Cholinized polymer, process for its preparation and use thereof

Abstract

The application discloses a choline polymer and a preparation method and application thereof, and the copolymer comprises an ethylenic unsaturated monomer unit with choline and / or a derivative thereof and a hydrophilic (methyl) acrylic acid monomer unit. The application introduces choline and a derivative monomer 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 a drug-loaded nanoparticle (such as a lipid nanoparticle), or a cation-drug complex can be formed through the cationic unit of choline and a drug molecule, so that drug leakage in the storage process and early release of the drug in blood circulation are greatly reduced, the toxic and side effects of the drug are reduced, and the drug is efficiently targeted to the liver.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of drug delivery, in particular to a cholinized polymer and a preparation method and application thereof. BACKGROUND

[0002] Traditional small molecule drugs can also achieve certain curative effect in some liver diseases, but they have poor selectivity, low efficiency and other problems, which bring great toxic side effects to patients during the entire treatment course and greatly reduce the quality of life of patients. In addition, the problem of low drug delivery efficiency will lead to drug resistance, which brings greater challenges to conventional treatment.

[0003] With the rapid development of nanotechnology, the combination of nanotechnology and medicine brings new strategies for the treatment of liver diseases. At present, researchers have creatively developed various types of multifunctional nanofactories to meet the needs of clinical diagnosis and treatment of liver diseases, especially liver cancer. Up to now, nanofactories applied in clinical practice, including liposomal doxorubicin, paclitaxel albumin nanofactories, etc., all show better performance than traditional small molecule drugs, to some extent, solving the problems of poor water solubility, short circulation half-life and large toxic side effects of traditional small molecule chemotherapy drugs, which undoubtedly greatly promotes the development of nanomedicine. Subsequent generations of lipid nanocarriers, such as lipid nanoparticles, are currently attracting attention as an important component of COVID-19 mRNA vaccines, showing more complex structures and stronger physical stability.

[0004] Although polymeric micelles, vesicles and liposomes and other multifunctional drug materials such as organic nanoparticles have been successfully developed, many nanomedicines still 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.), low delivery efficiency (C.W. Chan, Warren, et al. Nat. Mater. 2020, 19, 566.) and other problems, which are still important obstacles in 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 the stability of drugs and improve the delivery efficiency of nanomedicines and the specificity of lesion targeting is still a great challenge and a problem to be solved. SUMMARY

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

[0007] To achieve the above-mentioned object, the technical solution adopted by the present application is:

[0008] In a first aspect of the present application, a copolymer is provided, comprising an ethylenically unsaturated monomer unit with choline and / or derivatives thereof and a hydrophilic (meth)acrylic monomer unit.

[0009] In the present application, by introducing choline and / or its derivatives into the copolymer, the copolymer has both drug loading function and liver targeting. On the other hand, the choline cation in the copolymer can form a cation-π complex with the π structure in the drug to achieve the loading of hydrophilic / hydrophobic drugs containing aromatic structures; the hydrophobic group in the copolymer can also wrap and load hydrophobic drugs to achieve drug loading of hydrophobic drugs; the hydrophilic cavity of the vesicle structure formed by self-assembly can be used for wrapping of hydrophilic drugs. Choline and / or its derivatives can also interact with nanomaterials, allowing the copolymer to be modified on the surface of drug-loaded nanomaterials.

