Triptolide-polypeptide conjugate and preparation method of long-acting hydrogel preparation of triptolide-polypeptide conjugate

Abstract

The invention relates to a preparation method of a triptolide-polypeptide conjugate and a long-acting hydrogel preparation of the triptolide-polypeptide conjugate, the triptolide-polypeptide conjugate is formed by coupling triptolide and a polypeptide sequence with assembly performance through a degradable group, and the triptolide-polypeptide conjugate is obtained. The degradable group comprises a carboxylic ester bond formed by a hydroxyl group of triptolide and a carboxylic acid derivative. An alkyl chain can be further introduced into the N end of the polypeptide sequence with the self-assembly performance, so that the stability of an assembly is improved, and the drug release period is regulated and controlled through the length of the alkyl chain. The triptolide-polypeptide conjugate is self-assembled in an aqueous medium to form a nanostructure, and the nanostructure is subjected to solution-gel conversion under physiological conditions to form a hydrogel preparation. The hydrogel preparation can form a medicine storage cavern at a focus part, long-term and controllable medicine release is realized, and the safety of the medicine is improved.

Description

A method for preparing a triptolide-peptide conjugate and its long-acting hydrogel formulation. Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing a triptolide-peptide conjugate and its long-acting hydrogel formulation. Background Technology

[0002] Triptolide (TP) is a compound derived from the traditional Chinese medicine Tripterygium wilfordii. Since its isolation and identification in the 1970s, triptolide has been extensively studied due to its significant anti-inflammatory, immunosuppressive, and antitumor effects. Abundant pharmacological and molecular biological evidence suggests that triptolide can exert broad-spectrum biological effects by inhibiting key signaling pathways such as NF-κB and JAK / STAT, thereby regulating the activation of inflammatory factors and immune cells. However, despite its clear pharmacological potential, its clinical application has been limited by its unfavorable physicochemical properties and significant systemic toxicity. Triptolide is a highly hydrophobic molecule with extremely low solubility in aqueous solution (less than 0.02 mg / mL), resulting in severely inefficient drug release and absorption in oral or injectable formulations. Furthermore, triptolide is rapidly metabolized in vivo, with a plasma half-life typically less than one hour, making it easily eliminated and difficult to maintain effective blood drug concentrations. Furthermore, triptolide exhibits a lack of selectivity in its distribution, readily accumulating in vital organs such as the liver, kidneys, and heart after entering circulation, causing significant hepatotoxicity, nephrotoxicity, myocardial damage, and reproductive toxicity. These issues severely limit the therapeutic window of triptolide, significantly restricting its widespread clinical use.

[0003] To address these challenges, researchers have explored various improvement strategies, including chemical prodrug modification (such as the phosphate derivative Minnelide), liposomes, polymer nanoparticles, solid lipid nanoparticles, and cyclodextrin inclusion complexes. These methods have improved the solubility and in vivo distribution of triptolide to some extent and reduced acute toxicity, but they generally suffer from problems such as complex preparation, low synthesis efficiency, limited drug loading, and uneven release. Furthermore, most systems rely on organic solvents or complex polymers, increasing the difficulty of production and clinical translation.

[0004] In the prior art, Chinese invention patent CN120168394A discloses an immunomodulatory hydrogel formulation of triptolide prodrug responsive to reactive oxygen species, its preparation method, and its application. This technology involves multi-step chemical coupling of triptolide with ROS-sensitive groups, further linking it to a polypeptide sequence, and simultaneously incorporating immunomodulatory components to construct a composite gel formulation. Although the system can release drugs in the inflammatory microenvironment by relying on ROS triggering, which improves the local delivery safety of triptolide, it still has the following limitations: (1) The construction process of triptolide prodrug involves multiple chemical reactions and complicated purification processes, which require strict reaction conditions, and the overall synthesis route is complex and the yield is low (the actual yield is less than 15%). In addition, triptolide is expensive and the low yield is difficult to meet the needs of clinical large-scale preparation; (2) The activity of the ROS triggering mechanism varies greatly in different pathological tissues, and its response level is limited by the degree of local oxidative stress, resulting in insufficient release stability and universality; (3) The release time of the hydrogel preparation is fixed and the release rate or cycle cannot be controlled, which makes it difficult to meet the differentiated needs of drug release rate and duration in different treatment stages of chronic diseases such as arthritis.

