Novel polymer compound and composition for delivering nucleic acid, comprising same

Abstract

The present invention relates to a novel polymer compound and a composition for delivering nucleic acids, comprising same. More specifically, the novel polymer compound has a high binding force to nucleic acids, and effectively protects nucleic acids from nucleases, thereby improving nucleic acid delivery effects and stability.

Description

Novel polymer compound and composition for nucleic acid delivery comprising the same

[0001] The present invention relates to an amphiphilic polymer compound and a composition for nucleic acid delivery comprising the same.

[0002] mRNA vaccines, attracting attention as next-generation vaccines, artificially synthesize and inject mRNA containing the virus's genetic information, stimulating cells to produce the spike protein and activating immunity. Because mRNA vaccines utilize viral genes, they can be developed quickly once the viral genetic structure is identified. Furthermore, they eliminate the need for external antigen or antibody cultivation, allowing for rapid production. mRNA vaccines have been used globally to combat the COVID-19 pandemic, and various research and development efforts are currently underway to address not only infectious diseases but also various other diseases, including cancer and rare diseases.

[0003] Meanwhile, mRNA can be easily damaged by enzymes in the human body, so nanocarriers are attracting attention as a technology for safely delivering mRNA to cells. Currently, the most widely used nanocarrier is the lipid nanoparticle (LNP), and the COVID-19 vaccines recently developed by Pfizer and Moderna also use LNP carriers. However, lipid nanoparticles are prone to rancidity, making them difficult to store and distribute at room temperature. Furthermore, side effects, such as hypersensitivity reactions due to the LNP's own toxicity, have been reported.

[0004] To overcome this, active research is being conducted on synthetic polymer nanocarriers. Synthetic polymer nanocarriers are made by repeatedly combining small units, allowing for flexible design of their structure, size, ionization properties, and stimulus-responsive drug release. They could also be utilized in gene therapy, potentially enabling gene editing. Furthermore, by incorporating other components into polymer nanocarriers, the properties of polymers can be maintained while simultaneously achieving additional benefits. Furthermore, the composition can be controlled through multi-polymer systems, such as polymer-lipid and polymer-inorganic hybrid delivery nanoplatforms, and polymer-based hybrid systems.

[0005] mRNA-loaded polymeric nanoparticles have diverse structures, including polyplexes, polymer micelles, polymersomes, and core-shell polymer-lipid hybrids. These polymeric nanoparticle carriers exhibit low toxicity, excellent biodegradability, and biocompatibility, reducing toxicity and immune responses. They can also provide fundamental data for the development of next-generation vaccines and pharmaceuticals, where mRNA delivery using LNPs is challenging. Furthermore, polymer-based mRNA delivery technology can be applied not only to mRNA vaccines but also to the development of other RNA drugs, demonstrating its potential for use in the treatment of various diseases.

[0006] As an example of polymer nanoparticle carriers, cationic dendrimers have been used in nonclinical trials against various viruses, including Ebola, H1N1 influenza, and Zika. However, due to the structural characteristics of the highly branched molecules themselves, they are difficult to decompose in vivo, resulting in toxicity, and thus require improvement. Polymer nanoparticles have been reported in numerous publications, either alone or in combination with lipids as lipid-polymer complexes. However, compared to the research results, accurate property analysis and structural elucidation are difficult, making clinical entry somewhat difficult, and thus, commercialization has not yet been achieved.

[0007] The present invention has been devised to solve the above problems, and its purpose is to provide a high-performance polymer carrier and a nucleic acid delivery composition comprising the same.

[0008] The present invention provides a compound represented by the following chemical formula 1.

[0009] Chemical Formula 1

[0010]

[0011] {In the above chemical formula 1,

[0012] R 1 Silver C1~C 10 Alkyl group of; -(CH2)3-O-CH3; or -(CH2) x -[(CH2)2-O] y -CH3; and

[0013] x is 0 or 1, y is an integer from 1 to 45,

[0014] However, if y is 1, x is 1, and if y is 2 or greater, x is 0.

[0015] m and n are integers from 1 to 100, independently of each other,

[0016] However, m+n≥10,

[0017] a is an integer from 0 to 2,

[0018] R 2 and R 3 are independently represented by the following chemical formula 2,

[0019] Chemical Formula 2

[0020]

[0021] In the above chemical formula 2,

[0022] 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 of; C1~C 20Heteroaryl group of; C1~C 20 Heterocyclic group of; -NH-C1~C 20 Alkenyl group-NH2; -NH-C1~C 20 Hydroxyalkyl group of; -N(C1~C 20 alkyl group)(C1~C 20 alkyl group); -N(C1~C 20 alkyl group)(C1~C 20 alkenyl group-NH2); -(OH)(C1~C 20 alkyl group); and -(C1~C 20 Hydroxyalkyl group of (C1~C) 20 is selected from the group consisting of a hydroxyalkyl group);

[0023] z is an integer from 0 to 45.

[0024] The above R 1 can be represented by any one of the following chemical formulas 1-1 to 1-3.

[0025] Chemical Formula 1-1 Chemical Formula 1-2 Chemical Formula 1-3

[0026]

[0027] {In the above chemical formulas 1-1 to 1-3,

[0028] y' is an integer from 2 to 45,

[0029] indicates the position of the bond.}

[0030] The above R 2 or R 3 can be represented by any one of the following chemical formulas 2-1 to 2-20.

