Polyzwitterionic cholic acid, its synthesis, and its application in drug delivery

Abstract

This invention provides poly(zwitterionic) cholic acid, its synthesis, and its application in drug delivery. Specifically, this invention provides a polymer formed by covalently linking polycholic acid and one or more zwitterionic polymers, wherein the polycholic acid forms the backbone (backbone) of the polymer; and nanomedicines formed from said polymer as carriers of micelles or particles.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a polyzwitterionic cholic acid, its synthesis, and its application in drug delivery. Background Technology

[0002] Due to their high specificity and therapeutic efficacy, the US FDA has approved a large number of biological macromolecular drugs in recent years. These drugs have rapidly become one of the most important classes of drugs in the pharmaceutical field, accounting for nearly half of all drug sales annually. However, due to their unique structure, macromolecular drugs are easily damaged by adverse external environments. Furthermore, their large molecular weight makes them difficult to absorb in the gastrointestinal tract. Therefore, these drugs are rarely administered orally.

[0003] With social development and progress, the number of obese and diabetic patients is rapidly increasing in China and worldwide. Simultaneously, these diseases can cause many related complications, leading to numerous unresolved medical problems. Taking diabetes as an example, insulin is the most important drug for treating diabetes; however, on the one hand, because it is difficult to deliver orally and can only be administered via injection, patient compliance is low. On the other hand, insulin only controls blood sugar and does not improve related complications.

[0004] In summary, there is an urgent need in this field to develop a new class of carriers that are well-suited for delivering biopharmaceutical drugs, especially drug delivery carriers that possess certain pharmaceutical activity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a novel carrier that serves as a drug carrier, particularly suitable for oral delivery of macromolecular biological drugs. Furthermore, it can be used alone as a drug for the prevention or treatment of various diseases, including obesity, tumors, neurological disorders, enteritis, liver disease, stomach disease, kidney disease, pneumonia, bacterial and viral infections, diabetes, and non-alcoholic fatty liver disease. Specifically, this invention provides a polyzwitterionic polycholic acid, its preparation method, and its applications, as well as methods for preparing micelles and carriers from the said polyzwitterionic polycholic acid.

[0006] In a first aspect of the invention, a polymer is provided, the polymer comprising polycholic acid and a zwitterionic polymer covalently bonded to the polycholic acid.

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

[0008] In another preferred embodiment, the polycholic acid has a degree of polymerization of an integer from 1 to 100.

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

[0010] In another preferred embodiment, the degree of polymerization of the polycholic acid is an integer from 2 to 15; more preferably, it is an integer from 2 to 10, and even more preferably, it is 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, more preferably 600-10000; and even more preferably 600-5000.

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

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

[0014] In another preferred embodiment, the zwitterionic polymer is covalently bonded to the ends and / or repeating unit structures of the polycholic acid.

[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, the polycholic acid is terminally connected to a zwitterionic polymer, and each unit structure is independently connected to 0, 1, or 2 zwitterionic polymers.

[0017] In another preferred embodiment, the zwitterionic polymers may be the same or different.

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

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

[0020] In another preferred embodiment, the degree of polymerization of the zwitterionic polymer is an integer from 1 to 100; more preferably, n is an integer from 2 to 100; even more preferably, n is an integer from 2 to 50; and most preferably, n is an integer from 10 to 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 an integer from 1 to 100 (preferably, n is an integer from 2 to 100; more preferably, n is an integer from 2 to 50; best of all, it is an integer from 10 to 50), and n1 is an integer from 1 to 10 (preferably, it is an integer from 1 to 5; best of all, it is 1, 2, or 3).

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

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

[0028]

[0029] Among them, R 1 and R 2 Each is independently selected from the following groups: -H, -OH, C 1-6 Alkyl groups (e.g., -CH3).

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

[0031]

[0032] Among them, R 1 and R 2 As defined in equation 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 of A is an amphoteric or zwitterionic polymer moiety; and at least one A is a zwitterionic polymer moiety;

[0041] m is an integer between 1 and 100.

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

[0043] In another preferred embodiment, m is an integer from 2 to 10; more 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 term derived from the zwitterionic polymer refers to the process in which the active groups (such as -COOH) in the zwitterionic polymer react with the active groups (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 is independently selected from the following groups: -H, -OH, C 1-6 Alkyl groups (such as -CH3), zwitterionic polymers; and, A 1 A 2 and A 3 At least one is an amphoteric 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 example, A 1 and A 2 Each is an independent -H or zwitterionic polymer.

[0053] In another preferred example, A 1 and A 2 Both are zwitterionic polymers.

[0054] In another preferred example, A 1 Selected from the following groups: -H, -OH, C 1-6 Alkyl groups (e.g., -CH3) (preferably A) 1 For) H and A 2 It is an amphoteric polymer.

[0055] In another preferred example, A 1 It is an amphoteric polymer and A 2 Selected from the following groups: -H, -OH, C 1-6 Alkyl groups (e.g., -CH3) (preferably A) 2 (For H).

[0056] In another preferred example, A 3 It is an amphoteric 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 Table A below:

[0060] Table A

[0061]

[0062]

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

[0064] In another preferred embodiment, in polycholic acid, some 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 invention, a method for preparing a polymer as described in the first aspect is provided, the method comprising the steps of:

[0066] (A) Provides polycholic acid;

[0067] (B) Provide zwitterionic polymers or their esterified forms; and

[0068] (C) Reaction of polycholic acid with a zwitterionic polymer or its esterified form, and optionally hydrolysis, to obtain the polymer.

