Novel composite self-assembly polypeptide hydrogel as well as preparation and application thereof

Abstract

The invention relates to a novel composite self-assembly polypeptide hydrogel and preparation and application thereof, and the novel composite self-assembly polypeptide hydrogel comprises an active peptide and a drug-loading peptide with a self-assembly function. According to the invention, the injectable characteristic of hydrogel is utilized, and active peptide without a self-assembly function and drug-loading peptide with a self-assembly function are creatively combined to form a brand-new composite polypeptide hydrogel supramolecular system while the tumor resistance is realized, so that the slow release of the active peptide and the reinforcement of a self-assembly peptide system are realized; and the defect of poor polypeptide stability is overcome.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a novel composite self-assembling polypeptide hydrogel and a preparation method and application thereof. Background Art

[0002] The number of deaths from cancer is increasing every year in every country. There are many factors that induce cancer, including genetic diseases, eating habits, occupational stress, exposure to radiation or ingestion of toxic substances, so the number of cancer cases and deaths is expected to grow rapidly, which also makes cancer treatment a difficult and hot topic in world medicine. With the continuous development of medicine, people have discovered many tumor treatment drugs, but when delivering anti-tumor drugs to the tumor site, it is inevitable that the drugs will have toxic side effects on other normal tissues and organs.

[0003] Chinese patent CN115317619A discloses a nucleic acid nano drug carrier that can be used for the delivery and efficacy enhancement of small molecule anticancer drugs platinum drugs. The nucleic acid nano drug carrier of the invention has a highly sensitized property and can quickly enter eukaryotic cells. The carrier can accurately target and deliver small molecule drugs platinum drugs into tumor cells, effectively kill tumor cells and regulate the expression of drug-resistant genes in tumor cells. However, due to the low stability of nucleic acids in the body and the rapid clearance by the extracellular host, it is challenging to transport nucleic acids to active sites in cells. In addition, due to the negative charge, high molecular weight and hydrophilicity of nucleic acids, their ability to penetrate cell membranes is very poor.

[0004] With the rapid development of biology, oncology, medicinal chemistry, materials science and other disciplines, combined treatment methods in various fields have received widespread attention. Various new types of biomaterials have become effective methods for drug delivery and localization, allowing the release of loaded drugs to be controlled spatially, temporally and cellularly. In recent years, supramolecular polypeptide hydrogels (SPH) have stood out in the field of tumor treatment. Polypeptides have outstanding chemical diversity, high biocompatibility and biorecognition capabilities. The use of polypeptides to construct self-assembled drug carriers has broad application scenarios and unlimited imagination space in the field of biomedicine.

[0005] Chinese patent CN113648277A discloses a pH-responsive polypeptide hydrogel, preparation method and application of anti-tumor combination drugs. The polypeptide self-assembles under physiological conditions to form a polypeptide hydrogel with a dense nano-network microstructure. The polypeptide hydrogel of the invention is used as a co-drug delivery system for anticancer drugs paclitaxel and gemcitabine, which has good pH responsiveness. After reaching the lesion site, it first releases gemcitabine in large quantities at one time, and then slowly releases paclitaxel under the stimulation of the tumor microenvironment to achieve a synergistic anti-cancer effect. Among various anticancer drugs, small molecule drugs such as paclitaxel (PTX) and cisplatin have been widely recognized. However, due to the poor water solubility, low bioavailability and strong systemic adverse reactions of small molecule chemotherapy drugs, their clinical application is limited. It is reported that 80% of patients will experience adverse reactions to varying degrees when receiving cancer treatment, which not only reduces the anti-tumor efficacy but also increases medical costs.