[0031] Chemical Formula 2-1 Chemical Formula 2-2 Chemical Formula 2-3

[0032]

[0033] Chemical Formula 2-4 Chemical Formula 2-5 Chemical Formula 2-6

[0034]

[0035] Chemical Formula 2-7 Chemical Formula 2-8

[0036]

[0037] Chemical Formula 2-9 Chemical Formula 2-10 Chemical Formula 2-11

[0038]

[0039] Chemical Formula 2-12 Chemical Formula 2-13 Chemical Formula 2-14

[0040]

[0041] Chemical Formula 2-15 Chemical Formula 2-16

[0042]

[0043] Chemical Formula 2-17 Chemical Formula 2-18

[0044]

[0045] Chemical Formula 2-19 Chemical Formula 2-20

[0046]

[0047] {In the above chemical formulas 2-1 to 2-20, c is an integer from 0 to 12.}

[0048] In another aspect, the present invention provides a composition for delivering nucleic acids comprising the compound.

[0049] 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), small hairpin RNA (shRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), antisense oligonucleotides, and mixtures thereof.

[0050] Additionally, the nucleic acid delivery composition may further comprise a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt.

[0051] In another aspect, the present invention provides a nanoparticle formed by combining the compound and a nucleic acid.

[0052] The nitrogen and phosphoric acid ratio (N / P ratio) of the above compound is 1:1 to 30:1.

[0053] In another aspect, the present invention provides a vaccine composition comprising the compound.

[0054] In addition, in another aspect, the present invention provides a method for producing a compound represented by the above chemical formula 1, comprising the steps of: 1) synthesizing a beta-amino acid N-thiocarboxyanhydrides (beta-NTAs) monomer; 2) synthesizing a polymer precursor by ring-opening polymerization of the beta-NTAs monomer using butylamine as an initiator; and 3) modifying a side chain of the polymer precursor by an aminolysis reaction.

[0055] In another aspect, the present invention provides a method for delivering a nucleic acid into a cell using the compound.

[0056] The above intracellular location is the cytoplasm or nucleus.

[0057] The amphiphilic polymer compound according to the present invention can be easily manufactured through a simple reaction, exhibits high binding capacity to therapeutic nucleic acids such as RNA drugs, and effectively protects nucleic acids from nucleases, thereby improving stability and delivery ability in blood as a nucleic acid delivery vehicle.

[0058] Figure 1 is a diagram showing Benzyl-β according to one embodiment of the present invention. 3 -LCHG monomer 1 This is the H NMR spectrum.

[0059] Figure 2 is Benzyl-β according to one embodiment of the present invention. 3-LCHA monomer 1 This is the H NMR spectrum.

[0060] Figure 3 is a monomer (a=2) according to one embodiment of the present invention. 1 This is the H NMR spectrum.

[0061] Figure 4 is a butyl-poly(benzyl-β) according to one embodiment of the present invention. 3 (A) SEC chart and (B) of the precursor polymer (LCHG) 1 H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt).

[0062] Figure 5 is a PEG1-poly(benzyl-β) according to one embodiment of the present invention. 3 (A) SEC chart and (B) of the precursor polymer (LCHG) 1 H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt).

[0063] Figure 6 is a PEG4-poly(benzyl-β) according to one embodiment of the present invention. 3 (A) SEC chart and (B) of the precursor polymer (LCHG) 1 H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt).

[0064] Figure 7 is a PEG according to one embodiment of the present invention. 12 -poly(benzyl-β 3 (A) SEC chart and (B) of the precursor polymer (LCHG) 1 H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt).

[0065] Figure 8 is a PEG according to one embodiment of the present invention. 24 -poly(benzyl-β 3 (A) SEC chart and (B) of the precursor polymer (LCHG) 1 H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt).

[0066] Figure 9 is a butyl-poly(benzyl-β) according to one embodiment of the present invention. 3 -LCHA) SEC chart of precursor polymer (A) and (B) 1 H NMR spectrum (5 mg / mL, DMSO-d6, 400 MHz, rt).

[0067] Figure 10 is a butyl-PGly (DET / CHE) converter according to one embodiment of the present invention. 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0068] Figure 11 is a diagram of a PEG1-PGly (DET / CHE) convertant according to one embodiment of the present invention. 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0069] Figure 12 is a diagram of a PEG4-PGly (DET / CHE) convertant according to one embodiment of the present invention. 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0070] Figure 13 is a PEG according to one embodiment of the present invention. 12 -PGly(DET / CHE) converter 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0071] Figure 14 is a PEG according to one embodiment of the present invention. 24 -PGly(DET / CHE) converter 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0072] Figure 15 is a diagram of a Butyl-PGly (DET / ADO) converter according to one embodiment of the present invention. 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0073] Figure 16 is a diagram of a PEG1-PGly (DET / ADO) convertant according to one embodiment of the present invention.1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0074] Figure 17 is a diagram of a PEG4-PGly (DET / ADO) convertant according to one embodiment of the present invention. 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0075] Figure 18 is a diagram of a Butyl-PGly (AEP / ADO) converter according to one embodiment of the present invention. 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0076] Figure 19 is a diagram of a Butyl-PGly (HED / ADO) converter according to one embodiment of the present invention. 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0077] Figure 20 is a diagram of a Butyl-PGly (MDE / ADO) converter according to one embodiment of the present invention. 1 H NMR spectrum (5 mg / mL, D2O, 400 MHz, rt).

[0078] Figure 21 is a poly(benzyl-b) according to one embodiment of the present invention. 3 -LCHG) The results of size and surface charge measurements of polymer nanoparticles of mRNA and the transformant are shown.

[0079] Figure 22 shows TEM images (A-C) and size distribution histograms (E-G) of polymer nanoparticles. (A,E) butyl-PGly(DET / CHE), (B,F) PEG4-PGly(DET / CHE), and (C,G) PEG 24 -PGly(DET / CHE).