[0069] In another preferred embodiment, the esterified product refers to the esterification of the acid radical portion in a zwitterionic polymer.

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

[0071] In another preferred embodiment, the polycholic acid is prepared by a cholic acid polymerization step, which includes: providing cholic acid monomers and causing the cholic acid monomers to undergo a polymerization reaction to obtain polycholic acid.

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

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

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

[0075] In another preferred embodiment, the polymerization reaction of the cholic acid monomer takes place for 0.5 to 5 hours, more preferably 1 to 3 hours, and even more preferably 2 hours.

[0076] In another preferred embodiment, the polymerization reaction of the cholic acid monomer is carried out at a reaction temperature of 30–100°C, more preferably 30–50°C, and even more preferably 35–45°C.

[0077] In another preferred embodiment, the method for preparing polycholic acid includes the steps of: dissolving cholic 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; most preferably, 5:1); stirring under nitrogen protection at 30–100°C (optimally 40±5°C); then adding diisopropylcarbodiimide to the resulting reaction mixture and reacting for 0.5–5 hours (preferably 1–3 hours, more preferably 2 hours); after the reaction is complete, precipitating the resulting product in cold anhydrous methanol, centrifuging to collect the precipitate, and drying it to obtain polycholic acid.

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

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

[0080] Table A1.1

[0081]

[0082] Among them, R E It 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 includes a RAFT reagent; preferably, the RAFT reagent is 4-cyano-4-(dodecylthiothiocarbonyl)thioalkylpentanoic acid.

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

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

[0086] In another preferred embodiment, the polymerization reaction takes 1-24 hours; more preferably, 4-24 hours.

[0087] In another preferred embodiment, the concentration of zwitterions or their esterified monomers in the polymerization system is 0.2-1 g / mL.

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

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

[0090] In another preferred embodiment, a synthesis step of the zwitterionic monomer or its esterified monomer is included prior to the zwitterionic polymerization step, which includes: reacting N,N-dimethylaminopropylacrylamide with... The reaction proceeds to obtain the zwitterion or its esterified monomer.

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

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

[0093] In another preferred embodiment, the drug delivery carrier is capable of delivering drugs.

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

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

[0096] In another preferred embodiment, the biopharmaceutical includes: protein and polypeptide drugs, nucleic acid drugs, poorly soluble drugs, antibody drugs, antibody-drug conjugates (ADCs), vaccine drugs, or combinations thereof.

[0097] In another preferred embodiment, the protein-peptide drugs include: insulin, glargine insulin, lispro insulin, aspart insulin, detemir insulin, protamine human insulin, degludec insulin, exenatide, liraglutide, semaglutide, dulaglutide, liximab, benaglutide, loxenatide, semaglutide, abiglutide, growth hormone, glatiramer acetate, lupron (leuprolide acetate), texipatide, motixafortide (BL-8040, BKT140), pemoxatide (EPO-018B), parathyroid hormone (PTH), trofinetide, interferon, TNF-α, C5, coagulation factor IX, interleukin (IL-2, I... Recombinant human coagulation factor VIII, recombinant human erythropoietin, recombinant human epidermal growth factor, recombinant human interferon α1b, recombinant human granulocyte colony-stimulating factor, recombinant insulin, recombinant interferon, recombinant coagulation factor, recombinant erythropoietin, recombinant granulocyte colony-stimulating factor, enzyme-replaced recombinant protein drugs, recombinant growth hormone, recombinant human prourokinase, 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 combinations thereof.

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

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

[0100] In another preferred embodiment, the poorly soluble drug includes: a Bcl-xl protein degrader (e.g., DT2216), an androgen receptor degrader (e.g., ARV-110, ARV-766, AR-LDD), an estrogen receptor degrader (e.g., ARV-471), an IRAK4 degrader (e.g., KT-474, KT-413), a STAT3 degrader (e.g., KT-333), a BTK degrader (e.g., NX-2127, NX-5948), a TRK degrader (e.g., CG001419), a BRD9 degrader (e.g., CFT-8634, FHD-609), thalidomide, lenalidomide, pomalidomide, or combinations 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, cimiplimab, toripalimab, tislelizumab, camrelizumab, prolgolimab, penpulimab, dostarlimab, zimberelimab, serplulimab, atezolizumab, and durvalumab. (Durvalumab), Avelumab, Sugemalimab, Envafolimab, Cadonilimab, Bamlanivimab, Tebentafusp, Rituximab, Catuximab, 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), catutoxab, moxetumomabpasudotox, obinutuzumab, mogamulizumab, edrecolomab, nebacumab, benralizumab, inebilizumab, or combinations thereof.

[0103] In another preferred embodiment, the vaccine drug includes: polio vaccine, cholera vaccine, typhoid live vaccine, SARS-CoV-2 vaccine, HPV vaccine, hepatitis vaccine, tumor vaccine, Japanese encephalitis vaccine, shingles vaccine, influenza vaccine, BCG vaccine, measles vaccine, plague vaccine, fowlpox virus vaccine, monkeypox virus vaccine, adenovirus vaccine, herpesvirus vaccine, salmonella vaccine, Shigella vaccine, DPT vaccine, pneumococcal vaccine, avian influenza vaccine, pneumonia vaccine, Asian swine fever vaccine, or combinations 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) Provide 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 is mixed with an optional drug, and the polymer is then formed into micelles or particles encapsulating the optional drug by direct dissolution or dialysis.