[0006] RADA16 is a short peptide that has been proven to be self-assembled, biodegradable, non-toxic, and sustained-release. It can be used for injection into specific tissues for local sustained-release drug delivery therapy. On the other hand, tumor angiogenesis is mainly caused by the binding of abnormally expressed vascular endothelial growth factor (VEGF) to its receptor vascular endothelial growth factor receptors (VEGFRs). It is one of the most important anti-tumor targets. Tumor angiogenesis can be inhibited by blocking the interaction between the two. The corresponding small molecule targeted drugs (sunitinib, etc.) and monoclonal antibody drugs (bevacizumab, etc.) are currently important clinical therapeutic drugs. However, small molecule drugs have poor selectivity and large toxic side effects, and monoclonal antibody drugs have high production costs and are prone to antibody resistance after long-term use. Therefore, improving or developing new treatments remains a daunting challenge and a long-term task. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a novel composite self-assembling polypeptide hydrogel and its preparation and application. By utilizing the injectable characteristics of the hydrogel, while carrying out anti-tumor and other treatments, the active peptides with pharmacological functions and the drug-carrying peptides with self-assembly functions are creatively combined to form a new composite polypeptide hydrogel supramolecular system, thereby achieving the sustained release of the active peptides and the strengthening of the self-assembling peptide system, thus overcoming the disadvantage of poor polypeptide stability.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0009] A novel composite self-assembling polypeptide hydrogel comprises active peptides and drug-carrying peptides with self-assembly function.

[0010] Preferably, the active peptide is an active peptide with pharmacodynamic function, preferably an active peptide with self-assembly function, and more preferably a cyclic peptide inhibitor, and the active peptide is selected from one or more of B-cL1, insulin, thymopentin, octreotide, cetrorelix, canagliflozin, semaglutide and liraglutide;

[0011] Preferably, the drug-carrying peptide is selected from RADA16 and / or EAK16.

[0012] Preferably, the weight ratio of the active peptide to the drug-carrying peptide is 1:10 to 10:1.

[0013] In order to solve the above technical problems, another technical solution adopted by the present invention is:

[0014] A method for preparing the novel composite self-assembling polypeptide hydrogel as described above comprises the following steps:

[0015] (1) adding the drug-loaded peptide and the active peptide to a first solution to form a mixed solution, wherein the first solution includes a solution A suitable for physiological applications and an adjuvant;

[0016] (2) The mixed solution is allowed to stand for self-assembly to obtain a novel composite self-assembling polypeptide hydrogel.

[0017] Preferably, the specific operation of step (1) is: dissolving the drug-loaded peptide and the active peptide in a solution A suitable for physiological applications, and mixing or ultrasonically treating to form a mixed solution;

[0018] Preferably, the ultrasonic time is 0-30 min.

[0019] Preferably, the concentration of the active peptide in the mixed solution of step (1) is 0.01-100 mg / mL, and the concentration of the drug-loaded peptide is 0.01-100 mg / mL.

[0020] Preferably, the solution A suitable for physiological applications in step (1) is selected from one or more of ultrapure water, physiological saline, and phosphate buffer.

[0021] Preferably, the standing time in step (2) is 0-24 h, preferably 0-2 h.

[0022] Preferably, the standing temperature in step (2) is 0-65°C.

[0023] In order to solve the above technical problems, another technical solution adopted by the present invention is:

[0024] The novel composite self-assembling polypeptide hydrogel as described above is used as a drug delivery system in the preparation of anti-tumor drugs and drugs for the treatment of other diseases.

[0025] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0026] 1. The present invention utilizes the injectable feature of hydrogels, and while fighting against tumors, it also pioneered the concept of sustained-release drug delivery by synergistic self-assembly of different functional peptides. It combines active peptides with drug-carrying peptides with self-assembly function to form a new composite polypeptide hydrogel supramolecular system. VEGFR inhibitor B-cL1 and RADA16 are co-dissolved in the mother liquor and can self-assemble to form a hydrogel, thereby achieving sustained release of active peptides and strengthening of the self-assembly peptide system, overcoming the disadvantage of poor stability of polypeptides.

[0027] 2. The present invention can reduce the loss of drugs during the delivery process by local administration of self-assembling polypeptide hydrogels, and prolong the concentration and action time of drugs at the lesion site;

[0028] 3. The novel composite polypeptide hydrogel formed by self-assembly of the VEGFR inhibitor B-cL1 and RADA16 in the present invention can effectively solve the drawbacks of small molecule and polypeptide anticancer drugs and other drug carriers, better carry drugs and reach specific tumor sites, and the cyclic peptide inhibitor B-cL1 can inhibit angiogenesis, and the novel composite polypeptide has high safety, good biocompatibility, low toxicity and side effects, and excellent anticancer effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The scanning electron microscope images of the self-assembled hydrogels of Examples 1-2 and Comparative Example 1;

[0030] Figure 2 TEM images of the self-assembled hydrogels of Examples 1-2 and Comparative Example 1;