[0005] Therefore, it is necessary to develop a triptolide drug delivery system with a simpler synthetic route, higher yield, and easier large-scale preparation, which adopts a triggering mode that is more universal in the tissue environment and can adjust the drug release cycle according to treatment needs, so as to achieve long-term, stable and safe delivery of triptolide to meet the clinical drug needs of chronic diseases. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of complex preparation, low product synthesis efficiency, uneven release of triptolide, and inability to achieve long-term release of triptolide in existing triptolide delivery systems, and to provide a method for preparing triptolide-peptide conjugates and their long-acting hydrogel formulations.

[0007] The triptolide-peptide conjugate prepared by this invention is based on the hydroxyl sites in the triptolide molecule that can be modified by esterification. By constructing groups including carboxylic acid ester bonds, the chemical conjugation of the prodrug is achieved, which significantly simplifies the construction steps of the triptolide prodrug, improves the overall yield, and facilitates large-scale preparation.

[0008] Based on this, by introducing alkyl chains of different lengths at the N-terminus of peptide sequences with self-assembly properties, the molecular self-assembly behavior of the conjugate can be regulated, thereby changing the release kinetics of triptolide. This allows the drug release cycle to be controllably adjusted between short and long-term release, adapting to the medication needs of different treatment regimens and making it more suitable for the design and translation of long-acting clinical formulations.

[0009] The long-acting triptolide-peptide conjugate formulation prepared in this invention is a functional hydrogel formulation based on triptolide prodrug modification and self-assembled peptides. The triptolide-peptide conjugate can self-assemble into a nanostructure in an aqueous medium. After injection in solution form, it can undergo a phase transition in response to the physiological environment at the injection site, forming an in-situ hydrogel. This gel formulation can form a local drug reservoir, achieving long-term, controllable drug release and improving drug safety.

[0010] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of this invention is to provide a triptolide-peptide conjugate, which is formed by coupling triptolide with a polypeptide sequence having self-assembly properties through a degradable group, wherein the degradable group includes a carboxylic acid ester bond formed by the hydroxyl group of triptolide and a carboxylic acid derivative.

[0011] In some specific embodiments, the carboxylic acid derivative is a hydroxybutyric acid or hydroxypropionic acid derivative, which belongs to aliphatic carboxylic acids. Thus, the carboxylic acid ester bond is an aliphatic carboxylic acid ester bond formed by hydroxybutyric acid or hydroxypropionic acid derivative and an alcohol derived from triptolide. Compared with aromatic esters, aliphatic esters are easier to hydrolyze and have better biocompatibility.

[0012] In this invention, the carboxylic acid ester bond can be hydrolyzed by endogenous esterases under physiological conditions, thereby releasing triptolide. Since esterases are widely expressed in tissues, this hydrolysis method has good stability and applicability, which is beneficial for obtaining a stable and controllable local drug release effect.

[0013] In some specific embodiments, the self-assembling peptide sequences include, but are not limited to, FFYGKRGD (SEQ ID NO.1), FFYKRGD (SEQ ID NO.2), FFYRGD (SEQ ID NO.3), FFYYGKRGD (SEQ ID NO.4), FFYYKRGD (SEQ ID NO.5), FFYYRGD (SEQ ID NO.6), GFFYYGKRGD (SEQ ID NO.7), GFFYYKRGD (SEQ ID NO.8), CGFFYYGKRGD (SEQ ID NO.9), CFFYYGKRGD (SEQ ID NO.10), CFFYYKRGD (SEQ ID NO.11), VVAARGD (SEQ ID NO.12), VVVAARGD (SEQ ID NO.13), KFKFEFKFE (SEQ ID NO.14), AEAEAKAKAEAEAKAKA (SEQ ID NO.15), and RADARADARADA (SEQ ID NO.16).