[0108] In a fifth aspect of the invention, a nanomedicine formulation is provided, comprising:

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

[0110] (ii) Optionally, a pharmaceutically acceptable carrier.

[0111] In another preferred embodiment, the nanomedicine formulation further includes a drug optionally encapsulated in the micelles or particles.

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

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

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

[0115] In another preferred embodiment, the nanomedicine formulation is an oral formulation or an injectable formulation; preferably, the nanomedicine formulation is an oral formulation.

[0116] In another preferred embodiment, the nanomedicine formulation is a formulation selected from the group consisting of capsules, tablets, pills, powders, granules, emulsions, solutions, suspensions, syrups, tinctures, or combinations thereof.

[0117] In a sixth aspect of the invention, a nano-drug-carrying composition is provided, the nano-drug-carrying composition comprising the polymer as described in the first aspect.

[0118] In another preferred embodiment, the nano-drug-loaded composition may also include an optional drug.

[0119] In another preferred embodiment, the polymer exists either in the form of micelles or particles, or in the form of no 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 biological macromolecular drug.

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

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

[0124] In another preferred embodiment, the nano-drug-loaded composition is used to prepare nanomedicine formulations.

[0125] In a seventh aspect of the invention, a use of the polymer as described in the first aspect is provided, wherein the use is one or more uses selected from the group consisting of:

[0126] a. Used for delivering medicine;

[0127] b. Used in the preparation of nanodelivery carriers; and

[0128] c. Used in the preparation of nanomedicines or particulate drugs.

[0129] In another preferred embodiment, the nanomedicine may be either unencapsulated with any other drug (such as a biopharmaceutical) or encapsulated with a biopharmaceutical.

[0130] In an eighth aspect of the invention, there is provided the use of the polymer as described in the first aspect in the preparation of a medicament for treating or preventing a disease selected from the group consisting of: 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 combinations thereof.

[0131] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0132] Figures 1-4 The 1H NMR spectra of PCBBA and key intermediates in the synthesis process shown in Examples 1, 2 and 6 are displayed 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 the PBA prepared in Example 5 is shown.

[0135] Figure 7 The changes in particle size, PDI, and Zeta potential of polybetaine / cholic acid micelles are shown. Transmission electron microscopy was used to observe the morphology of PCBBA and PEGBA micelles. The results demonstrate that the micelles are smooth, uniform spheres with excellent stability.

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

[0137] Figure 9 The results of the study on the penetration rate of micelles in mucus in Example 10 (n=3) are shown, where the left figure shows the amount of drug accumulated in the lower chamber at different times, and the right figure shows the apparent permeability coefficient calculated based on the penetration rate at the fourth hour.

[0138] Figure 10 The image shows the uptake of micelles by Caco-2 cells in Example 11, where PCBBA micelles were taken up by cells more quickly and exhibited higher fluorescence intensity.

[0139] Figure 11 The body weights of type 2 diabetic mice (n=3) after treatment in Example 12 are shown.

[0140] Figure 12 The results of the glucose tolerance test (n=3) in type 2 diabetic mice after treatment in Example 12 are shown. The left figure shows the changes in blood glucose levels in the mice at the corresponding time points, and the right figure shows the area under the curve.

[0141] Figure 13The insulin test (n=3) of type 2 diabetic mice after treatment in Example 12 is shown. The left figure shows the changes in blood glucose in the mice at the corresponding time points, and the right figure shows the area under the curve.

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

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

[0144] Figure 16 The images show oil red staining (first row), H&E staining (second row), and H&E staining of white adipose tissue in liver sections of type II diabetic mice treated in Example 13.

[0145] Figure 17 The diagram shows the preparation and function of drug-loaded micelles. Detailed Implementation

[0146] Through long-term and in-depth research, the inventors have, for the first time, prepared micelles using a zwitterionic polymer as the hydrophilic group and polycholic acid as the hydrophobic group. These micelles exhibit better permeability compared to other micelles, such as those using PEG as the hydrophilic group. Furthermore, experimental verification has shown that these micelles also possess excellent drug loading and delivery capabilities, making them suitable as delivery carriers for biopharmaceutical drugs. Moreover, this carrier can be used alone as a drug for the prevention and treatment of diseases such as diabetes, obesity, and non-alcoholic fatty liver disease. Based on these findings, the inventors have completed this invention.

[0147] the term

[0148] Unless otherwise defined, 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 pertains.

[0149] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. 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 terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed 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] In this article, "zwitterionic polymer", "zwitterionic material" and "zwitterionic polymer" can be used interchangeably, referring to polymers formed by the polymerization of molecules containing the same number of positively and negatively charged functional groups in chemistry.

[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 are characterized by high dipole moments and highly charged groups, while possessing equimolar amounts of cations and anions, maintaining overall electroneutrality and high hydrophilicity. Zwitterions can form a super-strong hydration shell with water through ion-dipole interactions, thus zwitterionic polymers can reduce the adsorption of non-specific proteins and bacterial adhesion, thereby endowing carriers with superior mucus permeability. In addition to excellent antifouling properties, zwitterionic materials can also enhance biocompatibility, reduce immune responses, and prolong cycle time.

