Polyzwitterion-cholic acid, synthesis thereof and application of polyzwitterion-cholic acid in drug delivery

Abstract

The invention provides poly-zwitterion-cholic acid, synthesis thereof and application of the poly-zwitterion-cholic acid in drug delivery. Specifically, the invention provides a polymer, the polymer is formed by connecting polycholic acid and one or more zwitterionic polymers through covalent bonds, and the polycholic acid is a main chain (skeleton) of the polymer; and a nano-drug using a micelle or particle formed by the polymer as a carrier.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a polyzwitterion-cholic acid and its synthesis and application in drug delivery. Background Art

[0002] Due to the good specificity and therapeutic effects of biomacromolecule drugs, the US FDA has approved a large number of biomacromolecule drugs in recent years. Biomacromolecule drugs have quickly become the most important class of drugs in the pharmaceutical field, with annual sales close to half of the total sales of all drugs. However, due to their special structure, macromolecule drugs are easily damaged by adverse external environments. In addition, biomacromolecule drugs are difficult to absorb in the gastrointestinal tract due to their large molecular weight. Therefore, it is difficult to administer such drugs orally.

[0003] With the development and progress of society, the number of obese and diabetic patients has increased rapidly in China and even around the world. At the same time, such diseases can cause many related complications, and the many medical problems caused by them have been difficult to solve so far. Taking diabetes as an example, insulin is the most important drug for the treatment of diabetes, but on the one hand, it is difficult to deliver orally and can only be administered by injection, resulting in lower patient compliance. On the other hand, insulin can only control blood sugar and has no improvement effect on related complications.

[0004] In summary, there is an urgent need in the art to develop a new class of carriers that are very suitable for delivering biomacromolecule drugs, especially drug delivery carriers that have certain pharmaceutical activities themselves. Summary of the invention

[0005] In order to solve the deficiencies in the prior art, the present invention provides a new carrier, which can be used as a drug carrier, especially suitable for oral delivery of biomacromolecule drugs; on the other hand, it can be used alone as a drug for the prevention or treatment of a variety of diseases including obesity, tumors, neurological diseases, tumors, enteritis, liver disease, stomach disease, kidney disease, pneumonia, bacterial and viral infections, diabetes and non-alcoholic fatty liver. Specifically, the present invention provides a polyzwitterion-polycholic acid and a preparation method and application thereof, as well as a method for preparing micelles and carriers from the polyzwitterion-polycholic acid.

[0006] In a first aspect of the present invention, a polymer is provided, the polymer comprising polycholic acid and a zwitterionic polymer linked to the polycholic acid via a covalent bond.

[0007] In another preferred embodiment, the polycholic acid is the main chain (skeleton) of the polymer.

[0008] In another preferred embodiment, the degree of polymerization of the polycholic acid is an integer of 1-100.

[0009] In another preferred embodiment, the degree of polymerization of the polycholic acid is an integer of 2-100; preferably, an integer of 2-50; more preferably, an integer of 2-30; most preferably, an integer of 2-20.

[0010] In another preferred embodiment, the degree of polymerization of the polycholic acid is an integer of 2-15; preferably, an integer of 2-10, and more preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0011] In another preferred embodiment, the molecular weight of the polycholic acid is 600-20000, preferably, 600-10000; more preferably, 600-5000.

[0012] In another preferred embodiment, the molecular weight of the polycholic acid is 1000-5000, preferably, 1000-3000.

[0013] In another preferred embodiment, the zwitterionic polymer is covalently linked to the polycholic acid backbone.

[0014] In another preferred embodiment, the zwitterionic polymer is connected to the terminal and / or the repeating unit structure of the polycholic acid via a covalent bond.

[0015] In another preferred embodiment, each unit structure of the polycholic acid is independently connected to 0, 1 or 2 zwitterionic polymers.

[0016] In another preferred embodiment, a zwitterionic polymer is connected to the end of the polycholic acid, and each unit structure is independently connected to 0, 1 or 2 zwitterionic polymers.

[0017] In another preferred embodiment, each zwitterionic polymer is the same or different.

[0018] In another preferred embodiment, in the zwitterionic polymer, each unit structure includes an acid anion group and a quaternary ammonium cation group.

[0019] In another preferred embodiment, the acid anion group and the quaternary ammonium cation group are located in the side chain structure of the zwitterionic polymer.

[0020] In another preferred embodiment, the degree of polymerization of the zwitterionic polymer is an integer of 1-100; preferably, n is an integer of 2-100; more preferably, n is an integer of 2-50; most preferably, it is an integer of 10-50.

[0021] In another preferred embodiment, the molecular weight of the zwitterionic polymer is 1000-10000.

[0022] In another preferred embodiment, the zwitterionic polymer is selected from Table A1;

[0023] Table A1

[0024]

[0025] wherein n is each independently an integer of 1-100 (preferably, n is an integer of 2-100; more preferably, n is an integer of 2-50; most preferably, an integer of 10-50), and n1 is each independently an integer of 1-10 (preferably, an integer of 1-5, more preferably, 1, 2 or 3).

[0026] In another preferred embodiment, the monomer of the polycholic acid is free bile acid.

[0027] In another preferred embodiment, the monomer of polycholic acid is as shown in formula A

[0028]

[0029] Among them, R 1 and R 2 Each independently selected from the following group: -H, -OH, C 1-6 Alkyl (such as -CH3).

[0030] In another preferred embodiment, the monomer of polycholic acid is as shown in formula A-1

[0031]

[0032] Among them, R 1 and R 2 As defined in formula A;

[0033] In another preferred embodiment, the monomer of the polycholic acid is one or more cholic acid monomers selected from Table A2;

[0034] Table A2

[0035]

[0036] In another preferred embodiment, the polymer is as shown in Formula I;

[0037]

[0038] in,

[0039] P represents the unit structure of polycholic acid;

[0040] Each A is independently none or a zwitterionic polymer moiety; and at least one A is a zwitterionic polymer moiety;

[0041] m is an integer from 1 to 100.

[0042] In another preferred embodiment, m is an integer of 2-100; more preferably, m is an integer of 2-50; most preferably, m is an integer of 2-30; most preferably, m is an integer of 2-20.

[0043] In another preferred embodiment, m is an integer of 2-10; preferably, it is 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0044] In another preferred embodiment, the zwitterionic polymer portion is a moiety derived from the zwitterionic polymer.

[0045] In another preferred embodiment, the derivative from the zwitterionic polymer refers to a process in which an active group (such as -COOH) in the zwitterionic polymer reacts with an active group (such as -OH) in polycholic acid to form a monovalent group of the zwitterionic polymer.

[0046] In another preferred embodiment, the polymer is as shown in Formula II;

[0047]

[0048] in,

[0049] A 1 , A 2 and A 3 Each independently selected from the group consisting of: -H, -OH, C 1-6 Alkyl (such as -CH3), zwitterionic polymer; and, A 1 , A 2 and A 3 at least one is a zwitterionic polymer;

[0050] m is an integer from 1 to 100.

[0051] In another preferred embodiment, the zwitterionic polymer is as defined above.

[0052] In another preferred embodiment, A 1 and A 2 Each is independently -H or a zwitterionic polymer.

[0053] In another preferred embodiment, A 1 and A 2 All are zwitterionic polymers.

[0054] In another preferred embodiment, A 1 Selected from the group consisting of -H, -OH, C 1-6 Alkyl (such as -CH3) (preferably A 1 H and A 2 It is a zwitterionic polymer.

[0055] In another preferred embodiment, A 1 is a zwitterionic polymer and A 2 is selected from the group consisting of: -H, -OH, C 1-6 Alkyl (such as -CH3) (preferably A 2 for H).

