Active oxygen responsive triptolide prodrug immunoregulation hydrogel preparation as well as preparation method and application thereof

Abstract

The invention relates to an active oxygen responsive triptolide prodrug immunoregulation hydrogel preparation as well as a preparation method and application thereof. Triptolide is coupled with an active oxygen responsive group to synthesize a triptolide derivative; then, the triptolide derivative is coupled with polypeptide, and a polypeptide-reactive oxygen responsive group-triptolide conjugate is obtained; then dissolving the polypeptide-active oxygen responsive group-triptolide conjugate in a solvent, standing, and carrying out self-assembly to obtain a triptolide prodrug nanofiber solution; finally, an immunomodulator is added into the triptolide prodrug nanofiber solution for incubation, and the active oxygen responsive triptolide prodrug immunoregulation hydrogel preparation is obtained. The hydrogel preparation disclosed by the invention can responsively release the medicine triptolide and the immunomodulator at the focus part of rheumatoid arthritis, so that the medicine accumulation of non-target organs is reduced, the safety and effectiveness of treatment are improved, and the progress of diseases is effectively controlled.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation, a preparation method thereof, and an application thereof. Background Art

[0002] Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease with a complex pathogenesis, involving abnormal activation of various immune cells such as dendritic cells (DCs), T cells, B cells, macrophages, and over-secretion of various inflammatory factors such as tumor necrosis factor-α, interleukin-1, and interleukin-6. The abnormal local immune microenvironment in inflamed joints not only leads to continuous amplification of local inflammatory responses but also triggers systemic immune disorders. Currently, the clinical treatment of RA mainly relies on systemic administration of disease-modifying antirheumatic drugs, which can inhibit autoimmune responses and control disease progression. However, systemic administration has problems such as uneven drug distribution, poor targeting, and large side effects, and long-term use may cause adverse reactions such as liver and kidney function damage, gastrointestinal reactions, and immunosuppression. There are high levels of reactive oxygen species (ROS) in the diseased joints of RA. ROS is not only a by-product of inflammatory responses but also directly participates in joint tissue damage and disease progression, and can promote inflammatory responses, induce cell damage, and inhibit oxidative defense mechanisms, and has become an important target for RA treatment research in recent years. Therefore, developing a drug delivery system that can locally and in-situ treat RA and regulate the local immune microenvironment has important clinical significance.

[0003] Triptolide (TP) is a diterpenoid lactone compound extracted from the traditional Chinese medicine Tripterygium wilfordii, and has significant anti-inflammatory and immunosuppressive effects. Research shows that TP can effectively relieve the inflammatory response and immune damage of RA through multiple mechanisms such as inhibiting the NF-κB signaling pathway, reducing the secretion of inflammatory factors, and inducing apoptosis of T cells. However, the poor water solubility, low bioavailability, and strong systemic toxicity of TP limit its clinical application. How to overcome the problems of poor efficacy, non-target organ accumulation, and large toxic and side effects existing in the treatment of RA with tripterygium preparations is crucial for the development of in-situ treatment of RA. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problems of poor efficacy, non-target organ accumulation, and large toxic and side effects existing in the treatment of RA with tripterygium preparations, and thus provide a reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation, a preparation method thereof, and an application thereof.

[0005] The present invention provides a reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation, which is easy to prepare, has good safety, high stability, and high drug loading. As a reactive oxygen species-responsive delivery system, it can form a drug reservoir in situ after locally delivering triptolide and immunomodulators, and achieve drug release in response to the disease tissue microenvironment for a long time, thereby realizing in-situ treatment of rheumatoid arthritis, effectively controlling the disease progression, and improving the safety of treatment.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] The technical solution of the present invention is to provide a reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation, which includes a triptolide prodrug nanofiber solution and an immunomodulator added to the triptolide prodrug nanofiber solution;

[0008] The triptolide prodrug nanofiber solution is obtained by self-assembling a polypeptide-reactive oxygen species-responsive group-triptolide conjugate into nanofibers in a solvent, and the polypeptide-reactive oxygen species-responsive group-triptolide conjugate is composed of a polypeptide containing a self-assembling sequence, a reactive oxygen species-responsive linker molecule, and the drug triptolide;

[0009] The immunomodulator is selected from one or more combinations of cytokines or antibodies.

