Carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte compound and preparation method thereof

Abstract

The invention relates to the technical field of medical polymer materials, in particular to a carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte compound and a preparation method thereof. Comprising the following steps: S1, preparing peptidomimetic polyelectrolyte; the peptidomimetic polyelectrolyte comprises an aminated peptidomimetic electrolyte, an aminated poly (2-oxazoline) electrolyte, an acid responsive peptidomimetic electrolyte or an acid responsive poly (2-oxazoline) electrolyte; and S2, mixing the peptidomimetic polyelectrolyte with the carboxyl-containing triterpenoids by adopting a direct aqueous solution assembly method or a cosolvent-assisted assembly method, and obtaining the carboxyl-containing triterpenoids-peptidomimetic polyelectrolyte compound after 2-48 hours. Compared with a traditional delivery system and method, the triterpenoid delivery system has the advantages of simple synthesis steps, high operability and the like, the bioavailability of the triterpenoid is remarkably improved, and the triterpenoid delivery system has the characteristics of high drug loading capacity, good stability and the like; and the hydrogel has excellent in-vivo stability, biocompatibility and excellent anti-tumor and antibacterial properties.

Description

Technical Field

[0001] The present invention relates to the technical field of medical polymer materials, and particularly relates to a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex and a preparation method thereof. Background Art

[0002] Triterpenoid compounds, as a class of natural active substances widely distributed in plants, fruits and vegetables, have attracted much attention due to their significant pharmacological activities such as anti - inflammation, antioxidant and anti - tumor effects. At the same time, natural amino acid derivatives have shown broad application prospects in the fields of bionics, nano - drug carriers and tissue engineering because they can effectively mimic the structural and functional characteristics of proteins. Among them, polypeptides have become one of the research hotspots in biomedical materials due to their excellent biocompatibility and biodegradability. However, traditional polypeptide materials still face two major challenges in practical applications: poor solubility and easy degradation by proteases in the body. To overcome these limitations, researchers have carried out structural modification on their molecular skeletons through chemical modification means, and developed a variety of novel peptidomimetic polymer systems, including clustered peptides and poly(2 - oxazoline). These peptidomimetic polymers not only maintain good biocompatibility and biological activity, but also significantly improve enzyme stability. More importantly, the flexible tunability of intramolecular and intermolecular interactions provides an important molecular design basis for constructing multifunctional drug carriers.

[0003] In the research of drug delivery systems, liposomes show unique advantages as carriers of triterpenoid compounds. They form nanoscale vesicles by encapsulating drugs with phospholipid bilayers, which can effectively improve the solubility and stability of drugs. However, this delivery system still has technical defects such as being easily recognized and cleared by the body's immune system, poor physical stability, easy drug leakage and unsatisfactory encapsulation efficiency. Another delivery strategy is to use amphiphilic block copolymers to self - assemble into micelles and encapsulate drugs in their hydrophobic cores. Although this method improves the drug delivery effect to a certain extent, it still faces limitations such as complex preparation processes, high production costs and low drug loading efficiency. The existence of these problems has prompted researchers to continuously explore more efficient and stable novel delivery systems to fully exert the therapeutic potential of triterpenoid compounds.

[0004] Polyelectrolyte complexes (PECs) have attracted much attention due to their unique advantages. PECs are formed by the electrostatic interaction of oppositely charged polyelectrolytes in water. The preparation process avoids the addition of organic solvents and has mild conditions, which is beneficial to maintaining the drug activity. The large number of charges rich in PECs is conducive to loading charged biological macromolecules (such as proteins, nucleic acid drugs, etc.), and at the same time has high loading efficiency and release rate, showing great potential in the field of drug delivery. It is particularly noteworthy that the triterpenoid molecules with carboxyl groups contain conjugated groups at the same time, and can be assembled with peptidomimetic polyelectrolytes containing amino groups through electrostatic and hydrogen bond interactions, which can not only significantly improve the water solubility of the compound, but also facilitate the formation of the assembly. In addition, by chemically modifying the peptidomimetic polyelectrolyte, an intelligent delivery carrier can be constructed, which is of great significance for maintaining the drug efficacy and achieving targeted delivery.

[0005] Therefore, as a class of polymer materials with good biocompatibility and controllability, peptidomimetic polyelectrolytes can form PECs with drug molecules through electrostatic interaction, effectively improving the solubility, stability and targeting of drugs. Therefore, combining natural carboxyl-containing triterpenoid compounds with peptidomimetic polyelectrolytes to construct a new drug delivery system not only has important theoretical research value, but also shows broad application prospects. This research direction is expected to provide new solutions for the clinical application of natural active substances. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex and its preparation method.

[0007] The first object of the present invention is to provide a preparation method of a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare peptidomimetic polyelectrolytes; the peptidomimetic polyelectrolytes include amino-functionalized clustered peptide electrolytes, amino-functionalized poly(2-oxazoline) electrolytes, acid-responsive clustered peptide electrolytes or acid-responsive poly(2-oxazoline) electrolytes; S2. Using the direct aqueous solution assembly method or the co-solvent assisted assembly method, mix the peptidomimetic polyelectrolytes prepared in step S1 with carboxyl-containing triterpenoid compounds, and obtain carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complexes after 2 - 48 h.

[0008] Preferably, the structural formula of the acid-responsive clustered peptide electrolyte is:

[0009] In the structural formula, m is 45 or 113; n is any number between 3 - 200; x is any number between 0 - 0.9.

[0010] Preferably, the preparation method of the acid-responsive clustered peptide electrolyte specifically includes the following steps: S101. Preparation and polymerization of N-substituted carboxylic anhydride: React primary amines containing different groups with glyoxylic acid to prepare N-substituted glycine; Protect the secondary amine in N-substituted glycine using di-tert-butyl dicarbonate; Through a ring-forming reaction with phosphorus trichloride, convert N-substituted glycine into N-substituted carboxylic anhydride; Use a small molecule or PEG containing a primary amine as an initiator to prepare a polymer through ring-opening polymerization; The structural formula of the polymer is:

[0011] In the structural formula, m is 45 or 113; n is any number from 3 to 200; S102. Amino-functionalization modification: Perform a post-modification reaction on the polymer prepared in S101 with mercaptoethylamine to prepare an amino-functionalized clustered peptide electrolyte; The structural formula of the amino-functionalized clustered peptide electrolyte is:

[0012] In the structural formula, m is 45 or 113; n is any number from 3 to 200; S103. Acid-responsive modification: Use 2,3-dimethylmaleic anhydride to partially modify the amino-containing clustered peptide electrolyte prepared in S102 to finally obtain an acid-responsive clustered peptide electrolyte.

[0013] Preferably, the structural formula of the acid-responsive poly(2-oxazoline) electrolyte is:

[0014] In the structural formula, n is any number from 3 to 200; x is any number between 0 and 0.9.