[0056] In another preferred embodiment, A 3 It is a zwitterionic polymer.

[0057] In another preferred embodiment, the polymer is as shown in formula II-1;

[0058]

[0059] In another preferred embodiment, the polymer is selected from the following Table A:

[0060] Table A

[0061]

[0062]

[0063] Wherein, m is an integer of 1-100, n is an integer of 1-100 independently, and n1 is an integer of 1-10 independently; and the covalent bond is formed by the carboxyl group of the zwitterionic polymer and the hydroxyl group in the polycholic acid.

[0064] In another preferred embodiment, in the polycholic acid, part or all of the terminal hydroxyl groups and the hydroxyl groups in the unit structure form covalent bonds with the carboxyl groups of the zwitterionic polymer.

[0065] In a second aspect of the present invention, a method for preparing the polymer as described in the first aspect is provided, the method comprising the steps of:

[0066] (A) providing polycholic acid;

[0067] (B) providing a zwitterionic polymer or an ester thereof; and

[0068] (C) reacting polycholic acid with a zwitterionic polymer or an ester thereof, and optionally hydrolyzing the polymer to obtain the polymer.

[0069] In another preferred embodiment, the esterified product refers to the zwitterionic polymer in which the acid group is esterified.

[0070] In another preferred embodiment, the esterified product is tert-butyl ester.

[0071] In another preferred embodiment, the polycholic acid is prepared by a bile acid polymerization step, and the bile acid polymerization step comprises: providing bile acid monomers, and causing the bile acid monomers to undergo polymerization reaction, thereby obtaining polycholic acid.

[0072] In another preferred embodiment, the polymerization reaction of bile acid monomers is carried out in a mixed solvent of dichloromethane and pyridine.

[0073] In another preferred embodiment, in the polymerization reaction of bile acid monomers, the volume ratio of dichloromethane to pyridine in the mixed solvent is 1 to 10:1; preferably, 3 to 7:1; more preferably, 4 to 6:1, and most preferably, 5:1.

[0074] In another preferred embodiment, in the polymerization reaction of the bile acid monomer, the dichloromethane is anhydrous dichloromethane, and / or the pyridine is anhydrous pyridine.

[0075] In another preferred embodiment, in the polymerization reaction of bile acid monomers, the reaction time of the polymerization reaction is 0.5 to 5 hours, preferably, 1 to 3 hours, and more preferably, 2 hours.

[0076] In another preferred embodiment, in the polymerization reaction of bile acid monomers, the reaction temperature of the polymerization reaction is 30-100°C, preferably 30-50°C, and more preferably 35-45°C.

[0077] In another preferred embodiment, the preparation method of polycholic acid includes the steps of: dissolving bile acid monomer, p-toluenesulfonic acid and dimethylaminopyridine in a mixed solution of anhydrous dichloromethane and anhydrous pyridine (1-10:1; preferably, 3-7:1; more preferably, 4-6:1, and optimally, 5:1); stirring at 30-100°C (optimally 40±5°C) under nitrogen protection, and then adding diisopropylcarbodiimide to the obtained reaction mixture, reacting for 0.5-5 hours (preferably, 1-3 hours, and more preferably, 2 hours); after the reaction is completed, precipitating the obtained product into cold anhydrous methanol, collecting the precipitate by centrifugation and drying to obtain polycholic acid.

[0078] In another preferred embodiment, the zwitterionic polymer or its ester is prepared by a zwitterionic polymerization step, and the zwitterionic polymerization includes the following steps: providing a polymerization system containing zwitterionic or its ester monomers, allowing the zwitterionic or its ester monomers to undergo a polymerization reaction through RAFT polymerization (reversible addition-fragmentation chain transfer polymerization), thereby obtaining the zwitterionic polymer or its ester.

[0079] In another preferred embodiment, the zwitterion or its ester monomer is as follows:

[0080] Table A1.1

[0081]

[0082] Among them, R E is H or C1-6 alkyl (such as tert-butyl), and n1 is as defined above.

[0083] In another preferred embodiment, the polymerization system further comprises a RAFT agent; preferably, the RAFT agent is 4-cyano-4-(dodecylthiocarbonylthio)sulfanylpentanoic acid.

[0084] In another preferred embodiment, the polymerization system further comprises the presence of an initiator; preferably, the initiator is selected from the group consisting of azobisisobutyronitrile and 4,4'-azo(4-cyanovaleric acid).

[0085] In another preferred embodiment, the reaction temperature of the polymerization reaction is 65-95°C.

[0086] In another preferred embodiment, the reaction time of the polymerization reaction is 1-24 hours; preferably, 4-24 hours.

[0087] In another preferred embodiment, in the polymerization system, the concentration of the zwitterion or its ester monomer is 0.2-1 g / mL.

[0088] In another preferred embodiment, in the polymerization system, the concentration of the RAFT agent is 10-100 mg / mL.

[0089] In another preferred embodiment, in the polymerization system, the concentration of the initiator is 1-5 mg / mL.

[0090] In another preferred embodiment, before the zwitterion polymerization step, a step of synthesizing zwitterion or ester monomers thereof is further included, which comprises: reacting N,N-dimethylaminopropyl acrylamide with The reaction is carried out to obtain the zwitterion or its ester monomer.

[0091] In another preferred embodiment, in the synthesis step of the zwitterion or its ester monomer, the reaction temperature is 40-80°C (preferably 50-70°C; more preferably 55-65°C); and / or the reaction time is 4-24h (preferably 8-16h; more preferably 10-14h).

[0092] In the third aspect of the present invention, a drug delivery carrier is provided, wherein the drug delivery carrier comprises micelles or particles formed by the polymer as described in the first aspect.

[0093] In another preferred embodiment, the drug delivery carrier can be used to deliver drugs.

[0094] In another preferred embodiment, the drug is encapsulated in the micelles or particles.

[0095] In another preferred embodiment, the drug is a biomacromolecule drug.

[0096] In another preferred embodiment, the biomacromolecule drugs include: protein polypeptide drugs, nucleic acid drugs, poorly soluble drugs, antibody drugs, antibody-drug conjugates (ADCs), vaccine drugs, or a combination thereof.

[0097] In another preferred embodiment, the protein polypeptide drugs include: insulin, glargine insulin, lispro insulin, aspart insulin, detemir insulin, protamine human insulin, degludec insulin, exenatide, liraglutide, semaglutide, dulaglutide, lixisenatide, benaglutide, loxenatide, semaglutide, albiglutide, growth hormone, glatiramer acetate, Lupron (leuprolide acetate), tirzepatide (Tirzepatide), Motixafortide (BL-8040, BKT140), pemosapatide (EPO-018B), parathyroid hormone (PTH), Trofinetide, interferon, TNF-α, C5, coagulation factor IX, interleukin (IL-2, I L-3, IL-15, IL-7, IL-6R, IL-23, IL-17A, IL-1, etc.), recombinant human coagulation factor VIII, recombinant human erythropoietin, recombinant human epidermal growth factor, recombinant human interferon α1b, recombinant human granulocyte stimulating factor, recombinant insulin, recombinant interferon, recombinant coagulation factor, recombinant erythropoietin, recombinant granulocyte colony stimulating factor, enzyme replacement recombinant protein drug, recombinant growth hormone, recombinant human urokinase, recombinant hirudin, lumbrokinase, recombinant human bone morphogenetic protein, recombinant human granulocyte macrophage colony stimulating factor, fibroblast growth factor 1 (icvFGF1), fibroblast growth factor 4 (icvFGF4), fibroblast growth factor, or a combination thereof.

[0098] In another preferred embodiment, the nucleic acid drugs include: DNA drugs, mRNA drugs, RNAi interference drugs, gene editing drugs, siRNA drugs, circular RNA drugs, or a combination thereof.

