Choline polymer as well as preparation method and application thereof

Abstract

The invention discloses a choline polymer as well as a preparation method and application thereof. The copolymer comprises an ethylenically unsaturated monomer unit with choline and / or a derivative thereof and a hydrophilic (methyl) acrylic acid monomer unit. According to the invention, choline and a derivative monomer thereof are introduced into the copolymer, so that the copolymer has cationic polymer properties and liver efficient specific targeting properties. Meanwhile, the choline polymer can be modified on the surface of drug-loaded nanoparticles (such as lipid nanoparticles), and can also form a cation-drug compound through a cation unit of choline and drug molecules, so that drug leakage in the storage process and early release of the drug in blood circulation are greatly reduced, and the bioavailability of the drug is improved. Toxic and side effects of the medicine are reduced, and the medicine is efficiently targeted and delivered to the liver.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, and in particular to a choline polymer and a preparation method and application thereof. Background Art

[0002] Although traditional small molecule drugs can achieve certain therapeutic effects in certain liver diseases, their poor selectivity and low efficiency have brought significant toxic side effects to patients during the entire treatment process, greatly reducing their quality of life. Secondly, the problem of low drug delivery efficiency can lead to drug resistance, which brings greater challenges to conventional treatment.

[0003] With the rapid development of nanotechnology, the combination of nanotechnology and medicine has brought new strategies for the treatment of liver diseases. At present, researchers have creatively developed a variety of multifunctional nanoformulations to meet the clinical diagnosis and treatment needs of liver diseases, especially liver cancer. So far, nanoformulations that have been used in clinical practice include liposome doxorubicin, paclitaxel albumin nanoformulations, etc., which have shown superior properties to traditional small molecule drugs, and to a certain extent solved the problems of poor water solubility, short circulation half-life and large toxic side effects of traditional small molecule chemotherapy drugs. This has undoubtedly greatly promoted the development of nanomedicine. Subsequent generations of lipid nanocarriers, such as lipid nanoparticles, are currently receiving attention as an important component of the COVID-19 mRNA vaccine, showing more complex structures and stronger physical stability.

[0004] Although multifunctional drug materials such as polymer micelles, vesicles, and organic nanoparticles such as liposomes have been successfully developed, many nanodrugs have poor stability (Serre, Christian, et al. Adv. Mat. 2018, 30, 1707365.), aggregation and drug leakage caused by long-term storage (Fairen-Jimenez, David, et al. J. Am. Chem. Soc. 2021, 143, 13557.), and low delivery efficiency (CW Chan, Warren, et al. Nat. Mater. 2020, 19, 566.) and other problems are still important obstacles to the design of current drug and gene delivery systems.

[0005] Obviously, the existence of the above problems greatly restricts the therapeutic effect and clinical transformation of nanomedicines. How to conveniently and effectively improve drug stability, as well as nanomedicine delivery efficiency and lesion targeting specificity, is still a huge challenge and a problem that needs to be solved urgently. Summary of the invention

[0006] The present invention aims to solve at least one of the above technical problems existing in the prior art. To this end, the present invention aims to provide a choline polymer and a preparation method and application thereof.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect of the present invention, a copolymer is provided, comprising ethylenically unsaturated monomer units containing choline and / or its derivatives and hydrophilic (meth) acrylic acid monomer units.

[0009] In the present invention, by introducing choline and / or its derivatives into the copolymer, the copolymer has both drug-carrying function and liver-targeting property. On the other hand, the choline cation in the copolymer can realize the loading of hydrophilic / hydrophobic drugs containing aromatic structure by forming a cation-π complex with the π structure in the drug; the hydrophobic group in the copolymer can also encapsulate and load the hydrophobic drug to realize the loading of the hydrophobic drug; the hydrophilic cavity of the vesicle structure formed by self-assembly can be used for the encapsulation of hydrophilic drugs. Choline and / or its derivatives can also interact with nanomaterials, so that the copolymer is modified on the surface of the drug-carrying nanomaterial.

[0010] In some embodiments of the present invention, the choline and / or its derivatives include at least one of choline, acetylcholine, phosphorylcholine, and glycerophosphocholine.

[0011] In some embodiments of the present invention, the ethylenically unsaturated monomer includes at least one of hydroxyethyl acrylate, hydroxypropyl acrylate, methyl 2-(hydroxymethyl)acrylate, ethyl 2-(hydroxymethyl)acrylate, 2-methyl-2-acrylic acid-2-hydroxybutyl ester, hydroxypropyl methacrylate, N-hydroxymethyl acrylamide, N-(2-hydroxyethyl) acrylamide, 2-tert-butoxycarbonylaminomethyl acrylate, methyl 3-aminoacrylate, and isocyanoethyl acrylate.

[0012] In some embodiments of the present invention, the hydrophilic (meth) acrylic monomer includes at least one of polyethylene glycol methyl ether methacrylate, acrylic acid, methacrylic acid, acrylamide, N-isopropylacrylamide or N,N-dimethylaminoethyl methacrylate.