[0015] Preferably, the preparation method of the acid-responsive poly(2-oxazoline) electrolyte specifically includes the following steps: S111. React 4-pentenoic acid, N-hydroxysuccinimide, and EDC·HCl to generate N-succinimidyl 4-pentenoate; React N-succinimidyl 4-pentenoate with 2-chloroethylamine hydrochloride and sodium hydroxide to generate N-(2-chloroethyl)-4-pentenamide, and react with potassium hydroxide in methanol to obtain the monomer 2-(3-butenyl)-2-oxazoline; S112. Use methyl trifluoromethanesulfonate as an initiator to initiate the ring-opening polymerization of the monomer 2-(3-butenyl)-2-oxazoline to prepare a poly(2-oxazoline) containing an allyl side group; The structural formula of the poly(2-oxazoline) containing an allyl group is:

[0016] In the structural formula, n is any number between 3 and 200; S113. Amino functional modification: The poly(2-oxazoline) containing allyl side groups prepared in S202 is subjected to post-modification reaction with mercaptoethylamine to prepare an aminated poly(2-oxazoline) electrolyte; the aminated poly(2-oxazoline) electrolyte has the structural formula:

[0017] In the structural formula, n is any number between 3 and 200; S114. The amino-modified poly(2-oxazoline) electrolyte prepared in S113 was partially modified with 2,3-dimethylmaleic anhydride to obtain an acid-responsive poly(2-oxazoline) electrolyte.

[0018] Preferably, the carboxyl-containing triterpenoid compound is ursolic acid, oleanolic acid, betulinic acid, glycyrrhetinic acid or arjunic acid.

[0019] Preferably, step S2 adopts a direct aqueous solution assembly method, which specifically comprises the following steps: mixing the peptidomimetic polyelectrolyte and the carboxyl-containing triterpenoid compound in an aqueous solution at a molar ratio of 1-10:10-1, and continuously stirring for 2-48 hours to complete the assembly.

[0020] Preferably, step S2 adopts a solvent-assisted assembly method and specifically comprises the following steps: dissolving the triterpenoid compound in a solvent, and then mixing it with a peptidomimetic polyelectrolyte in a molar ratio of 1-10:10-1, and the assembly can be completed after stirring or standing at 20-80 degrees Celsius for 2-48 hours.

[0021] The second object of the present invention is to provide a carboxyl-containing triterpenoid compound-peptoid polyelectrolyte complex, which is prepared by a method for preparing a carboxyl-containing triterpenoid compound-peptoid polyelectrolyte complex.

[0022] The third purpose of the present invention is to provide an application of a carboxyl triterpenoid compound-peptidomimetic polyelectrolyte complex in the preparation of nanomedicines. The carboxyl triterpenoid compound is dissolved in a cosolvent and then slowly added to water to prepare a solution; a peptidomimetic polyelectrolyte is added in a molar ratio of 1 to 10:10 to 1 and mixed, wherein the molar ratio of the carboxyl triterpenoid compound to the peptidomimetic polyelectrolyte is 1 to 10:10 to 1; a functional protein solution is added at the same time, mixed, and stirred or allowed to stand for 2 to 48 hours; centrifuged, and the assembly is collected to obtain the nanomedicine.

[0023] Compared with the prior art, the present invention can achieve the following beneficial effects: In view of the technical problem of solving the inherent defects in the prior art, such as poor solubility and low bioavailability of carboxyl triterpenoids, which severely restrict the therapeutic effect and the effective accumulation in diseased tissues, the present invention innovatively develops a carboxyl triterpenoid-peptidomimetic polyelectrolyte complex and a drug delivery system based on this complex. The amino-functionalized peptidomimetic polyelectrolyte (peptoid / poly(2-oxazoline)) combines with natural carboxyl triterpenoids (ursolic acid, oleanolic acid, betulinic acid, glycyrrhetinic acid, arjunolic acid, etc.) to form a polyelectrolyte complex with a large number of charges. The biocompatibility of the peptidomimetic polyelectrolyte synergistically acts with the natural activity of triterpenoids. In terms of application, by using the large number of charges inside the complex, efficient loading of functional proteins can be directly achieved during the assembly process, avoiding the damage to protein activity caused by traditional chemical coupling. The complex realizes precise drug release at the tumor microenvironment and the site of bacterial infection, thereby simultaneously exerting high-efficiency anti-tumor and antibacterial effects.

[0024] Compared with traditional delivery systems and methods, this system has advantages such as simple synthesis steps and strong operability, significantly improving the bioavailability of triterpenoids. Specifically, the present invention successfully constructs a novel nano-drug by complexing a peptidomimetic polyelectrolyte with a triterpenoid. This polyelectrolyte complex not only has a simple preparation process but also has characteristics such as high drug loading capacity and good stability. More importantly, the peptidomimetic polymer used has excellent in vivo stability, biocompatibility, and excellent anti-tumor and antibacterial activities, providing a safe and reliable new strategy for the efficient delivery of triterpenoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the 1H NMR spectrum of poly(N-allylglycine) PNAG and amino-functionalized peptoid electrolyte PNAG- g -NH2 provided according to an embodiment of the present invention; in the figure, (a) represents PNAG in DMSO- 1 6, (b) represents PNAG- d -NH2 in D2O, g represents DMSO, represents H2O. represents H2O.

[0026] Figure 2 is the 1H NMR spectrum of the acid-responsive peptoid electrolyte in D2O provided according to an embodiment of the present invention; in the figure, 1 represents H2O. represents H2O.

[0027] Figure 3It is a negative staining TEM image of the carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex (1 mg / mL PNAG-g-NH2 + UA) provided according to an embodiment of the present invention after stirring in an aqueous solution for 48 h.

[0028] Figure 4 It is a negative staining TEM image of the carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex (1 mg / mL complex of poly(2-oxazoline) with amino group and ursolic acid, PBuOxz- g -NH2 + UA) after stirring in an aqueous solution for 48 h.

[0029] Figure 5 It is a negative staining TEM image of the carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex (1 mg / mL complex of amino-functionalized clustered peptide electrolyte and oleanolic acid, PNAG-g-NH2 + OA) after stirring in an aqueous solution for 48 h. Detailed implementation manners

[0030] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are denoted by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, their detailed descriptions will not be repeated.

[0031] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation to the present invention.