[0099] In another preferred embodiment, the poorly soluble drug includes: PROTAC, molecular glue, or a combination thereof.

[0100] In another preferred embodiment, the poorly soluble drugs include: targeted Bcl-xl protein degraders (such as DT2216), targeted androgen receptor degraders (such as ARV-110, ARV-766, AR-LDD), targeted estrogen receptor degraders (such as ARV-471), targeted IRAK4 degraders (such as KT-474, KT-413), targeted STAT3 degraders (such as KT-333), targeted BTK degraders (such as NX-2127, NX-5948), targeted TRK degraders (such as CG001419), targeted BRD9 degraders (such as CFT-8634, FHD-609), thalidomide, lenalidomide, pomalidomide, or a combination thereof.

[0101] In another preferred embodiment, the antibody drug includes: PD-1 monoclonal antibody.

[0102] In another preferred embodiment, the antibody drugs include: adalimumab, corticotropin, bevacizumab, pembrolizumab, nivolumab, sintilimab, cemiplimab, toripalimab, tislelizumab, camrelizumab, prolgolimab, penpulimab, dostarlimab, zimberelimab, serplulimab, atezolizumab, durvalumab Durvalumab, Avelumab, Sugemalimab, Envafolimab, Cadonilimab, Bamlanivimab, Tebentafusp, Rituximab, Catumaxomab, Emicizumab, Amivantamab, Faricimab, Mosunetuzumab, Blinatumomab, Cadonilimab, moxetumomab pasudotox), Iodine 131derlotuximab biotin, Iodine 131Tositumomab, Iodine 131Metuximab, Fc fusion protein, scFv antibody and TCR fusion protein (Tebentafusp), and antibody and enzyme fusion protein (Pabinafusp alfa), Caplacizumab, Envafolimab, Adalimumab, chimeric antibody I131-Metuximab, loncastuximabtesirine), Catumaxomab, Moxetumomab, Pasudotox, Obinutuzumab, Mogamulizumab, Edrecolomab, Nebacumab, Benralizumab, Inebilizumab, or a combination thereof.

[0103] In another preferred embodiment, the vaccine drugs include: polio vaccine, cholera vaccine, live typhoid vaccine, SARS-CoV-2 vaccine, HPV vaccine, hepatitis vaccine, tumor vaccine, Japanese encephalitis vaccine, herpes zoster vaccine, influenza vaccine, BCG vaccine, measles vaccine, plague vaccine, fowlpox virus vaccine, monkeypox virus vaccine, adenovirus vaccine, herpes virus vaccine, Salmonella vaccine, Shigella vaccine, diphtheria and pertussis vaccine, pneumonia vaccine, avian influenza vaccine, pneumonia vaccine, Asian swine fever vaccine, or a combination thereof.

[0104] In a fourth aspect of the present invention, a method for preparing a drug delivery carrier is provided, comprising the steps of:

[0105] (1) providing the polymer as described in the first aspect; and

[0106] (2) The polymer is formed into micelles or particles by direct dissolution or dialysis.

[0107] In another preferred embodiment, the polymer and the optional drug are mixed, and then the polymer is formed into micelles or particles encapsulating the optional drug by direct dissolution method or dialysis method.

[0108] In a fifth aspect of the present invention, there is provided a nano drug preparation comprising:

[0109] (i) micelles or particles formed from the polymer of the first aspect; and

[0110] (ii) optionally a pharmaceutically acceptable carrier.

[0111] In another preferred embodiment, the nano drug preparation further comprises a drug optionally encapsulated in the micelle or particle.

[0112] In another preferred embodiment, the drug is as defined in the third aspect.

[0113] In another preferred embodiment, the drug is a biomacromolecule drug.

[0114] In another preferred embodiment, the drug is an antibody drug.

[0115] In another preferred embodiment, the nano drug preparation is an oral preparation or an injection; preferably, the nano drug preparation is an oral preparation.

[0116] In another preferred embodiment, the nano drug preparation is a preparation selected from the following group: capsules, tablets, pills, powders, granules, emulsions, solutions, suspensions, syrups, tinctures, or a combination thereof.

[0117] In the sixth aspect of the present invention, a nano drug carrier composition is provided, wherein the nano drug carrier composition comprises the polymer as described in the first aspect.

[0118] In another preferred embodiment, the nano drug carrier composition further comprises an optional drug.

[0119] In another preferred embodiment, the polymer exists in the form of micelles or particles or does not form micelles or particles.

[0120] In another preferred embodiment, the drug is as defined in the third aspect.

[0121] In another preferred embodiment, the drug is a biomacromolecule drug.

[0122] In another preferred embodiment, the drug is an antibody drug.

[0123] In another preferred embodiment, the nano drug carrier composition is used to prepare a drug delivery carrier.

[0124] In another preferred embodiment, the nano drug carrier composition is used to prepare nano drug preparations.

[0125] In the seventh aspect of the present invention, there is provided a use of the polymer according to the first aspect, wherein the use is one or more uses selected from the following group:

[0126] a. For drug delivery;

[0127] b. for preparing nano-delivery carriers; and

[0128] c. Used to prepare nano drugs or granular drugs.

[0129] In another preferred embodiment, the nano drug may not encapsulate any other drug (such as a biomacromolecule drug), or may encapsulate a biomacromolecule drug.

[0130] In the eighth aspect of the present invention, there is provided a use of the polymer as described in the first aspect in the preparation of a medicament for treating or preventing a disease, wherein the disease is selected from the following group: obesity, diabetes, tumors, enteritis, diseases caused by bacterial or viral infections, non-alcoholic fatty liver disease, liver disease, stomach disease, kidney disease, neuropathy, pneumonia, or a combination thereof.

[0131] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1-Figure 4 The hydrogen nuclear magnetic resonance spectra of PCBBA and key intermediates in the synthesis process shown in Examples 1, 2 and 6 are shown respectively.

[0133] Figure 5 The size exclusion chromatogram (GPC) of PCBBA in Example 5 is shown.

[0134] Figure 6 The hydrogen nuclear magnetic resonance spectrum of PBA prepared in Example 5 is shown.

[0135] Figure 7 The particle size, PDI, and Zeta potential curves of polybetaine / cholic acid micelles stored at room temperature and 4°C are shown; the morphology of PCBBA micelles and PEGBA micelles was observed by transmission electron microscopy. It was proved that the micelles were smooth and uniform spheres with good stability.

[0136] Figure 8 The results of the PCBBA micelle hemolysis experiment (n=3) are shown, indicating that the micelles have good biocompatibility.

[0137] Fig. 9 The results of the study on the permeation rate of micelles in mucus in Example 10 (n=3) are shown, wherein the left figure shows the accumulation of drugs diffused into the lower chamber at different times, and the right figure shows the apparent permeability coefficient calculated based on the permeation rate in the fourth hour.

[0138] Fig.10 The uptake of micelles by Caco-2 cells in Example 11 is shown, wherein PCBBA micelles can be taken up by cells faster and show higher fluorescence intensity.

[0139] Fig.11 The body weight of type 2 diabetic mice after treatment in Example 12 (n=3) is shown.

[0140] Fig.12 The results of the glucose tolerance test (n=3) of type II diabetic mice after treatment in Example 12 are shown. The left figure shows the changes in blood glucose of mice at corresponding time points, and the right figure shows the area under the curve.

[0141] Fig.13The figure shows the insulin test (n=3) of type II diabetic mice after treatment in Example 12. The left figure shows the changes in blood glucose of mice at corresponding time points, and the right figure shows the area under the curve.

