds, including aromatic rings formed by the bonding or reaction of adjacent substituents. For example, the aryl group may be a phenyl group, a biphenyl group, a fluorene group, or a spirofluorene group.

[0028] The term "aliphatic ring" as used in this invention refers to an aliphatic hydrocarbon ring.

[0029] The term "aromatic ring" as used in this invention refers to an aromatic system consisting of hydrocarbons containing one or more rings, and examples include benzene and naphthalene.

[0030] Furthermore, the definitions described in this invention may be expanded to form chemically related combinations, such as "arylalkyl," "alkylcarbonyl," and "arylcarbonyl." When the term "alkyl" is used as a suffix, as in the following terms, "phenylalkyl" or "hydroxyalkyl," it means an alkyl group substituted with a substituent selected from another clearly named group. For example, "phenylalkyl" means an alkyl group having a phenyl substituent, and therefore includes benzyl, phenylethyl, and biphenyl. "Alkylaminoalkyl" means an alkyl group having an alkylamino substituent.

[0031] Essential elements for the production of high-performance polymer nanoparticles for nucleic acid delivery include the pK of the polymer. a These include hydrophobicity, the length and density of PEG on the nanoparticle surface, polymer biodegradability, and nanoparticle size.

[0032] pK of polymers a , in other words, the pK of the amino group a The pK value is responsible for binding to or associating with RNA and determines the endosome escape ability and surface charge of nanoparticles. a When the surface charge is between 5 and 7, it becomes highly charged within endosomes (pH 5.5) upon entering cells, facilitating the extrusion of the contained nucleic acids into endosomes and resulting in excellent nucleic acid delivery efficiency within the body. Furthermore, when the surface charge is high (+) or in the neutral range, safety and nucleic acid delivery rates can be improved.

[0033] The hydrophobicity of polymers can regulate the stability of nanoparticles in hydrophilic solvents, and the surface charge of nanoparticles can be neutralized by adjusting the PEG length and density.

[0034] The present invention relates to a poly(β-amino acid) converter, which is a cationic amphiphilic polymer capable of delivering RNA drugs, including mRNA, small interfering RNA (siRNA), and antisense oligonucleotides (ASOs), to cells, and a method for producing the same.

[0035] First, benzyl-β 3 -LCHG monomer and benzyl-β 3 -LCHA monomers are synthesized, and then the precursor polymer poly-(β 3 -benzyl-L-carboxyhomoglycine)[hereinafter, poly(benzyl-β) 3 -LCHG)] and poly-(β) 3 -benzyl-L-carboxyhomoalanine)[hereinafter, butyl-poly(benzyl-β 3 After synthesizing -LCHA), various pK a Poly(β-amino acid) converters of various structures were produced through secondary conversion using hydrophobic compounds. Nanoparticles were then produced using the synthesized polymers in conjunction with mRNA, and mRNA delivery capabilities were evaluated by in vitro transfection.

[0036] The present invention relates to the following formula (1) JPEG0007842172000005.jpg49170[wherein, R 1 C1~C 10 Alkyl alkyl group;-(CH2)3-O-CH3; or-(CH2) X -[(CH2)2-O] y -CH3(where X is 0 or 1, and y is an integer from 1 to 45, where if y is 1, X is 1, and if y is 2 or greater, X is 0); m and n are independent integers between 1 and 100, where m + n ≥ 10. a is an integer between 0 and 2. R 2 and R 3 These are independent of each other, and each is given by the following equation (2) JPEG0007842172000006.jpg26170{where, R 4 is hydrogen; amino group; hydroxyl group; C1~C 20 Alkoxy group of C3~C 20 aliphatic ring group; C6~C 20 aryl group; C1~C 20 heteroaryl group; C1~C 20 heterocyclic group; -NH-C1~C20 Alkenyl group -NH2;-NH-C1~C 20 hydroxyalkyl groups;-N(C1~C 20 (Alkyl group of C1~C) 20 alkyl groups);-N(C1~C 20 (Alkyl group of C1~C) 20 The alkenyl group -NH2);-(OH)(C1~C 20 alkyl groups); and -(C1~C 20 (Hydroxyalkyl groups) (C1~C 20 Selected from the group consisting of hydroxyalkyl groups; The compound is represented by} where z is an integer between 0 and 45.