[0032] The present invention provides a preparation method of a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the peptidomimetic polyelectrolyte; Specifically, the peptidomimetic polyelectrolyte includes an amino-functionalized clustered peptide electrolyte (PNAG-g-NH2), an amino-functionalized poly(2-oxazoline) electrolyte, an acid-responsive clustered peptide electrolyte or an acid-responsive poly(2-oxazoline) electrolyte; The acid-responsive clustered peptide electrolyte is prepared through three-step reactions; specifically, it includes the following sub-steps: S101. Preparation and polymerization of N-substituted carboxylic anhydride (NNCA): React a primary amine containing different groups with glyoxylic acid to prepare N-substituted glycine; subsequently, protect the secondary amine in N-substituted glycine with di-tert-butyl dicarbonate; convert N-substituted glycine into N-substituted carboxylic anhydride (NNCA) through a ring-forming reaction with phosphorus trichloride; use a small molecule or PEG containing a primary amine as an initiator to prepare a polymer through ring-opening polymerization reaction; Brief principle: First, react primary amines (R-NH2) containing different groups with glyoxylic acid (HOOC-CHO). The amino group (-NH2) of the primary amine undergoes a nucleophilic addition reaction with the aldehyde group (-CHO) of glyoxylic acid to form an intermediate, and then the intermediate rearranges to finally form N-substituted glycine (R-CH(NH2)-COOH); this step selectively reacts with glyoxylic acid through the nucleophilicity of the primary amine to introduce a carboxyl functional group. Next, use di-tert-butyl dicarbonate (Boc2O) to protect the secondary amine (-NH2) in N-substituted glycine. Boc2O is a commonly used amino protecting group, and it reacts with the secondary amine to form a tert-butoxycarbonyl (Boc)-protected amino group (-NHBoc). The purpose of this step is to prevent the secondary amine from being oxidized or undergoing other side reactions in subsequent reactions and ensure the specificity of the reaction. Finally, through the cyclization reaction of phosphorus trichloride (PCl3), N-substituted glycine is converted into N-substituted carboxylic anhydride (NNCA). Under the action of phosphorus trichloride, the carboxyl group (-COOH) and the amino group (-NH2) undergo a dehydration condensation reaction to form a cyclic N-substituted carboxylic anhydride. This step is a key cyclization reaction, providing an active intermediate for subsequent polymerization reactions. Ring-opening polymerization reaction: Use a small molecule containing a primary amine or polyethylene glycol (PEG) as an initiator to polymerize NNCA into a long-chain polymer through a ring-opening polymerization reaction. The primary amine group of the initiator can attack the cyclic structure of NNCA to initiate the ring-opening polymerization reaction and generate a polymer with repeating units. This step realizes the transformation from monomer to polymer and provides a basis for subsequent functionalization modification.

[0033] Specifically, the structural formula of the polymer (i.e., the clustered peptide) is:

[0034] In the structural formula, m is 45 or 113; n is any number from 3 to 200, and preferably 30 to 120.

[0035] S102. Amino-functionalization modification: React the polymer prepared in S101 with mercaptoethylamine for post-modification reaction to prepare a clustered peptide electrolyte containing amino groups; Specifically, the structural formula of the amino-functionalized clustered peptide electrolyte is:

[0036] In the structural formula, m is 45 or 113; n is any number from 3 to 200, and preferably 30 to 120.

[0037] S103. Acid-responsive modification: Use 2,3-dimethylmaleic anhydride to partially modify the amino-functionalized clustered peptide electrolyte prepared in S102 to finally obtain an acid-responsive clustered peptide electrolyte; Specifically, the structural formula of the acid-responsive poly(2-oxazoline) electrolyte is as follows:

[0038] In the structural formula, m is 45 or 113; n is any number from 3 to 200, optimally 30 to 120; x is any number between 0 and 0.9.

[0039] The preparation method of the amino-functionalized poly(2-oxazoline) electrolyte specifically includes the following steps: S111. React 4-pentenoic acid, N-hydroxysuccinimide (NHS), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) to form N-succinimidyl 4-pentenoate; react N-succinimidyl 4-pentenoate with 2-chloroethylamine hydrochloride and sodium hydroxide to form N-(2-chloroethyl)-4-pentenamide, and react it with potassium hydroxide in methanol to obtain the monomer 2-(3-butenyl)-2-oxazoline (BuOxz); Brief principle description: (1) Synthesis of N-succinimidyl 4-pentenoate: EDC·HCl activates the carboxyl group of 4-pentenoic acid, enabling it to undergo a coupling reaction with NHS to form N-succinimidyl 4-pentenoate. This step is achieved through the activation of EDC·HCl, causing the carboxyl group to couple with NHS to generate an intermediate with an active ester. (2) Synthesis of N-(2-chloroethyl)-4-pentenamide: React N-succinimidyl 4-pentenoate with 2-chloroethylamine hydrochloride and sodium hydroxide. Under the alkaline condition of sodium hydroxide, the amino group (-NH2) of 2-chloroethylamine hydrochloride attacks the active ester of N-succinimidyl 4-pentenoate, undergoing a nucleophilic substitution reaction to form N-(2-chloroethyl)-4-pentenamide. This step is accomplished by the nucleophilicity of the amino group, selectively reacting with the active ester to introduce the chloroethyl group. (3) Synthesis of the monomer 2-(3-butenyl)-2-oxazoline (BuOxz): React N-(2-chloroethyl)-4-pentenamide with potassium hydroxide in methanol. Under the alkaline condition of potassium hydroxide, the chloroethyl group undergoes a nucleophilic substitution reaction to form 2-(3-butenyl)-2-oxazoline (BuOxz). This step completes the conversion from amide to oxazoline through a nucleophilic substitution reaction under alkaline conditions, generating the target monomer.

[0040] S112. Use methyl trifluoromethanesulfonate as an initiator to initiate the ring-opening polymerization of the monomer 2-(3-butenyl)-2-oxazoline (BuOxz) to prepare poly(2-oxazoline) containing allyl side groups; Specifically, the structural formula of the poly(2-oxazoline) containing allyl side groups is as follows:

[0041] In the structural formula, n is any number from 3 to 200, and optimally it is from 30 to 120.

[0042] S113. Amino-functionalization modification: The poly(2-oxazoline) containing allyl side groups prepared in S202 is subjected to a post-modification reaction with mercaptoethylamine to prepare an amino-functionalized poly(2-oxazoline) electrolyte; Specifically, the structural formula of the amino-functionalized poly(2-oxazoline) electrolyte is:

[0043] In the structural formula, n is any number from 3 to 200, and optimally it is from 30 to 120.

[0044] S114. Acid-responsive modification: 2,3-Dimethylmaleic anhydride is used to partially modify the amino-functionalized poly(2-oxazoline) electrolyte prepared in S113 to finally obtain an acid-responsive poly(2-oxazoline) electrolyte; Specifically, the structural formula of the acid-responsive poly(2-oxazoline) electrolyte is:

[0045] In the structural formula, n is any number from 3 to 200, and optimally it is from 30 to 120. x is any number between 0 and 0.9.