[0010] In some specific embodiments, the polypeptide is selected from any of the following amino acid sequences: GFF, GFFY (SEQ ID NO.1), GFFYY (SEQ ID NO.2), GFFE (SEQ ID NO.3), GFFF (SEQ ID NO.4), VVA, VVAA (SEQ ID NO.5), VVVA (SEQ ID NO.6), VVVAA (SEQ ID NO.7), VVVAAA (SEQ ID NO.8), VVG, VVVG (SEQ ID NO.9), AAGG (SEQ ID NO.10), KFKFEFKFE (SEQ ID NO.11), KFKFKFEFE (SEQ ID NO.12), AEAEAKAKAEAEAKAKA (SEQ ID NO.13), FHFDFHFD (SEQ ID NO.14), IKVAV (SEQ ID NO.15), VKVKVKVKVKVKVKV (SEQ ID NO.16), RADARADARADARADA (SEQ ID NO.17), CGFFYGKRGD (SEQ ID NO.18).

[0011] In the present invention, the polypeptide is a self-assembling sequence, where S = serine, V = valine, A = alanine, E = glutamic acid, I = isoleucine, L = leucine, F = phenylalanine, R = arginine, D = aspartic acid, Q = glutamine, P = proline, G = glycine, K = lysine, C = cysteine, W = tryptophan, Y = tyrosine.

[0012] In some specific embodiments, the reactive oxygen species-responsive group is selected from any one of thioketals, ketothiolates, diselenide groups, monoselenide groups, oxalate groups, and borate groups.

[0013] In some specific embodiments, the cytokine is selected from any one of IL-10, TGF-β, IL-22, IL-35, IL-37, IL-38, and IL-1Ra.

[0014] In some specific embodiments, the antibody is selected from antibodies targeting any one of CD20, TNF-α, IL-6R, IL-17A, IL-23, CD52, CD3, IL-2, C5, CD19 / CD3, and CD20 / CD3.

[0015] In some specific embodiments, the antibody is selected from any one of rituximab, ofatumumab, infliximab, adalimumab, tocilizumab, secukinumab, ixekizumab, ustekinumab, alemtuzumab, muromonab, basiliximab, eculizumab, blinatumomab, motavizumab, and gefitinib.

[0016] More preferably, the triptolide prodrug nanofiber solution is obtained by self-assembling a polypeptide-reactive oxygen species-responsive group-triptolide conjugate into nanofibers in a solvent, where the polypeptide is CGFFYGKRGD (SEQ ID NO.18) and the reactive oxygen species-responsive group is ketothiolate.

[0017] More preferably, the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation comprises a triptolide prodrug nanofiber solution and an immunomodulator added to the triptolide prodrug nanofiber solution. The triptolide prodrug nanofiber solution is obtained by self-assembling a polypeptide-reactive oxygen species-responsive group-triptolide conjugate into nanofibers in a solvent, where the polypeptide is CGFFYGKRGD (SEQ ID NO.18), the reactive oxygen species-responsive group is ketothiolate, and the immunomodulator is an antibody targeting CD20.

[0018] The second technical solution of the present invention is to provide a preparation method of the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation as described in any one of the above technical solutions, comprising the following steps:

[0019] S1. Couple triptolide with a reactive oxygen species-responsive group to synthesize a triptolide derivative.

[0020] S2. Couple the triptolide derivative obtained in step S1 with a polypeptide to obtain a polypeptide-reactive oxygen species-responsive group-triptolide conjugate.

[0021] S3. Dissolve the polypeptide-reactive oxygen species-responsive group-triptolide conjugate obtained in step S2 in a solvent, let it stand still, and self-assemble to obtain a triptolide prodrug nanofiber solution.

[0022] S4. Add an immunomodulator to the triptolide prodrug nanofiber solution obtained in step S3 and incubate to obtain a reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation.

[0023] In some specific embodiments, in step S3, the solvent is selected from any one of water or phosphate buffer; the temperature for standing still is room temperature, and the standing time is 0 - 2 days.

[0024] More preferably, the phosphate buffer selected as the solvent has a low concentration. Exemplarily, the concentration of the phosphate buffer is not higher than 0.01 M.