[0031] Figure 3 The figure shows the biocompatibility test results of hydrogels with different active peptide concentrations;

[0032] Figure 4 Figure 1 is a diagram of the tumor shape of the first batch of experiments on the anti-tumor performance of Examples 1-2 and Comparative Example 1;

[0033] Figure 5 Tumor volume graph for the first batch of experiments on antitumor performance;

[0034] Figure 6 Tumor weight graph for the first batch of experiments on anti-tumor properties;

[0035] Figure 7 Figure 1 shows the body weight of nude mice in the first batch of experiments on anti-tumor properties;

[0036] Figure 8 The shape of the tumor in the second batch of experiments for anti-tumor performance;

[0037] Fig. 9 Tumor volume graph for the second batch of experiments on antitumor performance;

[0038] Fig.10 The tumor weight graph of the second batch of experiments for antitumor performance;

[0039] Fig.11 The body weight of nude mice in the second batch of experiments for anti-tumor performance;

[0040] Fig.12 These are histopathological sections of different groups. DETAILED DESCRIPTION

[0041] In order to make the technical solutions and beneficial effects of the present invention more clearly understood, the following is a detailed description by listing specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0042] If no specific techniques or conditions are specified in the following examples, they are generally carried out according to conventional techniques or conditions described in the literature in the art, or according to the product instructions and the conditions recommended by the manufacturer.

[0043] Unless otherwise indicated, various starting materials, materials and reagents were either commercially available or synthesized according to known methods.

[0044] Explanation of terms

[0045] Active peptides, also known as functional peptides, refer to peptide compounds that are beneficial to the life activities of biological organisms or have physiological effects.

[0046] Drug carriers are a type of medical biomaterial product that has gradually emerged with the development of biomaterial science, clinical medicine and pharmacology. Their essential characteristic is that they enter the human body as a carrier of drugs, allowing the drugs to better exert their effectiveness. Drug carriers can make the location, speed and method of drug release selective and controllable.

[0047] Drug-carrying peptides are peptide compounds used as drug carrier materials. They are biologically active, non-toxic and have good compatibility.

[0048] Self-assembly refers to a technology in which basic structural units spontaneously form an ordered structure. Peptides are small protein fragments formed by amino acids through amide bonds. Since amino acid residues have different chemical structures, peptides can effectively achieve molecular self-assembly by utilizing hydrogen bonds between peptide bonds and hydrogen bonds between amino acid residues, electrostatic effects, hydrophobic effects, and π-π stacking effects.

[0049] The invention discloses a novel composite self-assembling polypeptide hydrogel, which comprises an active peptide and a drug-carrying peptide with a self-assembling function.

[0050] In certain specific embodiments, the active peptide is an active peptide having pharmacological function.

[0051] In certain specific embodiments, the active peptide is an active peptide with self-assembly function.

[0052] In certain specific embodiments, the active peptide is a cyclic peptide inhibitor.

[0053] In certain specific embodiments, the active peptide is selected from one or more of B-cL1, insulin, thymopentin, octreotide, cetrorelix, canagliflozin, semaglutide and liraglutide.

[0054] In a specific embodiment, the active peptide is B-cL1 (CTVELMGTVAKQLVPC), and the preparation method is described in J.Med.Chem.2017, 60, 6598-6606. The peptide is solid-phase synthesized on a microwave-assisted CEM-Liberty1 synthesizer using Fmoc chemistry and Rink amide MBHA polystyrene resin (0.1, 0.45 mmol / g) with HBTU / DIEA or DIC / OXYMA as coupling agents.

[0055] In certain specific embodiments, the drug-carrying peptide is selected from RADA16 and / or EAK16.

[0056] In a specific embodiment, the drug-loaded peptide is RADA16 (n-RADARADARADARADA-c), which was commercially synthesized and purified by Jiangsu GenScript Co., Ltd. (Nanjing, China). The purity of the peptide was confirmed by HPLC and it was used without further purification.

[0057] In certain specific embodiments, the weight ratio of the active peptide to the drug-loaded peptide is 1:10 to 10:1.

[0058] The method for preparing the novel composite self-assembling polypeptide hydrogel as described above comprises the following steps:

[0059] (1) adding the drug-loaded peptide and the active peptide to a first solution to form a mixed solution, wherein the first solution includes a solution A suitable for physiological applications and an adjuvant;

[0060] (2) The mixed solution is allowed to stand for self-assembly to obtain a novel composite self-assembling polypeptide hydrogel.