[0037] Furthermore, the present invention relates to the R 1 This provides a compound represented by any one of the following formulas (1-1) to (1-3): JPEG0007842172000007.jpg46170

[0038] Furthermore, the present invention relates to the above R 2 or R 3 This provides a compound represented by any one of the following formulas (2-1) to (2-20): JPEG0007842172000008.jpg171170JPEG0007842172000009.jpg124170

[0039] The aforementioned R 2 The pK of polymers a A functional group for controlling the R 3 This is a functional group used to control the hydrophobicity of polymers.

[0040] Specifically, the compound represented by formula (1) above can be any one of the following compounds, but is not limited to these. JPEG0007842172000010.jpg56170JPEG0007842172000011.jpg220170

[0041] In another embodiment, the present invention provides a nucleic acid delivery composition comprising the compound.

[0042] The nucleic acid is selected from the group consisting of DNA, RNA, PNA, interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), antisense oligonucleotide (ASO), and mixtures thereof, but is not limited thereto, and is preferably mRNA, siRNA, or ASO.

[0043] The compound can bind to nucleic acids to form polymer nanoparticles. Because the compound is a cationic amphiphilic polymer and readily binds to nucleic acids, the binding does not break down in the event of changes in the blood environment, allowing for effective gene delivery to the target site.

[0044] The nucleic acid delivery composition may further comprise a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt.

[0045] In another embodiment, the present invention provides nanoparticles formed by binding the compound with nucleic acid.

[0046] In the aforementioned nanoparticles, the ratio of protonable nitrogen to nucleic acid phosphate (N / P ratio) of the compound may be 1 to 30, preferably 3 to 10, and more preferably 5 to 10.

[0047] In another aspect, the present invention provides a vaccine composition comprising the compound.

[0048] Furthermore, in another aspect, the present invention provides a method for producing the compound represented by formula (1), comprising: 1) a step of synthesizing a β-amino acid N-thiocarboxyanhydride (β-NTAs) monomer; 2) a step of synthesizing a polymer precursor by ring-opening polymerization of the β-NTAs monomer using butylamine as an initiator; and 3) a step of modifying the side chain of the polymer precursor by an aminolysis reaction.

[0049] In another aspect, the present invention provides a method for delivering nucleic acids into a cell using the compound. In this case, the intracellular location may be the cytoplasm or the nucleus.

[0050] The following are preferred embodiments to aid in understanding the present invention. However, these embodiments are provided for the purpose of aiding understanding the present invention, and the present invention is not limited to these embodiments.

[0051] [Example of combination] Formula (1) (final compound) of the present invention can be produced by the reaction shown in Reaction Scheme 1 below, but is not limited thereto. <Reaction Scheme 1> JPEG0007842172000012.jpg43170

[0052] Example 1. Synthesis example of poly(β-amino acid) converters 1-1. Benzyl-β 3 - Synthesis of LCHG monomers benzyl-β 3 -LCHG monomers were synthesized using the same method as in previous research (M. Zhou Angew. Chem. Int. Ed. 2020, 59, 7240-7244). The synthesized monomers were: 1 The presence or absence of synthesis was confirmed by 1H-NMR (Avance III, 400 MHz, Bruker, Billerica, MA, USA) (Figure 1).

[0053] 1-2. Benzyl-β 3- Synthesis of LCHA monomers benzyl-β 3 -LCHA monomers were synthesized based on previous research (M. Zhou Angew. Chem. Int. Ed. 2020, 59, 7240-7244). The synthesized monomers were: 1 The presence or absence of synthesis was confirmed by 1H-NMR (Avance III, 400 MHz, Bruker, Billerica, MA, USA) (Figure 2).

[0054] 1-3. Synthesis of monomers (a=2) JPEG0007842172000013.jpg56170 The monomer (a=2) shown in the above formula was synthesized based on previous research (M. Zhou Angew. Chem. Int. Ed. 2020, 59, 7240-7244). The monomer after synthesis is, 1 The presence or absence of synthesis was confirmed by 1H-NMR (A Avance III, 400 MHz, Bruker, Billerica, MA, USA) (Figure 4).

[0055] 1-4. Poly(benzyl-β) 3 -LCHG) Synthesis of Precursor Polymers The precursor polymer was synthesized as described below, based on previous research (M. Zhou Angew. Chem. Int. Ed. 2020, 59, 7240-7244).