[0142] Fig.14 The liver weight of type 2 diabetic mice after treatment in Example 13 (n=3) is shown.

[0143] Fig.15 The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of type 2 diabetic mice after treatment in Example 13 (n=3) are shown.

[0144] Fig.16 Oil red staining (first row) H&E staining (second row) of liver sections of type 2 diabetic mice after treatment in Example 13 and H&E staining of white adipose tissue are shown.

[0145] Fig.17 The preparation of drug-loaded micelles and a schematic diagram of their action are shown. DETAILED DESCRIPTION

[0146] The inventors have studied for a long time and thoroughly, and for the first time prepared micelles with a zwitterionic polymer as a hydrophilic group and a polymer with polycholic acid as a hydrophobic group, and the micelles have a better penetration effect than other micelles with PEG as a hydrophilic group. In addition, it has been verified experimentally that the micelles also have excellent drug loading, drug delivery capabilities, etc., and whether they are suitable as delivery carriers for biomacromolecule drugs. And this carrier can be used alone as a drug to prevent and treat diseases such as diabetes, obesity and non-alcoholic fatty liver. Based on this, the inventors have completed the present invention.

[0147] the term

[0148] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0149] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0150] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0151] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0152] Zwitterionic Materials

[0153] As used herein, "polyzwitterion," "zwitterionic polymer," and "zwitterionic material" are used interchangeably to refer to polymers formed by the polymerization of molecules that chemically contain an equal number of positively and negatively charged functional groups.

[0154] In recent years, zwitterionic materials have attracted much attention in the field of biomaterials due to their unique advantages such as superhydrophilicity, biocompatibility, and antifouling properties. Zwitterionic materials have the characteristics of high dipole moment and highly charged groups, and have equimolar cationic and anionic parts, maintaining overall electrical neutrality and high hydrophilicity. Zwitterions can form a super strong hydration shell with water through ion-dipole interactions, so zwitterionic polymers can reduce nonspecific protein adsorption and bacterial adhesion, and thus zwitterions can give carriers super mucus penetration ability. In addition to excellent antifouling properties, zwitterionic materials can also enhance biocompatibility, reduce immune responses, and prolong circulation time.

[0155] Bile acid

[0156] Bile acid (BA), as an important component of bile, is synthesized from cholesterol in the liver and stored in the gallbladder. Bile acid is involved in the absorption, distribution, metabolism and excretion of nutrients in the human body. At the same time, bile acid is also a nutrient sensor and metabolic regulator, which can activate nuclear farnesoid X receptor (FXR) and G protein-coupled bile acid receptor (TGR) signal transduction, participate in glucose and energy metabolism, and maintain metabolic homeostasis. In addition, as a natural product, bile acid can be used as a hydrophobic group of micelles with good biocompatibility on the one hand; on the other hand, it can control energy homeostasis and has a certain anti-inflammatory effect.

[0157] Bile acids can be divided into two categories according to their structure: one is free bile acid, including cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid; the other is the product of free bile acid combined with glycine or taurine, called conjugated bile acid, mainly including glycocholic acid, glycochenodeoxycholic acid, taurocholic acid and taurochenodeoxycholic acid.

[0158] Polyzwitterion-cholic acid and preparation method thereof

[0159] One object of the present invention is to provide a polyzwitterion-polycholic acid and a synthesis route thereof. First, the compound (or polymer) is an amphiphilic molecule, the hydrophilic end is composed of a zwitterion polymer (or polyzwitterion), and the hydrophobic end is composed of polycholic acid. Secondly, the compound can be used to prepare drug-loaded nanoparticles for drug delivery.

[0160] As used herein, "polymer of the invention", "polyzwitterion-cholic acid", "polyzwitterion-polycholic acid", "polyzwitterion / cholic acid", "polyzwitterion / polycholic acid", "zwitterionic polymer / polycholic acid", "polyzwitterionic cholic acid molecule", "polyzwitterion-polycholic acid" are used interchangeably to refer to the polymer as defined in the first aspect of the invention.

[0161] In a specific embodiment, a novel structure of zwitterionic polymer / polycholic acid (also referred to as the polymer of the present invention) is provided.

[0162] In further embodiments, the polymer is as defined in the first aspect.

[0163] In further embodiments, the polymer is capable of forming a nanodelivery vehicle (specifically, a nanomicelle or particle).

[0164] In other embodiments, the micelle or particle is mainly composed of the polymer, wherein the zwitterionic polymer serves as the hydrophilic end and the polycholic acid serves as the hydrophobic end.

[0165] In some embodiments, the polymer has the structural formula:

[0166]

[0167] Wherein, A is -H, -OH, -CH3 and zwitterionic polymer molecules, P is a bile acid molecule, and optionally, m can be an integer within the range of 1-100.

[0168] In another embodiment, A is a zwitterionic polymer; more preferably, selected from the following Table A1:

[0169] Table A1

[0170]

[0171] Wherein, n is the degree of polymerization, and as an option, n can be an integer within the range of 1-100. n1 is an integer within the range of 1-10.

[0172] In other embodiments, P is cholic acid.

[0173] In other embodiments, the cholic acid monomers may be those selected from Table A2 below:

[0174] Table A2

[0175]

[0176] In other embodiments, the polycholic acid backbone can be selected from those in Table A2.1 below.

[0177]

[0178]

[0179] Here, m is an integer from 1 to 100.

[0180] In other embodiments, the polymer is selected from Table A.

[0181] In other embodiments, when all hydroxyl groups in polycholic acid are linked to zwitterionic polymers, the polymers are as described in Table C:

[0182] Table C

[0183]

[0184]

[0185]

[0186] It should be understood that in the polymer, the hydroxyl groups of the polycholic acid may be ungrafted or linked to a zwitterionic polymer.

[0187] In another embodiment, the polymer is used to prepare a nano drug delivery composition.

[0188] In another embodiment, the present invention also provides use of the polymer in treating diseases, preferably the diseases include but are not limited to obesity, diabetes and non-alcoholic fatty liver disease.

[0189] In another specific embodiment, the present invention also provides a method for preparing the polymer, comprising the steps of:

[0190] (A) providing polycholic acid;

[0191] (B) providing a zwitterionic polymer or an ester thereof; and

[0192] (C) reacting polycholic acid with a zwitterionic polymer or an ester thereof, and optionally hydrolyzing the polymer to obtain the polymer.

[0193] In another embodiment, the preparation method is as described in the second aspect.

[0194] In some specific embodiments, the method for preparing the zwitterionic polymer / polycholic acid provided by the present invention, wherein, when the selected zwitterion is betaine, the method comprises the following steps.

[0195] (1) Synthesis of CB-tBu: N,N-dimethylaminopropylacrylamide and tert-butyl bromoacetate were dissolved in acetonitrile. The reactants were stirred in an oil bath (60°C) for 12 h and then cooled to room temperature. The product was precipitated in ether and the white precipitate 1 was obtained by vacuum drying.

[0196] (2) Synthesis of PCB-tBu: 4-cyano-4-(dodecylthiocarbonylthio)sulfanylpentanoic acid, CB-tBu and azobisisobutyronitrile were dissolved in anhydrous N,N-dimethylformamide, and the reactants were placed in a round-bottom flask and sealed with a sealing film. Freeze-degassing cycles were repeated three times to remove oxygen, and then stirred in an oil bath at 70°C for 4 hours. After the reaction was completed, it was precipitated in cold ethyl acetate three times, and the precipitate was vacuum dried to obtain a light yellow solid. The product was dissolved in DMF with triethylamine, n-hexylamine, and tri(2-carboxyethyl) hydrochloride. After stirring at room temperature for 4 hours, the color of the reaction solution turned colorless. The mixture was precipitated in ice-cold ethyl acetate and dried under vacuum.