[0061] In certain specific embodiments, the specific operation of step (1) is: dissolving the drug-loaded peptide and the active peptide in a solution A suitable for physiological applications, and mixing or ultrasonically treating the solution to form a mixed solution.

[0062] In some specific embodiments, the ultrasound time is 0-30 min, and can also be 2-25 min, 2-20 min, 5-18 min, 8-15 min, and more specifically can be 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min.

[0063] In some specific embodiments, the concentration of the active peptide in the mixed solution of step (1) is 0.01-100 mg / mL, and can also be 1-80 mg / mL, 1-60 mg / mL, 1-50 mg / mL, and more specifically can be 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL.

[0064] In certain specific embodiments, the concentration of the drug-loaded peptide in the mixed solution of step (1) is 0.01-100 mg / mL, and can also be 1-80 mg / mL, 1-60 mg / mL, 1-50 mg / mL, and more specifically can be 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL.

[0065] In certain specific embodiments, the solution A suitable for physiological applications in step (1) is selected from one or more of ultrapure water, physiological saline, and phosphate buffer.

[0066] In a specific embodiment, the solution A suitable for physiological applications in step (1) is selected from ultrapure water.

[0067] In some specific embodiments, the standing time in step (2) is 0-24 h, and can also be 0-18 h, 0-12 h, 0-6 h, 0-4 h, 0-2 h, and more specifically can be 1 h, 2 h, 3 h, 4 h, 5 h, 6 h.

[0068] In some specific embodiments, the standing temperature in step (2) is 0-65° C., and can also be 0-65° C., 0-35° C., 0-20° C., 0-15° C., 0-10° C., and more specifically can be 1° C., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C. Temperature control can control the assembly speed.

[0069] The novel composite self-assembling polypeptide hydrogel as described above is used as a drug delivery system in the preparation of anti-tumor drugs and drugs for the treatment of other diseases.

[0070] Example 1

[0071] The peptide powders of the drug-carrying peptide RADA16 and the active peptide B-cL1 (CTVELMGTVAKQLVPC) were dissolved in ultrapure water (Milli-Q) and subjected to ultrasonic treatment for 10 minutes to prepare a mixed solution, in which the concentration of the active peptide was 15 mg / mL and the concentration of the drug-carrying peptide was 10 mg / mL. The mixed solution was placed in a 4°C refrigerator for 60 minutes for self-assembly to obtain a new composite self-assembling peptide hydrogel, named RADA@L1. RADA@L1 was diluted ten times and placed in a 4°C refrigerator for 6 hours for self-assembly to obtain a peptide solution for analysis in test examples 1 and 2, and RADA@L1 was directly used for analysis in test examples 3 and 4.

[0072] Example 2

[0073] The peptide powders of drug-loaded peptide RADA16 and active peptide B-cL1 were dissolved in ultrapure water (Milli-Q) and subjected to ultrasonic treatment for 10 minutes to prepare a mixed solution with a concentration of 10 mg / mL for RADA16 and 30 mg / mL for active peptide B-cL1. The mixed solution was placed in a 4°C refrigerator for 1-10 minutes for self-assembly to obtain a new composite self-assembling peptide hydrogel, named RADA@L2. RADA@L2 was diluted ten times and placed in a 4°C refrigerator for 6 hours for self-assembly to obtain a peptide solution for analysis in test examples 1 and 2, and RADA@L2 was directly used in test examples 3 and 4.

[0074] Example 3

[0075] The polypeptide powders of the drug-loaded peptide RADA16 and the active peptide B-cL1 were dissolved in ultrapure water (Milli-Q) and subjected to ultrasonic treatment for 10 min to prepare solutions with a RADA16 concentration of 1.0 mg / mL and a B-cL1 concentration of 1.0 mg / mL. The solutions were vortexed and mixed to obtain a mixed solution, which was allowed to stand at 37°C for 6 h for self-assembly to obtain a polypeptide layered solution, named RADA@L3.

[0076] Comparative Example 1

[0077] RADA hydrogel,The self-assembling peptide RADA16 (n-RADARADARADARADA-c) was commercially synthesized and purified by Jiangsu GenScript Co., Ltd. (Nanjing, China), and the purity of the peptide was confirmed by HPLC and used without further purification. RADA16 was dissolved in ultrapure water at 10 mg / mL to form a hydrogel.