[0025] In some specific embodiments, in step S3, the mass concentration of the triptolide prodrug nanofiber solution is 5 - 100 mg / mL.

[0026] In some specific embodiments, in step S4, the incubation temperature is 4 - 50 °C, and the incubation time is 0 - 2 days.

[0027] The third aspect of the technical solution of the present invention is to provide an application of the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation as described in one of the above technical solutions in the preparation of a drug for treating rheumatoid arthritis.

[0028] The reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation of the present invention adopts a polypeptide-reactive oxygen species-responsive group-drug conjugate system. This system can self-assemble in solution to form a triptolide prodrug nanofiber solution, and can be triggered in situ and rapidly gelate under physiological conditions. This system contains ROS-responsive groups, can form a drug reservoir at the inflamed joint, achieve precise drug release by responding to the local ROS level, and reduce the ROS level in the joint. After further loading an immunomodulator, this preparation can effectively regulate the local immune microenvironment, reduce drug accumulation in non-target organs, and achieve long-term, safe and efficient treatment.

[0029] After the injection of the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation of the present invention into the joint cavity in liquid form, it can trigger a phase change in situ in the joint to form a gel, constructing an in-situ drug reservoir to achieve the long-term release of triptolide and immunomodulators at the lesion site, while effectively reducing the exposure time of the drug in the blood circulation, thereby significantly reducing the occurrence of systemic toxic reactions. This responsive hydrogel can achieve the intelligent release of drugs according to the changes in the local microenvironment and is an ideal drug carrier for in-situ treatment of diseases.

[0030] In addition, it can also respond to the ROS level in the inflamed joint cavity, precisely regulate the release of triptolide and immunomodulators, and simultaneously achieve the clearance of ROS in the joint cavity. Triptolide can effectively alleviate the inflammatory response and immune damage of RA through multiple mechanisms such as reducing the secretion of inflammatory factors and inducing apoptosis of T cells. Combining with immunomodulators can long-term regulate the local immune microenvironment and establish autoimmune tolerance, effectively controlling the progression of rheumatoid arthritis.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention utilizes the "solution-gel" transition property of the self-assembly of the polypeptide-reactive oxygen species-responsive group-triptolide conjugate to form a reactive oxygen species-responsive hydrogel in the joint cavity after injection. This property can not only ensure the long-term release of immunomodulators and triptolide at the lesion site, but also significantly reduce the concentration of the drug in the blood, avoid the accumulation of the drug in non-target organs, and greatly improve the safety of drug use.

[0033] (2) The reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation prepared by the present invention can respond to the microenvironment of high reactive oxygen species level in the joint cavity of rheumatoid arthritis, achieve the intelligent release of drugs and immunomodulators matching special pathological conditions, reduce the reactive oxygen species level in the joint cavity while precisely releasing drugs, and effectively control the progression of the disease.

[0034] (3) The preparation method of the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation of the present invention is simple, the administration is convenient, and it is easy to transport and store, providing an effective solution to the technical problem of poor clinical druggability of triptolide, opening up a new way for its clinical application, and having important clinical transformation value. Brief Description of the Drawings

[0035] Figure 1 It is the structural formula of TP-TK-RGD.

[0036] Figure 2 It is the mass spectrometry analysis result of TP-TK-RGD.

[0037] Figure 3It is the transmission electron microscope image of TPF.

[0038] Figure 4 It is the rheological test result of TPF.

[0039] Figure 5 It is the degradation curve of TP-TK-RGD under in vitro reactive oxygen conditions.

[0040] Figure 6 It is the "solution-gel" transition image of TPF@aCD20.

[0041] Figure 7 It is the fluorescence confocal photo of TPF@Cy5-aCD20.

[0042] Figure 8 It is the in vitro drug release curve of TPF@aCD20.

[0043] Figure 9 It is the photo of TPF@aCD20 forming an in-situ gel in the joint cavity of mice.

[0044] Figure 10 It is the photo of joint swelling of CIA mice after treatment with TPF@aCD20.

[0045] Figure 11 It is the level of immune cells in the lymph nodes of CIA mice after treatment with TPF@aCD20.