[0080] Figure 23 shows the results of measuring the expression level of luciferase mRNA of polymer nanoparticles in C2C12 cells according to one embodiment of the present invention, showing (A) a state with FBS added and (B) a state without FBS added.

[0081] Figure 24A shows the intracellular uptake efficiency of polymer nanoparticles in C2C12 cells, and Figure 24B shows the results of stability analysis of polymer nanoparticles.

[0082] Figure 25 PEG in C2C12 cells n -Shows the results of cell viability analysis for PGly(DET / CHE) polymer nanoparticles.

[0083] Figure 26A shows a schematic diagram of the fluorescence expression design of HEK293-loxP-GFP-RFP cells, and Figure 26B shows the results of analyzing the amount of fluorescence expression in cells treated with polymer nanoparticles using Image J software.

[0084] Figure 27 shows CLSM images observed 48 hours after transfection of various samples containing Cre mRNA into HEK293-loxP-GFP-RFP cells.

[0085] Hereinafter, the present invention will be described in more detail.

[0086] The term “amphiphilic” in this specification means the property of having both a water-soluble region and a hydrophobic region within a single molecular structure.

[0087] In the present invention, the term “alkyl” or “alkyl group” means an aliphatic hydrocarbon radical, and refers to a radical of a saturated aliphatic functional group including a straight-chain alkyl group, a branched-chain alkyl group, a cycloalkyl (alicyclic) group, an alkyl-substituted cycloalkyl group, and a cycloalkyl-substituted alkyl group. For example, C1~C6 alkyl is an aliphatic hydrocarbon having 1 to 6 carbon atoms, and includes methyl, ethyl, propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and the like.

[0088] The term “alkenyl group” or “alkynyl group” as used in the present invention means a group in which at least two carbon atoms are formed by at least one carbon-carbon double bond or at least two carbon atoms are formed by at least one carbon-carbon triple bond, and includes, but is not limited to, a straight-chain or branched-chain group.

[0089] The term “alkoxy group” or “alkoxy group” used in the present invention, unless otherwise defined, means a radical in which a hydrogen atom of a hydroxy group is replaced with an alkyl, and for example, C1 to C6 alkoxy includes methoxy, ethoxy, propoxy, n-butoxy, n-pentyloxy, isopropoxy, sec-butoxy, tert-butoxy, neopentyloxy, isopentyloxy, etc.

[0090] The term “heterocycle” or “heterocyclic group” as used in the present invention includes, unless otherwise specified, a compound containing one or more heteroatoms or heteroatom groups such as SO2, and includes at least one of a single ring and a multi-ring, and includes a heteroaliphatic ring and a heteroaromatic ring. It may also be formed by bonding adjacent functional groups.

[0091] The term “aryl group” or “arylene group” as used in the present invention refers to a single-ring or multi-ring aromatic group, and includes an aromatic ring 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.

[0092] The term “aliphatic ring” used in the present invention means an aliphatic hydrocarbon ring.

[0093] The term “aromatic ring” used in the present invention means an aromatic system composed of hydrocarbons containing one or more rings, examples of which include benzene and naphthalene.

[0094] Additionally, the definitions described herein can be added to form chemically related combinations, such as “arylalkyl,” “alkylcarbonyl,” “arylcarbonyl,” etc. The term “alkyl” when used as a suffix, such as in another term below, “phenylalkyl” or “hydroxyalkyl,” means an alkyl group substituted with a substituent selected from another specifically named group. Thus, for example, “phenylalkyl” means an alkyl group having a phenyl substituent, and thus includes benzyl, phenylethyl, and biphenyl. “Alkylaminoalkyl” means an alkyl group having an alkylamino substituent.

[0095]

[0096] An essential element for the production of high-performance polymer nanoparticles for nucleic acid delivery is the pK of the polymer. a , hydrophobicity, length and density of PEG on the nanoparticle surface, polymer degradability, and nanoparticle size.

[0097] pK of polymers a , i.e. pK of the amino group a The value is responsible for binding or association with RNA, and determines the endosomal escape ability and surface charge of the nanoparticle. The pK of the polymer a When the value is 5-7, upon entering the cell, it becomes highly charged within the endosome (pH 5.5), facilitating the escape of the contained nucleic acid from the endosome, thereby improving the efficiency of nucleic acid delivery in the body. In addition, when the surface charge is in the high (+) or neutral range, safety and nucleic acid delivery rate can be improved.

[0098] The hydrophobicity of the polymer can control the stability of the nanoparticles in hydrophilic solvents, and the surface charge of the nanoparticles can be controlled to be neutral by controlling the PEG length and density.

[0099] The present invention relates to a poly(beta-amino acids) converting agent, which is a cationic amphiphilic polymer capable of delivering RNA drugs including mRNA, small interfering RNA (siRNA), and antisense oligonucleotide (ASO) to cells, and a method for producing the same.

[0100] First, Benzyl-β 3 -LCHG monomer and Benzyl-β 3 -Synthesize the LCHA monomer, 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(benzy-β 3 After synthesizing [LCHA], various pK a Poly(beta-amino acids) derivatives of various structures were prepared through secondary transformation into hydrophobic compounds. Nanoparticles containing mRNA were prepared using the synthesized polymers, and the mRNA delivery ability was evaluated through in vitro transfection.

[0101] The present invention provides a compound represented by the following chemical formula 1.