[0078] Comparative Example 2

[0079] The active peptide is B-cL1 (CTVELMGTVAKQLVPC), and the preparation method is described in J.Med.Chem.2017, 60, 6598-6606. The peptide was synthesized in solid phase on a microwave-assisted CEM-Liberty1 synthesizer using Fmoc chemistry and Rink amide MBHA polystyrene resin (0.1, 0.45 mmol / g) with HBTU / DIEA or DIC / OXYMA as coupling agents.

[0080] Test Case

[0081] 1. SEM analysis of hydrogel

[0082] The products of Examples 1-2 and Comparative Example 1 were observed by scanning electron microscopy. The results of the scanning electron microscopy are shown in the attached figure. Figure 1 The surface morphology of the material was observed by scanning electron microscope (SEM). According to the instructions, the products of Examples 1-2 and Comparative Example 1 were first deionized and stained, and then scanned in a suitable mode to observe and analyze the three-dimensional morphology of the hydrogel. Figure 1 It can be seen that the nanofibers formed by the self-assembly of a single RADA16 peptide in comparative example 1 are small and regularly entangled in alternation; after the addition of the B-cL1 cyclic peptide inhibitor, the formation of coarser nanofibers was observed in the RADA@L1 hydrogel sample of Example 1, indicating that the degree of assembly was enhanced and a wrinkled surface structure was formed; further increasing the ratio of the B-cL1 cyclic peptide inhibitor, the nanofibers in the RADA@L2 hydrogel system of Example 2 are coarser and more obvious, and smaller granular self-assembled aggregates are formed on the surface of the nanofibers. The above results show that the B-cL1 cyclic peptide inhibitor participates in and affects the self-assembly of the RADA polypeptide based on the β-Sheet structure, improves the degree of self-assembly, and forms a new nanofiber structure.

[0083] 2. Transmission electron microscopy analysis of hydrogel

[0084] The products of Examples 1-2 and Comparative Example 1 were observed by transmission electron microscopy, and the transmission electron microscopy results are shown in the attached figure. Figure 2 As shown. The morphological changes in the microstructure of the nanofibers before and after the cross-linking of the polypeptide hydrogel were observed using a transmission electron microscope. The RADA group of Comparative Example 1 had many filamentous fibers, which indicated that the stacking self-assembled the β-Sheet secondary structure into a mesh network. After adding the cyclic peptide inhibitor, it was found that the RADA@L1 group of Example 1 had dense and short fibers cross-linked to form a mesh. As the concentration of the cyclic peptide inhibitor increased, the nanofibers became more tightly entangled, the self-assembled fibers became more continuous, and the RADA@L2 group of Example 2 showed an obvious nanofiber network.

[0085] 3. Biocompatibility and safety experiments of hydrogels containing different active peptide concentrations

[0086] Methods: L6 cells were revived and cultured in DMEM medium. Plated and cultured. B-cL1 drugs with gradient concentrations containing RADA16 were prepared in PBS solution, where the concentration of RADA16 was 10 mg / mL, and the concentrations of B-cL1 were 5 mM, 1 mM, 200 μM, 40 μM, 8 μM, and 1.6 μM, respectively. The control RADA group was a RADA16 hydrogel with a concentration of 10 mg / mL, and the control blank group (control) was a pure PBS solution group. The cell culture medium was discarded, and the prepared drugs were added. Five replicates were made for each concentration and cultured in an incubator for 48 hours. After 48 hours, MTT (thiazolyl blue) 10 μL / well (5 mg / mL) was added in a dark place. After 4 hours, the supernatant was removed, DMSO 150 μL / well was added, and the absorbance at 490 nm was measured with an enzyme marker.

[0087] The experimental results are as attached Figure 3 As shown, the cell survival rate of the RADA16 hydrogel group (control RADA group) is close to 100%, indicating that RADA can be used as a good drug carrier and has almost no toxicity to cells. In addition, through the treatment results of gradient concentrations of B-cL1 cyclic peptide inhibitors, it was found that only at ultra-high administration concentrations above 1mM, the hydrogel showed a slight inhibitory effect on the growth of L6 cells. This may be caused by the dense nanofiber network composed of polypeptide hydrogels. As the concentration decreases, the survival rate of L6 cells increases, which is almost the same as the control blank group and the control RADA group.