[0046] Figure 12 It is the Micro-CT image of the joint bones of CIA mice after treatment with TPF@aCD20. Detailed implementation mode

[0047] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation mode and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0048] In the following embodiments, the descriptions of some raw materials are as follows:

[0049] (1) Tripterygium wilfordii Hook. f. was purchased from Chengdu Pufeide Reference Technology Co., Ltd.;

[0050] (2) Amino acids were purchased from GL Biochem (Shanghai) Ltd.;

[0051] (3) Anti-mouse CD20 antibody (aCD20) was purchased from Startech Co., Ltd.

[0052] For the remaining raw materials or processing technologies without special instructions, it means that they are all conventional commercially available raw material products or conventional processing technologies in the art.

[0053] Example 1

[0054] This example provides a method for preparing a reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation, which includes the following steps:

[0055] (1) Synthesis of polypeptide-reactive oxygen species-responsive group-triptolide conjugate

[0056] The reactive oxygen species-responsive group used in this example is ketothiol, and the polypeptide containing a self-assembly sequence used is CGFFYGKRGD (SEQ ID NO.18). The specific experimental steps are as follows:

[0057] (1-1) Mix 3-mercaptopropionic acid (4.26 mL) with anhydrous acetone (7.34 mL), stir for 10 min, then add 4 mL of concentrated hydrochloric acid. React for 6 h under nitrogen protection and then quickly cool in an ice bath. After white crystals appear in the reaction flask, filter with ice-cyclohexane and freeze-dry to obtain white powdery ketothiol (TK).

[0058] (1-2) Dissolve triptolide (1.0 g), ketothiol (1.33 g), DMAP (4-dimethylaminopyridine, 60 mg), and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1.0 g) in (25 mL) of ultradry dichloromethane. React for 12 hours under nitrogen protection. Extract the reaction solution with 1 M hydrochloric acid and further purify by column chromatography. The mobile phase is dichloromethane:methanol (DCM:MeOH) = 50:1 (v / v) to obtain triptolide-ketothiol conjugate TP-TK.

[0059] (1-3) Dissolve TP-TK (1.0 g), DMAP (63 mg), and EDCI (442 mg) in 10 mL of ultradry dichloromethane, stir and react for 30 min, then add maleimide (850 mg), and react overnight under nitrogen protection. After extracting the reaction solution with 1 M hydrochloric acid, dry it with anhydrous sodium sulfate and further purify by column chromatography. The mobile phase is dichloromethane:methanol (DCM:MeOH) = 225:1 (v / v) to obtain triptolide-ketothiol-maleimide conjugate TP-TK-Mal, that is, triptolide derivative.

[0060] (1-4) React polypeptide Ac-CGFFYGKRGD and triptolide derivative TP-TK-Mal at a molar ratio of 2:1 under nitrogen protection in dimethyl sulfoxide solution for 48 hours. After purification by high performance liquid chromatograph and drying by freeze dryer (vacuum less than 100 Pa), obtain polypeptide-reactive oxygen species-responsive group-triptolide conjugate TP-TK-RGD.

[0061] (2) Dissolve TP-TK-RGD (2.5 mg) in deionized water (100 μL), with a concentration of 25 mg / mL. After standing at room temperature for 2 - 4 hours, TP-TK-RGD undergoes self-assembly to obtain the triptolide prodrug nanofiber solution TPF.

[0062] (3) Add the immunomodulator aCD20 (4.7 mg) to the triptolide prodrug nanofiber solution TPF and incubate at 37 °C for 4 hours to obtain the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation TPF@aCD20.

[0063] The immunomodulator aCD20 selected here is an antibody targeting mouse CD20. For the experimental study of the mouse CIA model constructed in the subsequent test examples, it is used to simulate the role of clinically used antibodies targeting human CD20 such as rituximab in the treatment of RA.

[0064] Test Example 1

[0065] The structural formula of the polypeptide-reactive oxygen species-responsive group-triptolide conjugate TP-TK-RGD is as Figure 1 shown, and the mass spectrometry analysis results of TP-TK-RGD are as Figure 2 shown, confirming the successful synthesis of TP-TK-RGD.

[0066] Perform the degradation test of the prepared TP-TK-RGD under in vitro reactive oxygen species conditions:

[0067] Prepare a TP-TK-RGD solution with a concentration of 0.25 mM, mix it with PBS or 1 mM H2O2 solution in equal volume respectively, and measure the degradation of TP-TK-RGD at 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively, and plot the degradation curve of TP-TK-RGD.