[0046] The preparation method of the amino-functionalized poly(peptide) electrolyte (PNAG-g-NH2) specifically includes the following steps: S121. Synthesis of N-allyl-substituted glycine hydrochloride: Glyoxylic acid aqueous solution is added to a flask. Using dichloromethane as a solvent, under ice bath conditions, allylamine is added dropwise with stirring and the reaction is continuously stirred for 24 hours; after the reaction is completed, the dichloromethane solvent is removed by rotary evaporation; subsequently, 2M hydrochloric acid is added to the residue and refluxed at 110 °C for 20 hours; after the reflux ends, the water is removed by rotary evaporation; the obtained residue is dissolved in a mixed solvent of methanol and tetrahydrofuran and recrystallized three times at -20 °C; filtered and vacuum dried to obtain white solid N-allyl-substituted glycine hydrochloride; S122. Synthesis of N-tert-butoxycarbonyl-N-allyl-substituted glycine: N-allyl-substituted glycine hydrochloride is completely dissolved in deionized water; Boc2O and triethylamine are added in sequence and stirred for 24 hours; after the reaction ends, n-hexane is used for extraction to remove the unreacted Boc2O; the pH of the solution is adjusted to about 2 with 2M hydrochloric acid, and then the product is extracted with ethyl acetate; the organic phase is washed with saturated brine and dried with anhydrous magnesium sulfate for 12 hours; filtered and the solvent is removed by rotary evaporation to obtain white solid N-tert-butoxycarbonyl-N-allyl-substituted glycine; Synthesis of N-allyl-N-carboxylic anhydride (NAG-NNCA): Dissolve N-Boc-N-allyl-substituted glycine in 200 mL of anhydrous dichloromethane, stir under an ice bath, and slowly add phosphorus trichloride while maintaining nitrogen protection throughout the process. After reacting for 3 hours, remove the solvent by rotary evaporation. Subsequently, transfer the product into a glove box and precipitate it three times with a mixed solvent of tetrahydrofuran and n-hexane under nitrogen protection. After drying the solvent, a colorless transparent liquid, namely N-allyl-N-carboxylic anhydride monomer (NAG-NNCA), is obtained.

[0047] S124. Synthesis of poly(N-allylglycine) (PNAG): Dissolve the NAG-NNCA monomer in anhydrous tetrahydrofuran (THF) with a concentration controlled at 100 mg / mL. Under nitrogen protection, add benzylamine dissolved in anhydrous THF and stir at 55 °C for 24 hours. After the reaction is completed, precipitate the product in cold diethyl ether to obtain white solid poly(N-allylglycine) (PNAG). S125.PNAG- g -NH2 synthesis: Take PNAG and dissolve it in DMF, then add benzoin dimethyl ether (DMPA) and mercaptoethylamine. Before the reaction, thoroughly remove the oxygen in the system. Under ultraviolet light irradiation, continuously stir and react for 3 hours. After the reaction is completed, dialyze the product and freeze-dry it to finally obtain the polymer PNAG- g -NH2.

[0048] S2. Using the direct aqueous solution assembly method or the co-solvent assisted assembly method, mix the amino-functionalized peptidomimetic polyelectrolyte prepared in step S1 with the carboxyl-containing triterpenoid compound at a reaction temperature of 0 to 80 °C, and the optimal temperature is 20 to 50 °C; after 2 to 48 h, a carboxyl-containing triterpenoid compound-peptidomimetic polyelectrolyte complex is obtained. Specifically, the direct aqueous solution assembly method specifically includes the following steps: Mix the peptidomimetic polyelectrolyte and the carboxyl-containing triterpenoid compound at a molar ratio of 1 to 10:10 to 1 in an aqueous solution and continuously stir for 2 to 48 hours to complete the assembly. Specifically, the co-solvent assisted assembly method specifically includes the following steps: Dissolve the carboxyl-containing triterpenoid compound in a co-solvent, then fully mix it with the peptidomimetic polyelectrolyte, and stir or stand at 20 to 80 °C (20 to 50 °C is the best) for 2 to 48 hours to complete the assembly. Specifically, the carboxyl-containing triterpenoid compound is ursolic acid, oleanolic acid, betulinic acid, glycyrrhetinic acid or arjunolic acid, and the optimal ones are ursolic acid or oleanolic acid.

[0049] The present invention also provides a method for preparing a nano-drug, which specifically includes: dissolving a triterpenoid compound in a co-solvent, and then slowly adding it to water to prepare a solution; adding a peptidomimetic polyelectrolyte in a ratio of (10:1 - 1:10), mixing well, and simultaneously adding a functional protein solution. After mixing, stir or stand for 2 - 48 hours (optimally 12 - 48 h); centrifuge to collect the assembled body, which is the nano-drug. Specifically, the co-solvent is dimethyl sulfoxide (DMSO) with a concentration of 1 - 20 mg / mL. In a specific embodiment, stir or stand for 12, 24, or 48 hours.

[0050] Example 1 This example provides a method for preparing an amino-functionalized clustered peptide electrolyte, which specifically includes the following steps: taking PNAG-g-NH2 initiated by benzylamine with one amino group in the side chain as an example: Synthesis of N-allyl-substituted glycine hydrochloride: Add an aqueous glyoxylic acid solution (150 g, 1 mol, 50% mass fraction) to a flask, and then add 500 mL of dichloromethane as a solvent; under ice bath conditions, dropwise add allylamine (44.5 mL, 0.5 mol) with stirring, and continuously stir and react for 24 hours; after the reaction is completed, rotary evaporate to remove the dichloromethane solvent; subsequently, add 500 mL of 2M hydrochloric acid to the residue, and reflux at 110 °C for 20 hours; after the reflux is completed, rotary evaporate to remove water; the obtained residue is dissolved in a mixed solvent of methanol and tetrahydrofuran (volume ratio 1:10), and recrystallized three times in a -20 °C refrigerator; finally, obtain a white solid N-allyl-substituted glycine hydrochloride by filtration and vacuum drying.

[0051] Synthesis of N-tert-butoxycarbonyl-N-allyl-substituted glycine: Dissolve N-allyl-substituted glycine hydrochloride (15.1 g, 0.1 mol) in deionized water. After complete dissolution, successively add Boc2O (76.35 g, 0.2 mol) and triethylamine (45.3 g, 0.448 mol), and stir and react for 24 hours. After the reaction is completed, extract with n-hexane to remove the unreacted Boc2O. Then, adjust the pH of the solution to about 2 with 2M hydrochloric acid, and extract the product with ethyl acetate. After that, wash the organic phase with saturated brine and add anhydrous magnesium sulfate to dry for 12 hours. After filtration, rotary evaporate to remove the solvent to obtain a white solid N-tert-butoxycarbonyl-N-allyl-substituted glycine.

[0052] Synthesis of N-allyl-N-carboxylic anhydride (NAG-NNCA): Dissolve N-tert-butoxycarbonyl-N-allyl-substituted glycine (15 g, 0.067 mol) in 200 mL of anhydrous dichloromethane, stir under an ice bath, and slowly add phosphorus trichloride (14.54 mL, 0.168 mol) while maintaining nitrogen protection throughout the process. After reacting for 3 hours, remove the solvent by rotary evaporation. Subsequently, transfer the product into a glove box, and under nitrogen protection, precipitate it three times with tetrahydrofuran and n-hexane (volume ratio 1:10). After drying the solvent by suction, a colorless transparent liquid, namely N-allyl-N-carboxylic anhydride monomer (NAG-NNCA), is obtained.

[0053] Synthesis of poly(N-allylglycine) (PNAG): Dissolve NAG-NNCA monomer (1.4 g, 0.1 mol) in anhydrous tetrahydrofuran (THF) with a concentration controlled at 100 mg / mL. Under nitrogen protection, add benzylamine (2.1 mmol) dissolved in anhydrous THF and stir at 55 °C for 24 hours. After the reaction is completed, precipitate the product in cold diethyl ether to obtain white solid poly(N-allylglycine) (PNAG).