[0056] benzyl-β 3 -LCHG monomer (100 mg) was dissolved in DCM (2 mL), and then acetic acid (3.45 μL) was added (D. Siefker ACS Macro Lett. 2018, 7, 1272-1277). The initiator n-butylamine (1.1 mg), dissolved in DCM, was added to the monomer solution and the reaction was stirred at 35°C for 72 hours. The completed solution was precipitated in hexane / ethyl acetate (6:4 v / v), filtered, and dried under reduced pressure.

[0057] Synthesized butyl-poly(benzyl-β) 3-LCHG) precursor polymers were analyzed in a DMF solution containing 10 mM lithium chloride using gel permeation chromatography (HLC-8420, Tosoh Corporation) with two TSK columns (G3000HHR and G4000HHR) to determine the molecular weight distribution (M w / M n The degree of polymerization (DP) was calculated. 1 Analysis was performed using 1H-NMR. The results showed that butyl-poly(benzyl-β 3 -LCHG) Precursor polymer M w / M n The DP values ​​were confirmed to be 1.041 and 25, respectively (Figure 4).

[0058] In the same manner as described above, the initiator is PEG1-amine, PEG4-amine, PEG 12 -amine, PEG 24 -The amine was changed to produce different precursor polymers with different PEG lengths (Figures 5-7), and each of the poly(benzyl-β) polymers was produced. 3 -LCHG) Precursor polymer M w / M n The dynamic programming (DP) is shown in Table 1 below. JPEG0007842172000014.jpg46170

[0059] 1-5. Butyl-poly(benzyl-β) 3 -LCHA) Synthesis of Precursor Polymers The butyl-poly(benzyl-β) 3 -LCHG) precursor polymers are used in the same manner as butyl-poly(benzyl-β) 3 -LCHA) precursor polymer was synthesized.

[0060] benzyl-β 3 -LCHA monomer (100 mg) was dissolved in DCM (2 mL), and then acetic acid (3.28 μL) was added. The initiator n-butylamine (1.047 mg), dissolved in DCM, was added to the monomer solution and the reaction was stirred at 35°C for 72 hours. The completed solution precipitated in hexane / ethyl acetate (6:4 v / v), filtered, and dried under reduced pressure. Butyl-poly(benzyl-β 3-LCHA) The Mw / Mn and DP of the precursor polymer were confirmed to be 1.030 and 16, respectively (Figure 9).

[0061] 1-6. Poly(benzyl-β 3 -LCHG) Synthesis of the convertant The synthesized precursor polymer was converted into various poly(β-amino acid) convertants by aminolysis reaction with compounds having various pK a and hydrophobicity.

[0062] Synthesis example of butyl-PGly(DET / CHE) Butyl-poly(benzyl-β 3 -LCHG) precursor polymer (20 mg) was dissolved in N-methyl l-2-pyrrolidone (NMP, 1 mL) at 35 °C and then cooled to 4 °C. While maintaining the solution of NMP (1 mL) added to diethylenetriamine (DET, 0.26 mL) and cyclohexylethylamine (CHE, 0.93 mL) at 10 °C, the precursor polymer solution was slowly added in an argon gas state. The reaction solution was reacted at 10 °C for 24 hours. After the reaction was completed, the reaction solution was precipitated in ethyl ether to obtain a powder. This powder was dissolved in cold 0.01 M HCl and then dialyzed at 4 °C for 2 days using 0.01 M HCl. Finally, after dialysis with only water for 1-2 hours, the solution was lyophilized to obtain the final product of the powder. The produced convertant was 1 The introduction amounts of DET and CHE were calculated using 1H-NMR.