[0197] (3) Synthesis of PBA: Bile acid, p-toluenesulfonic acid and dimethylaminopyridine were dissolved in a mixed solution of anhydrous dichloromethane and anhydrous pyridine (5:1). Stirred at 30°C-100°C (optimal 40±5°C) under nitrogen protection. Then diisopropylcarbodiimide was added to the reaction mixture and reacted for 2 hours. After the reaction was completed, the product pBA was precipitated into cold anhydrous methanol, and the precipitate was collected by centrifugation and dried to obtain a white powder.

[0198] (4) Synthesis of PCBBA: PBA, PCB, EDCI and dimethylaminopyridine were dissolved in DMF. The mixture was stirred overnight under nitrogen protection. After the reaction, the crude product was precipitated into cold ethyl acetate, and the precipitate was collected by centrifugation and dried to obtain a white powder. The product was dissolved in dichloromethane, trifluoroacetic acid (TFA) was added and stirred at room temperature for 4 h, and the product was dropped into cold ethyl acetate for precipitation. The precipitate was collected by centrifugation and dried to obtain a white final product.

[0199] In another embodiment, in step (2), the reaction concentration of CB-tBu is 0.2-1 g / mL, the concentration of azobisisobutyronitrile is 1-5 mg / mL, and the concentration of 4-cyano-4-(dodecylthiocarbonylthio)sulfanylpentanoic acid is 10-100 mg / mL.

[0200] In another embodiment, in step (2), azobisisobutyronitrile can be replaced by 4,4'-azo(4-cyanovaleric acid).

[0201] In another embodiment, in step (2), the reaction temperature is 65-95° C., and / or the reaction time is 4-24 hours.

[0202] In another embodiment, in step (4), the molecular weight of PCB is 1000-10000 g / mol.

[0203] Nano delivery carrier, nano medicine and preparation method thereof

[0204] Another object of the present invention is to provide a nanocarrier (or nano delivery carrier) and a preparation method thereof. First, the nanocarrier has good biocompatibility and mucus permeability, and secondly, the carrier can improve symptoms such as obesity and insulin sensitivity in type 2 diabetic mice. In addition, the preparation method provided by the present invention is simple, the materials are readily available, the reaction conditions are mild, and it has industrial prospects.

[0205] In a specific embodiment, Fig.17 As shown, the nano delivery carrier is a nano drug-loaded micelle or particle formed by self-assembly of the polymer (i.e., polyzwitterion-cholic acid) of the present invention. The nano delivery carrier includes a hydrophilic shell containing zwitterions and a hydrophobic core of polycholic acid. The hydrophobic core is optionally loaded with drugs such as macromolecular drugs. The periphery of the drug-loaded micelle or particle is hydrophilic zwitterions, which can protect macromolecular drugs and promote the penetration rate of drugs in the intestine, thereby improving the oral bioavailability of macromolecular drugs. In addition, after the micelle or particle enters the blood, it can effectively resist nonspecific protein adsorption, so that the drug-loaded micelle or particle can achieve stable long circulation in the blood.

[0206] In one embodiment, a method for preparing the nano-delivery carrier is provided, comprising the steps of:

[0207] At least one polymer described in the present invention (ie, polyzwitterion-cholic acid) is mixed with an optional biomacromolecule drug, and the drug-loaded nanometer micelles or particles are prepared by a direct dissolution method or a dialysis method.

[0208] As an optional method, the method specifically comprises dissolving the amphiphilic polymer and insulin in PBS at the same time, stirring for 2 hours to make them dispersed evenly, and placing them in a 10K dialysis bag and dialyzing with PBS to obtain drug-loaded micelles.

[0209] As an optional manner, the method specifically comprises dissolving the amphiphilic polymer and insulin in PBS simultaneously, and stirring for 4-12 hours to allow them to self-assemble into uniform drug-loaded micelles or particles.

[0210] Alternatively, the micelle or particle has a size between 10-300 nm.

[0211] As an optional mode, in the above-mentioned nano drug-loaded micelles or particles, the drug contained in the hydrophobic core is one or more of polypeptide drugs (insulin, GLP-1, etc.), protein drugs, nucleic acid drugs, poorly soluble small molecule drugs and fluorescent molecules, preferably insulin and GLP-1.

[0212] As an optional manner, in the above-mentioned nano drug-loaded micelles or particles, the encapsulation rate ranges from 50% to 95%, and the drug loading amount ranges from 5% to 15%.

[0213] The successful preparation of micelles or particles was proved by particle size, Zeta potential and transmission electron microscopy, and the micelles or particles were smooth spheres. The blank micelles were placed in room temperature and 4°C refrigerators, and the changes in particle size and PDI were observed regularly. The results showed that the micelles had good stability. In vitro mucus penetration experiments and cell penetration experiments showed that the micelles had strong mucus permeability. In summary, the micelles are an excellent oral delivery carrier.

[0214] Pharmaceutical composition, nanopharmaceutical preparation and administration method

[0215] Since the polymer of the present invention and the micelles or particles formed therefrom have excellent prevention and treatment of diseases such as diabetes, obesity and non-alcoholic fatty liver disease, and have excellent drug loading and drug delivery capabilities and can be used as drug delivery carriers, the micelles or blank micelles encapsulating other drugs formed by the polymer of the present invention, as well as the pharmaceutical composition or nano drug preparation containing the micelle of the present invention as the main active ingredient can be used to treat, prevent and alleviate a variety of diseases or symptoms.

[0216] The pharmaceutical composition or nano drug preparation of the present invention comprises the micelle (or particle) of the present invention and a pharmaceutically acceptable carrier within a safe and effective amount. The "safe and effective amount" means that the amount of the micelle (or particle) or other drugs contained therein is sufficient to significantly improve the condition without causing serious side effects.

[0217] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds.

[0218] The administration mode of the micelle (or particle) or pharmaceutical composition of the present invention is not particularly limited, and representative administration modes include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. Considering the excellent delivery performance of the micelle or particle of the present invention, the micelle or particle of the present invention and the pharmaceutical composition are particularly suitable for oral administration. Therefore, the nano drug preparation of the present invention is preferably an oral preparation.

[0219] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.

[0220] The main advantages of the present invention include:

[0221] 1. The present invention uses non-immunogenic, easy-to-load drug polyzwitterion / polycholic acid (such as PCBBA) as the basic unit. The raw materials of the prepared PCBBA nano delivery carrier are cheap and easy to obtain, and have good biocompatibility. The synthesis conditions are mild, and it is an excellent oral nano carrier.

[0222] 2. The polyzwitterion-polycholic acid nano-delivery carrier of the present invention is a regular and uniform sphere with a high encapsulation rate and drug loading capacity, and can be loaded with a variety of biomacromolecule drugs including insulin and GLP-1.

[0223] 3. The polyzwitterion-polycholic acid (such as PCBBA) nano delivery carrier of the present invention can quickly penetrate into mucus and can also be taken up by cells in large quantities, and has a high potential for oral delivery.

[0224] 4. The polyzwitterion-polycholic acid nano-delivery carrier of the present invention can reduce the body weight of type 2 diabetic mice and improve the insulin sensitivity of mice.

[0225] In summary, the present invention synthesizes polyzwitterion / cholic acid, and uses it as a monomer to self-assemble into micelles. The polybetaine / polycholic acid (such as PCBBA) micelles in the present invention have excellent biocompatibility and stability, and can effectively load a variety of macromolecular drugs including insulin and genes, as well as poorly soluble small molecule drugs. PCBBA micelles have strong penetration ability in mucus and can be quickly taken up by cells. In addition, the carrier itself can improve the problems of obesity and insulin resistance in type 2 diabetic mice, and has obvious preventive and therapeutic effects.