[0054] Synthesis of PNAG- g -NH2: Take PNAG (1.2 g, 2.63×10 -4 mol) and dissolve it in DMF, then add benzoin dimethyl ether (DMPA, 158 mg, 6.18×10 -4 mol) and mercaptoethylamine (4.76 g, 6.18×10 -2 mol); before the reaction, thoroughly remove the oxygen in the system; under ultraviolet light irradiation, continuously stir and react for 3 hours; after the reaction is completed, dialyze the product and freeze-dry it to finally obtain the polymer PNAG- g -NH2 ( Figure 1 ).

[0055] Example 2 This example provides a preparation method of an acid-responsive cluster peptide electrolyte, which specifically includes the following sub-steps: S101. Preparation and polymerization of N-substituted carboxylic anhydride (NNCA): React primary amines containing different groups with glyoxylic acid to prepare N-substituted glycine; subsequently, protect the secondary amine in N-substituted glycine with di-tert-butyl dicarbonate; convert N-substituted glycine into N-substituted carboxylic anhydride (NNCA) through a ring-forming reaction with phosphorus trichloride; use small molecules or PEG containing primary amines as initiators to prepare polymers through ring-opening polymerization reactions. S102. Amino-functionalization modification: The polymer prepared in S101 is subjected to a post-modification reaction with mercaptoethylamine to prepare a clustered peptide electrolyte PNAG containing amino groups n - g -NH2; S103. Acid-responsive modification: Dissolve PNAG n - g -NH2 (100 mg) in 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, and adjust the pH of the system to about 8.5 using 1 M aqueous sodium hydroxide solution; add 2,3-dimethylmaleic anhydride (362 mg) and add it to the reaction system in three portions. After each addition, adjust the solution pH to 8 - 9 using 1 M aqueous sodium hydroxide solution, and stir at room temperature for 24 h; dialyze for two days, and after lyophilization, the obtained white solid is 2,3-dimethylmaleic anhydride-modified PNAGn-g-NH2, which is labeled as DA. The 1 1H NMR spectrum of DA in D2O is as Figure 2 shown.

[0056] Example 3 This example provides a method for preparing an amino-functionalized poly(2-oxazoline) electrolyte, which specifically includes the following steps: S111. React 4-pentenoic acid, N-hydroxysuccinimide (NHS), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) to generate N-succinimidyl 4-pentenoate; react N-succinimidyl 4-pentenoate with 2-chloroethylamine hydrochloride and sodium hydroxide to generate N-(2-chloroethyl)-4-pentenamide, and react with potassium hydroxide in methanol to obtain the monomer 2-(3-butenyl)-2-oxazoline (BuOxz); S112. Use methyl trifluoromethanesulfonate as an initiator to initiate the ring-opening polymerization of the monomer 2-(3-butenyl)-2-oxazoline (BuOxz) to prepare poly(2-oxazoline) (PBuOxz) containing allyl side groups; S113. Amino-functionalization modification: The poly(2-oxazoline) containing allyl side groups prepared in S202 is subjected to a post-modification reaction with mercaptoethylamine to prepare an amino-functionalized poly(2-oxazoline) electrolyte (PBuOxz- g -NH2).

[0057] Example 4 This example provides a method for preparing an acid-responsive poly(2-oxazoline) electrolyte, which specifically includes the following sub-steps: S112. Poly(2-oxazoline) with allyl side groups (PBuOxz-g-NH2): React 4-pentenoic acid with 2-chloroethylamine hydrochloride to prepare N-(2-chloroethyl)-4-pentenamide; react with potassium hydroxide in methanol to obtain the monomer 2-(3-butenyl)-2-oxazoline (BuOxz); use methyl trifluoromethanesulfonate as an initiator to prepare the polymer through ring-opening polymerization reaction; S113. Amino-functionalized modification: Perform a post-modification reaction on the polymer prepared in S112 with mercaptoethylamine to prepare the amino-functionalized poly(2-oxazoline) electrolyte PBuOxz- g -NH2; S103. Acid-responsive modification: Dissolve PBuOxz- g -NH2 in 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, and adjust the pH of the system to about 8.5 using 1 M aqueous sodium hydroxide solution; add 2,3-dimethylmaleic anhydride (362 mg) and add it to the reaction system in three portions. After each addition, adjust the solution pH to 8 - 9 using 1 M aqueous sodium hydroxide solution, and stir at room temperature for 24 h; dialyze for two days, and after lyophilization, the obtained white solid is 2,3-dimethylmaleic anhydride-modified PBuOxz-g-NH2.

[0058] Example 5 This example provides a method for preparing a carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the amino-functionalized peptidomimetic polyelectrolyte according to the method of Example 1; S2. Mix the amino-functionalized peptidomimetic polyelectrolyte and ursolic acid in an aqueous solution at a molar ratio of 10:1 - 1:10, continuously stir at 20 - 50 °C for 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain the carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex. The negative staining TEM image is shown in Figure 3 .

[0059] Example 6 This example provides a method for preparing a carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid-responsive peptidomimetic polyelectrolyte according to the method of Example 2; S2. Mix the acid-responsive peptidomimetic polyelectrolyte and ursolic acid at a ratio of (10:1 - 1:10) in an aqueous solution, continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain the carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex.

[0060] Example 7 This example provides a preparation method for a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an amino-functionalized poly(2-oxazoline) electrolyte according to the method of Example 3; S2. Mix the amino-functionalized poly(2-oxazoline) electrolyte and ursolic acid in an aqueous solution at a ratio of (10:1 - 1:10), stir at 20 - 50 °C for 2 - 48 h, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex. The negative staining TEM image is shown in Figure 4 .

[0061] Example 8 This example provides a preparation method for a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an acid-responsive poly(2-oxazoline) electrolyte according to the method of Example 4; S2. Mix the acid-responsive poly(2-oxazoline) electrolyte and ursolic acid in an aqueous solution at a ratio of (10:1 - 1:10), continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0062] Example 9 This example provides a preparation method for a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an amino-functionalized clustered peptide electrolyte according to the method of Example 1; S2. Dissolve ursolic acid in DMSO (1 - 20 mg / mL), then slowly add it to water to prepare a solution of 0.1 - 1 mg / mL; then add the aqueous solution of the clustered peptide electrolyte containing amino groups at a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0063] Example 10 This example provides a preparation method for a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an acid-responsive clustered peptide electrolyte according to the method of Example 2; S2. Dissolve ursolic acid in DMSO (1 - 20 mg / mL), then slowly add it to water to prepare a solution of 0.1 - 1 mg / mL; then add the aqueous solution of the acid-responsive clustered peptide electrolyte at a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0064] Example 11 This example provides a method for preparing a carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the amino-functionalized poly(2-oxazoline) electrolyte according to the method of Example 3; S2. Dissolve ursolic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL. Subsequently, add the aqueous solution of the amino-functionalized poly(2-oxazoline) electrolyte in a molar ratio of 10:1 - 1:10, and stir or leave it standing at 20 - 50 °C for 2 - 48 hours to obtain the carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex.

