Drug-loaded polypeptide hydrogel as well as preparation method and application thereof

Abstract

The invention relates to drug-loaded polypeptide hydrogel as well as a preparation method and application thereof, in particular to polypeptide hydrogel which is formed by self-assembling polypeptide containing a specific amino acid sequence and doxorubicin active drugs, the polypeptide hydrogel disclosed by the invention can realize rapid hemostasis in an operation, precise targeted drug delivery after the operation and long-acting drug sustained release, effectively reduces toxic and side effects, and has the advantages of safe and reliable components, simple preparation method, good biocompatibility and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a drug-loaded polypeptide hydrogel and a preparation method and application thereof. Background Art

[0002] The conventional treatment for solid tumors in clinical tumor surgery is surgical resection combined with radiotherapy and chemotherapy. Intraoperative bleeding causes cancer cells in the tumor site to enter the circulatory system after surgery, which will lead to the risk of tumor recurrence and metastasis. The effectiveness of tumor treatment is challenged by problems such as intraoperative bleeding, impaired postoperative recovery, and increased nonspecific toxicity of chemotherapy. In response to the above problems, many biological hemostatic materials (natural polysaccharide hemostatic powder, polymer hydrogel, gelatin sponge, medical embolic agent, etc.) and anti-tumor drug delivery systems (nanovesicles, gel scaffolds, etc.) have been developed for hemostasis and anti-tumor, respectively.

[0003] Doxorubicin (Adriamycin) belongs to the anthracycline class of drugs. Its main mechanism of action is to inhibit the synthesis of DNA and RNA, and its strongest inhibitory effect on RNA. Doxorubicin has effects on a variety of tumors. It is a broad-spectrum antitumor antibiotic and a non-specific drug for the cycle. Doxorubicin has a killing effect on tumor cells in various growth cycles. It is mainly suitable for acute leukemia and has therapeutic effects on myeloid leukemia and acute lymphocytic leukemia. It has certain therapeutic effects on various other cancers such as malignant lymphoma, sarcoma, lung cancer, breast cancer and bladder cancer. However, it can cause toxic and side effects such as hair loss, bone marrow suppression and cardiotoxicity. In particular, cardiotoxicity is the most serious toxic and side effect of this type of drug, which limits its clinical application. Although doxorubicin liposome nanoparticles can be enriched and retained in tumor tissues by using the tumor EPR effect (can only pass through the gaps between vascular endothelial cells of tumor tissues, 100-400nm), thus significantly improving the actual efficacy of doxorubicin and reducing toxic and side effects, doxorubicin liposomes still have significant hematotoxicity and cardiotoxicity.

[0004] Therefore, the development of a new drug-loaded polypeptide hydrogel that organically combines the two functions of hemostasis and anti-tumor is of great significance for postoperative recovery and chemotherapy treatment, as it can stop hemorrhage during solid tumor resection and deliver anti-tumor drugs in situ, effectively reduce the toxic side effects of doxorubicin and prevent the recurrence and metastasis of residual tumor cells. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a polypeptide hydrogel that can quickly stop bleeding during surgery, accurately target drug delivery after surgery, reduce toxic side effects, and release drugs for a long time. At the same time, all components of the system are safe and reliable and have good biocompatibility.

[0006] The present invention selects the model active drug doxorubicin (DOX) as the loaded anti-tumor drug, and uses the bifunctional peptide B-cL1 with VEGFR inhibitory activity and self-assembly function as the drug-loaded hydrogel peptide to prepare a type of DOX-loaded polypeptide hydrogel drug delivery system BDO, which is applied to the hemostasis and anti-tumor drug delivery at the tumor resection site by in situ injection. The present invention proves that the DOX-loaded polypeptide hydrogel can organically combine the hemostasis and anti-tumor functions and produce a synergistic effect. The effects of the hydrogel system in both surgical hemostasis and tumor inhibition are not inferior to those of commercially available hemostatic agents and anti-tumor drugs used alone. The new drug delivery system provided by the present invention opens up a new integrated surgical treatment and in situ chemotherapy scheme, which is of great significance for the treatment of solid tumors that are prone to recurrence and metastasis.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] In one aspect, the present invention provides a polypeptide hydrogel, which is formed by self-assembly of a polypeptide and a doxorubicin active drug, wherein the amino acid sequence of the polypeptide is selected from any one of the following:

[0009] (1) the amino acid sequence shown in SEQ ID NO: 1;

[0010] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 1, and which retains the biological function of the amino acid sequence shown in SEQ ID NO: 1;

[0011] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1, and which retains the biological function of the amino acid sequence shown in SEQ ID NO: 1.

[0012] Preferably, the mass ratio of the polypeptide to the doxorubicin active drug is (10-30):(1-4), preferably (15-25):(1-4), more preferably 20:(1-4), and most preferably 20:1.

[0013] Preferably, the polypeptide is a cyclic peptide, which is formed by cyclization of two cysteine ​​residues in the amino acid sequence of the polypeptide.

[0014] Preferably, the self-assembly is performed under neutral pH conditions.

[0015] Preferably, the doxorubicin active drug is doxorubicin or doxorubicin liposome nanoparticles.

[0016] In one aspect, the present invention provides a method for preparing the above-mentioned polypeptide hydrogel, comprising the following steps:

[0017] (1) preparing a polypeptide solution;

[0018] (2) The polypeptide solution of step (1) is mixed and dissolved with the active drug doxorubicin to obtain a polypeptide hydrogel solution, and the solution is allowed to stand to form a gel.