[0068] Figure 5 The degradation curve shown indicates that the TP-TK-RGD molecule can respondently break under the reactive oxygen species concentration simulating the pathological environment, releasing triptolide. Under reactive oxygen species conditions, after 24 h, 80% of the TP-TK-RGD molecules can undergo responsive cleavage. The cleavage of the TP-TK-RGD molecule is caused by the oxidation and cleavage of the ketothiol bond in the molecule by ROS, and ROS undergoes a reduction reaction and is consumed during this process. When the TP-TK-RGD molecule reacts with ROS in the joint cavity, it can reduce the ROS level in the joint cavity and regulate the immune microenvironment in the joint cavity.

[0069] Test Example 2

[0070] The transmission electron microscope photograph of the triptolide prodrug nanofiber solution TPF is as Figure 3As shown, a clear nanofiber structure of TPF can be seen.

[0071] The gelling properties of the prepared TPF were investigated:

[0072] The rheological properties of triptolide prodrug nanofiber solution TPF were tested using a rotary rheometer. At 70 seconds during the test, 0.1M PBS was added to examine the instantaneous gelling ability of TPF. In the rheological test, G′ represents the storage modulus of the material, which represents the ability of the material to maintain shape stability after being stressed; G″ represents the loss modulus of the material, which represents the ability of the material to rebound and deform. Generally, when G′ < G″, the material is in a liquid state, and when G′ > G″, the material is in a solid state.

[0073] Figure 4 For the rheological test results of TPF, it can be seen that the triptolide prodrug nanofiber solution quickly changes from a liquid state to a gel state after adding 0.1M PBS.

[0074] Test Example 3

[0075] Simulating the physiological environment in vivo, 0.1M PBS was added to the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation TPF@aCD20, and the "solution-gel" transition characteristics of TPF@aCD20 were tested using the inverted method. Figure 6 For the inverted experiment results, it can be seen that the TPF@aCD20 solution can quickly form a gel after adding 0.1M PBS, showing immediate "solution-gel" transition characteristics.

[0076] Using the method of replacing "immunomodulator aCD20" with "aCD20 labeled with fluorescent dye Cy5" and preparing the TPF@Cy5-aCD20 hydrogel according to the above method, fluorescence photographs of the TPF@Cy5-aCD20 hydrogel were taken using a fluorescence confocal microscope, as Figure 7 shown, it can be seen that Cy5-aCD20 is evenly distributed in the hydrogel.

[0077] Test Example 4

[0078] In vitro drug release test of the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation TPF@Cy5-aCD20:

[0079] 500 μL of 0.1M PBS was added above TPF@Cy5-aCD20 (500 μL) as the release medium and placed in an environment at 37°C. Samples of 400 μL were taken at 1, 3, 5, 7... 23 days respectively and an equal amount of fresh 0.1M PBS was added. HPLC was used to determine the cumulative release of the TP-TK-RGD molecule, and an enzyme-linked immunosorbent assay (ELISA) reader was used to determine the cumulative release of Cy5-aCD20, and the release curve was plotted.

[0080] Figure 8 The in vitro release curve shows the slow release process of TP-TK-RGD molecules and aCD20 from the triptolide prodrug immunomodulatory hydrogel. As shown in the figure, the in vitro drug release process is uniform and slow. By the 23rd day of the experiment, approximately 40% of the TP-TK-RGD molecules and 60% of the aCD20 had been released.

[0081] Test Example 5

[0082] In-situ formation test of the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation TPF@aCD20 in the joint cavity:

[0083] Inject the TPF@aCD20 solution into the knee joint cavity of mice. After 10 minutes, dissect the mice and observe the formation of TPF@aCD20 in the joint cavity and take pictures.

[0084] Figure 9 The results show that TPF@aCD20 has the "solution-gel" transition property in mice. It is injected in solution state and triggers the formation of the triptolide prodrug immunomodulatory hydrogel under physiological conditions.