[0155] bile acids

[0156] Bile acids (BA), important components of bile, are synthesized from cholesterol in the liver and stored in the gallbladder. Bile acids participate in the absorption, distribution, metabolism, and excretion of nutrients in the body. They also act as nutrient sensors and metabolic regulators, activating nuclear farnesoid X receptor (FXR) and G protein-coupled bile acid receptor (TGR) signaling to participate in glucose and energy metabolism and maintain metabolic homeostasis. Furthermore, as natural products, bile acids can function as hydrophobic groups in micelles, exhibiting good biocompatibility; they can also control energy homeostasis and possess certain anti-inflammatory effects.

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

[0158] Poly(zwitterionic)-cholic acid and its preparation method

[0159] One object of this invention is to provide a polyzwitterionic polycholic acid and its synthetic route. Firstly, the compound (or polymer) is an amphiphilic molecule, with its hydrophilic end composed of a zwitterionic polymer (or polyzwitterionic polymer) and its hydrophobic end composed of polycholic acid. Secondly, this compound can be used to prepare drug-loaded nanoparticles for drug delivery.

[0160] In this document, the terms “polymer of the present invention,” “poly-zwitterionic cholic acid,” “poly-zwitterionic cholic acid,” “poly-zwitterionic / cholic acid,” “poly-zwitterionic / polycholic acid,” “zwitterionic polymer / polycholic acid,” “poly-zwitterionic cholic acid molecule,” and “poly-zwitterionic cholic acid” are used interchangeably to refer to the polymer as defined in the first aspect of the present invention.

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

[0162] In another embodiment, the polymer is as defined in the first aspect.

[0163] In another embodiment, the polymer is capable of forming a nanodelivery carrier (specifically, nanomicelles or particles).

[0164] In another embodiment, the micelles or particles are 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 following structural formula:

[0166]

[0167] Where A is a -H, -OH, -CH3 and zwitterionic polymer molecules, P is a cholic acid molecule, and m can be an integer between 1 and 100.

[0168] In another embodiment, A is an amphoteric polymer; more preferably, it is selected from Table A1 below:

[0169] Table A1

[0170]

[0171] Where n is the aggregation degree, which is optional and can be an integer between 1 and 100. n1 is an integer between 1 and 10.

[0172] In other implementations, P is bile acid.

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

[0174] Table A2

[0175]

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

[0177]

[0178]

[0179] Where 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 the polycholic acid are linked to the zwitterionic polymer, the polymer is as described in Table C:

[0182] Table C

[0183]

[0184]

[0185]

[0186] It should be understood that in the polymer, the hydroxyl groups of polycholic acid can be ungrafted or connected to zwitterionic polymers.

[0187] In another embodiment, the polymer is used to prepare a nanoparticle-loaded drug composition.

[0188] In another embodiment, the present invention also provides the use of the polymer in the treatment of diseases, preferably including, but 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) Provides polycholic acid;

[0191] (B) Provide zwitterionic polymers or their esterified forms; and

[0192] (C) Reaction of polycholic acid with a zwitterionic polymer or its esterified form, and optionally hydrolysis, 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 includes the following steps when the selected zwitterion is betaine.

[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 diethyl ether and dried under vacuum to obtain a white precipitate 1.

[0196] (2) Synthesis of PCB-tBu: 4-cyano-4-(dodecylthiothiocarbonyl)thioalkylpentanoic acid, CB-tBu, and azobisisobutyronitrile were dissolved in anhydrous N,N-dimethylformamide. The reactants were placed in a round-bottom flask and sealed with a sealing film. Oxygen was removed by three cycles of cryogenic degassing, followed by stirring in an oil bath at 70°C for 4 hours. After the reaction was complete, the product was precipitated three times in cold ethyl acetate, and the precipitate was dried under vacuum to obtain a pale yellow solid. The product was dissolved in DMF with triethylamine, n-hexylamine, and tris(2-carboxyethyl) hydrochloride. After stirring at room temperature for 4 hours, the reaction solution became 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). The mixture was stirred at 30℃-100℃ (optimal 40±5℃) under nitrogen protection. Then, diisopropylcarbodiimide was added to the reaction mixture, and the reaction was allowed to proceed for 2 hours. After the reaction was completed, the product pBA was precipitated in cold anhydrous methanol, 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 was complete, the crude product was precipitated in cold ethyl acetate. The precipitate was collected by centrifugation and dried to obtain a white powder. The product was dissolved in dichloromethane, and trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 4 hours. The product was then added dropwise to cold ethyl acetate to precipitate. 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-(dodecylthiothiocarbonyl)thioalkylpentanoic acid is 10-100 mg / mL.

[0200] In another embodiment, in step (2), azobisisobutyronitrile can be replaced with 4,4'-azo (4-cyanopentanoic 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 the PCB is 1000-10000 g / mol.

[0203] Nanodelivery carriers, nanomedicines and their preparation methods

[0204] Another objective of this invention is to provide a nanocarrier (or nanodelivery carrier) and its preparation method. Firstly, this nanocarrier exhibits good biocompatibility and mucus permeability. Secondly, this carrier can improve symptoms such as obesity and insulin sensitivity in type II diabetic mice. Furthermore, the preparation method provided by this invention is simple, the materials are readily available, and the reaction conditions are mild, thus possessing promising industrialization potential.