[0013] In some embodiments of the present invention, the copolymer is synthesized from an ethylenically unsaturated monomer having a cationic functional group R1 and a hydrophilic (meth) acrylic monomer by a suitable polymerization technique, such as atom transfer radical polymerization, free radical polymerization, nitroxide polymerization, mediated polymerization, reversible addition fragmentation chain transfer polymerization, etc. In the present invention, reversible addition fragmentation chain transfer polymerization (RAFT) is particularly useful. In some cases, RAFT polymerization can form copolymers by independently and precisely controlling the molecular weight distribution and composition, thereby generating combinatorial polymer designs. Suitable RAFT polymerization CTA (chain transfer agent, chain transfer agent) includes 3,5-bis(2-dodecylthiothiocarbonylthio-1-oxopropoxy)benzoic acid, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid 3-butenyl ester, 4-chloro-3,5-dimethyl-1H-pyrazole-1-dithiocarboxylic acid 2-cyanobutane-2-yl ester, 3,5-dimethyl-1H-pyrazole-1-dithiocarboxylic acid 2-cyanobutyl-2-yl ester, 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanol, (3,5-dimethyl-1H-pyrazole)-dithiocarboxylic acid cyanomethyl ester, trithiocarbonic acid cyanomethyl dodecyl ester, [3-(trimethoxysilyl) [propyl] cyanomethyl trithiocarbonate, 2-cyano-2-propyl dodecyl trithiocarbonate, S,S-dibenzyl trithiocarbonate, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid 3-azido-1-propanol ester, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid N-hydroxysuccinimide ester, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid pentafluorophenyl ester, 2-(dodecylthiothiocarbonylthio) propionic acid, 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid methyl ester, pentaerythritol tetrakis[2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid ester], phthalimidomethyl butyl trithiocarbonate, 1,1,1-Tris[(dodecylthiothiocarbonylthio)-2-methylpropionate]ethane, 1H-pyrrole-1-dithiocarbamate phenyl, diphenyl dithiocarbamate cyanomethyl ester, methyl (phenyl) dithiocarbamate cyanomethyl ester, methyl (4-pyridyl) dithiocarbamate cyanomethyl ester, N-methyl-N-(pyridin-4-yl) dithiocarbamate 2-cyanopropane-2-yl ester, 2-[methyl (4-pyridyl) thiocarbamate thio] propionate methyl Ester, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)thiocarbamic acid thio] pentanoate, benzyl dithiobenzoate, cyanomethyl dithiobenzoate, 4-cyano-4-(phenylthiocarbonylthio) pentanoic acid, 4-cyano-4-(phenylthiocarbonylthio) pentanoic acid N-succinimidyl ester, 2-cyano-2-propyl dithiobenzoate, 2-cyano-2-propyl 4-cyanodithiobenzoate, 2-(4-methoxy ethyl 2-methyl-2-(phenylthiocarbonylthio)propanoate, ethyl 2-(phenylthiocarbonylthio)-2-phenylacetate, ethyl 2-(phenylthiocarbonylthio)propanoate, ethyl 2-(phenylthiocarbonylthio)propanoate, 1-(methoxycarbonyl)ethyl dithiobenzoate, 2-(4-methoxyphenylthiocarbonylthio)acetic acid, 2-nitro-5-(2-propynyloxy)benzyl 4-cyano-4-(phenylthiocarbonylthio)pentanoate, 2-(phenylthiocarbonylthio)pentanoate methyl (4-pyridyl) dithiocarbamate, 2-cyanopropane-2-yl N-methyl-N-(pyridin-4-yl) dithiocarbamate, methyl 2-[methyl (4-pyridyl) thiocarbamate thio] propionate, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl) thiocarbamate thio] pentanoate, etc.

[0014] When CAT is used for polymerization, the CAT fragment can remain as the terminal group of the polymer.

[0015] The fragmentation of CTA and the resulting fragment structures are well known in the art, and thus the structure of the terminal group can be determined by utilizing the prior art without using inventive techniques.

[0016] In some embodiments of the present invention, the number average molecular weight of the copolymer is 2000-200000 g / mol, such as 5000, 10000, 15000, 20000, 30000, 50000, 80000, 100000, 150000 g / mol, etc.

[0017] The second aspect of the present invention provides a copolymer of formula I or a pharmaceutically acceptable salt thereof:

[0018]

[0019] Wherein, A is a terminal group; B is a terminal group or does not exist;

[0020] L is a linker or is absent; the linker includes a substituted or unsubstituted -C1-C6 alkylene-, -C1-C6 ester-, which contains a functional group selected from the group consisting of acetal, imine, amide, hydrazine, carbonate or carbamate;

[0021] R1, R3 are independently selected from H, -CH3;

[0022] R2 is selected from choline and / or its derivatives;

[0023] R4 is selected from -OH, -NH2, -NH-C1~C6 alkyl, -O-C1~C6 alkylene-N(C1~C6 alkylene)2, -O-C1~C6 alkylene-PEG-O-C1~C6 alkyl;

[0024] m is a natural number ranging from 2 to 400; 0<x<1.