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

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

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

[0013] In some embodiments of the present application, the ethylenically unsaturated monomers having cationic functionality R1and the hydrophilic (meth)acrylic monomers are synthesized into copolymers by suitable polymerization techniques, such as atom transfer radical polymerization, free radical polymerization, nitroxide polymerization, mediated polymerization, reversible addition fragmentation chain transfer polymerization, and the like. Reversible addition fragmentation chain transfer polymerization (RAFT) is particularly useful in the present application. In certain instances, RAFT polymerization can form copolymers with independent and precise control of molecular weight distribution and composition, resulting in combinatorial polymer design. Suitable RAFT polymerization CTA (chain transfer agent) include 3,5-bis(2-dodecylthiuramylthiocarbonylthio-1-oxopropoxy)benzoic acid, 2-(dodecylthiuramylthiocarbonylthio)-2-methylpropionic acid 3-butenyl ester, 4-chloro-3,5-dimethyl-1H-pyrazole-1-dithioic acid 2-cyanobutane-2-yl ester, 3,5-dimethyl-1H-pyrazole-1-dithioic acid 2-cyanobutyl-2-yl ester, 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanoic acid, 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanol, (3,5-dimethyl-1H-pyrazole)-dithioic acid cyanomethyl ester, trithiocarbonic acid cyanomethyldodecyl ester, [3-(trimethoxysilyl)propyl]trithiocarbonic acid cyanomethyl ester, trithiocarbonic acid 2-cyano-2-propyldodecyl ester, trithiocarbonic acid S,S-dibenzyl ester, 2-(dodecylthiuramylthiocarbonylthio)-2-methylpropionic acid, 2-(dodecylthiuramylthiocarbonylthio)-2-methylpropionic acid 3-azido-1-propanol ester, 2-(dodecylthiuramylthiocarbonylthio)-2-methylpropionic acid N-hydroxysuccinimidyl ester, 2-(dodecylthiuramylthiocarbonylthio)-2-methylpropionic acid pentafluorophenyl ester, 2-(dodecylthiuramylthiocarbonylthio)propionic acid, 2-(dodecylthiuramylthiocarbonylthio)-2-methylpropionic acid methyl ester, pentaerythrityl tetrakis[2-(dodecylthiuramylthiocarbonylthio)-2-methylpropionate], trithiocarbonic acid phthalimidomethyl butyl ester, 1,1,1 -tris[(dodecylthiothiocarbonylthio)-2-methylpropionate]ethane, phenyl 1H-pyrrole-1 -dithioformate, cyanomethyl diphenyl dithio carbamate, cyanomethyl methyl(phenyl) dithio carbamate, cyanomethyl methyl(4-pyridyl) dithio carbamate, 2-cyanopropan-2-yl N-methyl-N-(pyridin-4-yl) dithio carbamate, methyl 2-[methyl(4-pyridyl)thiocarbamic acid thio]propanoate, 1 -succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)thiocarbamic acid thio]pentanoate, benzyl dithiobenzoate, cyanomethyl dithiobenzoate, 4-cyano-4-(phenylthiocarbonylthio)pentanoic acid, N-succinimidyl 4-cyano-4-(phenylthiocarbonylthio)pentanoate, 2-cyanopropan-2-yl 4-cyanodithiobenzoate, ethyl 2-(4-methoxyphenylthiocarbonylthio)acetate, ethyl 2-methyl-2-(phenylthiocarbonylthio)propanoate, ethyl 2-(phenylthiocarbonylthio)-2-phenylacetate, ethyl 2-(phenylthiocarbonylthio)propanoate, 1 -(methoxycarbonyl)ethyl dithiobenzoate, 2-(4-methoxyphenylthiocarbonylthio)acetic acid, 2-nitro-5-(2-propynoxy)benzyl 4-cyano-4-(phenylthiocarbonylthio)pentanoate, 2-(phenylthiocarbonylthio)propanoic acid, 2-phenyl-2-propyl dithiobenzoate, cyanomethyl methyl(4-pyridyl) dithio carbamate, 2-cyanopropan-2-yl N-methyl-N-(pyridin-4-yl) dithio carbamate, methyl 2-[methyl(4-pyridyl)thiocarbamic acid thio]propanoate, 1 -succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)thiocarbamic acid thio]pentanoate, and the like.

[0014] When the CAT is used in a polymerization reaction, the CAT fragment can be retained as an end group of the polymer.

[0015] CTA fragmentation and the resulting fragment structure are very well established in the art, so that the structure of the end group can be determined by using the prior art without the need to use inventive skill.

[0016] In some embodiments of the application, the number average molecular mass of the copolymer is from 2000 to 200000 g / mol, such as 5000, 10000, 15000, 20000, 30000, 50000, 80000, 100000, 150000 g / mol, and the like.