[0063] The synthesis reaction conditions of each convertant with different PEG lengths are shown in Table 2 below, and the introduction amounts of the produced convertants are shown in Table 3 below. JPEG0007842172000015.jpg140170JPEG0007842172000016.jpg51170

[0064] pK a Poly(β-amino acid) convertants with further converted pK a and hydrophobicity were synthesized in the same manner as the above synthesis method, and the chemical conversions of each pK and hydrophobic group are shown in Table 4 below. JPEG0007842172000017.jpg71170

[0065] Example 2. Measurement of binding between poly(benzyl-β 3 -LCHG) variant and mRNA Each variant was dissolved in 10 mM Hepes (pH 7.3) buffer at 2 mg / mL, and then rediluted in 10 mM Hepes (pH 7.3) buffer so that the molar concentration of the protonatable amino group became 0.624 mM. A solution prepared with luciferase mRNA (L-7202; TriLink Biotechnologies, San Diego, CA, USA) (100 ng / μL) was mixed so that the molar concentration of the protonatable amino group / molar concentration of phosphate group (N / P ratio) = 1 to 5, and then incubated at room temperature for about 1 hour. Then, after preparing a 1.0% agarose gel, the polymeric nanoparticles (10 μL) and glycerol solution (50% v / v) (2 μL) were mixed, and then electrophoresis (135 V, 20 minutes) was performed. The agarose gel after electrophoresis was observed with a gel Imager (Figure 21). As a result, it was confirmed that the binding force between the polymeric variant of the present invention and mRNA was excellent.

[0066] Example 3. Measurement of the size and surface charge of poly(benzyl-β 3 -LCHG) variant and mRNA polymeric nanoparticles After producing polymeric nanoparticles containing luciferase mRNA by the above method at N / P = 5, the nanoparticle diameter (D H ), polydispersity index (PDI), and surface charge (ζ-potential) were measured using a Zetasizer Pro (RED) (Malvern Instruments, Worcestershire, UK), and the measured values are shown in Table 5 below. JPEG0007842172000018.jpg87170

[0067] Similarly, after producing polymeric nanoparticles containing luciferase mRNA at N / P = 7, the nanoparticle diameter, polydispersity index (PDI), and surface charge (ζ-potential) were measured using a Zetasizer Pro (RED), and the measured values are shown in Table 6 below. JPEG0007842172000019.jpg50170

[0068] Example 4. Transmission electron microscope (TEM) measurement Luciferase mRNA and polymers of different lengths (butyl-PG) l y(DET / CHE), PEG4-PGly(DET / CHE), PEG 24 Polymer nanoparticles (N / P=5) were fabricated using butyl-PGly(DET / CHE). The fabricated polymer nanoparticles were placed on a 400-mesh copper grid (Electron Microscopy Sciences, Pennsylvania, USA), stained with UranyLess EMS staining solution (Electron Microscopy Sciences), and measured using a field emission transmission electron microscope (FE-TEM, JEM2100F; JEOL Ltd., Tokyo, Japan) (Figure 22 A-C). The average diameter of the nanoparticles was measured using ImageJ software (Figure 22 D-F). Butyl-PGly(DET / CHE), PEG4-PGly(DET / CHE), PEG 24 The average diameters of the PGly(DET / CHE) polymer nanoparticles were measured to be 50±9, 45±10, and 52±13 nm, respectively (n=70).

[0069] Example 5. Measurement of luciferase mRNA expression levels of high molecular weight nanoparticles in C2C12 cells High molecular weight nanoparticles containing luciferase mRNA were transfected into mouse myoblast cells, C2C12 cells, and the luciferase mRNA delivery capacity was measured. C2C12 cells were prepared using DMEM medium supplemented with 10% FBS. C2C12 cells were seeded at 8000 cells / well in 96-well plates and cultured in a CO2 chamber for 1 day. High molecular weight nanoparticles (N / P=5) were transfected at a concentration of 50 ng mRNA / well, and after 24 hours, the cells were lysed. The luminescence intensity was then measured using a luciferase assay system (Promega, Madison, WI, USA) and a luminescence microplate reader (Mithras LB940; Berthold Technologies, Bad Wildbad, Germany) with the cell lysate. Transfection with high molecular weight nanoparticles was measured separately with and without 10% FBS (Figure 23A) to further investigate the PEG effect. We confirmed that butyl-, PEG1-, and PEG4-PGly(DET / CHE) polymers exhibit high luciferase mRNA expression levels.