[0014] In the above sequence, the single-letter abbreviations of each amino acid are as follows: G = glycine (Gly), F = phenylalanine (Phe), Y = tyrosine (Tyr), K = lysine (Lys), R = arginine (Arg), A = alanine (Ala), V = valine (Val), E = glutamic acid (Glu), D = aspartic acid (Asp), C = cysteine ​​(Cys).

[0015] In some specific embodiments, an alkyl chain is further introduced at the N-terminus of the self-assembling polypeptide sequence; one end of the alkyl chain is connected to the amino group of the N-terminal amino acid of the self-assembling polypeptide sequence via an amide bond.

[0016] In some specific embodiments, the alkyl chain is C10. n A straight-chain alkyl group, wherein n is selected from any integer from 1 to 16.

[0017] Preferably, n can be 6, 12, or 16, such as a hexyl chain or a dodecyl chain. The introduction of an alkyl chain in this invention can regulate the self-assembly properties of the molecule, thereby controlling the drug release time.

[0018] In some specific embodiments, the triptolide-peptide conjugate self-assembles into a nanostructure in an aqueous medium.

[0019] The second technical solution of the present invention is to provide a method for preparing triptolide-peptide conjugate as described in one of the above technical solutions, comprising the following steps: S1, esterifying the hydroxyl group of triptolide to obtain triptolide derivative; S2, synthesizing a self-assembling functional peptide and introducing an alkyl chain at its N-terminus; S3, conjugating the triptolide derivative obtained in step S1 with the self-assembling peptide obtained in step S2 to obtain triptolide-peptide conjugate.

[0020] The third technical solution of the present invention is to provide a long-acting hydrogel preparation, which is prepared from the triptolide-peptide conjugate described in one of the above technical solutions.

[0021] The fourth technical solution of the present invention is to provide a method for preparing a long-acting hydrogel preparation as described in the third technical solution above, comprising the following steps: dissolving triptolide-peptide conjugate in an aqueous medium, allowing it to stand, and allowing it to self-assemble into a nano solution; adding the nano solution to a physiological medium to form a long-acting hydrogel preparation of triptolide.

[0022] The long-acting hydrogel formulation prepared by this invention can form a drug reservoir locally, enabling long-term and controllable release of triptolide and improving the systemic safety of the drug.

[0023] In some specific implementations, the standing conditions are: standing at room temperature for 1 to 48 hours.

[0024] In some specific embodiments, the physiological medium includes, but is not limited to, any one of phosphate buffer, bodily fluids or tissue culture media from animals, plants, and humans.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) The synthetic route of the present invention is simple, the structure is clear, and the yield is high. Compared with the problems of numerous steps and uneven structure in existing polymer modification or polymer systems, the present invention prepares triptolide-peptide conjugates by chemically conjugating peptide sequences with prodrugs, and the construction of the target molecule can be completed in only three steps. The reaction conditions of this synthetic process are mild and the operation is highly stable. The product obtained is a single and verifiable chemical entity, which is convenient for structural characterization and quality control using conventional analytical methods. The overall yield of the synthesis is about 60%, which is significantly higher than the effective yield of traditional multi-step polymerization systems and has good scale-up potential.

[0026] (2) This invention possesses both good biocompatibility and functional tunability, and the drug release cycle can be regulated by altering the molecular structure. This invention uses peptides with well-known origins and controllable sequences as coupling units, exhibiting good biocompatibility and suitability for local drug delivery systems. By introducing alkyl chains into the coupling molecule, the self-assembly characteristics of the coupling can be regulated, thereby altering the release kinetics of triptolide. By adjusting the length of the alkyl chain, this invention allows for controllable switching between short-term and long-term drug release cycles to adapt to the release duration requirements of different treatment regimens.