[0017] In a second aspect of the application, there is provided 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 is absent;

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

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

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

[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 from 2 to 400; 0 < x < 1.

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

[0026] When CAT is used in a polymerization reaction, the CAT fragment can remain as a terminal group of the polymer. As used herein, CTA 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 breaking of one or more covalent bonds of a chain transfer agent molecule.

[0028] CTA fragmentation and the resulting fragment structures are well established in the art, and thus the structure of A can be determined by utilizing the prior art without the need for using inventive skill.

[0029] Commonly, CTA fragments include: and the like.

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

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

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

[0033] In some embodiments of the present application, m is a natural number from 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 application, the copolymer of formula I is selected from the following compounds:

[0035]

[0036] wherein x is defined as described above.

[0037] In a third aspect of the present application, a method for preparing a copolymer is provided, comprising the following steps: polymerizing an ethylenically unsaturated monomer with choline and / or derivatives thereof, a hydrophilic (meth)acrylic monomer, a chain transfer agent, and an initiator to obtain the copolymer or the copolymer of formula I.

[0038] In some embodiments of the present application, the ethylenically unsaturated monomer with choline and / or derivatives thereof is prepared by catalyzing the ethylenically unsaturated monomer with choline and / or derivatives thereof, and then halogenating the catalyzed product. The catalyst includes any one of dibutyltin dilaurate, dibutyltin diacetate, and dibutyltin dilaurate.

[0039] In some embodiments of the present application, the halogenating agent used in the halogenation includes any one of iodomethane, dichloromethane, and trichloromethane.

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

[0041] In a fourth aspect of the present application, a nanoparticle is provided, comprising the copolymer and / or the copolymer of formula I and a pharmaceutically active ingredient.

[0042] In some embodiments of the present application, the nanoparticle has a positive charge on its surface.

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

[0044] In some embodiments of the present application, the copolymer and / or the copolymer of formula I load and / or encapsulate the pharmaceutically active ingredient.

[0045] In some embodiments of the present application, the mass ratio of the pharmaceutical active ingredient to the copolymer and / or the copolymer of Formula I is 1:1-100, 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 application, the nanoparticle further comprises a lipid; preferably, the lipid loads and / or encapsulates the pharmaceutical active ingredient to form a drug-loaded lipid, and the copolymer and / or the copolymer of Formula I is modified on the surface of the drug-loaded lipid.

[0047] In some embodiments of the present application, the lipid comprises a steroidal lipid, a phospholipid.

[0048] In some embodiments of the present application, the steroidal lipid is selected from one or more of avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, fecosterol, epicholesterol, ergosterol, fucosterol, hexahydroergosterol, hydroxycholesterol, and cholesterol modified by a polypeptide; lanosterol, mycoceroid, mycosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid, preferably cholesterol.

[0049] In some embodiments of the present application, the phospholipid is selected from one or more of 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), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine (POPE).

[0050] In some embodiments of the present application, the mass ratio of the pharmaceutical active ingredient to the lipid is 1:1-100; and 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 application, the hydrated particle size of the nanoparticles is 5-500 nm, such as 10 nm, 50 nm, 100 nm, 120 nm, 130-140 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 monomer polymerization degree in the copolymer, the molecular weight of the active pharmaceutical ingredient, and whether the lipid is contained. The greater the molecular weight of the copolymer, the greater the molecular weight of the active pharmaceutical ingredient, and the more the lipid contained, the relatively larger the hydrated particle size.

[0052] In some embodiments of the present application, 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 application, the active pharmaceutical ingredient includes a hydrophobic drug and / or a hydrophilic drug.

[0054] In some embodiments of the present application, the active pharmaceutical ingredient includes 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 application, the active pharmaceutical ingredient includes at least one of doxorubicin hydrochloride, cisplatin, mitoxantrone, paclitaxel, camptothecin, sorafenib, lenvatinib, regorafenib, gefitinib, ganetespib, curcumin, rhein, berberine

[0056] In a fifth aspect of the present application, a preparation method of the nanoparticles is provided, comprising the following steps: mixing and reacting the copolymer and / or the copolymer of formula I with the active pharmaceutical ingredient in an organic solvent and a buffer solution, and preparing the nanoparticles after dialysis.