[0070] Example 6. Analysis of cellular uptake and serum stability of high molecular weight nanoparticles in C2C12 cells. PEG nTo investigate the effect of PEG length on the PGly(DET / CHE) polymer in more detail, the amount of macromolecular nanoparticles taken up by C2C12 cells was analyzed. Using the Label IT Cy5 labeling kit (Mirus Bio Corporation, Madison, WI, USA), luciferase mRNA labeled with Cy5 was prepared by reacting it according to the protocol provided by the kit (Cy5-mRNA). Macromolecular nanoparticles containing Cy5-mRNA (N / P=5, 125 ng mRNA / well) were transfected into C2C12 cells seeded in a 48-well plate (50,000 cells / well). After incubation for 4 hours, cells were detached using trypsin-EDTA, and the intracellular uptake of macromolecular nanoparticles was compared by comparing fluorescence expression levels using flow cytometry (Attune CytPiX, ThermoFisher Scientific) (Figure 24A). This confirmed that a considerable amount of Cy5-mRNA was taken up by C2C12 cells for macromolecules with PEG lengths of 0-4. In contrast, we confirmed that in the case of macromolecules with PEG lengths of 12 and 24, intracellular uptake hardly occurs.

[0071] To investigate in more detail the effect of PEG length on the amount of cellular uptake of high molecular weight nanoparticles, the particle stability of the same high molecular weight nanoparticles against serum albumin was measured using fetal bovine albumin (FBS). High molecular weight nanoparticles (N / P=5, 100ng mRNA, 10μL) were mixed with a PBS solution containing 10% FBS (PBS / FBS, 90μL) and reacted at 37°C for 1 hour. After 1 hour, mRNA was purified using the RNeasy Mini kit (Qiagen). After agarose gel electrophoresis (1wt% agarose gel, 0.5X TAE buffer, 135V, 15 minutes), the bands were observed using a gel imager (WSE-5300 Printgraph CMOS I) (Figure 24B). In the case of butyl-, PEG1-, and PEG4-PGly (DET / CHE) high molecular weight nanoparticles, bands of intact mRNA were shown in FBS. This indicates that high molecular weight nanoparticles are stable in FBS. On the other hand, PEG12 -, PEG 24 - In the case of PGly(DET / CHE) polymer nanoparticles, no intact mRNA band was shown. This means that the two polymer nanoparticles were degraded within 1 hour in PBS / FBS, which simulates cell culture. This result confirms that, in the case of PGly(DET / CHE) polymers, when the PEG length is 12 or greater, the nanoparticles are unstable in the cell culture environment, degrade quickly, and are unable to deliver mRNA into the cell.

[0072] Example 7. Analysis of viability of C2C12 cells (Viability assay) C2C12 cells were seeded into 96-well plates (8,000 cells / well), and the following day, PEG containing luciferase mRNA was added. n -PGly(DET / CHE) polymer nanoparticles (N / P=5, 50 ng / well) were transfected. After incubation for 24 hours, 10 μμL of CCK-8 solution was applied to each well, and the absorbance at 450 nm was measured after 1.5 hours using a microplate reader (Spark, Tecan Group Ltd., Mδnnedorf, Switzerland) (Figure 25). In this experimental environment, PEG n -PGly(DET / CHE) polymer nanoparticles did not show serious cytotoxicity to C2C12.

[0073] Example 8. PEG in HEK293-loXP-GFP-RFP cells n - Confirmation of the gene editing effect of PGly(DET / CHE) polymers HEK293-loXP-GFP-RFP cells (GenTarget Inc, SC018-Neo, San Diego, CA, USA) have a structure in which the loXP-GFP-stop-loXP-RFP cassette is located behind the CMV-promoter. Normally, they emit strong green fluorescence, but when loXP is cleaved by CRE recombinase, they emit red fluorescence (Figure 26A). This allowed us to measure how well high molecular weight nanoparticles containing Cre mRNA (L-7211; TriLink Biotechnologies) delivered Cre mRNA into the cells and how effectively they achieved gene editing efficiency. Cells were seeded in a 96-well optical bottom plate (8,000 cells / well) and PEG was used. n Cells were transfected with PEG-PGly(DET / CHE) polymer nanoparticles (N / P=5, 100 ng / μL). After 48 hours of incubation, green (GFP) and red (RFP) fluorescence were measured using a confocal laser microscope (CLSM) (ZEISS LSM980; Carl Zeiss, Oberkochen, Germany) (Figure 27). The fluorescence intensities were then analyzed and quantified using ImageJ software (Figure 26B). High RFP fluorescence was observed in cells treated with butyl-, PEG1-, and PEG4-PGly(DET / CHE) polymer nanoparticles, confirming the high Cre mRNA delivery capability of the three polymers. In particular, PEG1-PGly(DET / CHE) polymer nanoparticles were found to have the highest gene editing effect.