[0027] (3) This invention is well-suited for in-situ drug delivery applications. The triptolide prodrug-peptide conjugate of this invention can spontaneously form nanofibers in an aqueous medium and further transform into a stable three-dimensional gel network under physiological conditions. Compared with existing systems that require external cross-linking agents, physical stimulation, or organic solvents to gel, this invention can complete the phase transition from solution to gel under mild conditions without additional cross-linking steps. The gelation process is simple and reproducible. The resulting gel structure is stable and suitable for in-situ injection into sites such as joint cavities to improve local drug retention and significantly reduce the frequency of administration. Attached Figure Description

[0028] Figure 1 shows the reaction formula for synthesizing the intermediate 4-(2-pyridyldithio)butyric acid.

[0029] Figure 2 shows the mass spectrum of the intermediate 4-(2-pyridyldithio)butyric acid.

[0030] Figure 3 shows the reaction formula for synthesizing triptolide hydroxy derivatives.

[0031] Figure 4 shows the mass spectrum of the hydroxy derivative of triptolide.

[0032] Figure 5 shows the structural formula of TP-KRGD.

[0033] Figure 6 shows the mass spectrum of TP-KRGD.

[0034] Figure 7 shows the degradation curve of TP-KRGD under esterase conditions.

[0035] Figure 8 shows the structural formula of TP-C6KRGD.

[0036] Figure 9 shows the mass spectrum of TP-C6KRGD.

[0037] Figure 10 shows TP-C 12 The structural formula of KRGD.

[0038] Figure 11 shows TP-C 12 Mass spectrum of KRGD.

[0039] Figure 12 shows transmission electron microscope images of TF and TC6F.

[0040] Figure 13 shows the rheological test results of TF and TC6F.

[0041] Figure 14 shows the solution-gel transition of TF and TC6F.

[0042] Figure 15 shows TF, TC6F and TC 12 In vitro drug release curve of F hydrogel.

[0043] Figure 16 shows a photograph of TF forming an in situ gel in the joint cavity of a mouse and remaining there for a long time.

[0044] Figure 17 shows the degradation curve of TF in the knee joint cavity of mice.

[0045] Figure 18 is a graph showing the joint swelling in CIA mice after TF treatment.

[0046] Figure 19 shows a Micro-CT image of the joint bones of CIA mice after TF treatment.

[0047] Figure 20 shows the stained tissue section of the ankle joint of CIA mice after TF treatment. Detailed Implementation

[0048] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0049] In the following embodiments, some of the required materials are sourced as follows: (1) Tripterygium wilfordii was purchased from Chengdu Pufeed Reference Technology Co., Ltd.

[0050] (2) The amino acids were purchased from Jier Biochemical (Shanghai) Co., Ltd.

[0051] Unless otherwise specified, all chemical reagents, raw materials, and experimental conditions mentioned in this manual should be understood as commercially available products that can be directly obtained from conventional commercial channels under existing technical conditions. Furthermore, unless specifically described, all experimental steps and processing techniques involved should be considered as content that is well known to those skilled in the art and can be implemented in accordance with common technical practices.

[0052] Example 1 This example provides a method for preparing a long-acting formulation of triptolide-peptide conjugate, the steps of which are as follows: (1) Synthesis of triptolide-peptide conjugate (1-1) 2,2'-dithiopyridine (6.35 g) was weighed and dissolved in 20 mL of methanol. Hydroxybutyric acid (2 g) was slowly added dropwise under nitrogen protection and stirred overnight at room temperature. The reaction solution was purified by column chromatography, with hexane:ethyl acetate = 3:1 (volume ratio) and 1‰ acetic acid as the eluent, to obtain the intermediate 4-(2-pyridyldithio)butyric acid.