[0065] Example 12 This example provides a method for preparing a carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid-responsive poly(2-oxazoline) electrolyte according to the method of Example 4; S2. Dissolve ursolic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL. Subsequently, add the aqueous solution of the acid-responsive poly(2-oxazoline) electrolyte in a molar ratio of 10:1 - 1:10, and leave it standing at 20 - 80 °C (the optimal temperature is 20 - 50 °C) for 2 - 48 hours to obtain the carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex.

[0066] Example 13 This example provides a method for preparing a carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the amino-functionalized peptidomimetic polyelectrolyte according to the method of Example 1; S2. Mix the amino-functionalized peptidomimetic polyelectrolyte and oleanolic acid in an aqueous solution in a molar ratio of 10:1 - 1:10, continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain the carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex. The negative staining TEM image is shown in Figure 5 .

[0067] Example 14 This example provides a method for preparing a carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid-responsive peptidomimetic polyelectrolyte according to the method of Example 2; S2. Mix the acid-responsive clustered peptide electrolyte and oleanolic acid in an aqueous solution at a ratio of (10:1 - 1:10), continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0068] Example 15 This example provides a method for preparing a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the amino-functionalized poly(2-oxazoline) electrolyte according to the method of Example 3; S2. Mix the amino-functionalized poly(2-oxazoline) electrolyte and oleanolic acid in an aqueous solution at a ratio of (10:1 - 1:10), stir at 20 - 50 °C for 2 - 48 h, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0069] Example 16 This example provides a method for preparing a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid-responsive poly(2-oxazoline) electrolyte according to the method of Example 4; S2. Mix the acid-responsive poly(2-oxazoline) electrolyte and oleanolic acid in an aqueous solution at a ratio of (10:1 - 1:10), continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0070] Example 17 This example provides a method for preparing a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the amino-functionalized clustered peptide polyelectrolyte according to the method of Example 1; S2. Dissolve oleanolic acid in DMSO (1 - 20 mg / mL), then slowly add it to water to prepare a solution of 0.1 - 1 mg / mL; then add the aqueous solution of the clustered peptide electrolyte containing amino groups in a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0071] Example 18 This example provides a method for preparing a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid-responsive clustered peptide electrolyte according to the method of Example 2; S2. Dissolve oleanolic acid in DMSO (1 - 20 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL. Subsequently, add an acid-responsive clustered peptide electrolyte aqueous solution in a molar ratio of 10:1 - 1:10, and stir or let it stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl triterpenoid-peptidomimetic polyelectrolyte complex.

[0072] Example 19 This example provides a preparation method for a carboxyl triterpenoid-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an amino-functionalized poly(2-oxazoline) electrolyte according to the method of Example 3; S2. Dissolve oleanolic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL. Subsequently, add an aqueous solution of the amino-functionalized poly(2-oxazoline) electrolyte in a molar ratio of 10:1 - 1:10, and stir or let it stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl triterpenoid-peptidomimetic polyelectrolyte complex.

[0073] Example 20 This example provides a preparation method for a carboxyl triterpenoid-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an acid-responsive poly(2-oxazoline) electrolyte according to the method of Example 4; S2. Dissolve oleanolic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL. Subsequently, add an aqueous solution of the acid-responsive poly(2-oxazoline) electrolyte in a molar ratio of 10:1 - 1:10, and stir or let it stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl triterpenoid-peptidomimetic polyelectrolyte complex.

[0074] Example 21 This example provides a preparation method for a carboxyl triterpenoid-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an amino-functionalized clustered peptide polyelectrolyte according to the method of Example 1; S2. Mix the amino-functionalized clustered peptide polyelectrolyte and betulinic acid in an aqueous solution in a molar ratio of 10:1 - 1:10, continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl triterpenoid-peptidomimetic polyelectrolyte complex.

[0075] Example 22 This example provides a method for preparing a carboxyl group-containing triterpenoid compound-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an acid-responsive clustered peptide electrolyte according to the method of Example 2; S2. Mix the acid-responsive clustered peptide electrolyte and betulinic acid in an aqueous solution at a ratio of (10:1 - 1:10), continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl group-containing triterpenoid compound-peptidomimetic polyelectrolyte complex.

[0076] Example 23 This example provides a method for preparing a carboxyl group-containing triterpenoid compound-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an amino-functionalized poly(2-oxazoline) electrolyte according to the method of Example 3; S2. Mix the amino-functionalized poly(2-oxazoline) electrolyte and betulinic acid in an aqueous solution at a ratio of (10:1 - 1:10), stir at 20 - 50 °C for 2 - 48 h, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl group-containing triterpenoid compound-peptidomimetic polyelectrolyte complex.

[0077] Example 24 This example provides a method for preparing a carboxyl group-containing triterpenoid compound-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an acid-responsive poly(2-oxazoline) electrolyte according to the method of Example 4; S2. Mix the acid-responsive poly(2-oxazoline) electrolyte and betulinic acid in an aqueous solution at a ratio of (10:1 - 1:10), continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl group-containing triterpenoid compound-peptidomimetic polyelectrolyte complex.

[0078] Example 25 This example provides a method for preparing a carboxyl group-containing triterpenoid compound-peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an amino-functionalized clustered peptide polyelectrolyte according to the method of Example 1; S2. Dissolve betulinic acid in DMSO (1 - 20 mg / mL), then slowly add it to water to prepare a solution of 0.1 - 1 mg / mL; then add an aqueous solution of an amino-containing clustered peptide electrolyte at a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl group-containing triterpenoid compound-peptidomimetic polyelectrolyte complex.

[0079] Example 26 This embodiment provides a method for preparing a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid-responsive clustered peptide electrolyte according to the method of Example 2; S2. Dissolve betulinic acid in DMSO (1 - 20 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; then add the acid-responsive clustered peptide electrolyte aqueous solution in a molar ratio of 10:1 - 1:10, and stir or leave it standing at 20 - 50 °C for 2 - 48 hours to obtain the carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0080] Example 27 This embodiment provides a method for preparing a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the amino-functionalized poly(2-oxazoline) electrolyte according to the method of Example 3; S2. Dissolve betulinic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; then add the amino-functionalized poly(2-oxazoline) electrolyte aqueous solution in a molar ratio of 10:1 - 1:10, and stir or leave it standing at 20 - 50 °C for 2 - 48 hours to obtain the carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0081] Example 28 This embodiment provides a method for preparing a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid-responsive poly(2-oxazoline) electrolyte according to the method of Example 4; S2. Dissolve betulinic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; then add the acid-responsive poly(2-oxazoline) electrolyte aqueous solution in a molar ratio of 10:1 - 1:10, and stir or leave it standing at 20 - 50 °C for 2 - 48 hours to obtain the carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0082] Example 29 This embodiment provides a method for preparing a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the amino-functionalized clustered peptide polyelectrolyte according to the method of Example 1; S2. Mix the aminated clustered peptide polyelectrolyte and glycyrrhetinic acid in an aqueous solution at a molar ratio of 10:1 - 1:10, continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex.