[0074] In summary, the present invention synthesizes a novel poly-β-peptide precursor polymer that has not been conventionally used for RNA drug delivery, and through additional aminolysis conversion, various pKs are used. aWe synthesized a hydrophobic, PEG-length converted polymer. We also successfully bound the synthesized polymer to mRNA, producing nanoparticles with a size of 100-300 nm. Furthermore, from these results, we confirmed that the nanoparticles of the present invention successfully delivered luciferase mRNA to in vitro C2C12 cells, and by delivering Cre mRNA to HEK293-loXP-GFP-RFP cells and comparing and analyzing the gene editing efficiency, we confirmed the potential of polyβ-peptide polymers for RNA drug delivery.

[0075] The above description is merely illustrative, and a person with ordinary skill in the art relating to the present invention could make various modifications without departing from the essential characteristics of the present invention. Therefore, the examples disclosed herein are for illustrative purposes only, not to limit the present invention, and these examples do not limit the spirit or scope of the present invention. The scope of protection of the present invention should be interpreted by the following claims, and all art of an equivalent scope should be interpreted as being included within the scope of the present invention.

Claims

1. The following formula (1) [In the formula, R 1 is an alkyl group of C 1 to C 10 ; -(CH 2 ) 3 -O-CH 3 ; or -(CH 2 ) Ｘ -[(CH 2 ) 2 -O] y -CH 3 (where X is 0 or 1, y is an integer from 1 to 45, provided that when y is 1, X is 1, and when y is 2 or more, X is 0); and​ m and n are independent integers between 1 and 100, where m + n ≥ 10. a is an integer between 0 and 2. R 2 and R 3 These are independent of each other, and each is given by the following equation (2) {In the formula, R 4 is hydrogen; amino group; hydroxyl group; C 1 ~C 20 alkoxy group of; C 3 ~C 20 aliphatic ring group of; C 6 ~C 20 aryl group of; C 1 ~C 20 heteroaryl group of C 1 ~C 20 heterocyclic group; -NH-C 1 ~C 20 alkenyl group -NH 2 ;-NH-C 1 ~C 20 hydroxyalkyl groups; -N(C) 1 ~C 20 (Alkyl alkyl group) (C 1 ~C 20 (Alkyl alkyl group); -N(C 1 ~C 20 (Alkyl alkyl group) (C 1 ~C 20 alkenyl group -NH 2 ); -(OH)(C 1 ~C 20 (Alkyl alkyl group); and -(C 1 ~C 20 (Hydroxyalkyl group) (C 1 ~C 20 Selected from the group consisting of (hydroxyalkyl groups of); A compound represented by} where z is an integer between 0 and 45.

2. The aforementioned R 1 The compound according to claim 1 is characterized by being one of the following formulas (1-1) to (1-3):

3. The aforementioned R 2 or R 3 The compound according to claim 1 is characterized by being one of the following formulas (2-1) to (2-20):

4. A nucleic acid delivery composition comprising the compound described in any one of claims 1 to 3.

5. The nucleic acid delivery composition according to claim 4, characterized in that the nucleic acid is selected from the group consisting of DNA, RNA, PNA, interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), antisense oligonucleotides, and mixtures thereof.

6. The nucleic acid delivery composition according to claim 4, further comprising a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt.

7. Nanoparticles formed by bonding a compound according to any one of claims 1 to 3 with a nucleic acid.

8. The nanoparticle according to claim 7, characterized in that the ratio of protonable nitrogen to nucleic acid phosphate (N / P ratio) of the compound is 1 to 30.

9. A vaccine composition comprising the nanoparticles described in claim 7.

10. 1) A step to synthesize β-amino acid N-thiocarboxy anhydride (β-NTAs) monomers; 2) A step of synthesizing a polymer precursor by ring-opening polymerization of the β-NTAs monomer using butylamine as an initiator; and 3) A step of modifying the side chains of the polymer precursor by an aminolysis reaction; A method for producing the compound represented by formula (1) of claim 1, which includes [a specific element].

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