[0053] (1-2) 4-(2-pyridyldithio)butyric acid (63.63 mg), DMAP (4-dimethylaminopyridine) (5.08 mg), and EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) (53.19 mg) were dissolved in anhydrous dichloromethane (DCM) and reacted under nitrogen protection for 30 min. Tripterygium wilfordii (50 mg) was dissolved in anhydrous DCM and slowly added dropwise to the above reaction solution, stirred overnight at room temperature. The reaction solution was extracted with 1 M hydrochloric acid, separated, and purified by column chromatography. The eluent was n-hexane:ethyl acetate containing 1‰ acetic acid, eluted in gradients of 5:1, 4:1, and 3:1. The product, triptolide hydroxy derivative, was separated and collected at a 3:1 gradient.

[0054] (1-3) The synthesized polypeptide Ac-CGFFYYGKRGD (SEQ ID NO.9) and the triptolide hydroxy derivative were added to a reaction flask at a molar ratio of 2.5:1. An appropriate amount of dimethyl sulfoxide (DMSO) was added as a solvent, and the mixture was stirred at room temperature for 36 h under nitrogen protection. After purification by reversed-phase high-performance liquid chromatography (RP-HPLC) (the aqueous phase was pure water, and the organic phase was acetonitrile) and drying by freeze dryer, the triptolide prodrug-peptide conjugate TP-KRGD was obtained.

[0055] (2) TP-KRGD (1.8 mg) was dissolved in deionized water (100 μL) and left to stand at room temperature. After several hours, the molecules self-assembled to form nanofiber solution TF.

[0056] (3) Add 10 μL of 10×PBS to the above nanofiber solution TF. The solution undergoes a solution-gel transition to obtain a long-acting hydrogel formulation of triptolide prodrug.

[0057] Compared with Example 1, Example 2 differs in the polypeptide structure and its N-terminal modification method.

[0058] In this embodiment, the polypeptide Ac-CFFYYGKRGD (SEQ ID NO.10) was selected as the coupling matrix, and a C6 straight-chain alkyl chain was further introduced at the N-terminal amino acid to obtain the aliphatic chain modified polypeptide Ac-C6-CFFYYGKRGD. The introduction of the aliphatic chain enhances the hydrophobic interaction of the molecule and the side chain stacking interaction between molecules, thereby improving its self-assembly ability in aqueous solution and further prolonging the release time of triptolide. The remaining synthesis steps are the same as in Example 1. The aliphatic chain modified polypeptide Ac-C6-CFFYYGKRGD was reacted with the triptolide hydroxyl derivative under the same coupling conditions as in Example 1 to obtain the triptolide-polypeptide conjugate TP-C6KRGD. This conjugate can spontaneously form a nanofiber structure after dissolving in water, and its self-assembled solution is designated as TC6F.

[0059] Compared to Example 2, Example 3 changed the straight-chain alkyl chain introduced at the N-terminal amino acid of the polypeptide from C6 to C4. 12 Thus, the fatty acid chain modified polypeptide Ac-C was obtained. 12 -CFFYYGKRGD further prolongs the release time of triptolide. The remaining synthetic steps are the same as in Example 1, with the fatty acid chain-modified polypeptide Ac-C... 12 -CFFYYGKRGD reacted with the triptolide hydroxy derivative under the same coupling conditions as in Example 1 to obtain the triptolide-peptide conjugate TP-C. 12 KRGD. This conjugate, when dissolved in water, can spontaneously form nanofiber structures; its self-assembled solution is denoted as TC. 12 F.

[0060] Test Example 1: Figure 1 shows the synthesis reaction of the intermediate 4-(2-pyridyldithio)butyric acid, with a yield of up to 92.4%. Figure 2 shows the mass spectrometry analysis of 4-(2-pyridyldithio)butyric acid, verifying the correctness of its molecular synthesis.

[0061] Figure 3 shows the synthetic reaction formula of triptolide hydroxy derivative, and the yield of triptolide hydroxy derivative can reach 65.2%. Figure 4 shows the mass spectrometry analysis of triptolide hydroxy derivative, which verifies that the molecular synthesis of triptolide hydroxy derivative is correct.