[0025] In some embodiments of the present invention, A is a CAT fragment; B is H, -CH3 or absent.

[0026] When CAT is used in a polymerization reaction, the CAT fragment can remain as the terminal group of the polymer. The CTA used herein can be any material suitable for initiating a polymerization reaction known in the art.

[0027] The term "fragment" refers to a compound formed as a result of the cleavage of one or more covalent bonds forming a chain transfer agent molecule.

[0028] The fragmentation of CTA and the resulting fragment structures are well established in the art, and thus the structure of A can be determined by utilizing existing techniques without using creative techniques.

[0029] Common CTA snippets include: wait.

[0030] In some embodiments of the present invention, L is -O-C1~C6 alkylene-NH-CO-.

[0031] In some embodiments of the present invention, R2 is selected from

[0032] In some embodiments of the present invention, -CO-R4 is selected from

[0033] In some embodiments of the present invention, m is a natural number of 5 to 350, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, etc.

[0034] In some embodiments of the present invention, the copolymer of formula I is selected from the following compounds:

[0035]

[0036] Where x is defined as above.

[0037] The third aspect of the present invention provides a method for preparing a copolymer, comprising the following steps: subjecting an ethylenically unsaturated monomer having choline and / or its derivatives, a hydrophilic (meth) acrylic acid monomer, a chain transfer agent, and an initiator to a polymerization reaction to obtain the copolymer or the copolymer of formula I.

[0038] In some embodiments of the present invention, choline and / or its derivatives are subjected to a catalytic reaction with an ethylenically unsaturated monomer and then halogenated to obtain an ethylenically unsaturated monomer containing choline and / or its derivatives; the catalyst includes any one of dibutyltin dilaurate, dibutyltin diacetate, and dibutyltin dilaurate.

[0039] In some embodiments of the present invention, the halogenation agent used in the halogenation comprises any one of iodomethane, dichloromethane and chloroform.

[0040] In some embodiments of the present invention, the initiator includes any one of azobisisobutyronitrile, azobisisoheptanenitrile, cyclohexanone peroxide, dibenzoyl peroxide, and tert-butyl hydroperoxide.

[0041] The fourth aspect of the present invention provides a nanoparticle comprising the copolymer and / or the copolymer of formula I and a pharmaceutically active ingredient.

[0042] In some embodiments of the present invention, the surface of the nanoparticles carries a positive charge.

[0043] In the nanoparticles of the present invention, choline and / or its derivatives in the copolymer are distributed on the surface thereof, so that the surface of the nanoparticles carries a positive charge and has choline activity.

[0044] In some embodiments of the present invention, the copolymer and / or the copolymer of formula I loads and / or encapsulates the pharmaceutically active ingredient.

[0045] In some embodiments of the present invention, the mass ratio of the pharmaceutical active ingredient to the copolymer and / or the copolymer of formula I is 1:1 to 100, such as 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:70, 1:80, 1:90, etc.

[0046] In some embodiments of the present invention, the nanoparticles further include lipids; preferably, the lipids load and / or encapsulate the active pharmaceutical ingredient to form drug-loaded lipids, and the copolymer and / or copolymer of formula I is modified on the surface of the drug-loaded lipids.

[0047] In some embodiments of the present invention, the lipids include steroidal lipids and phospholipids.

[0048] In some embodiments of the present invention, the steroidal lipids are selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol and cholesterol modified by polypeptide; one or more combinations of lanosterol, luminosterol, alginasterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol.

[0049] In some embodiments of the present invention, the phospholipid is selected from: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and one or more combinations thereof.

[0050] In some embodiments of the present invention, the mass ratio of the active pharmaceutical ingredient to the lipid is 1:1-100; the mass ratio of the drug-loaded lipid to the copolymer and / or the copolymer of formula I is 1:1-100.

[0051] In some embodiments of the present invention, the hydrated particle size of the nanoparticles is 5 to 500 nm, such as 10 nm, 50 nm, 100 nm, 120 nm, 130 to 140 nm, 150 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, etc. The hydrated particle size of the nanoparticles is related to the degree of polymerization of the monomers in the copolymer, the molecular weight of the active pharmaceutical ingredient, and whether lipids are contained. The larger the molecular weight of the copolymer, the larger the molecular weight of the active pharmaceutical ingredient, and the more lipids contained, the larger the hydrated particle size.

[0052] In some embodiments of the present invention, the encapsulation efficiency of the drug in the nanoparticles is at least 60%, such as at least 65%, at least 68%.

[0053] In some embodiments of the present invention, the pharmaceutically active ingredient includes a hydrophobic drug and / or a hydrophilic drug.

[0054] In some embodiments of the present invention, the active pharmaceutical ingredient comprises at least one of an anti-tumor chemotherapy drug, an anti-tumor immunotherapy drug, an anti-tumor targeted drug or an anti-inflammatory drug.

[0055] In some embodiments of the present invention, the active pharmaceutical ingredients include doxorubicin hydrochloride, cisplatin, mitoxantrone, paclitaxel, camptothecin, sorafenib, lenvatinib, regorafenib, gefitinib, ganiserti, curcumin, rhein, berberine

[0056] At least one of them.