[0226] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0227] General method for preparing polymers

[0228] Each polymer in the embodiment can be synthesized by the following method:

[0229] (1) Synthesis of CB-tBu: N,N-dimethylaminopropylacrylamide and tert-butyl bromoacetate were dissolved in acetonitrile. The reactants were stirred in an oil bath (60°C) for 12 h and then cooled to room temperature. The product was precipitated in diethyl ether and the obtained white precipitate (Compound 1) was dried in vacuo.

[0230]

[0231] (2) Synthesis of PCB-tBu:

[0232] (2.1) 4-Cyano-4-(dodecylthiothiocarbonyl)sulfanylpentanoic acid, CB-tBu and azobisisobutyronitrile (AIBN) were dissolved in anhydrous N,N-dimethylformamide (DMF), and the reactants were placed in a round-bottom flask and sealed with a sealing film. Freeze degassing cycles were repeated three times to remove oxygen, and then stirred in an oil bath at 70°C for 4 hours. After the reaction was completed, it was precipitated in cold ethyl acetate three times, and the precipitate was vacuum dried to obtain a light yellow solid (i.e., compound 2).

[0233] (2.2) The product was dissolved in DMF with triethylamine, n-hexylamine, and tri(2-carboxyethyl) hydrochloride. After stirring at room temperature for 4 h, the color of the reaction solution turned colorless. The mixture was precipitated in ice-cold ethyl acetate and dried under vacuum to obtain PCB-tBu (i.e., compound 3).

[0234]

[0235] (3) Synthesis of PBA: Ursodeoxycholic acid, p-toluenesulfonic acid and dimethylaminopyridine were dissolved in a mixed solution of anhydrous dichloromethane and anhydrous pyridine (5:1). Stirred at 40°C under nitrogen protection. Then diisopropylcarbodiimide was added to the reaction mixture and reacted for 2 hours. After the reaction was completed, the product pBA was precipitated into cold anhydrous methanol, and the precipitate was collected by centrifugation and dried to obtain PBA in the form of white powder.

[0236]

[0237] (4) Synthesis of PCBBA: PBA, PCB, EDCI and dimethylaminopyridine were dissolved in DMF. The mixture was stirred overnight under nitrogen protection. After the reaction, the crude product was precipitated into cold ethyl acetate, and the precipitate was collected by centrifugation and dried to obtain a white powder. The product was dissolved in dichloromethane, trifluoroacetic acid (TFA) was added and stirred at room temperature for 4 h, and the product was dropped into cold ethyl acetate for precipitation. The precipitate was collected by centrifugation and dried to obtain a white final product 5.

[0238]

[0239] Synthesis of PEGBA

[0240] (1) Synthesis of PEGBA: PBA, PEG, EDCI and dimethylaminopyridine were dissolved in DMF. The mixture was stirred overnight under nitrogen protection. After the reaction, the crude product was precipitated into cold ether, and the precipitate was collected by centrifugation and dried to obtain a white powder, namely PEGBA.

[0241]

[0242] Optionally, in step (2), the reaction concentration of CB-tBu is 0.2-1 g / mL, the concentration of azobisisobutyronitrile is 1-5 mg / mL, and the concentration of 4-cyano-4-(dodecylthiocarbonylthio)sulfanylpentanoic acid is 10-100 mg / mL.

[0243] Optionally, in step (2), azobisisobutyronitrile can be replaced by 4,4'-azo(4-cyanovaleric acid).

[0244] Optionally, in step (2), the reaction temperature is 65-95° C. and the reaction time is 4-24 hours.

[0245] Optionally, in step (3), ursodeoxycholic acid can be replaced by other free bile acids or the bile acid monomers described above.

[0246] Optionally, in step (4), the molecular weight of PCB is 1000-10000 g / mol.

[0247] Optionally, in step (5), the molecular weight of PEG is 1000-10000 g / mol.

[0248] Example 1. Synthesis of CB-tBu

[0249] Synthesis of CB-tBu: Dissolve N,N-dimethylaminopropyl acrylamide (6 g, 38.4 mmol, 1 eq) and tert-butyl bromoacetate (8.5 mL, 57.6 mmol, 1.5 eq) in acetonitrile (35 mL). Stir the reactants in an oil bath (60 ° C) for 12 h and then cool to room temperature. The product is precipitated in ether and the obtained white precipitate is vacuum dried. The H NMR spectrum is shown in Figure 1 As shown: 1 HNMR (400 MHz, chloroform-d) δ 8.26 (t, J = 5.8 Hz, 1H), 6.49 (dd, J = 17.1, 10.2 Hz, 1H), 6.30 (dd, J = 17.1, 1.7 Hz, 1H), 5.63 (dd, J = 10.2, 1.7 Hz, 1H), 4.40 (s, 2H), 4.17–4.07 (m, 2H), 3.53–3.44 (m, 8H), 2.17 (m, 2H), 1.50 (s, 9H).

[0250] Example 2: Synthesis of PCB-tBu (5000)

[0251] Synthesis of PCB-tBu: 4-cyano-4-(dodecylthiothiocarbonyl)sulfanylpentanoic acid (108 mg, 0.27 mmol, 1 eq), CB-tBu (1.5 g, 5.4 mmol, 20 eq.), azobisisobutyronitrile (AIBN, 8.9 mg, 0.032 mmol, 0.1 eq) were dissolved in anhydrous N,N-dimethylformamide (DMF, 35 mL), and the reactants were placed in a 100 mL round-bottom flask and sealed with a sealing film. Freeze degassing cycle 3 times to remove oxygen, and then stirred in an oil bath at 70 ° C for 4 hours. After the reaction was completed, it was precipitated in cold ethyl acetate three times, and the precipitate was vacuum dried overnight to obtain a light yellow solid (i.e., compound 2). Its nuclear magnetic resonance hydrogen spectrum is shown as ( Figure 2 ) as shown: 1 H NMR (400MHz, chloroform-d) δ 0.83-0.93, 1.2-1.3, 1.4-1.7, 1.9-2.4, 3.1-3.7, 3.7-4.2, 4, 3-4.8. According to the H NMR spectrum ( Figure 2 ) determined that the degree of polymerization of the obtained compound 2 was 24.

[0252] Triethylamine (TEA, 375 μL, 2.7 mmol, 10 eq), n-hexylamine (357 μL, 2.7 mmol, 10 eq), 2 (1.5 g, 0.27 mmol, 1 eq) and tris(2-carboxyethyl) hydrochloride (TCEP, 115 mg, 0.4 mmol, 1.5 eq) were dissolved in DMF (10 mL). After stirring at room temperature for 4 h, the color of the reaction solution turned colorless. Then, the mixture was precipitated in ice-cold ethyl acetate and dried under vacuum to obtain a white solid. The H NMR spectrum is shown in ( Figure 3 ) as shown.

[0253] Example 3. Synthesis of PCB-tBu (2000)

[0254] Synthesis of PCB-tBu: 4-cyano-4-(dodecylthiothiocarbonyl)sulfanylpentanoic acid (108 mg, 0.27 mmol, 1 eq), CB-tBu (0.725 g, 2.7 mmol, 10 eq.), azobisisobutyronitrile (AIBN, 8.9 mg, 0.032 mmol, 0.1 eq) were dissolved in anhydrous N,N dimethylformamide (DMF, 35 mL), and the reactants were placed in a 100 mL round-bottom flask and sealed with a sealing film. Freeze degassing cycles were repeated 3 times to remove oxygen, and then stirred in an oil bath at 70 ° C for 4 hours. After the reaction was completed, it was precipitated in cold ethyl acetate three times, and the precipitate was vacuum dried overnight to obtain a light yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 0.83-0.93, 1.2-1.3, 1.4-1.7, 1.9-2.4, 3.1-3.7, 3.7-4.2, 4, 3-4.8. The polymer with the desired degree of polymerization was obtained according to the hydrogen nuclear magnetic resonance spectrum.