[0102] Chemical Formula 1

[0103]

[0104] {In the above chemical formula 1,

[0105] R 1 Silver C1~C 10 Alkyl group of; -(CH2)3-O-CH3; or -(CH2) x -[(CH2)2-O] y -CH3; and

[0106] x is 0 or 1, y is an integer from 1 to 45,

[0107] However, if y is 1, x is 1, and if y is 2 or greater, x is 0.

[0108] m and n are integers from 1 to 100, independently of each other,

[0109] However, m+n≥10,

[0110] a is an integer from 0 to 2,

[0111] R 2 and R 3 are independently represented by the following chemical formula 2,

[0112] Chemical Formula 2

[0113]

[0114] In the above chemical formula 2,

[0115] 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 of; C1~C 20 Heteroaryl group of; C1~C 20 Heterocyclic group of; -NH-C1~C 20 Alkenyl group-NH2; -NH-C1~C 20 Hydroxyalkyl group of; -N(C1~C 20 alkyl group)(C1~C 20 alkyl group); -N(C1~C 20 alkyl group)(C1~C 20 alkenyl group-NH2); -(OH)(C1~C 20 alkyl group); and -(C1~C 20 Hydroxyalkyl group of (C1~C) 20 is selected from the group consisting of a hydroxyalkyl group);

[0116] z is an integer from 0 to 45.

[0117]

[0118] In addition, the present invention provides the R 1Provides a compound represented by any one of the following chemical formulae 1-1 to 1-3.

[0119] Chemical Formula 1-1 Chemical Formula 1-2 Chemical Formula 1-3

[0120]

[0121] {In the above chemical formulas 1-1 to 1-3,

[0122] y' is an integer from 2 to 45, preferably y' is an integer from 2 to 25,

[0123] indicates the position of the bond.}

[0124]

[0125] In addition, the present invention provides the R 2 or R 3 Provides a compound represented by any one of the following chemical formulas 2-1 to 2-20.

[0126] Chemical Formula 2-1 Chemical Formula 2-2 Chemical Formula 2-3

[0127]

[0128] Chemical Formula 2-4 Chemical Formula 2-5 Chemical Formula 2-6

[0129]

[0130] Chemical Formula 2-7 Chemical Formula 2-8

[0131]

[0132] Chemical Formula 2-9 Chemical Formula 2-10 Chemical Formula 2-11

[0133]

[0134] Chemical Formula 2-12 Chemical Formula 2-13 Chemical Formula 2-14

[0135]

[0136] Chemical Formula 2-15 Chemical Formula 2-16

[0137]

[0138] Chemical Formula 2-17 Chemical Formula 2-18

[0139]

[0140] Chemical Formula 2-19 Chemical Formula 2-20

[0141]

[0142] {In the above chemical formulas 2-1 to 2-20, c is an integer from 0 to 12.}

[0143] The above R 2 is the pK of the polymer a is a functional group for controlling the R 3 is a functional group for controlling the hydrophobicity of polymers.

[0144]

[0145] Specifically, the compound represented by the above chemical formula 1 may be represented by any one of the following compounds, but is not limited thereto.

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] In another aspect, the present invention provides a composition for delivering nucleic acids comprising the compound.

[0154] 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), small hairpin RNA (shRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), antisense oligonucleotides (ASOs) and mixtures thereof, but is not limited thereto, and preferably may be mRNA, siRNA or ASO.

[0155] The above compound can bind to nucleic acids to form polymer nanoparticles. As a cationic amphiphilic polymer, the compound binds well to nucleic acids, preventing the bond from breaking even under changes in the blood environment, thereby effectively delivering genes to the target site.

[0156] The above nucleic acid delivery composition may further comprise a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt.

[0157] In another aspect, the present invention provides a nanoparticle formed by combining the compound and a nucleic acid.

[0158] In the above nanoparticles, the protonable nitrogen / phosphate ratio of the nucleic acid (N / P ratio) of the compound may be 1 to 30, preferably 3 to 10, and more preferably 5 to 10.

[0159] In another aspect, the present invention provides a vaccine composition comprising the compound.

[0160] In addition, in another aspect, the present invention provides a method for producing a compound represented by the above chemical formula 1, comprising the steps of: 1) synthesizing a beta-amino acid N-thiocarboxyanhydrides (beta-NTAs) monomer; 2) synthesizing a polymer precursor by ring-opening polymerization of the beta-NTAs monomer using butylamine as an initiator; and 3) modifying a side chain of the polymer precursor by an aminolysis reaction.

[0161] In another aspect, the present invention provides a method for delivering a nucleic acid into a cell using the compound. The intracellular location may be the cytoplasm or the nucleus.

[0162]

[0163] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to aid in understanding the present invention, and the present invention is not limited to the following examples.

[0164] [Synthesis example]

[0165] The chemical formula 1 (final compounds) of the present invention can be prepared by a reaction as shown in the following reaction formula 1, but is not limited thereto.

[0166] <Reaction Scheme 1>

[0167]

[0168]

[0169] Example 1. Synthesis of poly(beta-amino acids) transformants

[0170] 1-1. Benzyl-β 3 -LCHG monomer synthesis

[0171] Benzyl-β 3-LCHG monomer was synthesized using the same method based on a previous study (M. Zhou Angew. Chem. Int. Ed. 2020, 59, 7240-7244). The synthesized monomer was 1 Synthesis was confirmed using H-NMR (Avance III, 400 MHz, Bruker, Billerica, MA, USA) (Fig. 1).

[0172] 1-2. Benzyl-β 3 -LCHA monomer synthesis

[0173] Benzyl-β 3 -LCHA monomer was synthesized based on previous research (M. Zhou Angew. Chem. Int. Ed. 2020, 59, 7240-7244). The synthesized monomer was 1 Synthesis was confirmed using H-NMR (Avance III, 400 MHz, Bruker, Billerica, MA, USA) (Fig. 2).