[0057] The fifth aspect of the present invention provides a method for preparing nanoparticles, comprising the following steps: mixing the copolymer and / or the copolymer of formula I with the active pharmaceutical ingredient in an organic solvent and a buffer solution, and obtaining the nanoparticles after dialysis.

[0058] In some embodiments of the present invention, the method for preparing the nanoparticles further comprises mixing the copolymer and / or the copolymer of formula I, lipids and active pharmaceutical ingredients in an organic solvent with a buffer solution, and obtaining the nanoparticles after dialysis.

[0059] In some embodiments of the present invention, the organic solvent includes at least one of N,N-dimethylformamide, dioxane, acetone, anhydrous ethanol or tetrahydrofuran.

[0060] The sixth aspect of the present invention provides a pharmaceutical composition comprising the nanoparticles and a pharmaceutically acceptable medium or excipient.

[0061] In some embodiments of the invention, the medium comprises an aqueous solution; the nanoparticles are dispersed in the aqueous solution and stored in liquid form until needed, or they can be dried and introduced and dispersed in an aqueous solution prior to administration to a subject.

[0062] Pharmaceutically acceptable media or excipients include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; astragalus powder; malt; gelatin; lubricants, such as magnesium stearate, sodium lauryl sulfate and talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols, such as propylene glycol, glycerol, ... diols; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol (PEG); esters, such as ethyl oleate, ethyl laurate; agar; buffers, such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; fillers, such as polypeptides and amino acid serum components, such as serum albumin, HDL and LDL; C2-C12 alcohols, such as ethanol; and other non-toxic compatible substances used in pharmaceutical formulations.

[0063] Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, aromas, preservatives and antioxidants may also be present in the pharmaceutical composition.

[0064] The aqueous solution contains, for example, water, cell culture medium, buffer (e.g., phosphate buffered saline), polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), or a suitable mixture thereof. In some embodiments, the aqueous solution can be a buffered solution (e.g., PBS).

[0065] The seventh aspect of the present invention provides a use of the copolymer and / or the copolymer of formula I, the nanoparticles and / or the pharmaceutical composition in the preparation of a drug for treating liver diseases.

[0066] Pharmaceutical composition of the present invention can be in suitable form, and described suitable form depends on the required method that it is given to the patient.Comprise pharmaceutical composition of the present invention can be configured to oral administration, for example, the form of liquid dispersion or aqueous or oily suspension, or it can be configured to parenteral administration, for example, for subcutaneous, intravenous, intramuscular, intrasternal, intraperitoneal, intradermal, transdermal or other infusion techniques.Comprise pharmaceutical composition of the present invention can also be configured to by giving aerosol or solution form suction administration with inhaler or sprayer.Preferred transdermal, subcutaneous, intranasal, intravenous, intramuscular, tumor or by suction administration object of pharmaceutical composition of the present invention.In any given case, the most suitable route of administration will depend on the physical condition of the concrete therapeutic agent, object, the nature and severity of the disease and the object present in the pharmaceutical composition of the present invention.

[0067] In some embodiments of the present invention, the liver disease includes hepatitis, liver fibrosis, and liver cancer.

[0068] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C 1～4 The term "alkyl" refers to an alkyl group containing 1 to 4 carbon atoms.

[0069] The "alkyl" described in the present invention refers to a saturated hydrocarbon group having a specified number of carbon atoms. For example, C1-8 alkyl refers to an alkyl group having 1 to 8 carbon atoms, for example, preferably 1 to 4 carbon atoms. The alkyl group may be linear or branched. Representative branched alkyl groups have one, two or three branches. The alkyl group may also be optionally substituted with one or more substituents as defined herein. Specific examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0070] The term "alkylene" as used herein refers to a divalent saturated aliphatic hydrocarbon group having a specified number of carbon atoms. a ~ b "Alkylene" refers to an alkylene group having a to b carbon atoms. The alkylene group can be straight chain or branched. For example, "C1-6 alkylene" is intended to include methylene, ethylene, propylene, 2-methylpropylene, dimethylethylene, pentylene, and the like. Thus, the term "propylene" can be exemplified by the following structures: Likewise, the term "dimethylpropylene" can be exemplified, for example, by any of the following structures:

[0071] The beneficial effects of the present invention are as follows: the present invention introduces choline and its derivative monomers into the copolymer, so that the copolymer has cationic polymer properties and liver-specific high-efficiency targeting properties. At the same time, the choline polymer can be modified on the surface of drug-loaded nanoparticles (such as lipid nanoparticles), and can also form a cation-drug complex through the cationic unit of choline and the drug molecule, which greatly reduces the drug leakage during storage and the premature release of the drug in the blood circulation, reduces the toxic side effects of the drug, and efficiently delivers the drug to the liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the copolymer prepared in Example 1 of the present invention;

[0073] Figure 2 is the gel permeation chromatography determination of the copolymer prepared in Example 1 of the present invention;