[0062] Figure 5 shows the structural formula of the triptolide prodrug-peptide conjugate TP-KRGD. The yield of TP-KRGD can reach 51%. The molecular weight was detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the results are shown in Figure 6, which verifies that the TP-KRGD molecule was synthesized correctly.

[0063] The in vitro degradation capacity of its esterase response was verified using porcine liver esterase (PLE).

[0064] The 0.25 mM TP-KRGD working solution was thoroughly mixed with an equal volume of PBS buffer or an enzyme solution containing 10 U / mL PLE. Samples were taken at predetermined time points (30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours) to analyze the molecular degradation process, and degradation kinetic curves were generated based on the data, as shown in Figure 7. It can be seen that within 24 hours, 62.5% of the TP-KRGD molecules were cleaved by esterases, and the molecules remained stable in the PBS environment. This test result ensures that the molecules can release the drug and exert their effects under physiological conditions in vivo.

[0065] Under the conditions of esterase response release experiments, the molecular concentration is low, and the molecules exist in the system as unimolecular rather than assembled forms. At this point, the ester bonds are in the same chemical microenvironment, and changes in alkyl chain length do not substantially affect esterase recognition and hydrolysis behavior. Therefore, selecting TP-KRGD as a representative compound for esterase response release verification can reflect the common esterase response characteristics of this type of molecule.

[0066] Test Example 2, Figure 8 shows the structural formula of the triptolide prodrug-peptide conjugate TP-C6KRGD. The molecular weight was detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the results are shown in Figure 9, verifying the correct synthesis of TP-C6KRGD.

[0067] Test Example 3, Figure 10, shows the triptolide prodrug-peptide conjugate TP-C. 12 The structure of KRGD was determined, and its molecular weight was detected using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results are shown in Figure 11, validating TP-C. 12 The KRGD molecule was synthesized correctly.

[0068] In Test Example 4, the triptolide prodrug nanofibers (denoted as TF) formed by the self-assembly of TP-KRGD in water were observed using transmission electron microscopy (TEM). The image is shown in Figure 12a, exhibiting a uniform and regular nanofiber structure. Under the same conditions, nanofibers (denoted as TC6F) formed by the self-assembly of TP-C6KRGD also formed a well-morphologically good fibrous structure, and its TEM image is shown in Figure 12b.

[0069] Further analysis using a rotational rheometer was conducted to investigate the mechanical properties and solution-gel transition behavior under physiological conditions. Figure 13a shows that after adding an equal volume of 0.1 M PBS, the concentration of 10 mM TF rapidly increased from G′ to G″, indicating that the system transitioned from a solution state to an elastically dominant gel structure. Figure 13b shows the rheological curves of TC6F under the same conditions, showing that its gelation rate and network stability were both enhanced compared to TF, indicating that the introduction of fatty acid chains further improved the gel strength of the self-assembled system.

[0070] Test Example 5 was conducted to verify the solution-gel transition characteristics of the self-assembled system. An equal volume of 10 mM TF was mixed with 0.1 M PBS, and the gel formation was assessed using the inverted test tube method. As shown in Figure 14a, TF rapidly stopped flowing after the addition of PBS, forming a structurally stable gel. The same procedure was applied to TC6F, and the results are shown in Figure 14b. The solution-gel transition also occurred quickly. There was no significant difference in gel formation time between TF and TC6F; both gelled immediately after the addition of PBS.

[0071] In Test Example 6, 500 μL of a 10 mM TF solution was added, and an equal volume of 0.1 M PBS was added to induce the formation of a hydrogel (denoted as TF hydrogel). 500 μL of 0.1 M PBS was added to the surface of the prepared TF hydrogel sample as a release buffer, and the experiment was conducted at a constant temperature of 37°C. At specified time points, 400 μL of supernatant was aspirated each time, and an equal volume of fresh PBS was added to maintain a constant total volume. The concentration of TP-KRGD in the release solution was determined by high-performance liquid chromatography (HPLC) to calculate the cumulative release ratio. Following the same method, the same procedure was applied to the gel formed by TP-C6KRGD (denoted as TC6F hydrogel) and TP-C... 12 KRGD-formed gel (denoted as TC) 12 The F hydrogel was subjected to in vitro release testing.