[0174] 1-3. Monomer (a=2) synthesis

[0175]

[0176] The monomer (a = 2) represented by the above chemical formula was synthesized based on a previous study (M. Zhou Angew. Chem. Int. Ed. 2020, 59, 7240-7244). The synthesized monomer 1 Synthesis was confirmed using H-NMR (Avance III, 400 MHz, Bruker, Billerica, MA, USA) (Fig. 4).

[0177] 1-4. poly(benzyl-β 3 -LCHG) precursor polymer synthesis

[0178] The precursor polymer was synthesized as follows based on a previous study (M. Zhou Angew. Chem. Int. Ed. 2020, 59, 7240-7244).

[0179] benzyl-β 3 -LCHG monomer (100 mg) was dissolved in DCM (2 mL), and acetic acid (3.45 μL) was added (D. Siefker ACS Macro Lett. 2018, 7, 1272-1277). The initiator n-butylamines (1.1 mg) dissolved in DCM was added to the monomer solution, and the mixture was stirred at 35°C for 72 h. The resulting solution was precipitated in hexane / ethyl acetate (6:4 v / v), filtered, and dried under reduced pressure.

[0180] Synthetic Butyl-poly(benzyl-β) 3 -LCHG) precursor polymer was analyzed in DMF solution containing 10 mM lithium chloride by gel permeation chromatography (HLC-8420, Tosoh corporation) equipped with two TSK columns (G3000HHR and G4000HHR) to determine molecular weight distribution (M w / M n ) was calculated. In addition, the degree of polymerization (DP) was 1 It was analyzed by H-NMR. As a result, Butyl-poly(benzyl-β 3 -LCHG) M of precursor polymer w / M n And DP was confirmed to be 1.041 and 25 (Fig. 4).

[0181] In the same manner as above, the initiator was PEG1-amine, PEG4-amine, PEG 12 -amine, PEG 24 -amine was used to prepare precursor polymers with different PEG lengths (Figs. 5 to 7), and each poly(benzyl-β 3 -LCHG) M of precursor polymer w / M n And DP is shown in Table 1 below.

[0182] Precursor polymer initiator DPM w / M n Yield (%)butyl-poly(benzyl-β 3 -LCHG)butylamine251.04194.4PEG1-poly(benzyl-β 3 -LCHG)PEG1-amine251.03185.6PEG4-poly(benzyl-β 3 -LCHG)PEG4-amine251.03286.0PEG 12 -poly(benzyl-β 3 -LCHG)PEG 12 -amine221.04190.0PEG 24 -poly(benzyl-β 3 -LCHG)PEG 24 -amine261.02965.4

[0183] 1-5. Butyl-poly(benzy-β 3 -LCHA) precursor polymer synthesis

[0184] The above butyl-poly(benzyl-β 3 Butyl-poly(benzyl-β) precursor polymer in the same manner as -LCHG 3 -LCHA) precursor polymer was synthesized.

[0185] Benzyl-β 3 -LCHA monomer (100 mg) was dissolved in DCM (2 mL), and acetic acid (3.28 μL) was added. Initiator n-butylamines (1.047 mg) dissolved in DCM was added to the monomer solution, and the mixture was stirred at 35°C for 72 hours. The solution was precipitated in hexane / ethyl acetate (6:4 v / v), filtered, and dried under reduced pressure. Butyl-poly(benzyl-β) 3 -LCHA) M of precursor polymer w / M n And DP was confirmed to be 1.030 and 16 (Fig. 9).

[0186] 1-6. Poly(benzyl-β 3 -LCHG) converter synthesis

[0187] The synthesized precursor polymers have various pK a And it was converted into various poly(beta amino acids) transformants through aminolysis reaction with hydrophobic compounds.

[0188] Synthesis example of butyl-PGly(DET / CHE)

[0189] butyl-poly(benzyl-β 3 -LCHG) precursor polymer (20 mg) was dissolved in N-Methyl-2-pyrrolidone (NMP, 1 mL) at 35°C and cooled to 4°C. The precursor polymer solution was slowly added under argon gas to a solution of NMP (1 mL) in diethylenetriamine (DET, 0.26 mL) and cyclohexylethylamine (CHE, 0.93 mL) while maintaining the temperature at 10°C. 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 dialyzed against 0.01 M HCl for two days at 4°C. Finally, the solution was freeze-dried after dialyzing with water only for 1-2 hours to obtain the final product in the form of a powder. The manufactured converter 1 The amounts of DET and CHE introduced were calculated using H-NMR.

[0190] The synthesis reaction conditions for each transformant with different PEG lengths are presented in Table 2 below, and the introduction amounts of the manufactured transformants are shown in Table 3 below.