[0205] In one specific implementation, such as Figure 17 As shown, the nanodelivery carrier is a drug-loaded micelle or particle formed by the self-assembly of the polymer (i.e., polyzwitterionic-cholic acid) of the present invention. The nanodelivery carrier comprises a hydrophilic shell containing zwitterions and a hydrophobic core of polycholic acid. Optionally, a drug, such as a macromolecular drug, is loaded into the hydrophobic core. The periphery of the drug-loaded micelles or particles is a hydrophilic zwitterion, which can protect the macromolecular drug and promote its penetration rate in the intestine, thereby improving the oral bioavailability of the macromolecular drug. Furthermore, after entering the bloodstream, the micelles or particles can effectively resist non-specific protein adsorption, enabling the drug-loaded micelles or particles to achieve stable long-term circulation in the blood.

[0206] In one specific embodiment, a method for preparing the nanodelivery carrier is provided, comprising the steps of:

[0207] At least one of the polymers described in this invention (i.e., polyzwitterionic-cholic acid) is mixed with an optional biomolecular drug, and drug-loaded nanomicelles or particles are prepared by direct dissolution or dialysis.

[0208] Alternatively, the method involves simultaneously dissolving the amphiphilic polymer and insulin in PBS and stirring for 2 hours to ensure uniform dispersion. The mixture is then placed in a 10K dialysis bag and dialyzed with PBS to obtain drug-loaded micelles.

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

[0210] Alternatively, the particle size of micelles or particles can be between 10 and 300 nm.

[0211] Alternatively, in the aforementioned drug-loaded nanomicelles or particles, the drug encapsulated in the hydrophobic core is one or more of the following: peptide drugs (insulin, GLP-1, etc.), protein drugs, nucleic acid drugs, poorly soluble small molecule drugs, and fluorescent molecules. Insulin and GLP-1 are preferred.

[0212] Alternatively, the encapsulation efficiency of the aforementioned drug-loaded nanomicelles or particles ranges from 50% to 95%, and the drug loading is from 5% to 15%.

[0213] The successful preparation of micelles or particles, exhibiting smooth spherical shapes, was confirmed by particle size analysis, zeta potential measurement, and transmission electron microscopy. Blank micelles were placed at room temperature and 4°C, and changes in particle size and PDI were observed periodically, demonstrating good stability. In vitro mucus permeation and cell permeation experiments confirmed the strong mucus permeability of the micelles. In conclusion, these micelles represent an excellent oral delivery carrier.

[0214] Pharmaceutical compositions, nanomedicine formulations and methods of administration

[0215] Since the polymers of the present invention and the micelles or particles formed therefrom have excellent preventive and therapeutic effects on diseases such as diabetes, obesity and non-alcoholic fatty liver, and have excellent drug loading and drug delivery capabilities, they can be used as drug delivery carriers. Therefore, micelles or blank micelles encapsulating other drugs formed by the polymers of the present invention, as well as pharmaceutical compositions or nanomedicine formulations containing the polymers 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 compositions or nanomedicine formulations of the present invention comprise micelles (or particles) of the present invention and pharmaceutically acceptable carriers within a safe and effective range. "Safe and effective range" refers to a amount of micelles (or particles) or other drugs contained therein 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 that 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 and with the compounds of the present invention without significantly reducing the efficacy of the compounds.

[0218] The administration method of the micelles (or particles) or pharmaceutical compositions of the present invention is not particularly limited, and representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and local administration. Considering the excellent delivery properties of the micelles or particles of the present invention, the micelles or particles and pharmaceutical compositions of the present invention are particularly suitable for oral administration. Therefore, the nanomedicine formulations of the present invention are preferably oral formulations.

[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 this invention include:

[0221] 1. This invention uses non-immunogenic, readily drug-loadable polyzwitterionic / polycholic acid (e.g., PCBBA) as the basic unit. The prepared PCBBA nanocarrier raw material is inexpensive, readily available, and has excellent biocompatibility. The synthesis conditions are mild, making it an excellent oral nanocarrier.

[0222] 2. The polyzwitterionic-polycholic acid nanocarrier of the present invention is a regular and uniform sphere with high encapsulation efficiency and drug loading capacity, and can load a variety of biological macromolecular drugs including insulin and GLP-1.

[0223] 3. The polyzwitterionic-polycholic acid (such as PCBBA) nanocarrier of the present invention can rapidly penetrate into mucus and can also be taken up in large quantities by cells, showing high potential for oral delivery.

[0224] 4. The polyzwitterionic-polycholic acid nanocarrier of the present invention can reduce the body weight of type II diabetic mice and improve the insulin sensitivity of mice.

[0225] In summary, this invention synthesizes polyzwitterionic / cholic acid micelles and uses them as monomers to self-assemble into micelles. The polybetaine / polycholic acid (e.g., PCBBA) micelles of this invention exhibit excellent biocompatibility and stability, and can effectively load various macromolecular drugs, including insulin and gene therapies, as well as poorly soluble small molecule drugs. PCBBA micelles have strong permeability in mucus and can be rapidly taken up by cells. Furthermore, the carrier itself can improve obesity and insulin resistance in type 2 diabetic mice, demonstrating significant preventative and therapeutic effects.

[0226] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0227] General preparation methods for polymers

[0228] The polymers in the examples can be synthesized by the following methods:

[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 dried under vacuum to obtain a white precipitate (compound 1).

[0230]

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

[0232] (2.1) 4-Cyano-4-(dodecylthiothiocarbonyl)thioalkylpentanoic acid, CB-tBu, and azobisisobutyronitrile (AIBN) were dissolved in anhydrous N,N-dimethylformamide (DMF). The reactants were placed in a round-bottom flask and sealed with sealing film. The mixture was degassed three times under freeze-drying conditions to remove oxygen, and then stirred in an oil bath at 70°C for 4 hours. After the reaction was complete, the mixture was precipitated three times in cold ethyl acetate. The precipitates were dried under vacuum to give a pale yellow solid (i.e., compound 2).