[0057] In some embodiments of the present application, the preparation method of the nanoparticles further comprises mixing and reacting the copolymer and / or the copolymer of formula I, the lipid, and the active pharmaceutical ingredient in an organic solvent and a buffer solution, and preparing the nanoparticles after dialysis.

[0058] In some embodiments of the present application, the organic solvent includes at least one of N, N-dimethylformamide, dioxane, acetone, anhydrous ethanol, or tetrahydrofuran.

[0059] In a sixth aspect of the present application, a pharmaceutical composition is provided, comprising the nanoparticles and a pharmaceutically acceptable medium or adjuvant.

[0060] ​In some embodiments of the application, the medium comprises an aqueous solution; the nanoparticles are dispersed in the aqueous solution and stored in liquid form until needed, or can be dried and introduced and dispersed in an aqueous solution prior to administration to a subject.

[0061] Pharmaceutically acceptable media or excipients include: sugars, such as lactose, dextrose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); esters, such as ethyl oleate, ethyl laureate; agar; buffering agents, such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; fillers, such as polypeptides and amino acids 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.

[0062] Wetting agents, coloring agents, release agents, coating agents, sweetening, flavoring, perfuming agents, preservatives, and antioxidants can also be present in the pharmaceutical compositions.

[0063] The aqueous solution contains, for example, water, cell culture media, buffers (e.g., phosphate buffered saline), polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycols, and the like), suitable mixtures thereof. In some embodiments, the aqueous solution can be a buffered solution (e.g., PBS).

[0064] In a seventh aspect of the application, there is provided use of the copolymer and / or the copolymer of Formula I, the nanoparticle and / or the pharmaceutical composition in the manufacture of a medicament for treating a liver disease.

[0065] The pharmaceutical compositions of the present application can be in a form suitable for oral administration, for example, in the form of liquid dispersions or aqueous or oily suspensions, or they can be in a form suitable for parenteral administration, for example, for subcutaneous, intravenous, intramuscular, intrasternal, intraperitoneal, intradermal, transdermal, or other intmdse techniques. The pharmaceutical compositions comprising the present application can also be in a form suitable for administration by inhalation, in the form of an aerosol or solution, by administration with an inhaler or a nebulizer. The pharmaceutical compositions of the present application are preferably administered transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, or by inhalation to a subject. The most suitable administration route in any given case will depend on the particular therapeutic agent present in the pharmaceutical composition of the present application, the subject, the nature and severity of the disease, and the physical condition of the subject.

[0066] In some embodiments of the present application, the liver disease comprises hepatitis, liver fibrosis, liver cancer.

[0067] The minimum and maximum number of carbon atoms in a hydrocarbon group is indicated by a prefix, e.g., the prefix C a～b Alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Thus, for example, "C 1～4 Alkyl" means an alkyl group containing 1 to 4 carbon atoms.

[0068] "Alkyl" as used herein refers to a saturated hydrocarbon group having the indicated number of carbon atoms. For example, C1-8alkyl refers to an alkyl group having from 1 to 8 carbon atoms, e.g., preferably specifically from 1 to 4 carbon atoms. The alkyl group can be straight or branched. Representative branched alkyl groups have one, two, or three branches. The alkyl group can also be optionally substituted with one or more substituents as defined herein. Specific examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, i-hexyl, 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, and the like.