[0191] Precursor polymer Precursor polymer Reaction amount (mg) Molar ratio (pK a Molecule: Hydrophobic molecule)pK a분자 및 투입량소수성 분자 및 투입량수득량 (mg)butyl-poly(benzyl-β 3 -LCHG)2025:65DET, 263.5 ㎕CHE, 927.4 ㎕14PEG1-poly(benzyl-β 3 -LCHG)2025:65DET, 263.5 ㎕CHE, 927.4 ㎕17PEG4-poly(benzyl-β 3 -LCHG)2025:65DET, 263.5 ㎕CHE, 927.4 ㎕17PEG 12 -poly(benzyl-β 3 -LCHG)18.825:70DET, 221.4 ㎕CHE, 839.2 ㎕13PEG 24 -poly(benzyl-β 3 -LCHG)20.225:70DET, 223.7 ㎕CHE, 847.94 ㎕7butyl-poly(benzyl-β 3 -LCHG)2025:80DET, 263.5 ㎕ADO, 1.35 g15PEG1-poly(benzyl-β 3 -LCHG)2025:65DET, 263.5 ㎕ADO, 1.1 g15PEG4-poly(benzyl-β 3 -LCHG)1225:80DET, 158.1 ㎕ADO, 0.81 g10butyl-poly(benzyl-β 3 -LCHG)20100:160AEP, 1281 ㎕CHE, 2283 ㎕9butyl-poly(benzyl-β 3 -LCHG)2025:65HED, 493 ㎕CHE, 927.4 ㎕8butyl-poly(benzyl-β 3 -LCHG)2025:65MDE, 314.1 ㎕CHE, 927.4 ㎕17

[0192] 고분자Ethylene glycol repeating number도입양DET moietyCHE moietyButyl-PGly(DET / CHE)01213PEG1-PGly(DET / CHE)11016PEG4-PGly(DET / CHE)41312PEG 12-PGly(DET / CHE)121111PEG 24 -PGly(DET / CHE)241214

[0193] pK a Poly(beta-amino acids) convertants with additional hydrophobicity were synthesized in the same manner as the above synthetic method, and each pK a The chemical transformations of the hydrophobic group are shown in Table 4 below.

[0194] Polymer pK a Chemical groupHydrophobic Chemical groupChemical groupIntroduction amountChemical groupIntroduction amountHydrophobicity controlButyl-PGly(DET / ADO)diethylenetriamine (DET)1310-amino-1-decanol (ADO)12PEG1-PGly(DET / ADO)diethylenetriamine (DET)1410-amino-1-decanol (ADO)11PEG4-PGly(DET / ADO)diethylenetriamine (DET)1410-amino-1-decanol (ADO)11pKa controlButyl-PGly(AEP / CHE)2-aminoethylpiperazine (AEP)8cyclohexylethylamine (CHE)17Butyl-PGly(HED / CHE)(2-hydroxyethyl)ethylenediamine (HED)10cyclohexylethylamine (CHE)15Butyl-PGly(MDE / CHE)2,2'-diamino-N-methyldiethylamine (MDE)13cyclohexylethylamine (CHE)12

[0195]

[0196] Example 2. Poly(benzyl-β) 3 -LCHG) Measurement of binding of transformants and mRNA

[0197] Each transformant was dissolved in 10 mM Hepes (pH 7.3) buffer at 2 mg / mL and then diluted again in 10 mM Hepes (pH 7.3) buffer so that the molar concentration of the protonable amino group was 0.624 mM. The 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 protonable amino group / the molar concentration of the phosphate group (N / P ratio) = 1–5, and incubated at room temperature for approximately 1 hour. After preparing a 1.0% agarose gel, polymer nanoparticles (10 μL) and glycerol solution (50% v / v) (2 μL) were mixed and electrophoresis (135 V, 20 min) was performed. The agarose gel after electrophoresis was observed using a gel imager (Fig. 21). As a result, it was confirmed that the polymer transformant of the present invention had excellent binding affinity with mRNA.

[0198]

[0199] Example 3. poly(benzyl-β 3 -LCHG) Size and surface charge measurement of polymer nanoparticles of mRNA and transformants

[0200] After manufacturing polymer nanoparticles containing luciferase mRNA at N / P=5 by the above method, the size of the nanoparticles (D H ), polydispersity index (PDI), and surface charge (ξ-potential) were measured using Zetasizer Pro (RED) (Malvern Instruments, Worcestershire, UK), and the measured values ​​are shown in Table 5 below.

[0201] Polymer N / P ratioD H(nm)PDIξ-potential (mV)-Butyl-PGly(DET / CHE)5119±70.12±0.0122.16PEG length controlPEG1-PGly(DET / CHE)5106±50.16±0.0328.65PEG4-PGly(DET / CHE)5120±120.14±0.0327PEG 12 -PGly(DET / CHE)5202±190.22±0.0116.95PEG 24 -PGly(DET / CHE)5130±120.17±0.0426.13Hydrophobicity controlButyl-PGly(DET / ADO)5171±100.10±0.0129.2 1PEG1-PGly(DET / ADO)5187±60.09±0.0125.4PEG4-PGly(DET / ADO)5316±190.20±0.0120.54pKa ControlButyl-PGly(AEP / CHE)5191±250.26±0.0936.74Butyl-PGly(HED / CHE)5145±40.13±0.0344.08Butyl-PGly(MDE / CHE)5176±170.21±0.0443.83

[0202] <Hydrodynamic diameter (D) of polymer nanoparticles H ), PDI, surface charge (N / P=5)>

[0203]

[0204] In addition, similarly, polymer nanoparticles containing luciferase mRNA were prepared at N / P=7, and the size, polydispersity index (PDI), and surface charge (ξ-potential) of the nanoparticles were measured using Zetasizer Pro (RED), and the measured values ​​are shown in Table 6 below.