[0233] (2.2) The product was dissolved in DMF with triethylamine, n-hexylamine, and tris(2-carboxyethyl) hydrochloride. After stirring at room temperature for 4 h, 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). The mixture was stirred at 40°C under nitrogen protection. Then, diisopropylcarbodiimide was added to the reaction mixture, and the reaction was allowed to proceed for 2 hours. After the reaction was complete, the product pBA was precipitated in cold anhydrous methanol, the precipitate was collected by centrifugation and dried to obtain a white powder of PBA.

[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 was complete, the crude product was precipitated in cold ethyl acetate. The precipitate was collected by centrifugation and dried to obtain a white powder. The product was dissolved in dichloromethane, and trifluoroacetic acid (TFA) was added and stirred at room temperature for 4 h. The product was then added dropwise to cold ethyl acetate to precipitate. 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 was completed, the crude product was precipitated in cold diethyl ether, 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-(dodecylthiothiocarbonyl)thioalkylpentanoic acid is 10-100 mg / mL.

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

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

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

[0246] Optionally, in step (4), the molecular weight of the 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: N,N-dimethylaminopropylacrylamide (6 g, 38.4 mmol, 1 eq) and tert-butyl bromoacetate (8.5 mL, 57.6 mmol, 1.5 eq) were dissolved in acetonitrile (35 mL). The reaction mixture was 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 resulting white precipitate was dried under vacuum. The 1H NMR spectrum is shown below. Figure 1 As shown: 1 ¹H NMR (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)thioalkylpentanoic acid (108 mg, 0.27 mmol, 1 eq), CB-tBu (1.5 g, 5.4 mmol, 20 eq.), and azobisisobutyronitrile (AIBN, 8.9 mg, 0.032 mmol, 0.1 eq) were dissolved in anhydrous N,N-dimethylformamide (DMF, 35 mL). The reactants were placed in a 100 mL round-bottom flask and sealed with sealing film. Oxygen was removed by three cycles of cryogenic degassing, followed by stirring in an oil bath at 70 °C for 4 hours. After the reaction, the mixture was precipitated three times in cold ethyl acetate. The precipitate was dried under vacuum overnight to give a pale yellow solid (compound 2). Its 1H NMR spectrum is shown below. Figure 2 As shown in the image: 1 ¹H 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. Based on the ¹H NMR spectrum (… Figure 2 The degree of polymerization of the obtained compound 2 was determined to be 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 reaction solution turned colorless. The mixture was then precipitated in ice-cold ethyl acetate and dried under vacuum to give a white solid. The 1H NMR spectrum is shown below. Figure 3 As shown in the figure.

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

[0254] Synthesis of PCB-tBu: 4-Cyano-4-(dodecylthiothiocarbonyl)thioalkylpentanoic acid (108 mg, 0.27 mmol, 1 eq), CB-tBu (0.725 g, 2.7 mmol, 10 eq.), and azobisisobutyronitrile (AIBN, 8.9 mg, 0.032 mmol, 0.1 eq) were dissolved in anhydrous N,N-dimethylformamide (DMF, 35 mL). The reactants were placed in a 100 mL round-bottom flask and sealed with sealing film. The mixture was degassed three times under freeze-drying to remove oxygen, and then stirred in an oil bath at 70 °C for 4 hours. After the reaction was complete, the mixture was precipitated three times in cold ethyl acetate. The precipitate was dried under vacuum overnight to give a pale yellow solid. 1 ¹H 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. The desired degree of polymerization was determined based on the ¹H NMR 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 reaction solution became colorless. The mixture was then precipitated in ice-cold ethyl acetate and dried under vacuum to give a white solid.

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

[0257] Synthesis of PCB-tBu: 4-Cyano-4-(dodecylthiothiocarbonyl)thioalkylpentanoic acid (108 mg, 0.27 mmol, 1 eq), CB-tBu (3 g, 10.8 mmol, 40 eq.), and azobisisobutyronitrile (AIBN, 8.9 mg, 0.032 mmol, 0.1 eq) were dissolved in anhydrous N,N-dimethylformamide (DMF, 35 mL). The reactants were placed in a 100 mL round-bottom flask and sealed with sealing film. The mixture was degassed three times under freeze-drying to remove oxygen, and then stirred in an oil bath at 70 °C for 4 hours. After the reaction was complete, the mixture was precipitated three times in cold ethyl acetate. The precipitate was dried under vacuum overnight to give a pale yellow solid. 1 ¹H 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. The desired degree of polymerization was determined based on the ¹H NMR 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 reaction solution became colorless. The mixture was then precipitated in ice-cold ethyl acetate and dried under vacuum to give a white solid.

[0259] Example 5: Synthesis of PBA

[0260] Ursodeoxycholic acid (UDCA) (5.4 mmol), p-toluenesulfonic acid (0.652 mmol), and dimethylaminopyridine (0.652 mmol) were dissolved in 60 mL of a mixture of anhydrous dichloromethane and anhydrous pyridine (5:1). The mixture was stirred at 40 °C under nitrogen protection. Then, diisopropylcarbodiimide (DIC, 6.92 mmol) was added to the reaction mixture, and the reaction was allowed to proceed for 2 hours. After the reaction was complete, the product pBA was precipitated in 400 mL of cold anhydrous methanol. The precipitate was collected by centrifugation and dried to obtain a white powder. Its 1H NMR spectrum is shown below. Figure 6 As shown. Based on GPC and NMR characterization results, the weight-average molecular weight (MW) of the product was determined to be 2880, and the degree of polymerization was approximately 4.