[0069] "Alkylene" as used herein refers to a divalent saturated aliphatic hydrocarbon group having the indicated number of carbon atoms. For example, "C a b Alkylene" refers to an alkylene group having a to b carbon atoms. The alkylene group can be straight or branched. For example, "C1-6alkylene" 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 one of the following structures:

[0070] The present application has the beneficial effect that the present application introduces choline and its derivative monomers into the copolymer, so that the copolymer has cationic polymer properties and liver efficient specific targeting properties. At the same time, the choline polymer can be modified on the surface of the drug-loaded nanoparticles (such as lipid nanoparticles), or form a cation-drug complex through the cationic unit of choline and the drug molecule, greatly reducing the drug leakage during storage and the premature release of the drug in the blood circulation, reducing the toxic side effects of the drug, and efficiently targeting the drug delivery to the liver. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the copolymer prepared in Example 1 of the present application;

[0072] Figure 2 is the gel permeation chromatography determination of the copolymer prepared in Example 1 of the present application;

[0073] Figure 3 is the nuclear magnetic resonance hydrogen spectrum of the copolymer prepared in Example 2 of the present application;

[0074] Figure 4 is the particle size diagram of the copolymer drug-loaded lipid nanoparticles prepared in the comparative example (left) and Example 3 (right) of the present application;

[0075] Figure 5 is the transmission electron microscopy diagram of the copolymer drug-loaded lipid nanoparticles prepared in the comparative example (left) and Example 3 (right) of the present application;

[0076] Figure 6 is the particle size diagram of the copolymer drug-loaded micelles prepared in Example 4 of the present application;

[0077] Figure 7 is the transmission electron microscopy diagram of the copolymer drug-loaded micelles prepared in Example 4 of the present application;

[0078] Figure 8 is the cytotoxicity detection diagram of the copolymer drug-loaded lipid nanoparticles prepared in Example 3 and the comparative example of the present application;

[0079] Figure 9 is the liver targeting delivery in vivo imaging diagram of the copolymer drug-loaded lipid nanoparticles prepared in Example 3 (upper diagram) and the comparative example (lower diagram) of the present application. DETAILED DESCRIPTION

[0080] The application will be further described in details by specific examples. The raw materials, reagents or devices used in the examples and comparative examples are commercially available or can be obtained by prior art methods unless otherwise specified. The test or test method is the conventional method in the art unless otherwise specified.

[0081] The reagents, methods and devices used in the application are the conventional reagents, methods and devices in the art unless otherwise specified.

[0082] The raw materials used in the following examples, comparative examples or test examples are as follows:

[0083] L02, LX-2 cells were purchased from Shanghai ATCC Cell Bank; C57 mice were purchased from N, N-dimethyl ethanolamine and isocyanatoethyl methacrylate, rhein were purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.; Iodomethane, camptothecin were purchased from Shanghai Jizhishe Biochemical Technology Co., Ltd.; Doxorubicin hydrochloride, curcumin were purchased from Shanghai Mayreer Chemical Technology Co., Ltd.; 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Shanghai Pangshuo Biological Technology Co., Ltd.; Distearoyl phospholipid polyethylene glycol 2000 was purchased from Guangzhou Carbohydrate Biological Technology Co., Ltd.; Cholesterol was purchased from Shanghai Yuanye Biological Co., Ltd.; Poly (ethylene glycol) methyl methacrylate ether (average molecular weight 320, OEGMA) was purchased from Sigma-Aldrich; L-CTA was purchased from Daraykan Biological Technology Co., Ltd.; Curcumin was purchased from Tianjin Xiness Biochemical Technology Co., Ltd.; Rhein was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; Carbon tetrachloride was purchased from Guangzhou Kutaide Trade Co., Ltd.

[0084] Example 1

[0085] A copolymer was prepared in this example, the structural formula of which is as follows, and the specific process is as follows:

[0086]

[0087] 1) Preparation of TAMA, the synthetic route is as follows:

[0088]

[0089] S1. 1.8 g of N, N-dimethyl ethanolamine (20.0 mmol) was dissolved in 40 mL of anhydrous tetrahydrofuran, then a catalytic amount of dibutyltin dilaurate (40 μL) was added, stirred for 15 min, then 3.8 g of isocyanatoethyl methacrylate (24.0 mmol) was slowly added. After stirring at room temperature for 4 h, the reaction mixture was concentrated under reduced pressure, and purified by basic aluminum oxide column to obtain 4.2 g of colorless oil (yield: 85%).