[0083] Example 30 This example provides a preparation method of a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid - responsive clustered peptide electrolyte according to the method of Example 2; S2. Mix the acid - responsive clustered peptide electrolyte and glycyrrhetinic acid in an aqueous solution at a ratio of (10:1 - 1:10), continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex.

[0084] Example 31 This example provides a preparation method of a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the aminated poly(2 - oxazoline) electrolyte according to the method of Example 3; S2. Mix the aminated poly(2 - oxazoline) electrolyte and glycyrrhetinic acid in an aqueous solution at a ratio of (10:1 - 1:10), stir at 20 - 50 °C for 2 - 48 h, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex.

[0085] Example 32 This example provides a preparation method of a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid - responsive poly(2 - oxazoline) electrolyte according to the method of Example 4; S2. Mix the acid - responsive poly(2 - oxazoline) electrolyte and glycyrrhetinic acid in an aqueous solution at a ratio of (10:1 - 1:10), continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and save the supernatant to obtain a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex.

[0086] Example 33 This example provides a preparation method of a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the aminated clustered peptide polyelectrolyte according to the method of Example 1; S2. Dissolve glycyrrhetinic acid in DMSO (1 - 20 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; then add an aqueous solution of amino - containing cluster peptide electrolyte in a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl - containing triterpenoid - peptidomimetic polyelectrolyte complex.

[0087] Example 34 This example provides a method for preparing a carboxyl - containing triterpenoid - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an acid - responsive cluster peptide electrolyte according to the method of Example 2; S2. Dissolve glycyrrhetinic acid in DMSO (1 - 20 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; then add an aqueous solution of acid - responsive cluster peptide electrolyte in a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl - containing triterpenoid - peptidomimetic polyelectrolyte complex.

[0088] Example 35 This example provides a method for preparing a carboxyl - containing triterpenoid - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an amino - functionalized poly(2 - oxazoline) electrolyte according to the method of Example 3; S2. Dissolve glycyrrhetinic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; then add an aqueous solution of amino - functionalized poly(2 - oxazoline) electrolyte in a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl - containing triterpenoid - peptidomimetic polyelectrolyte complex.

[0089] Example 36 This example provides a method for preparing a carboxyl - containing triterpenoid - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an acid - responsive poly(2 - oxazoline) electrolyte according to the method of Example 4; S2. Dissolve glycyrrhetinic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; then add an aqueous solution of acid - responsive poly(2 - oxazoline) electrolyte in a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl - containing triterpenoid - peptidomimetic polyelectrolyte complex.

[0090] Example 37 This example provides a method for preparing a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the amino-functionalized clustered peptide polyelectrolyte according to the method of Example 1; S2. Mix the amino-functionalized clustered peptide polyelectrolyte and arjunolic acid in an aqueous solution at a molar ratio of 10:1 - 1:10, continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and preserve the supernatant to obtain the carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0091] Example 38 This example provides a method for preparing a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid-responsive clustered peptide electrolyte according to the method of Example 2; S2. Mix the acid-responsive clustered peptide electrolyte and arjunolic acid in an aqueous solution at a molar ratio of 10:1 - 1:10, continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and preserve the supernatant to obtain the carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0092] Example 39 This example provides a method for preparing a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the amino-functionalized poly(2-oxazoline) electrolyte according to the method of Example 3; S2. Mix the amino-functionalized poly(2-oxazoline) electrolyte and arjunolic acid in an aqueous solution at a molar ratio of 10:1 - 1:10, stir at 20 - 50 °C for 2 - 48 h, centrifuge to remove the precipitate, and preserve the supernatant to obtain the carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0093] Example 40 This example provides a method for preparing a carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare the acid-responsive poly(2-oxazoline) electrolyte according to the method of Example 4; S2. Mix the acid-responsive poly(2-oxazoline) electrolyte and arjunolic acid in an aqueous solution at a molar ratio of 10:1 - 1:10, continuously stir at 20 - 50 °C for 2 - 48 hours, centrifuge to remove the precipitate, and preserve the supernatant to obtain the carboxyl-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0094] Example 41 This example provides a method for preparing a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an amino-functionalized clustered peptide polyelectrolyte according to the method of Example 1; S2. Dissolve arjunolic acid in DMSO (1 - 20 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; subsequently, add an aqueous solution of the amino-functionalized clustered peptide polyelectrolyte in a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0095] Example 42 This example provides a method for preparing a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an acid-responsive clustered peptide electrolyte according to the method of Example 2; S2. Dissolve arjunolic acid in DMSO (1 - 20 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; subsequently, add an aqueous solution of the acid-responsive clustered peptide electrolyte in a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0096] Example 43 This example provides a method for preparing a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an amino-functionalized poly(2-oxazoline) electrolyte according to the method of Example 3; S2. Dissolve arjunolic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL; subsequently, add an aqueous solution of the amino-functionalized poly(2-oxazoline) electrolyte in a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex.

[0097] Example 44 This example provides a method for preparing a carboxyl group-containing triterpenoid compound - peptidomimetic polyelectrolyte complex, which specifically includes the following steps: S1. Prepare an acid-responsive poly(2-oxazoline) electrolyte according to the method of Example 4; S2. Dissolve arjunolic acid in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL. Subsequently, add an acid-responsive poly(2-oxazoline) electrolyte aqueous solution in a molar ratio of 10:1 - 1:10, and stir or stand at 20 - 50 °C for 2 - 48 hours to obtain a carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complex.

[0098] Example 45 Application Example Complex of acid-responsive peptidomimetic polyelectrolyte and oleanolic acid loaded with functional protein, specifically including: dissolve OA in DMSO (10 mg / mL), and then slowly add it to water to prepare a solution with a concentration of 0.1 - 1 mg / mL. Subsequently, add an acid-responsive peptidomimetic in a molar ratio of 10:1 - 1:10 for mixing, and at the same time add a protein solution. After mixing evenly, stand for 2 - 48 hours; centrifuge to collect the assembly.

[0099] Natural triterpenoids have significant antibacterial and antitumor activities. In the tumor microenvironment or bacterial infection environment, oxidative stress and acidic conditions can effectively promote the responsive release of the complex, thereby realizing the controlled release of peptidomimetic polyelectrolyte and carboxyl-containing triterpenoids. Add protein synchronously during the polyelectrolyte mixing stage, and use electrostatic adsorption to achieve the loading of the protein. The complex after protein loading can achieve efficient intracellular delivery and significantly enhance its therapeutic efficacy.