[0074] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the copolymer prepared in Example 2 of the present invention;

[0075] Figure 4 Graphs showing the particle sizes of copolymer drug-loaded lipid nanoparticles prepared in the comparative example (left) and Example 3 (right);

[0076] Figure 5 1 is a transmission electron micrograph of the copolymer drug-loaded lipid nanoparticles prepared in the comparative example (left figure) and Example 3 (right figure) of the present invention;

[0077] Figure 6 is a particle size diagram of the copolymer drug-loaded micelles prepared in Example 4 of the present invention;

[0078] Figure 7 is a transmission electron microscopy image of the copolymer drug-loaded micelles prepared in Example 4 of the present invention;

[0079] Figure 8 This is a cytotoxicity test diagram of the copolymer drug-loaded lipid nanoparticles prepared in Example 3 and the comparative example of the present invention;

[0080] Fig. 9 These are in vivo imaging images of liver-targeted delivery of copolymer drug-loaded lipid nanoparticles prepared in Example 3 of the present invention (upper figure) and the comparative example (lower figure). DETAILED DESCRIPTION

[0081] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0082] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0083] The raw materials used in the following examples, comparative examples or test examples are as follows:

[0084] L02 and LX-2 cells were purchased from Shanghai ATCC Cell Bank; C57 mice were purchased from N,N-dimethylethanolamine and isocyanoethyl methacrylate, and rhein were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; iodomethane and camptothecin were purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.; doxorubicin hydrochloride and curcumin were purchased from Shanghai Myrrel Chemical Technology Co., Ltd.; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Shanghai Pangshuo Biotechnology Co., Ltd.; distearoyl phosphate polyethylene glycol 2000 was purchased from Guangzhou Carbon Water Biotechnology Co., Ltd.; cholesterol was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; poly(ethylene glycol) methyl methacrylate (average molecular weight 320, OEGMA) was purchased from Sigma-Aldrich; L-CTA was purchased from Darukon Biotechnology Co., Ltd.; curcumin was purchased from Tianjin Xiensi Biochemical Technology Co., Ltd.; rhein was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; and carbon tetrachloride was purchased from Guangzhou Kutai Trading Co., Ltd.

[0085] Example 1

[0086] This example prepares a copolymer, the structural formula of which is as follows, and the specific process is as follows:

[0087]

[0088] 1) Preparation of TAMA, the synthetic route is as follows:

[0089]

[0090] S1. 1.8 g N, N-dimethylethanolamine (20.0 mmol) was dissolved in 40 mL of anhydrous tetrahydrofuran, and then a catalytic amount of dibutyltin dilaurate (40 μL) was added. After stirring for 15 min, 3.8 g of isocyanoethyl methacrylate (24.0 mmol) was slowly added dropwise. After stirring at room temperature for 4 h, the reaction mixture was concentrated under reduced pressure and separated and purified by a basic alumina column to obtain 4.2 g of a colorless oily product (yield: 85%).

[0091] S2. The product of step S1 (4.2 g, 17.2 mmol) was dissolved in anhydrous tetrahydrofuran (30 mL). After ice bathing for 15 min, 2.7 g of iodomethane (18.9 mmol) was slowly added dropwise. After stirring for 30 min, stirring was continued at room temperature for 2 h. A large amount of white precipitate was produced in the reaction system. The reaction mixture was filtered and washed with tetrahydrofuran (50 mL). It was dried in vacuo at room temperature to obtain 5.9 g of white TAMA solid (yield: 88%).

[0092] 2) LbP (TAMA) 0.15 -co-OEG 0.85 ) 33 The preparation route is as follows:

[0093]

[0094] S3. 50 mg TAMA (0.13 mmol), 312 mg OEGMA (0.97 mmol), 23 mg L-CTA (0.025 mmol) and 1 mg azobisisobutyronitrile (AIBN, 0.006 mmol) were dissolved in 900 μL of a mixed solution (ethanol: dichloromethane: n-hexane = 1:1:1), then transferred to a polymerization tube, degassed through three freeze-thaw cycles, and finally sealed under vacuum. The polymerization tube was then immersed in a preheated 65°C oil bath. After stirring for 12 h, the polymerization tube was placed in liquid nitrogen for a while, and the lid was opened to terminate the polymerization. The reaction mixture was transferred to a dialysis bag (MWCO: 7000 Da), dialyzed with pure water for 12 h at room temperature and freeze-dried. The resulting product was 288.7 mg of a yellow oily liquid (yield: 75%).

[0095] Example 2

[0096] This embodiment provides a copolymer, P(OEG 0.61 -CO-TAMA 0.39 ) 66 The preparation route is as follows:

[0097]

[0098] 200 mg TAMA (0.514 mmol), 462.6 mg OEGMA (1.028 mmol), 4.75 mg CTA (0.017 mmol) and 1.5 mg azobisisobutyronitrile (AIBN, 0.009 mmol) were dissolved in 600 μL N, N-dimethylformamide, then transferred to a polymerization tube, degassed by three freeze-thaw cycles, and finally sealed under vacuum. The polymerization tube was then immersed in a preheated 65°C oil bath. After stirring for 6 h, the polymerization tube was placed in liquid nitrogen for a while, and the lid was opened to terminate the polymerization. The reaction mixture was transferred to a dialysis bag (MWCO: 7000 Da), dialyzed with pure water for 12 h at room temperature and freeze-dried. The resulting product was 441.45 mg of a yellow viscous solid (yield: 66%).