[0205] Polymer N / P ratioD H(nm)PDIξ-potential (mV)-Butyl-PGly(DET / CHE)787±40.13±0.0228.7PEG length controlPEG1-PGly(DET / CHE)7129±30.12±0.0436.4PEG4-PGly(DET / CHE)777±90.13±0.0127.1PEG 12 -PGly(DET / CHE)789±60.16±0.0218.7PEG 24 -PGly(DET / CHE)787±190.15±0.0521.7

[0206] <Hydrodynamic diameter (D) of polymer nanoparticles H ), PDI, surface charge (N / P=7)>

[0207]

[0208] Example 4. Transmission Electron Microscope (TEM) Measurements

[0209] Luciferase mRNA and polymers of different lengths (butyl-PGly(DET / CHE), PEG4-PGly(DET / CHE), PEG 24 Polymer nanoparticles (N / P=5) were prepared using -PGly(DET / CHE)). The prepared polymer nanoparticles were mounted on 400 mesh copper grids (Electron Microscopy Sciences, Pennsylvania, USA), stained with UranyLess EM Stain solution (Electron Microscopy Sciences), and measured using a field emission transmission electron microscope (FE-TEM, JEM2100F, JEOL Ltd., Tokyo, Japan) (Fig. 22A to C). The measured images were analyzed using ImageJ software, and the average diameter of the nanoparticles was measured (Fig. 22D to F). butyl-PGly(DET / CHE), PEG4-PGly(DET / CHE), PEG 24The average diameters of -PGly(DET / CHE) polymer nanoparticles were measured as 50 ± 9, 45 ± 10, and 52 ± 13 nm, respectively (n = 70).

[0210]

[0211] Example 5. Measurement of luciferase mRNA expression levels of polymer nanoparticles in C2C12 cells.

[0212] Luciferase mRNA-containing polymer nanoparticles were transfected into mouse myoblast C2C12 cells, and the luciferase-luciferase mRNA delivery ability was measured. C2C12 cells were prepared using DMEM medium supplemented with 10% FBS. C2C12 cells were seeded at 8,000 cells / well in a 96-well plate and cultured in a CO2 chamber for 1 day. After transfection with polymer nanoparticles (N / P=5) at a concentration of 50 ng mRNA concentration / well, cells were lysed 24 hours later, and the cell lysate was used for the Luciferase Assay System (Promega, Madison, WI, USA). The luminescence intensity was measured using a luminescence microplate reader (Mithras LB940, Berthold technologies, Bad Wildbad, Germany). Transfection of polymer nanoparticles was measured in the presence of 10% FBS (Fig. 23A) and absence of 10% FBS (Fig. 23B) to further investigate the PEG effect. We confirmed that butyl-, PEG1-, and PEG4-PGly(DET / CHE) polymers exhibited high luciferase mRNA expression levels.

[0213]

[0214] Example 6. Analysis of cellular uptake and serum stability of polymer nanoparticles in C2C12 cells.

[0215] PEG n To further investigate the PEG length effect of the -PGly(DET / CHE) polymer, we analyzed the intracellular uptake of polymer nanoparticles into C2C12 cells. Cy5-labeled luciferase mRNA was prepared (Cy5-mRNA) using the Label IT Cy5 labeling kit (Mirus Bio Corporation, Madison, WI, USA) according to the protocol provided in the kit. Polymer 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 4 h of incubation, cells were detached using trypsin-EDTA, and the fluorescence expression was compared using flow cytometry (Attune CytPix, ThermoFisher Scientific) to compare the intracellular uptake of polymer nanoparticles (Fig. 24A). Through this, we confirmed that a significant amount of Cy5-mRNA was taken up by C2C12 cells for polymers with PEG lengths of 0-4. In contrast, we confirmed that almost no cellular uptake occurred for polymers with PEG lengths of 12 and 24.

[0216] To examine the effect of PEG length on cellular uptake of polymeric nanoparticles in more detail, the particle stability of the same polymeric nanoparticles against serum albumin was measured using fetal bovine albumin (FBS). Polymer nanoparticles (N / P=5, 100 ng mRNA, 10 μL) were mixed with PBS solution containing 10% FBS (PBS / FBS, 90 μL) and incubated at 37°C for 1 hour. After 1 hour, mRNA was purified using the RNeasy Mini kit (Qiagen). After agarose gel electrophoresis (1 wt% agarose gel, 0.5X TAE buffer, 135 V, 15 min), the bands were observed using a gel imager (WSE-5300 Printgraph CMOS I) (Fig. 24B). In the case of butyl-, PEG1-, and PEG4-PGly(DET / CHE) polymer nanoparticles, intact mRNA bands were observed in the solution in FBS. This indicates that the polymer nanoparticles are stable in FBS. On the other hand, PEG 12 -, PEG 24 - In the case of PGly(DET / CHE) polymer nanoparticles, no intact mRNA band was observed. This indicates that both polymer nanoparticles degrade within 1 hour in PBS / FBS, which mimics cell culture. These results confirm that in the case of PGly(DET / CHE) polymers, when the PEG length is 12 or longer, the nanoparticles are unstable in the cell culture environment, degrade quickly, and cannot deliver mRNA into cells.

[0217]

[0218] Example 7. Viability assay for C2C12 cells

[0219] C2C12 cells were seeded in a 96-well plate (8,000 cells / well), and the next day, PEG containing luciferase mRNA n-PGly(DET / CHE) polymer nanoparticles (N / P=5, 50 ng / well) were transfected. After 24 h of incubation, 10 μL of CCK-8 solution was added to each well, and the absorbance at 450 nm was measured 1.5 h later using a microplate reader (Spark, Tecan Group Ltd., Mδnnedorf, Switzerland) (Fig. 25). In this experimental environment, PEG n -PGly(DET / CHE) polymer nanoparticles did not show significant cytotoxicity against C2C12.