[0072] The cumulative release curves of the three gels are shown in Figure 15. The TF hydrogel exhibited a continuous and relatively stable release process throughout the testing period, with a cumulative release rate of approximately 40% by day 24; the TC6F hydrogel showed a significantly lower cumulative release rate than the TF hydrogel, with a cumulative release rate of less than 20% by day 24; TC...12 The release rate of the F hydrogel was further slowed down, with the lowest cumulative release ratio within the same time period, less than 10%, showing a more significant delayed release characteristic. These results indicate that the introduction of alkyl chains can effectively enhance the gel network strength and prolong the drug release time, and that the release time is dependent on the carbon chain length.

[0073] In Test Example 7, a 10 mM aqueous solution of triptolide was injected into the knee joint cavity of 8-week-old female BALB / c mice, with an injection volume of 10 μL per knee. Mice were sacrificed at 30 min post-injection and on days 5, 10, 20, and 30. The knee joints were dissected and exposed, and images of the local joint appearance were taken to observe local changes. As shown in Figure 16, the TF aqueous solution rapidly formed a hydrogel after injection into the mouse knee joint, and gel remained in the knee joint until day 30. The knee joint gel was removed, dissolved in acetonitrile, and quantified using HPLC. The curve is shown in Figure 17, indicating that 16% triptolide hydrogel remained in situ in the mouse knee joint 30 days after injection.

[0074] Because rheumatoid arthritis is a chronic autoimmune disease, clinical treatment often requires long-term medication. The presence of gel residue in the knee joints of mice 30 days after injection indicates that the gel can establish a long-term drug reservoir in the body, continuously releasing the drug in situ and providing sustained control of disease progression. Looking towards clinical applications, in-situ gels capable of long-acting sustained release hold promise for reducing dosing frequency and improving patient adherence.

[0075] Example 8 used healthy DBA / 1 mice (6–8 weeks old) to establish a collagen-induced arthritis (CIA) model. The specific method was as follows: On Day 0, 100 μL of a mixture of type II collagen (CAS No. 9007-34-5) and complete Freund's adjuvant (CAS No. 9007-81-2) emulsion was administered subcutaneously at the base of the mouse tail. On Day 21, the procedure was repeated, but an equal volume emulsion of type II collagen (CAS No. 9007-34-5) and incomplete Freund's adjuvant (Chondrex, Catalog #7002) was used to trigger arthritis attacks. One week after induction, the animals were randomly divided into three groups: an untreated control, a free triptolide group, and a triptolide prodrug hydrogel preparation group. 10 μL of the corresponding preparation was injected into the bilateral posterior knee joint cavity of each group of mice. Tripterygium wilfordii was administered at a uniform dose of 10 μg (the dose with verified efficacy). Healthy mice that were not modeled or treated were set up as controls. The specific grouping is detailed in Table 1.