[0099] Example 3

[0100] This embodiment provides a copolymer drug-loaded lipid nanoparticle, comprising the copolymer prepared in Example 1, a lipid, and a pharmaceutically active ingredient, wherein the pharmaceutically active ingredient is curcumin. The method for preparing the copolymer drug-loaded lipid nanoparticle comprises the following steps:

[0101] 4.3 mg LbP (TAMA) prepared in Example 1 0.15 -co-OEG 0.85 )3 and 1.6 mg curcumin, 2.5 mg cholesterol, and 18 mg 1,2-distearoyl-sn-glycero-3-phosphocholine were co-dissolved in 1 mL of a suitable organic solvent (anhydrous ethanol), and quickly added to vigorously stirred PBS (9 mL, 10 mM, pH 7.4). After stirring for 7 minutes, the sample was transferred to a dialysis bag and dialyzed in water to remove the organic solvent and unloaded drug.

[0102] Comparative Example

[0103] This comparative example provides a copolymer-free drug-loaded lipid nanoparticle, comprising lipid and a pharmaceutically active ingredient, wherein the pharmaceutically active ingredient is curcumin. The method for preparing the copolymer-free drug-loaded lipid nanoparticle comprises the following steps:

[0104] 4.3 mg of distearoyl phosphate polyethylene glycol 2000, 1.6 mg of curcumin, 2.66 mg of cholesterol, and 18 mg of 1,2-distearoyl-sn-glycero-3-phosphocholine were dissolved in 1 mL of a suitable organic solvent (anhydrous ethanol) and quickly added to vigorously stirred PBS (9 mL, 10 mM, pH 7.4). After stirring for 7 minutes, the sample was transferred to a dialysis bag and dialyzed in water to remove the organic solvent and unloaded drug.

[0105] Example 4

[0106] This embodiment provides a copolymer drug-loaded micelle, comprising the copolymer prepared in Example 2 and a pharmaceutically active ingredient, wherein the pharmaceutically active ingredient is rhein. The preparation method of the copolymer drug-loaded micelle comprises the following steps:

[0107] 6 mg P(OEG prepared in Example 2 0.61 -CO-TAMA 0.39 ) 66 It was dissolved in 1 mL of a suitable organic solvent (N,N-dimethylformamide) with 0.6 mg of rhein, and quickly added to vigorously stirred PBS (9 mL, 10 mM, pH 7.4). After stirring for 7 minutes, the sample was transferred to a dialysis bag and dialyzed in water to remove the organic solvent and unloaded drug.

[0108] Test Example 1

[0109] The nuclear magnetic resonance test and gel permeation chromatography (GPC) of Example 1 were measured:

[0110] The LbP (TAMA) prepared in Example 1 0.15 -co-OEG 0.85 ) 33 The results were confirmed by H NMR and GPC. Figure 1-2 ,from Figure 1 The results show that the composition ratio of the two was calculated based on the characteristic peak of polyethylene glycol (near 3.6 ppm) and the characteristic peak of TAMA (the chemical shift of the methyl hydrogen of the quaternary ammonium group is around 3.4 ppm), and the polymer composition was determined to be LbP (TAMA 0.15 -co-OEG 0.85 ) 33 The polydispersity index (PDI) of the copolymer was determined to be 1.34 by gel permeation chromatography (GPC), and the number average molecular weight (Mn) was 5700 g / mol.

[0111] Nuclear magnetic resonance test of Example 2:

[0112] The P(OEG prepared in Example 2 0.61 -CO-TAMA 0.39 ) 66 The results were confirmed by H NMR and GPC. Figure 3 From the results, the composition ratio of the two was calculated based on the characteristic peak of polyethylene glycol (near 3.65 ppm) and the characteristic peak of TAMA (the chemical shift of methyl hydrogen and methylene hydrogen of the quaternary ammonium group was near 3.5 ppm), and the polymer composition was determined to be P(OEG 0.61 -CO-TAMA 0.39 ) 66 .

[0113] Particle size determination and transmission electron microscopy determination of copolymer-modified drug-loaded lipid nanoparticles and copolymer-loaded drug-loaded micelles of Examples 3-4 and Comparative Examples:

[0114] Test method: Take LbP (TAMA) prepared in Example 3 0.15 -co-OEG 0.85 ) 33 1.5 mL of the aqueous solution of drug-loaded lipid nanoparticles and the copolymer-free drug-loaded lipid nanoparticles prepared in the comparative example was placed in an unopened dynamic light scattering culture dish and the particle size of the solution was tested; a small amount of lipid nanoparticles was negatively stained and subjected to conventional transmission electron microscopy test operation, and the results are shown in FIG. Figures 4-5 Similarly, take the P(OEG obtained in Example 4 0.61 -CO-TAMA 0.39 ) 66 1.5 mL of drug-loaded micelle aqueous solution was placed in an unopened dynamic light scattering culture dish and the particle size of the solution was tested; a small amount of copolymer drug-loaded micelles was taken for conventional transmission electron microscopy testing. The results are shown in Figures 6-7 .