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

[0262] PBA (1 mmol), PCB (1 mmol, degree of polymerization approximately 24), EDCI (1.2 mmol), and dimethylaminopyridine (0.652 mmol) were dissolved in DMF. The mixture was stirred overnight under nitrogen protection. After the reaction was complete, the crude product was precipitated in 400 mL of cold ethyl acetate. The precipitate was collected by centrifugation and dried to obtain a white powder. The product was dissolved in dichloromethane, and trifluoroacetic acid (TFA, 10 eq) was added. The mixture was stirred at room temperature for 4 h, and the product was added dropwise into cold ethyl acetate to precipitate. The precipitate was collected by centrifugation and dried to obtain a white final product. The 1H NMR spectrum is shown below. Figure 4 As shown in the figure, GPC ( Figure 5 This 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 was complete, the crude product was precipitated in cold diethyl ether, the precipitate was collected by centrifugation and dried to obtain a white powder.

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

[0266] 1. Preparation of blank micelles: 15 mg of PCBBA was dissolved in 1 mL of PBS and stirred at room temperature for 2 h. After thorough mixing, larger particles were removed by passing the solution through a 0.22 μm filter membrane, and smaller particles were removed by dialysis using a 10 kDa dialysis bag. The particle size, PDI, and zeta potential of the nanoparticles were then measured using a Malvern Zetasizer Nano.

[0267] 2. Preparation of drug-loaded micelles: 15 mg of PCBBA was dissolved in 1 mL of PBS, and then 1 mL of insulin PBS solution (1 mg / mL) was added to the polymer solution. The mixture was stirred at room temperature for 2 h. After homogenization, larger particles were removed by passing the solution through a 0.22 μm filter membrane, and smaller particles were removed by dialysis using a 10 kDa dialysis bag. The particle size, PDI, and zeta potential of the nanoparticles were then measured using a Malvern Zetasizer Nanoparticle. The prepared drug-loaded nanoparticles were then ultrafiltered through a 50 kDa ultrafiltration tube to remove free insulin. Free insulin was detected by BCA.

[0268] 3. Analysis and observation of the micelles prepared in step 2 using dynamic light scattering and transmission electron microscopy revealed that the micelle size was approximately 120 nm, and the micelles were smooth and uniform spheres. Free insulin was separated by ultrafiltration, and the content of free insulin was determined by BCA protein quantification. The results showed that the encapsulation efficiency of insulin by these micelles could reach up to 92%, with a drug loading of 9.1%. This experiment demonstrates that insulin can be encapsulated in self-assembled micelles with a high encapsulation efficiency.

[0269] Example 9: Stability study of a polybetaine-cholic acid (PCBBA) nanocarrier:

[0270] Blank PCBBA micelles were dispersed in PBS. Part of the resulting micelle solution was stored at room temperature (25°C), and the other 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 days 0, 10, and 20.

[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] Fresh mouse blood was collected and centrifuged at 1500 rpm for 5 minutes in a centrifuge tube containing anticoagulant. The supernatant was slowly aspirated. A small amount of PBS was added to the centrifuge tube and mixed well. The tube was then centrifuged at 1500 rpm for 5 minutes. The PBS washing step was repeated two to three times. The erythrocyte pellet was gently dispersed with PBS. Different concentrations of micelles were added to the PBS solution of erythrocytes to prepare erythrocyte PBS solutions with different micelle concentrations (50, 100, 200, 500, and 1000 μg / mL). Deionized water and PBS were used as positive and negative groups, respectively. All groups were incubated at 37°C for 4 hours, followed by centrifugation. The UV absorbance of the supernatant at 540 nm was measured, and the hemolysis rate of the sample was calculated using the formula.

[0274] Hemolysis (%) = ((Sample A - Negative A) / (Positive A - Negative A)) × 100%

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

[0276] Example 11, Permeation rate of polybetaine-cholic acid (PCBBA) nanocarrier in mucus:

[0277] 100 μL of 6% (m / v) mucin solution was added to the upper chamber of the Transwell plate and gently shaken until the solution leveled. The chambers were divided into two groups: a PEGBA / FITC-insulin group and a PCBBA / FITC-insulin group. 200 μL of the drug solution was carefully added above the mucin solution, while 800 μL of PBS solution was added to the lower chamber. The Transwell chambers, along with the plate, were placed in a shaker at 37°C and protected from light. At predetermined time intervals, 200 μL of the solution was aspirated from the lower chamber to detect fluorescence intensity, and an equal volume of PBS solution was added. After the experiment, the apparent permeability coefficient Papp was calculated using the corresponding formula, in cm / s, where Q is the drug permeation volume (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 time.

[0278]

[0279] Experimental results show that ( Figure 9 (Left figure) In the first two hours, the permeation rate of zwitterionic micelles was much greater than that of PEGBA micelles, with a cumulative permeation volume 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 and entered the lower chamber. In the fourth hour, 80.9% of the PCBBA micelles had diffused into the lower chamber. The apparent permeability coefficient was calculated based on the permeation data over the four hours. Figure 9 (See right figure). The apparent permeability coefficient of PCBBA micelles is 1.96 times that of PEGBA micelles. Overall, PCBBA micelles exhibit extremely strong mucus penetration and diffusion properties, which can shorten the time for drugs to pass through the gastrointestinal mucus layer and improve the bioavailability of macromolecular drugs in practical applications.