[0076] Table 1. Injectable substances in different groups of mouse models in Test Example 7 After treatment began, the thickness of the hind paws of mice was measured every 4 days, and a thickness trend graph was generated to evaluate the intervention effect of TF gel on rheumatoid arthritis. Figure 18 shows the dynamic curves of hind paw thickness in each group of mice. Compared with the untreated group and the free TP group, triptolide in situ hydrogel significantly alleviated the swelling of the paws in CIA mice. After the condition of the untreated group stabilized and the efficacy of the treated group tended to stabilize, the experimental endpoint was confirmed. The hind paws of mice were collected, and the ankle joint bone destruction was analyzed by Micro-CT scanning to evaluate the protective effect of TF on the joint bone structure. Histological sections of the mouse ankle joints were stained (H&E staining and Safranin-Fix-Green staining) to evaluate the effect of TF on protecting the synovium and cartilage of the mouse joints. Figure 19 shows the Micro-CT results, indicating significant bone erosion in the ankle joints of the untreated group and the Free TP group, while no significant bone damage was observed in the ankle joint of the hydrogel treatment group (TF), and its bone structure was similar to that of the healthy controls, demonstrating its bone-protective effect in rheumatoid arthritis. Figure 20 shows the tissue section staining results, indicating significant synovial damage and a significant reduction in cartilage in the untreated group and the Free TP group, while the synovial structure of the hydrogel treatment group (TF) remained intact, the cartilage structure was clear, and no significant erosion was observed, demonstrating its significant synovial protection and cartilage repair effects.

[0077] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A triptolide-polypeptide conjugate, characterized in that, It is formed by coupling triptolide with a polypeptide sequence with self-assembly properties through a degradable group, wherein the degradable group includes a carboxylic acid ester bond formed by the hydroxyl group of triptolide and a carboxylic acid derivative.

2. The triptolide-polypeptide conjugate according to claim 1, characterized in that, The carboxylic acid derivative is a hydroxybutyric acid or hydroxypropionic acid derivative.

3. The triptolide-polypeptide conjugate according to claim 1, characterized in that, The self-assembling peptide sequences include, but are not limited to, FFYGKRGD (SEQ ID NO.1), FFYKRGD (SEQ ID NO.2), FFYRGD (SEQ ID NO.3), FFYYGKRGD (SEQ ID NO.4), FFYYKRGD (SEQ ID NO.5), FFYYRGD (SEQ ID NO.6), GFFYYGKRGD (SEQ ID NO.7), GFFYYKRGD (SEQ ID NO.8), CGFFYYGKRGD (SEQ ID NO.9), CFFYYGKRGD (SEQ ID NO.10), CFFYYKRGD (SEQ ID NO.11), VVAARGD (SEQ ID NO.12), VVVAARGD (SEQ ID NO.13), KFKFEFKFE (SEQ ID NO.14), AEAEAKAKAEAEAKAKA (SEQ ID NO.15), and RADARADARADA (SEQ ID NO.16).

4. The triptolide-polypeptide conjugate according to claim 1, characterized in that, The self-assembly polypeptide sequence is further connected to an alkyl chain at its N-terminus, and one end of the alkyl chain is connected to the amino group of the N-terminal amino acid of the self-assembly polypeptide sequence via an amide bond.

5. The triptolide-polypeptide conjugate according to claim 4, characterized in that, The alkyl chain is C. n Straight-chain alkyl group, where n is any integer from 1 to 16.

6. The triptolide-polypeptide conjugate according to claim 1, characterized in that, The triptolide-peptide conjugate self-assembles into a nanostructure in an aqueous medium.

7. A method for preparing the triptolide-peptide conjugate as described in any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Esterify the hydroxyl group of triptolide to obtain a triptolide derivative; S2. Synthesize a self-assembling functional polypeptide and introduce an alkyl chain at its N-terminus; S3. Couple the triptolide derivative obtained in step S1 with the self-assembling polypeptide obtained in step S2 to obtain a triptolide-polypeptide conjugate.

8. A long-acting hydrogel formulation, characterized in that, It is prepared from the triptolide-polypeptide conjugate according to any one of claims 1 to 6.

9. A method for preparing a long-acting hydrogel formulation as described in claim 8, characterized in that, The process includes the following steps: dissolving triptolide-peptide conjugate in an aqueous medium, allowing it to stand, and allowing it to self-assemble into a nano solution; adding the nano solution to a physiological medium to form a long-acting hydrogel formulation of triptolide.

10. The preparation method according to claim 9, characterized in that, The conditions for standing are: standing at room temperature for 1 to 48 hours; the physiological medium is selected from any one of phosphate buffer, animal, plant and human body fluids or tissue culture media.

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