[0115] from Figure 4 Look, LbP (TAMA 0.15 -co-OEG 0.85 ) 33 The drug-loaded lipid nanoparticles and the copolymer-free drug-loaded lipid nanoparticles are both nanoparticles with a hydrated particle size of about 130-140 nm, and a small PDI (less than 0.2), indicating that the nanoparticle size is relatively uniform. From the transmission electron microscopy results of representative copolymer-free drug-loaded lipid nanoparticles ( Figure 5 ), after negative staining, the lipid nanoparticles loaded with curcumin were all membrane-structured nanoparticles of uniform size under electron microscopy, from which it can be inferred that LbP (TAMA 0.15 -co-OEG 0.85 ) 33 The drug was made into lipid nanoparticles.

[0116] from Figure 6 Look, P(OEG 0.61 -CO-TAMA 0.39 ) 66 The hydrated particle size of drug-loaded micelles is about 10 nm. From the transmission electron microscopy results of representative block polymer drug-loaded micelles ( Figure 7 ), the block polymer micelles loaded with rhein were solid and uniform particles, proving that the block polymer and rhein were successfully self-assembled.

[0117] The performance tests of the copolymer drug-loaded micelles prepared in Examples 3-4 and Comparative Examples are shown in Table 1:

[0118] Table 1 Data of copolymer drug-loaded micelles of Examples 3 to 6

[0119]

[0120] Wherein, NP1-LNP / Cur refers to lipid nanoparticles formed by the copolymer, lipid and curcumin of Example 1;

[0121] Ctrl-LNP / Cur refers to lipid nanoparticles formed by the lipid without copolymer described in the comparative example and curcumin;

[0122] CP2-NPs / Rh refers to micelles formed by the copolymer of Example 2 and rhein;

[0123] Particle size and zeta potential test method: Take 1 ml of the drug-loaded micelle aqueous solution prepared in Examples 3 to 4 and the comparative example respectively, put it into a special sample cell for potential measurement, and use a Malvern particle size analyzer to perform a potential test on the solution.

[0124] Loading rate and encapsulation rate test method: prepare drug-loaded lipid nanoparticles according to the steps described in the embodiment or comparative example, take 1 mL of the solution, use 4 times the volume of DMSO to destroy the structure of the lipid nanoparticles, perform UV-visible absorption spectrum detection, substitute into the standard curve, and calculate the amount of loaded drug.

[0125] Prepare drug-loaded micelles according to the steps described in the examples or comparative examples, take 1 mL of the solution, use 4 times the volume of DMF to destroy the structure of the micelles, perform UV-visible absorption spectrum detection, substitute into the standard curve, and calculate the amount of loaded drug. Calculation formula: drug loading (%) = drug amount in micelles / (drug mass in micelles + micelle mass) × 100%; encapsulation efficiency (%) = drug mass in micelles / total drug amount added × 100%.

[0126] Test Example 2: Toxicity test of copolymer drug-loaded lipid nanoparticles on L02 cells and LX-2 cells

[0127] Test method: LbP (TAMA 0.15 -co-OEG 0.85 ) 33 The drug-loaded lipid nanoparticles, the copolymer-free drug-loaded lipid nanoparticles prepared in the comparative example, and the free small molecule drugs were tested for cytotoxicity. The standard MTT test method was used, and the cells used were L02 cells and LX-2 cells. First, 10 cells were seeded on a 96-well cell culture plate. 5 100 cells / mL L02 cells and LX-2 cells were cultured for 24 h to allow them to adhere to the wall. The upper culture medium was then discarded and LbP (TAMA 0.15 -co-OEG 0.85 )33 Drug-loaded lipid nanoparticles, copolymer-free drug-loaded lipid nanoparticles, and free small molecule drug curcumin were added to make the drug concentration in the 4T1 cell culture medium reach the corresponding concentration. After incubation for 24 hours, the 96-well cell culture plate was taken out and the cytotoxicity test was performed according to the MTT standard operation method. The results are as follows Figure 8 shown.

[0128] The results showed that in LX-2 cells, LbP (TAMA 0.15 -co-OEG 0.85 ) 33 Compared with the drug-loaded lipid nanoparticles, the copolymer-free drug-loaded lipid nanoparticles, and the free small molecule drugs, the LbP (TAMA 0.15 -co-OEG 0.85 ) 33 Drug-loaded lipid nanoparticles showed a significant toxicity enhancement phenomenon; in L02 cells, LbP (TAMA 0.15 -co-OEG 0.85 ) 33 Drug-loaded lipid nanoparticles and free small molecule drugs showed no significant cytotoxicity, while copolymer-free drug-loaded lipid nanoparticles caused about 25% cell killing to L02 cells. This shows that after the drug was delivered to the two cells by copolymer-modified drug-loaded lipid nanoparticles, it had no significant effect on normal liver cells L02 cells, but had a significant effect on hepatic stellate cells LX-2 cells, one of the culprits of liver fibrosis. 0.15 -co-OEG 0.85 ) 33 The drug-loaded lipid nanoparticles showed significantly enhanced toxicity, demonstrating the high efficiency and safety of drug delivery by choline copolymer-loaded lipid nanoparticles.