[0280] Example 12: Study on the uptake rate of polybetaine-cholic acid (PCBBA) nanocarriers by cells:

[0281] Free FITC-Insulin, insulin-loaded polybetaine-polycholic acid micelles (PCBBA / F-Insulin), and insulin-loaded polyethylene glycol / cholic acid micelles (PEGBA / F-Insulin) were co-cultured with Caco-2 cells. After 4 hours, the drugs were aspirated, and the cells were washed 3-5 times with PBS, followed by fixation with 4% paraformaldehyde for 15 minutes. Cell nuclei were stained with DAPI under light-protected conditions, and the fluorescence signal density of FITC in the cells was observed under a laser confocal microscope. The results showed that ( Figure 10 The cells showed the highest uptake of zwitterionic nanosystems, demonstrating that Caco-2 cells have a strong uptake of zwitterionic drug-loaded nanosystems.

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

[0283] db / db mice were administered PCBBA-loaded insulin micelles (PCBBA: 150 mg / kg) for five consecutive days, while an oral PBS group (Control) and a subcutaneous insulin injection group (Insulin 5 IU / kg) group (SC) served as controls. Three weeks after the end of administration, mouse body weight was measured, and glucose tolerance and insulin tolerance tests were performed.

[0284] Oral glucose tolerance test (OGTT): db / db mice were fasted overnight (20:00-8:00), but water was allowed. Note that the bedding should be changed before fasting. Fasting blood glucose levels were measured before administration, and then each mouse was orally administered a 20% glucose solution (1g / kg). Blood glucose levels in the tail vein were measured at 30 min, 60 min, and 120 min after administration.

[0285] Insulin tolerance test (ITT), db / db mice were fasted for 4 hours but allowed free access to water. The bedding was changed before fasting. Blood glucose levels were measured after fasting, followed by subcutaneous injection of insulin (2 IU / kg). Blood glucose levels were measured at 30, 60, 90, and 120 minutes post-injection.

[0286] Experimental results show that ( Figure 11-13 PCBBA significantly reduced the weight of obese mice (by an average of about 10g per mouse). Furthermore, based on changes in blood glucose levels and the area under the curve, PCBBA improved glucose tolerance and insulin tolerance in mice, and also showed some improvement in insulin sensitivity.

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

[0288] For five consecutive days, db / db mice were administered PCBBA-loaded insulin micelles (PCBBA: 150 mg / kg), while an oral PBS group (Control) and a subcutaneous insulin injection group (SC) served as controls. Three weeks after the end of administration, mouse serum was collected for biochemical analysis, and mouse liver sections were collected for H&E staining and Oil Red O staining.

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

[0290] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A polymer, characterized in that, The polymer comprises polycholic acid and a zwitterionic polymer covalently bonded to the polycholic acid; and, The polymer is as shown in Formula II; in, A 1 A 2 and A 3 Each is independently selected from the following groups: -H, -OH, C 1-6 Alkyl, zwitterionic polymers; and, A 1 A 2 and A 3 At least one is an amphoteric polymer; m is an integer from 3 to 50; The zwitterionic polymer is selected from the group consisting of: Where n is an integer from 1 to 100, and n1 is an integer from 1 to 10.

2. The polymer according to claim 1, characterized in that, The polymers mentioned are selected from Table A below: Table A Where m is an integer from 3 to 50, n is an integer from 1 to 100, and n1 is an integer from 1 to 10; and the covalent bond is formed by the carboxyl group of the zwitterionic polymer and the hydroxyl group of polycholic acid.

3. A method for preparing the polymer as described in claim 1, characterized in that, The preparation method includes the following steps: (A) Provides polycholic acid; (B) Provide zwitterionic polymers or their esterified forms; and (C) Reaction of polycholic acid with a zwitterionic polymer or its esterified form, and optionally hydrolysis, to obtain the polymer.

4. A drug delivery carrier, characterized in that, The drug delivery carrier is a micelle or particle formed from the polymer as described in claim 1, or a solution containing the polymer as described in claim 1 or micelles or particles formed therefrom.

5. A method for preparing a drug delivery carrier, characterized in that, Including the following steps: (1) The polymer as claimed in claim 1; and (2) The polymer is formed into micelles or particles by direct dissolution or dialysis.

6. A nanomedicine formulation, comprising: (i) micelles or particles formed from the polymer as described in claim 1; and (ii) optionally a pharmaceutically acceptable carrier.

7. A nano-drug-loaded composition, characterized in that, The nano-drug-carrying composition comprises the polymer as described in claim 1.

8. Use of the polymer as claimed in claim 1, characterized in that, The use described is one or more uses selected from the group consisting of: a. Used for delivering medicine; b. Used in the preparation of nanodelivery carriers; and c. Used in the preparation of nanomedicines.

9. Use of the polymer as described in claim 1 in the preparation of a medicament for treating or preventing a disease, characterized in that, The diseases mentioned are selected from the following group: obesity, diabetes, tumors, enteritis, diseases caused by bacterial or viral infections, liver disease, stomach disease, kidney disease, neuropathy, pneumonia, or a combination thereof.

10. The use as described in claim 9, characterized in that, The liver diseases mentioned include: non-alcoholic fatty liver disease.

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