[0085] Test Example 6

[0086] Regulation of the immune cell level in the lymph nodes of rheumatoid arthritis mice by the reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation TPF@aCD20

[0087] Establish a CIA model of rheumatoid arthritis using healthy DBA / 1 mice: Select female DBA / 1 mice at 6-8 weeks old. Subcutaneously inject 100 μL of a mixed emulsion of type II collagen and complete Freund's adjuvant (1:1, v:v) at the base of the mouse tail on day 0, and subcutaneously inject 100 μL of a mixed emulsion of type II collagen and incomplete Freund's adjuvant (1:1, v:v) at the base of the mouse tail on day 21 to construct a CIA model of rheumatoid arthritis.

[0088] On the 7th day after model establishment, randomly divide the mice into 6 groups (saline group, free triptolide group, free triptolide + aCD20 group, blank gel + aCD20 group, triptolide prodrug nanofiber group, triptolide prodrug nanofiber + aCD20 group), and inject the corresponding therapeutic drugs into the knee joint cavities of the hind legs on both sides of the mice (see Table 1 for details. Inject 10 μL of the therapeutic agent into each joint cavity of each mouse. The dosing dose of aCD20 is 100 μg, and the dosing dose of triptolide is 10 μg. The dosing doses of aCD20 and triptolide are both effective therapeutic dosing doses).

[0089] Table 1 Therapeutic agents for different groups of the CIA mouse model

[0090] Group Therapeutic drugs injected into mice Saline group (Untreated) Saline Free triptolide group (Free TP) Triptolide Free triptolide + aCD20 group (Free TP / aCD20) Triptolide, aCD20 Blank gel + aCD20 group (C8-RGD@aCD20) C8-RGD solution, aCD20 Triptolide prodrug nanofiber group (TPF) TPF Triptolide prodrug nanofiber + aCD20 group (TPF@aCD20) TPF@aCD20

[0091] Wait until the onset of the untreated group is stable and the treatment effect of the treatment group is stable, then collect mouse lymph node cells and label CD80 with an antibody. + CD86 + DC cells, CD3 + T cells, CD19 + B cells and Foxp3 + CD4 + CD3 + T cells, and analyze their proportions in the lymph nodes.

[0092] Figure 11 A shows CD80 + CD86 + The proportion of DC cells in the lymph nodes, Figure 11 B, Figure 11 C respectively show CD3 + T cells, CD19 + The proportion of B cells in the lymph nodes, Figure 11 D shows Foxp3 + CD4 + CD3 + The proportion of T cells. The results of the immune investigation show that TPF@aCD20 can effectively inhibit the maturation of DC cells in CIA mice, reduce the levels of their T cells and B cells, and increase their Treg levels, which plays an effective role in inhibiting autoimmunity and establishing autoimmune tolerance.

[0093] Test Example 7

[0094] The reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel preparation TPF@aCD20 controls the disease progression of rheumatoid arthritis in mice

[0095] As in Test Example 6, use DBA / 1 mice to establish a CIA model of rheumatoid arthritis and group the mice for treatment. After starting the administration, monitor the paw thickness of the mice every other day, draw the paw thickness curve of the mice, and evaluate the effect of TPF@aCD20 in treating rheumatoid arthritis.

[0096] Figure 10 It is the effect diagram of TPF@aCD20 in treating rheumatoid arthritis in CIA mice. Compared with the untreated group and the control group, the triptolide prodrug immunomodulatory hydrogel TPF@aCD20 can effectively relieve the paw swelling of CIA mice and significantly control the disease progression.

[0097] Test Example 8

[0098] ROS-responsive triptolide prodrug immunomodulatory hydrogel preparation TPF@aCD20 protects the joint bones of rheumatoid arthritis mice from erosion

[0099] As described in Test Example 6, a CIA model of rheumatoid arthritis was established using DBA / 1 mice, and the mice were grouped for treatment. After the untreated group developed the disease stably and the treatment effect of the treatment group was stable, the mouse feet were taken and Micro-CT was used to identify the degree of bone erosion at the ankle joints of the mice to evaluate the effect of TPF@aCD20 in protecting the joint bones of the mice.

[0100] As Figure 12 shown in the Mirco CT images, compared with the untreated group, the joints and foot bones of TPF@aCD20 were intact and no deformities were seen, demonstrating that TPF@aCD20 can significantly protect the joint bones of mice from erosion and confirming its effective therapeutic effect on rheumatoid arthritis.

[0101] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention as disclosed should be within the protection scope of the present invention.