[0255] Triethylamine (TEA, 375 μL, 2.7 mmol, 10 eq), n-hexylamine (357 μL, 2.7 mmol, 10 eq), 2 (1.5 g, 0.27 mmol, 1 eq) and tris (2-carboxyethyl) hydrochloride (TCEP, 115 mg, 0.4 mmol, 1.5 eq) were dissolved in DMF (10 mL). After stirring at room temperature for 4 h, the color of the reaction solution became colorless. Then, the mixture was precipitated in ice-cold ethyl acetate and dried under vacuum to obtain a white solid.

[0256] Example 4, PCB-tBu (8000)

[0257] Synthesis of PCB-tBu: 4-cyano-4-(dodecylthiothiocarbonyl)sulfanylpentanoic acid (108 mg, 0.27 mmol, 1 eq), CB-tBu (3 g, 10.8 mmol, 40 eq.), azobisisobutyronitrile (AIBN, 8.9 mg, 0.032 mmol, 0.1 eq) were dissolved in anhydrous N,N dimethylformamide (DMF, 35 mL), and the reactants were placed in a 100 mL round-bottom flask and sealed with a sealing film. Freeze degassing cycles were repeated 3 times to remove oxygen, and then stirred in an oil bath at 70 ° C for 4 hours. After the reaction was completed, it was precipitated in cold ethyl acetate three times, and the precipitate was vacuum dried overnight to obtain a light yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 0.83-0.93, 1.2-1.3, 1.4-1.7, 1.9-2.4, 3.1-3.7, 3.7-4.2, 4, 3-4.8. The polymer with the desired degree of polymerization was obtained according to the hydrogen nuclear magnetic resonance spectrum.

[0258] Triethylamine (TEA, 375 μL, 2.7 mmol, 10 eq), n-hexylamine (357 μL, 2.7 mmol, 10 eq), 2 (1.5 g, 0.27 mmol, 1 eq) and tris (2-carboxyethyl) hydrochloride (TCEP, 115 mg, 0.4 mmol, 1.5 eq) were dissolved in DMF (10 mL). After stirring at room temperature for 4 h, the color of the reaction solution became colorless. Then, the mixture was precipitated in ice-cold ethyl acetate and dried under vacuum to obtain a white solid.

[0259] Example 5, Synthesis of PBA

[0260] Dissolve ursodeoxycholic acid (UDCA) (5.4 mmol), p-toluenesulfonic acid (0.652 mmol) and dimethylaminopyridine (0.652 mmol) in 60 mL of a mixed solution of anhydrous dichloromethane and anhydrous pyridine (5:1). Stir at 40 °C under nitrogen protection. Then add diisopropylcarbodiimide (DIC, 6.92 mmol) to the reaction mixture and react for 2 hours. After the reaction, precipitate the product pBA into 400 mL of cold anhydrous methanol, collect the precipitate by centrifugation and dry it to obtain a white powder. Its hydrogen nuclear magnetic resonance spectrum is as follows Figure 6 According to the GPC and NMR characterization results, the product weight average molecular weight MW is 2880 and the degree of polymerization is about 4.

[0261] Example 6. Synthesis of PCBBA (P2A1)

[0262] Dissolve PBA (1 mmol), PCB (1 mmol, degree of polymerization is about 24), EDCI (1.2 mmol) and dimethylaminopyridine (0.652 mmol) in DMF. Stir overnight under nitrogen protection. After the reaction is completed, precipitate the crude product into 400 mL of cold ethyl acetate, collect the precipitate by centrifugation and dry to obtain a white powder. Dissolve the product in dichloromethane, add trifluoroacetic acid (TFA, 10 eq) and stir at room temperature for 4 h, and drop the product into cold ethyl acetate for precipitation. Collect the precipitate by centrifugation and dry to obtain a white final product. The hydrogen nuclear magnetic resonance spectrum is shown as ( Figure 4 ), GPC ( Figure 5 ) indicates that its molecular weight is around 5000.

[0263] Example 7. Synthesis of PEGBA

[0264] Synthesis of PEGBA: PBA, PEG (5000), EDCI and dimethylaminopyridine were dissolved in DMF. The mixture was stirred overnight under nitrogen protection. After the reaction, the crude product was precipitated into cold ether, and the precipitate was collected by centrifugation and dried to obtain a white powder.

[0265] Example 8: Preparation of a polybetaine-cholic acid (PCBBA) nano-delivery carrier

[0266] 1. Preparation of blank micelles: Dissolve 15 mg of PCBBA in 1 mL of PBS and stir at room temperature for 2 h. After stirring evenly, remove larger particles through a 0.22 μm filter membrane and dialyze with a 10 kDa dialysis bag to remove small particles. Then use Malvern Zetasizer Nano to measure the particle size, PDI and zeta potential of the nanoparticles.

[0267] 2. Preparation of drug-loaded micelles: Dissolve 15 mg of PCBBA in 1 mL of PBS, then add 1 mL of insulin PBS solution (1 mg / mL) to the polymer solution and stir at room temperature for 2 hours. After stirring evenly, remove larger particles through a 0.22 μm filter membrane, and dialyze with a 10 kDa dialysis bag to remove small particles. Then use Malvern Zetasizer Nano to measure the particle size, PDI and zeta potential of the nanoparticles. Ultrafilter the prepared drug-loaded nanoparticles through a 50 kDa ultrafiltration tube to remove free insulin. Detect free insulin by BCA.

[0268] 3. The micelles prepared in step 2 were analyzed and observed by dynamic light scattering and transmission electron microscopy, and it was found that the micelle particle size was about 120nm, and the micelles were smooth and uniform spheres. The free insulin was separated by ultrafiltration, and the content of free insulin was determined by BCA protein quantitative method. The results showed that the micelles had an encapsulation rate of up to 92% for insulin and a drug loading of 9.1%. This experiment proved that insulin can be encapsulated in self-assembled micelles with a high encapsulation rate.

[0269] Example 9, a polybetaine-cholic acid (PCBBA) nano delivery carrier stability study:

[0270] PCBBA blank micelles were dispersed in PBS, and part of the obtained micelle solution was placed at room temperature (25°C), and part was stored in a refrigerator at 4°C. The particle size, PDI and Zeta potential of the micelles were measured using a Malvern Zetasizer Nano on day 0, day 10 and day 20, respectively.

[0271] like Figure 7 As shown, the particle size and potential of the micelles did not change significantly within 20 days, indicating that the PCBBA micelles have good stability.

[0272] Example 10: In vitro hemolysis test

[0273] Take fresh blood from mice and place it in a centrifuge tube containing anticoagulant and centrifuge at 1500rpm for 5 minutes, then slowly remove the supernatant. Add a little PBS to the centrifuge tube and mix well, then centrifuge at 1500rpm for 5 minutes in a centrifuge. Repeat the PBS washing step two or three times. The erythrocyte precipitate is gently dispersed with PBS, and micelles of different concentrations are added to the PBS solution of erythrocytes to prepare erythrocyte PBS solutions with different micelle concentrations (50, 100, 200, 500 and 1000μg / mL), and deionized water and PBS are used as positive and negative groups, respectively. Incubate all groups at 37°C for 4 hours and centrifuge after incubation. Determine the ultraviolet absorption value of the supernatant at 540nm, and calculate the hemolysis rate of the sample according to the formula.