[0090] S2. The product of step S1 (4.2 g, 17.2 mmol) was dissolved in anhydrous tetrahydrofuran (30 mL), and after ice-bath for 15 min, 2.7 g of methyl iodide (18.9 mmol) was slowly added dropwise. After stirring for 30 min, the reaction was continued to stir at room temperature for 2 h. A large amount of white precipitate was generated in the reaction system. The reaction mixture was filtered and washed with tetrahydrofuran (50 mL). It was dried under vacuum at room temperature to obtain white TAMA solid 5.9 g (yield: 88%).

[0091] 2) L-b-P(TAMA 0.15 -co-OEG 0.85 ) 33 Preparation of P(OEG 0.61 -CO-TAMA 0.39 ) 66 , the synthetic route is as follows:

[0092]

[0093] S3. 50 mg of TAMA (0.13 mmol), 312 mg of OEGMA (0.97 mmol), 23 mg of L-CTA (0.025 mmol) and 1 mg of azobisisobutyronitrile (AIBN, 0.006 mmol) were dissolved in 900 μL of a mixed solution (ethanol: dichloromethane: n-hexane = 1:1:1), then transferred into a polymerization tube, degassed by three freeze-thaw cycles, and finally sealed under vacuum. Then the polymerization tube was immersed in a preheated 65°C oil bath. After stirring for 12 h, the polymerization tube was placed in liquid nitrogen for a moment, and then the cap was opened to terminate the polymerization. The reaction mixture was transferred to a dialysis bag (MWCO: 7000 Da), dialyzed with pure water at room temperature for 12 h and lyophilized. The resulting product was 288.7 mg of yellow oily liquid (yield: 75%).

[0094] Example 2

[0095] This example provides a copolymer, P(OEG 0.61 -CO-TAMA 0.39 ) 66 , the synthetic route is as follows:

[0096]

[0097] A solution of 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) was prepared in 600 μL of N,N-dimethylformamide and then transferred into a polymerization tube, degassed by three freeze-thaw cycles and finally sealed under vacuum. The polymerization tube was then immersed in a pre-heated 65 °C oil bath. After stirring for 6 h, the polymerization was terminated by opening the cap after the polymerization tube was placed in liquid nitrogen for a few minutes. The reaction mixture was transferred into a dialysis bag (MWCO: 7000 Da) and dialyzed against pure water for 12 h at room temperature and lyophilized. The resulting product was 441.45 mg of a yellow viscous solid (yield: 66%).

[0098] Example 3

[0099] This example provides a copolymer drug-loaded lipid nanoparticle, which includes the copolymer prepared in Example 1, lipids, and a pharmaceutically active ingredient, which is curcumin. The preparation method of the copolymer drug-loaded lipid nanoparticle includes the following steps:

[0100] A solution of 4.3 mg L-b-P(TAMA 0.15 -co-OEG 0.85 )3 and 1.6 mg curcumin, 2.5 mg cholesterol, 18 mg 1,2-distearoyl-sn-glycero-3-phosphocholine was prepared in 1 mL of a suitable organic solvent (absolute ethanol), which was quickly added to vigorously stirred PBS (9 mL, 10 mM, pH 7.4). After stirring for 7 min, the sample was transferred into a dialysis bag and dialyzed against water to remove the organic solvent and unloaded drugs.

[0101] Comparative Example

[0102] This comparative example provides a copolymer-free drug-loaded lipid nanoparticle, which includes lipids and a pharmaceutically active ingredient, which is curcumin. The preparation method of the copolymer-free drug-loaded lipid nanoparticle includes the following steps:

[0103] A solution of 4.3 mg distearoyl phospho lipopolyethylene glycol 2000 and 1.6 mg curcumin, 2.66 mg cholesterol, 18 mg 1,2-distearoyl-sn-glycero-3-phosphocholine was prepared in 1 mL of a suitable organic solvent (absolute ethanol), which was quickly added to vigorously stirred PBS (9 mL, 10 mM, pH 7.4). After stirring for 7 min, the sample was transferred into a dialysis bag and dialyzed against water to remove the organic solvent and unloaded drugs.