Claims

1. An active oxygen-responsive triptolide prodrug immunomodulatory hydrogel preparation, characterized in that: It includes a triptolide prodrug nanofiber solution and an immunomodulator added to the triptolide prodrug nanofiber solution; The triptolide prodrug nanofiber solution is obtained by self-assembly of a polypeptide-active oxygen responsive group-triptolide conjugate in a solvent to form nanofibers, and the polypeptide-active oxygen responsive group-triptolide conjugate is composed of a polypeptide containing a self-assembly sequence, an active oxygen responsive linker molecule and the drug triptolide; The immunomodulator is selected from one or more combinations of cytokines or antibodies.

2. The active oxygen responsive triptolide prodrug immunomodulatory hydrogel preparation according to claim 1, characterized in that: The polypeptide is selected from any of the following amino acid sequences: GFF, GFFY (SEQ ID NO.1), GFFYY (SEQ ID NO.2), GFFE (SEQ ID NO.3), GFFF (SEQ ID NO.4), VVA, VVAA (SEQ ID NO.5), VVVA (SEQ ID NO.6), VVVAA (SEQ ID NO.7), VVVAAA (SEQ ID NO.7) NO.8), VVG, VVVG (SEQ ID NO.9), AAGG (SEQ ID NO.10), KFKFEFKFE (SEQ ID NO.11), KFKFKFEFE (SEQ ID NO.12), AEAEAKAKAEAEAKAKA (SEQ ID NO.13), FHFDFHFD (SEQ ID NO.14), IKVAV (SEQ ID NO.15), VKVKVKVKVKVKVKV (SEQ ID NO.16), RADARADARADARADA (SEQ ID NO.17), CGFFYGKRGD (SEQ ID NO.18).

3. The active oxygen responsive triptolide prodrug immunomodulatory hydrogel preparation according to claim 1, characterized in that: The active oxygen responsive group is selected from any one of thioketal, thioketal, diselenide group, monoselenide group, oxalate group and borate group.

4. The active oxygen responsive triptolide prodrug immunomodulatory hydrogel preparation according to claim 1, characterized in that: The cytokine is selected from any one of IL-10, TGF-β, IL-22, IL-35, IL-37, IL-38, and IL-1Ra; The antibody is selected from antibodies targeting any one of CD20, TNF-α, IL-6R, IL-17A, IL-23, CD52, CD3, IL-2, C5, CD19 / CD3, and CD20 / CD3.

5. The active oxygen responsive triptolide prodrug immunomodulatory hydrogel preparation according to claim 4, characterized in that: The antibody is selected from any one of rituximab, obinutuzumab, infliximab, adalimumab, tocilizumab, secukinumab, ixekizumab, ustekinumab, alemtuzumab, muromonab, basiliximab, eculizumab, blinatumomab, motuzumab, and gefuzumab.

6. A method for preparing the active oxygen responsive triptolide prodrug immunomodulatory hydrogel preparation according to any one of claims 1 to 5, characterized in that: The steps include: S1, coupling triptolide with an active oxygen responsive group to synthesize a triptolide derivative; S2, coupling the triptolide derivative obtained in step S1 with a polypeptide to obtain a polypeptide-active oxygen responsive group-triptolide conjugate; S3, dissolving the polypeptide-active oxygen responsive group-triptolide conjugate obtained in step S2 in a solvent, allowing it to stand and self-assemble to obtain a triptolide prodrug nanofiber solution; S4, adding the immunomodulator to the triptolide prodrug nanofiber solution obtained in step S3 and incubating the solution to obtain an active oxygen responsive triptolide prodrug immunomodulatory hydrogel preparation.

7. The preparation method according to claim 6, characterized in that: In step S3, the solvent is selected from any one of water or phosphate buffer; the standing temperature is room temperature, and the standing time is 0-2 days.

8. The preparation method according to claim 6, characterized in that: In step S3, the mass concentration of the triptolide prodrug nanofiber solution is 5-100 mg / mL.

9. The preparation method according to claim 6, characterized in that: In step S4, the incubation temperature is 4-50° C., and the incubation time is 0-2 days.

10. Use of the active oxygen-responsive triptolide prodrug immunomodulatory hydrogel preparation according to any one of claims 1 to 5 in the preparation of a drug for treating rheumatoid arthritis.

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