[0088] These results indicate that the composite self-assembled polypeptide hydrogel has good biocompatibility and has no obvious cytotoxicity to L6 cells.

[0089] 4. Analysis of anti-tumor performance of Examples 1-2 and Comparative Example 1

[0090] Methods: To culture SGC-7901 gastric cancer cells, 45 18-20g 3-5 week old BALB / c nude mice were purchased and raised in the same environment for 3-5 days to allow the nude mice to adapt to the growth environment. SGC-7901 gastric cancer cells were digested and collected in advance, resuspended in serum-free 1640 medium, and injected into the right armpit of the nude mice with 2×10 6 After injection, the animals were fed normally and the tumor size was detected every day.

[0091] First batch of experiments: Nude mice were divided into 5 groups, 5 mice in each group. The drugs for each group were as follows: the control group (control) was injected with 100μL sterile PBS peritumorally every 5 days; the experimental groups were divided into RADA group, RADA@L15 day group, RADA@L17 day group, and RADA@L27 day group. Among them, the RADA group was injected with 100μL RADA peritumorally every 5 days, the RADA@L15 day group was injected with 100μL RADA@L1 peritumorally every 5 days, the RADA@L17 day group was injected with 100μL RADA@L1 peritumorally every 7 days, and the RADA@L27 day group was injected with 100μL RADA@L2 peritumorally every 7 days. The injection solvents were all sterile PBS. All nude mice were raised in the same environment, and the weight and tumor diameter of nude mice were measured regularly. The whole drug administration process lasted for two weeks. After 14 days, the mice were euthanized and the tumors were removed and weighed.

[0092] The experimental results are as attached Figure 4-7 As shown in the figure, compared with the control group, the RADA@L17-day group can reduce 40% of tumor weight and 50% of tumor volume. The RADA@L27-day group can reduce 70% of tumor weight and 75% of tumor volume. Importantly, the body weight of all nude mice maintained a stable growth, indicating that high-concentration composite self-assembling polypeptide hydrogels can maintain efficient tumor growth inhibition without causing obvious toxic side effects to nude mice.

[0093] In addition, the applicant also found that the inhibitory effect of the RADA@L15-day group was better than that of the RADA@L17-day group, and the inhibitory effect of the RADA@L27-day group was better than that of the RADA@L15-day group. The applicant speculated that the higher the concentration of the B-cL1 cyclic peptide drug, the denser the nanofibers of the composite self-assembling polypeptide hydrogel, the slower the drug release, and the higher drug release level could be maintained during peritumoral injection. On the other hand, the composite self-assembling polypeptide hydrogel has a high drug loading capacity and excellent biocompatibility.

[0094] The second batch of experiments: The anti-tumor effects of the composite polypeptide hydrogel, bevacizumab and cyclic peptide inhibitor were used to evaluate the sustained-release drug ability and anti-tumor activity of the composite polypeptide hydrogel.

[0095] Methods: Nude mice were divided into 4 groups, 5 mice in each group. The drugs for each group were as follows: the control group (control) was divided into three groups, among which, the control group (PBS) was injected with 100 μL sterile PBS peritumorally every 5 days, the control group BcL1 was injected with 100 μL peritumorally every 7 days (the concentration of B-cL1 was equivalent to that of B-cL1 in the RADA@L2 group), and the control group bevacizumab was injected into the tail vein at a rate of 5 mg / kg of mouse body weight every 14 days; the experimental group RADA@L27-day group was injected with 100 μL RADA@L2 peritumorally every 7 days, and the injection solvents were all sterile PBS. When the tumor volume reached 100-120 mm 3 The nude mice were injected and administrated at 1:00 pm. All the nude mice were kept in the same environment, and their weight and tumor diameter were measured regularly. The whole administration process lasted for two weeks. The mice were euthanized after 14 days, and the tumors were removed and weighed.

[0096] The experimental results are as attached Figure 8-11 As shown, compared with the control group, the BcL1 group can reduce 28% of tumor weight and 38% of tumor volume, while the RADA@L2 group can reduce 50% of tumor weight and 64% of tumor volume, showing significant differences. On the other hand, bevacizumab (5 mg / kg) reduced 55% of tumor weight and 63% of tumor volume with only one administration. In these results, the RADA@L2 group showed similar antitumor activity to bevacizumab.