[0129] Test Example 3: Detection of the liver-targeted drug delivery effect of copolymer-loaded lipid nanoparticles

[0130] Test method: After carbon tetrachloride was used to induce liver fibrosis in mice, the mice with liver fibrosis were randomly divided into two groups and injected with 100 μL of 0.2 g / L DiD-labeled LbP (TAMA 0.15 -co-OEG 0.85 ) 33 Drug-loaded lipid nanoparticles and copolymer-free drug-loaded lipid nanoparticles. After injection, fluorescence imaging (excitation wavelength: 630 nm, emission wavelength: 700 nm) was performed using a small animal in vivo imaging system at 10 min, 30 min, 2 h, 6 h, 12 h, and 24 h, respectively. Fig. 9 shown.

[0131] The results showed that the choline copolymer-modified drug-loaded lipid nanoparticles were rapidly (less than 10 minutes) and specifically (almost 100%) enriched in the liver lesions of mice, and the delivery efficiency was much higher than that of the drug-loaded lipid nanoparticles without choline modification. Therefore, choline copolymer-modified nanoparticles have great application prospects in liver-targeted delivery.

[0132] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A copolymer, characterized in that: The polyol comprises ethylenically unsaturated monomer units containing choline and / or its derivatives and hydrophilic (meth)acrylic acid monomer units.

2. A copolymer of formula I, or a pharmaceutically acceptable salt thereof: in, A is a terminal group; B is a terminal group or does not exist; L is a linker or is absent; the linker includes a substituted or unsubstituted -C1-C6 alkylene-, -C1-C6 ester-, which contains a functional group selected from the group consisting of acetal, imine, amide, hydrazine, carbonate or carbamate; R1, R3 are independently selected from H, -CH3; R2 is selected from choline and / or its derivatives; R4 is selected from -OH, -NH2, -NH-C1~C6 alkyl, -O-C1~C6 alkylene-N(C1~C6 alkylene)2, -O-C1~C6 alkylene-PEG-O-C1~C6 alkyl; m is a natural number ranging from 2 to 400; 0＜x＜1。 3. The copolymer of formula (I) according to claim 2, characterized in that: R2 is selected from Preferably, A is a CAT fragment; B is H, -CH3 or absent.

4. The copolymer of formula (I) according to claim 2, characterized in that: The copolymer of formula I is selected from the following compounds: Wherein, the definition of x is as described in claim 2.

5. A method for preparing a copolymer, characterized in that: The following steps are involved: The copolymer according to claim 1 or the copolymer of formula I according to any one of claims 2 to 4 is prepared by polymerizing an ethylenically unsaturated monomer containing choline and / or its derivatives, a hydrophilic (meth) acrylic monomer, a chain transfer agent and an initiator.

6. A nanoparticle, characterized in that: The invention comprises the copolymer according to claim 1 and / or the copolymer of formula I according to any one of claims 2 to 4 and a pharmaceutical active ingredient.

7. The nanoparticle according to claim 6, characterized in that: The nanoparticles at least meet any of the following conditions: (1) The surface of the nanoparticles has a positive charge; (2) the copolymer and / or the copolymer of formula I loads and / or encapsulates the active pharmaceutical ingredient; (3) The mass ratio of the pharmaceutical active ingredient to the copolymer and / or the copolymer of formula I is 1:1 to 100; (4) The nanoparticles further include lipids; preferably, the lipids load and / or encapsulate the active pharmaceutical ingredient to form drug-loaded lipids, and the copolymers and / or copolymers of formula I are modified on the surface of the drug-loaded lipids; (5) The hydrated particle size of the nanoparticles is 5 to 500 nm; (6) The encapsulation efficiency of the drug in the nanoparticles is at least 60%; (7) The active pharmaceutical ingredient includes a hydrophobic drug and / or a hydrophilic drug.

8. A method for preparing nanoparticles, comprising the following steps: The copolymer according to claim 1 and / or the copolymer of formula I according to any one of claims 2 to 4 is mixed with a pharmaceutical active ingredient in an organic solvent and a buffer solution for reaction, and the nanoparticles are obtained after dialysis.

9. A pharmaceutical composition comprising the nanoparticles according to claim 6 or 7 and a pharmaceutically acceptable medium or excipient.

10. Use of the copolymer according to claim 1 and / or the copolymer of formula I according to any one of claims 2 to 4, the nanoparticles according to claim 6 or 7 and / or the pharmaceutical composition according to claim 9 in the preparation of a drug for treating liver diseases.

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