[0104] Example 4

[0105] 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 method for preparing the copolymer drug-loaded micelle comprises the following steps:

[0106] 6 mg of P(OEG prepared in Example 2 0.61 -CO-TAMA 0.39 ) 66 The drug was dissolved in 1 mL of a suitable organic solvent (N,N-dimethylformamide) with 0.6 mg of rhein. The mixture was 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.

[0107] Test Example 1

[0108] The nuclear magnetic resonance test and gel permeation chromatography (GPC) of Example 1 were measured:

[0109] The LbP (TAMA) prepared in Example 1 0.15 -co-OEG 0.85 ) 33 The results were confirmed by H NMR and GPC. Figures 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 near 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.

[0110] Nuclear magnetic resonance test of Example 2:

[0111] 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 3.65ppm) and the characteristic peak of TAMA (chemical shift of methyl hydrogen and methylene hydrogen of quaternary ammonium group near 3.5ppm), and the polymer composition was determined to be P(OEG 0.61 -CO-TAMA 0.39 ) 66 .

[0112] Particle size determination and transmission electron microscopy determination of the copolymer-modified drug-loaded lipid nanoparticles and copolymer-loaded micelles of Examples 3-4 and Comparative Examples:

[0113] 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 measured; a small amount of copolymer drug-loaded micelles was taken for conventional transmission electron microscopy test operation, and the results are shown in Figures 6-7 .

[0114] 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 and copolymer-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.

[0115] 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 are solid and uniform particles, proving that the block polymer and rhein are successfully self-assembled.

[0116] The performance tests of the copolymer drug-loaded micelles prepared in Examples 3-4 and Comparative Examples are shown in Table 1:

[0117] Table 1 Data of copolymer drug-loaded micelles of Examples 3-6

[0118]

[0119] NP1-LNP / Cur refers to the lipid nanoparticles formed by the copolymer of Example 1, lipids and curcumin;

[0120] Ctrl-LNP / Cur refers to the lipid nanoparticles formed by the lipids and curcumin without the copolymer of the copolymer in the comparative example;

[0121] CP2-NPs / Rh refers to the micelles formed by the copolymer of Example 2 and rhein;

[0122] Particle size and zeta potential test method: 1 mL of the drug-loaded micelle aqueous solution prepared in Examples 3-4 and the comparative example was taken into a sample cell for zeta potential test and the solution was tested by a Malvern particle size analyzer.

[0123] Loading rate and encapsulation efficiency test method: the drug-loaded lipid nanoparticles were prepared according to the steps described in the examples or comparative examples, 1 mL of the solution was taken, and the structure of the lipid nanoparticles was destroyed by 4 times the volume of DMSO, and ultraviolet-visible absorption spectrum detection was performed, and the standard curve was substituted to calculate the amount of loaded drug.

[0124] The drug-loaded micelles were prepared according to the steps described in the examples or comparative examples, 1 mL of the solution was taken, and the structure of the micelles was destroyed by 4 times the volume of DMF, and ultraviolet-visible absorption spectrum detection was performed, and the standard curve was substituted to calculate the amount of loaded drug. The calculation formula is: drug loading amount (%) = drug amount in micelles / (drug mass in micelles + micelle mass) x 100%; encapsulation efficiency (%) = drug mass in micelles / total drug added x 100%.

[0125] Test Example 2: Toxicity test of copolymer drug-loaded lipid nanoparticles on L02 cells and LX-2 cells

[0126] Test method: L-b-P(TAMA 0.15 -co-OEG 0.85 ) 33 The drug-loaded lipid nanoparticles, the drug-loaded lipid nanoparticles without copolymer prepared in the comparative example, and the free small molecule drug were tested for cytotoxicity. The standard MTT test method was used, and the cells used were L02 cells and LX-2 cells. First, 10 5 thousand / mL of L02 cells and LX-2 cells were seeded on a 96-well cell culture plate and cultured for 24 h until they adhered. Then the upper culture solution was discarded and L-b-P(TAMA 0.15 -co-OEG 0.85 )33 Drug-loaded lipid nanoparticles, drug-loaded lipid nanoparticles without copolymer, free small molecule drug curcumin, the drug concentration in 4T1 cell culture medium reaches the corresponding concentration, after 24h incubation, remove the 96-well cell culture plate and perform cytotoxicity detection according to the standard MTT operation method, and the results are shown in Figure 8 .