[0274] Hemolysis (%) = ((A sample - A negative) / (A positive - A negative)) × 100%

[0275] The experimental results are as follows Figure 8 As shown, the carrier itself does not cause obvious hemolysis, indicating that the carrier has good biocompatibility.

[0276] Example 11, penetration rate of polybetaine-cholic acid (PCBBA) nano delivery carrier in mucus:

[0277] 100 μL of 6% (m / v) mucin solution was added to the upper chamber of the Transwell and shaken steadily until the solution became flat. The chambers were evenly divided into two groups, namely the PEGBA / FITC-insulin group and the PCBBA / FITC-insulin group. 200 μL of drug solution was carefully added above the mucin solution, and 800 μL of PBS solution was added to the lower chamber. The Tranwell chambers together with the well plate were placed in a shaker at 37°C and kept away from light. At predetermined time intervals, 200 μL of solution was drawn from the lower chamber to detect the fluorescence intensity, and an equal volume of PBS solution was added. After the experiment, the apparent permeability coefficient Papp was calculated according to the corresponding formula in cm / s, where Q is the drug permeation amount (mg), A is the bottom area of ​​the Transwell 12-well plate membrane, C0 is the initial drug concentration on the AP side (μg·mL-1), and t is the time.

[0278]

[0279] The experimental results show that ( Fig. 9 , left), in the first two hours, the penetration rate of polyzwitterionic micelles was much greater than that of PEGBA micelles, and the cumulative penetration was three times that of PEGBA micelles. By the time of sampling in the second hour, more than half of the PCBBA micelles had passed through the mucus layer into the lower chamber. In the fourth hour, 80.9% of the PCBBA micelles had diffused into the lower chamber. The apparent permeability coefficient ( Fig. 9 , right), the apparent permeability coefficient of PCBBA micelles is 1.96 times that of PEGBA micelles. In general, PCBBA micelles have a strong mucus permeation and diffusion effect. In practical applications, they can shorten the time for drugs to pass through the gastrointestinal mucus layer and improve the bioavailability of macromolecular drugs.

[0280] Example 12: Study on the Cellular Uptake Rate of Polybetaine-Cholic Acid (PCBBA) Nano-Delivery Carriers:

[0281] Free FITC-Insulin, polybetaine-polycholic acid micelles loaded with insulin (PCBBA / F-Insulin), and polyethylene glycol / cholic acid micelles loaded with insulin (PEGBA / F-Insulin) were co-cultured with Caco-2 cells, and the drugs were removed after 4 hours and washed with PBS for 3-5 times, and then fixed with 4% paraformaldehyde for 15 minutes. The cell nuclei were stained with DAPI under light-proof conditions, and the fluorescence signal density of FITC in the cells was observed under a laser confocal microscope. The results showed that ( Fig.10 ), the cells took up the most zwitterionic nanosystem, which proved that Caco-2 cells had a strong uptake of zwitterionic drug-loaded nanosystem.

[0282] Example 13: Use of polybetaine-cholic acid (PCBBA) micelles in the treatment and prevention of type 2 diabetes

[0283] PCBBA-loaded insulin micelles (PCBBA: 150 mg / kg) were administered to db / db mice for five consecutive days, and the oral PBS group (Control) and the subcutaneous insulin injection group (SC) (Insulin 5 IU / kg) were used as controls. In the third week after the administration, the body weight of the mice was measured and glucose tolerance test and insulin tolerance test were performed.

[0284] Glucose tolerance test (OGTT): The db / db mice were fasted overnight (20:00-8:00), but water was not allowed. Note that the mice's bedding needs to be changed before fasting. The fasting blood glucose of the mice was measured before administration, and then each mouse was orally administered with 20% glucose solution (1g / kg). The blood glucose level of the tail vein blood was measured at 30min, 60min and 120min after administration.

[0285] Insulin tolerance test (ITT), db / db mice were fasted for 4 hours, but not water. The mice's bedding was changed before fasting. The blood glucose level of the mice was measured after the fasting, and then insulin (2 IU / kg) was injected subcutaneously. The blood glucose level was measured at 30min, 60min, 90min and 120min after injection.

[0286] The experimental results show that ( Figure 11-13 ), PCBBA can significantly reduce the body weight of obese mice (the average weight of each mouse decreased by about 10g). At the same time, according to the changes in blood sugar and the area under the curve, it can be found that PCBBA can improve the glucose tolerance and insulin tolerance of mice, and has a certain improvement on the insulin sensitivity of mice.

[0287] Example 14: Use of polybetaine-cholic acid (PCBBA) micelles in the treatment of non-alcoholic fatty liver disease

[0288] PCBBA-loaded insulin micelles (PCBBA: 150 mg / kg) were administered to db / db mice for five consecutive days, and the oral PBS group (Control) and subcutaneous insulin injection (Insulin 5 IU / kg) group (SC) were used as controls. In the third week after the end of administration, the mouse serum was collected for biochemical analysis, and the mouse liver sections were taken for H&E staining and Oil Red O staining.

[0289] The experimental results show that ( Figure 14-16 ), PCBBA can improve the liver and kidney function of db / db mice, and significantly reduce the cavitation phenomenon and fat content in the liver of mice, reflecting a good therapeutic effect on non-alcoholic fatty liver disease.

[0290] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A polymer, characterized in that The polymer includes polycholic acid and a zwitterionic polymer connected to the polycholic acid by a covalent bond.

2. The polymer according to claim 1, characterized in that (a) the zwitterionic polymer is selected from Table A1; Table A1 wherein n is each independently an integer of 1-100, and n1 is each independently an integer of 1-10; and (b) The monomer of the polycholic acid is shown in formula A Among them, R 1 and R 2 Each independently selected from the following group: -H, -OH, C 1-6 alkyl.

3. The polymer according to claim 1, characterized in that The polymer is as shown in formula II; in, A 1 , A 2 and A 3 Each is independently selected from the following group: -H, -OH, C 1-6 Alkyl, zwitterionic polymer; and, A 1 , A 2 and A 3 at least one is a zwitterionic polymer; m is an integer from 1 to 100.

4. The polymer according to claim 1, characterized in that The polymer is selected from the following Table A: Table A Wherein, m is an integer of 1-100, n is an integer of 1-100 independently, and n1 is an integer of 1-10 independently; and the covalent bond is formed by the carboxyl group of the zwitterionic polymer and the hydroxyl group in the polycholic acid.

5. A method for preparing a polymer as claimed in claim 1, characterized in that: The preparation method comprises the steps of: (A) providing polycholic acid; (B) providing a zwitterionic polymer or an ester thereof; and (C) reacting polycholic acid with a zwitterionic polymer or an ester thereof, and optionally hydrolyzing the polymer to obtain the polymer.

6. A drug delivery carrier, characterized in that: The drug delivery carrier is a micelle or particle formed by the polymer described in the first aspect, or a solution containing the polymer described in the first aspect or micelles or particles formed thereby.

7. A nano drug preparation comprising: (i) micelles or particles formed from the polymer of the first aspect; and (ii) optionally a pharmaceutically acceptable carrier.

8. A nano drug-carrying composition, characterized in that: The nano drug delivery composition comprises the polymer as claimed in claim 1.

9. Use of the polymer according to claim 1, characterized in that: The use is one or more uses selected from the following group: a. For drug delivery; b. for preparing nano-delivery carriers; and c. Used to prepare nanomedicines.

10. Use of the polymer according to claim 1 in preparing a drug for treating or preventing a disease, characterized in that: The disease is selected from the following group: obesity, diabetes, tumors, enteritis, diseases caused by bacterial and viral infections, non-alcoholic fatty liver disease, liver disease, stomach disease, kidney disease, neuropathy, pneumonia or a combination thereof.

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