[0100] The present invention has the following remarkable advantages: (1) Outstanding raw material advantages. The peptidomimetic polymer has the characteristics of strong designability and simple synthesis route, while triterpenoid compounds are widely sourced and low-cost; (2) Simple and environmentally friendly preparation process. The controlled construction of polyelectrolyte complexes can be achieved through aqueous self-assembly; (3) Significant functional characteristics. The obtained complex not only has the ability to release drugs in response to the tumor microenvironment and can achieve precise delivery of active ingredients, but also has a synergistic antitumor effect; (4) Diversified drug-loading functions. The complex can not only carry triterpenoid compounds and peptidomimetic polyelectrolytes to exert synergistic effects, but also serve as a co-delivery system to transport chemotherapeutic drugs and biologic macromolecule drugs (such as therapeutic proteins). This technical solution provides an innovative solution for the development of antitumor therapy and combined drug delivery systems, and has important translational application value in the field of biomedicine.

[0101] It should be understood that various forms of processes shown above can be used, reordering, adding or deleting steps. For example, the steps recorded in the disclosure of the present invention can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and no limitation is made herein.

[0102] The above specific embodiments do not constitute a limitation to the protection scope of the present invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex, wherein: Specifically, it includes the following steps: S1. Prepare peptidomimetic polyelectrolytes; the peptidomimetic polyelectrolytes include amino-functionalized clustered peptide electrolytes, amino-functionalized poly(2-oxazoline) electrolytes, acid-responsive clustered peptide electrolytes, or acid-responsive poly(2-oxazoline) electrolytes; S2. Using the direct aqueous solution assembly method or the co-solvent-assisted assembly method, mix the peptidomimetic polyelectrolytes prepared in step S1 with carboxyl-containing triterpenoids, and after 2 - 48 h, obtain carboxyl-containing triterpenoid-peptidomimetic polyelectrolyte complexes.

2. The preparation method of a carboxyl triterpenoid compound-peptide mimetic polyelectrolyte complex according to claim 1, characterized in that: The structural formula of the acid-responsive clustered peptide electrolyte is: In the structural formula, m is 45 or 113; n is any number from 3 to 200; x is any number between 0 and 0.

9.

3. The preparation method of a carboxyl triterpenoid compound-peptide mimetic polyelectrolyte complex according to claim 2, characterized in that: The preparation method of the acid-responsive clustered peptide electrolyte specifically includes the following steps: S101. Preparation and polymerization of N-substituted carboxylic anhydrides: React primary amines containing different groups with glyoxylic acid to prepare N-substituted glycines; use di-tert-butyl dicarbonate to protect the secondary amines in N-substituted glycines; through a ring-forming reaction with phosphorus trichloride, convert N-substituted glycines into N-substituted carboxylic anhydrides; use small molecules or PEG containing primary amines as initiators, and prepare polymers through ring-opening polymerization; the structural formula of the polymer is: In the structural formula, m is 45 or 113; n is any number from 3 to 200; S102. Amino-functionalization modification: Perform a post-modification reaction on the polymer prepared in S101 with mercaptoethylamine to prepare amino-functionalized clustered peptide electrolytes; the structural formula of the amino-functionalized clustered peptide electrolytes is: In the structural formula, m is 45 or 113; n is any number from 3 to 200; S103. Acid-responsive modification: Use 2,3-dimethylmaleic anhydride to partially modify the amino-containing clustered peptide electrolytes prepared in S102 to finally obtain acid-responsive clustered peptide electrolytes.

4. The preparation method of a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex according to claim 1, characterized in that: The structural formula of the acid-responsive poly(2-oxazoline) electrolyte is: In the structural formula, n is any number from 3 to 200; x is any number between 0 and 0.

9.

5. The preparation method of a carboxyl triterpenoid compound-peptidomimetic polyelectrolyte complex according to claim 4, characterized in that: The preparation method of the acid-responsive poly(2-oxazoline) electrolyte specifically includes the following steps: S111. React 4-pentenoic acid, N-hydroxysuccinimide, and EDC·HCl to generate N-succinimidyl-4-pentenoate; react N-succinimidyl-4-pentenoate with 2-chloroethylamine hydrochloride and sodium hydroxide to generate N-(2-chloroethyl)-4-pentenamide, and react with potassium hydroxide in methanol to obtain the monomer 2-(3-butenyl)-2-oxazoline; S112. Use methyl trifluoromethanesulfonate as an initiator to initiate the ring-opening polymerization of the monomer 2-(3-butenyl)-2-oxazoline to prepare poly(2-oxazoline) containing allyl side groups; the structural formula of the poly(2-oxazoline) containing allyl is: In the structural formula, n is any number from 3 to 200; S113. Amino functional modification: The poly(2-oxazoline) containing allyl side groups prepared in S202 is subjected to post-modification reaction with mercaptoethylamine to prepare an aminated poly(2-oxazoline) electrolyte; the aminated poly(2-oxazoline) electrolyte has the structural formula: In the structural formula, n is any number between 3 and 200; S114. The amino-modified poly(2-oxazoline) electrolyte prepared in S113 was partially modified with 2,3-dimethylmaleic anhydride to obtain an acid-responsive poly(2-oxazoline) electrolyte.

6. The preparation method of a carboxyl triterpenoid compound-peptidomimetic polyelectrolyte complex according to claim 1, characterized in that: The carboxyl-containing triterpenoid compound is ursolic acid, oleanolic acid, betulinic acid, glycyrrhetinic acid or arjunic acid.

7. The preparation method of a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex according to claim 6, characterized in that: The step S2 adopts a direct aqueous solution assembly method, which specifically includes the following steps: mixing the peptidomimetic polyelectrolyte and the carboxyl-containing triterpenoid compound in an aqueous solution at a molar ratio of 1-10:10-1, and continuously stirring for 2-48 hours to complete the assembly.

8. A method for preparing a carboxyl triterpenoid compound - peptidomimetic polyelectrolyte complex according to claim 6, characterized in that: The step S2 adopts a solvent-assisted assembly method and specifically includes the following steps: dissolving the carboxyl-containing triterpenoid compound in a solvent, and then fully mixing it with the peptidomimetic polyelectrolyte in a molar ratio of 1-10:10-1, and completing the assembly after stirring or standing at 20-80 degrees Celsius for 2-48 hours.

9. A carboxyl triterpenoid compound-peptidomimetic polyelectrolyte complex, characterized in that: The preparation method is based on the preparation method of a carboxyl-containing triterpenoid compound-peptidomimetic polyelectrolyte complex according to any one of claims 1 to 8.

10. Use of a carboxyl triterpenoid - peptidomimetic polyelectrolyte complex as described in claim 9 in the preparation of nano - drugs, characterized in that: The carboxyl triterpenoid compound is dissolved in a cosolvent, and then slowly added into water to prepare a solution; the peptidomimetic polyelectrolyte is added and mixed, wherein the molar ratio of the carboxyl triterpenoid compound to the peptidomimetic polyelectrolyte is 1-10:10-1; the functional protein solution is added at the same time, mixed, and stirred or allowed to stand for 2-48 hours; the assembly is collected by centrifugation to obtain the nanomedicine.

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