[0127] The results show that in LX-2 cells, L-b-P(TAMA 0.15 -co-OEG 0.85 ) 33 Drug-loaded lipid nanoparticles and drug-loaded lipid nanoparticles without copolymer, free small molecule drug, compared with the same concentration of L-b-P(TAMA 0.15 -co-OEG 0.85 ) 33 Drug-loaded lipid nanoparticles show a significant increase in toxicity; in L02 cells, L-b-P(TAMA 0.15 -co-OEG 0.85 ) 33 Drug-loaded lipid nanoparticles, free small molecule drug, and drug-loaded lipid nanoparticles without copolymer do not show significant cytotoxicity to L02 cells, which shows that the drug delivered by the copolymer-modified drug-loaded lipid nanoparticles into two kinds of cells has little effect on the normal liver cells L02 cells, but on the liver fibrosis culprit, liver stellate cells LX-2 cells, L-b-P(TAMA 0.15 -co-OEG 0.85 ) 33 Drug-loaded lipid nanoparticles show a significant increase in toxicity, proving the high efficiency and safety of choline-modified copolymer drug-loaded lipid nanoparticles for drug delivery.

[0128] Test Example 3: Copolymer drug-loaded lipid nanoparticle liver-targeted drug delivery effect detection

[0129] Test method: After inducing liver fibrosis in mice with carbon tetrachloride, the liver fibrosis mice were randomly divided into two groups, and 100 μL of DiD-labeled L-b-P(TAMA 0.15 -co-OEG 0.85 ) 33 Drug-loaded lipid nanoparticles and drug-loaded lipid nanoparticles without copolymer were injected into the tail vein. After injection, fluorescence imaging was performed at 10 min, 30 min, 2 h, 6 h, 12 h, and 24 h using a small animal live imaging system (excitation wavelength: 630 nm, emission wavelength: 700 nm), as shown in Figure 9 .

[0130] The results show that the choline copolymer modified drug-loaded lipid nanoparticles are rapidly (less than 10 min) and specifically (almost 100%) enriched in the liver lesion site of the mouse, and the delivery efficiency is much higher than that of the drug-loaded lipid nanoparticles without choline modification. Therefore, the choline copolymer modified nanoparticles have great application prospects in liver-targeted delivery.

[0131] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A copolymer comprising: m is 66, and x is 0.

39. ； 2. A nanoparticle comprising the copolymer of claim 1 and rhein.

2. A nanoparticle characterized by:

3. The nanoparticle of claim 2, wherein the nanoparticle surface is positively charged.

3. The nanoparticle of claim 2, wherein:

4. The copolymer of claim 1, wherein the copolymer loads and / or encapsulates the rhein.

4. The nanoparticle of claim 2, wherein:

5. The nanoparticle of claim 2, wherein the mass ratio of the rhein to the copolymer is 1:

10.

5. The nanoparticle of claim 2, wherein:

6. The nanoparticle of claim 2, wherein the hydrated particle size of the nanoparticle is 11.00±2.00 nm.

6. The nanoparticle of claim 2, wherein:

7. The nanoparticle of claim 2, wherein the encapsulation efficiency of the drug in the nanoparticle is at least 60%.

7. The nanoparticle of claim 2, wherein:

8. The nanoparticle of claim 2, wherein the nanoparticle is prepared by mixing the copolymer of claim 1 and the rhein in an organic solvent with a buffer solution and dialyzing after the reaction.

8. A method of producing the nanoparticle of any one of claims 2 to 7, comprising the steps of:

9. A pharmaceutical composition comprising the nanoparticle of any one of claims 2-7 and a pharmaceutically acceptable medium or excipient.

10. Use of the copolymer of claim 1, the nanoparticle of any one of claims 2-7, or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating liver disease. ​

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