[0097] It is worth pointing out that Figure 7 and 11 As shown, no death of nude mice was observed within 2 weeks of treatment, and the weight of nude mice increased reasonably, indicating that the peptide drug and the composite peptide hydrogel have extremely high safety.

[0098] 5. Analysis of tissue samples in anti-tumor treatment of Example 2

[0099] Methods: After 14 days of administration, the organs (heart, liver, spleen, lung, and kidney) of nude mice in the control group (PBS, control) and RADA@L27-day group were randomly taken; the tumor tissues of nude mice in the control group (PBS), RADA group, BcL1 control group, bevacizumab and RADA@L2 groups were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced ​​for H&E staining to observe the pathological sections of the tissues.

[0100] The results are as follows Fig.12As shown: Histological examination showed that there was no specific toxicity or tissue damage in the examined organs of the different treatment groups compared with the control group. Most of the H&E-stained sections of the tumor tissues of the control group (PBS) and the RADA@L2 group showed excessive proliferation of tumor cells and obvious nuclear polymorphism. In the H&E-stained tumor tissue sections, at the same B-cL1 dose as in Example 2, the tumor cells of tumor-bearing nude mice treated with B-cL1 alone decreased and the degree of tumor necrosis increased; however, the RADA@L2 group and the bevacizumab group had the least tumor cells and the highest degree of tumor necrosis. The degree of tumor necrosis from high to low is RADA@L2 group>bevacizumab group>BcL1 control group>RADA control group>PBS control group, which shows that the co-assembled polypeptide hydrogel has extremely high biosafety while being able to efficiently inhibit and kill tumor tissues.

[0101] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A novel composite self-assembling polypeptide hydrogel, characterized in that: It contains active peptides and drug-carrying peptides with self-assembly function.

2. The novel composite self-assembling polypeptide hydrogel according to claim 1, characterized in that: The active peptide is an active peptide with pharmacodynamic function, preferably an active peptide with self-assembly function, and more preferably a cyclic peptide inhibitor, wherein the active peptide is selected from one or more of B-cL1, insulin, thymopentin, octreotide, cetrorelix, canagliflozin, semaglutide and liraglutide; Preferably, the drug-carrying peptide is selected from RADA16 and / or EAK16.

3. The novel composite self-assembling polypeptide hydrogel according to claim 1 or 2, characterized in that: The weight ratio of the active peptide to the drug-carrying peptide is 1:10 to 10:

1.

4. A method for preparing a novel composite self-assembling polypeptide hydrogel as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: (1) adding the drug-loaded peptide and the active peptide to a first solution to form a mixed solution, wherein the first solution includes a solution A suitable for physiological applications and an adjuvant; (2) The mixed solution is allowed to stand for self-assembly to obtain a novel composite self-assembling polypeptide hydrogel.

5. The method for preparing the novel composite self-assembling polypeptide hydrogel according to claim 4, characterized in that: The specific operation of step (1) is: dissolving the drug-loaded peptide and the active peptide in a solution A suitable for physiological applications, and mixing or ultrasonically treating to form a mixed solution; Preferably, the ultrasonic time is 0-30 min.

6. The method for preparing the novel composite self-assembling polypeptide hydrogel according to claim 4, characterized in that: The concentration of the active peptide in the mixed solution of step (1) is 0.01-100 mg / mL, and the concentration of the drug-loaded peptide is 0.01-100 mg / mL.

7. The method for preparing the novel composite self-assembling polypeptide hydrogel according to claim 4, characterized in that: The solution A suitable for physiological applications in step (1) is selected from one or more of ultrapure water, physiological saline, and phosphate buffer.

8. The method for preparing the novel composite self-assembling polypeptide hydrogel according to claim 4, characterized in that: The standing time in step (2) is 0-24 hours, preferably 0-2 hours.

9. The method for preparing the novel composite self-assembling polypeptide hydrogel according to claim 4, characterized in that: The standing temperature in the step (2) is 0-65°C.

10. Use of the novel composite self-assembling polypeptide hydrogel according to any one of claims 1 to 3 as a drug delivery system in the preparation of anti-tumor drugs and drugs for the treatment of other diseases.

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