[0220]

[0221] Example 8. PEG in HEK293-loxP-GFP-RFP cells n -Confirmation of the gene editing effect of PGly(DET / CHE) polymer

[0222] 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-protomer. Normally, they show strong green fluorescence, but when loxP is cleaved by CRE recombinase, they show red fluorescence (Fig. 26A). Through this, we measured how well polymer nanoparticles containing Cre mRNA (L-7211, TriLink Biotechnologies) deliver Cre mRNA into cells to achieve gene editing efficiency. Cells were seeded (8,000 cells / well) in a 96-well optical bottom plate, and PEG n-PGly(DET / CHE) polymer nanoparticles (N / P=5, 100 ng / uL) were transfected. After 48 h of incubation, green (GFP) and red (RFP) fluorescence was measured (Fig. 27) using a confocal laser scanning microscope (CLSM) (ZEISS LSM 980, Carl Zeiss, Oberkochen, Germany), and each fluorescence intensity was analyzed and quantified using Image J software (Fig. 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 ability of the three polymers. In particular, PEG1-PGly(DET / CHE) polymer nanoparticles were confirmed to have the highest gene editing effect.

[0223]

[0224] In summary, the present invention synthesizes a novel poly beta peptide precursor polymer that has never been used for RNA drug delivery before, and produces various pKs through additional aminolysis conversion. a , hydrophobic, and PEG-length-adjusted polymers were synthesized. In addition, the synthesized polymers were successfully conjugated to mRNA, and nanoparticles having a size of 100-300 nm were manufactured. In addition, from the above results, it was confirmed that the nanoparticles of the present invention successfully delivered luciferase mRNA to C2C12 cells in vitro, and the gene editing efficiency was compared and analyzed by delivering Cre mRNA to HEK293-loxP-GFP-RFP cells, thereby confirming the possibility of RNA drug delivery of poly-beta peptide polymers.

[0225]

[0226] The above description is merely illustrative of the present invention, and those skilled in the art will appreciate that various modifications may be made without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in this specification are intended to illustrate, not limit, the present invention, and the spirit and scope of the present invention are not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all techniques within a scope equivalent thereto should be interpreted as being included within the scope of the present invention.

[0227] According to the present invention, an amphiphilic polymer compound having an easy manufacturing process, high binding capacity with therapeutic nucleic acids, excellent stability in blood, and delivery characteristics can be manufactured, and thus has industrial applicability.

Claims

1. A compound represented by the following chemical formula 1 Chemical Formula 1 {In the above chemical formula 1, R 1 Silver C1~C 10 Alkyl group of; -(CH2)3-O-CH3; or -(CH2) x -[(CH2)2-O] y -CH3; and x is 0 or 1, y is an integer from 1 to 45, However, if y is 1, x is 1, and if y is 2 or greater, x is 0. m and n are integers from 1 to 100, independently of each other, However, m+n≥10, a is an integer from 0 to 2, R 2 and R 3 are independently represented by the following chemical formula 2, Chemical Formula 2 In the above chemical formula 2, 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 of; C1~C 20 Heteroaryl group of; C1~C 20 Heterocyclic group of; -NH-C1~C 20 Alkenyl group-NH2; -NH-C1~C 20 Hydroxyalkyl group of; -N(C1~C 20 alkyl group)(C1~C 20 alkyl group); -N(C1~C 20 alkyl group)(C1~C 20 alkenyl group-NH2); -(OH)(C1~C 20 alkyl group); and -(C1~C 20 Hydroxyalkyl group of (C1~C) 20 is selected from the group consisting of a hydroxyalkyl group); z is an integer from 0 to 45.

2. In the first paragraph, the R 1 A compound characterized by any one of the following chemical formulas 1-1 to 1-3 Chemical Formula 1-1 Chemical Formula 1-2 Chemical Formula 1-3 {In the above chemical formulas 1-1 to 1-3, y' is an integer from 2 to 45, indicates the position of the bond.} 3. In the first paragraph, the R 2 or R 3 A compound characterized by any one of the following chemical formulas 2-1 to 2-20. Chemical Formula 2-1 Chemical Formula 2-2 Chemical Formula 2-3 Chemical Formula 2-4 Chemical Formula 2-5 Chemical Formula 2-6 Chemical Formula 2-7 Chemical Formula 2-8 Chemical Formula 2-9 Chemical Formula 2-10 Chemical Formula 2-11 Chemical Formula 2-12 Chemical Formula 2-13 Chemical Formula 2-14 Chemical Formula 2-15 Chemical Formula 2-16 Chemical Formula 2-17 Chemical Formula 2-18 Chemical Formula 2-19 Chemical Formula 2-20 {In the above chemical formulas 2-1 to 2-20, c is an integer from 0 to 12.} 4. A composition for nucleic acid delivery comprising a compound according to any one of claims 1 to 3.

5. A 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), small hairpin RNA (shRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), antisense oligonucleotide, and mixtures thereof.

6. A composition for nucleic acid delivery, characterized in that it further comprises a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt in accordance with paragraph 4.

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

8. Nanoparticles characterized in that in the 7th paragraph, the protonable nitrogen / phosphate ratio of the nucleic acid (N / P ratio) of the compound is 1 to 30.

9. A vaccine composition comprising nanoparticles according to Article 7 or 8. 10.1) A step of synthesizing a beta-amino acid N-thiocarboxyanhydrides (beta-NTAs) monomer; 2) A step of synthesizing a polymer precursor by ring-opening polymerization of the beta-NTAs monomer using butylamine as an initiator; and 3) A method for producing a compound represented by Chemical Formula 1 of claim 1, comprising a step of modifying the side chain of the polymer precursor through an aminolysis reaction; 11. Method for delivering nucleic acid into a cell using the compound of paragraph 1 12. A method according to claim 11, characterized in that the intracellular location is the cytoplasm or nucleus.

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