[0019] Preferably, the step (1) specifically comprises: completely dissolving the polypeptide under a neutral pH condition to obtain the polypeptide solution.

[0020] Preferably, the concentration of the polypeptide solution is 10-30 mg / mL, preferably 10-20 mg / mL.

[0021] Preferably, the concentration of the doxorubicin active drug in the polypeptide hydrogel solution is 1-4 mg / mL, preferably 1-3 mg / mL.

[0022] In one aspect, the present invention provides use of the polypeptide hydrogel according to any one of the above items or the polypeptide hydrogel obtained according to any one of the above preparation methods in the preparation of anti-tumor drugs and / or hemostatic drugs.

[0023] In one aspect, the present invention provides use of the polypeptide hydrogel according to any one of the above items or the polypeptide hydrogel obtained according to any one of the above preparation methods in preparing therapeutic agents for use during and / or after tumor surgery.

[0024] The present invention also discloses the application effects of the polypeptide hydrogel product in experiments such as hemostasis of SD rat half tail amputation, hemostasis of SD rat liver injury, and human colorectal cancer cell HCT-116 nude mouse tumor transplantation model.

[0025] The hemostatic hydrogel containing doxorubicin active drug provided by the present invention has an extremely high water absorption and swelling ratio, and successfully loads doxorubicin, and has the effect of slowly releasing the drug. In the simulated hemorrhage rat tail amputation and liver injury model, the strong gel aggregation characteristics and strong cell adhesion of the polypeptide hydrogel can be used to quickly enrich blood cells and coagulation factors, forming a physical embolism to reduce blood loss; in the tumor inhibition model, the polypeptide hydrogel slowly releases doxorubicin, directly delivers anti-tumor drugs to the remaining tumor tissue in situ, has a cytotoxic killing effect on the residual tumor cells, and forms a physical barrier to prevent the residual tumor cells from metastasizing to other surrounding organs and blood vessels.

[0026] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0027] The polypeptide hydrogel provided by the present invention stops bleeding quickly and in a short time, and can be precisely targeted for drug delivery after surgery, can effectively fight tumors, reduce the non-targeted toxicity of chemotherapeutic drugs, and can release drugs for a long time, reduce the frequency and dosage of drug delivery; the polypeptide hydrogel of the present invention has safe and reliable ingredients, good biocompatibility, hydrolyzable properties, good degradation performance and no immunogenicity; the preparation method provided by the present invention is simple, green, low-toxic, high in drug loading, high in drug encapsulation efficiency, no introduction of other chemical reagents, and safe to use. In addition, the polypeptide hydrogel provided by the present invention is easy to store and has a long shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The scanning electron micrographs of the anti-tumor hemostatic hydrogel products of Examples (1, 2, and 25-27);

[0029] Figure 2 Transmission electron micrographs of the anti-tumor hemostatic hydrogel products of Examples (1, 2, and 25-27);

[0030] Figure 3 Degradation curves of the anti-tumor hemostatic hydrogel products of Examples (2 and 25);

[0031] Figure 4 The in vitro hemostatic performance evaluation diagram of the anti-tumor hemostatic hydrogel products of Examples (1, 2 and 25-27) (*: P < 0.1, ***: P < 0.001), wherein: (a): in vitro coagulation experiment, (b): in vitro coagulation experiment time, (c): scanning electron microscope images of blood clots of different gels;

[0032] Figure 5 The SD rat half-tail model hemostatic performance evaluation diagram of the anti-tumor hemostatic hydrogel product of Examples (2, 26-27) (*: P<0.1, ****: P<0.0001), wherein: (a): rat tail-cut hemostasis experiment, (b): rat tail-cut hemostasis time, (c): rat tail-cut hemostasis blood loss;

[0033] Figure 6 The hemostatic performance evaluation diagram of the anti-tumor hemostatic microsphere product of Example (2, 26-27) in the SD rat liver injury model (*: P < 0.1, **: P < 0.01, ****: P < 0.0001), wherein: (a): rat liver injury hemostasis diagram, (b): liver injury coagulation time, (c): liver injury blood loss;

[0034] Figure 7These are the anti-tumor performance evaluation diagrams of the anti-tumor hemostatic hydrogel products of Examples (1, 2 and 26) in the nude mouse model, wherein: (a): nude mouse tumor comparison, (b): nude mouse tumor size change trend within 21 days, (c): nude mouse tumor weight comparison diagram, (d): nude mouse body weight change trend within 21 days, (e): nude mouse tumor H&E staining section. DETAILED DESCRIPTION

[0035] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0036] Through extensive and in-depth research, the inventors have developed a class of DOX-loaded polypeptide hydrogel drug delivery systems BDO for the first time. The hydrogel system has good hemostatic properties. After the introduction of DOX, the physical morphology and properties of the polypeptide hydrogel change. Through scanning electron microscopy and transmission electron microscopy, it is found that the diameter of the hydrogel nanofibers increases, the mechanical strength increases, and the stacking ability of the nanofibers increases, which further enhances the hemostatic properties of the hydrogel. In vitro and in vivo hemostasis experiments have shown that compared with the commercially available hydrogel hemostatic agent RADA-16, BDO has higher hemostatic efficiency and faster hemostasis speed (rat liver hemostasis model, RADA-16 hemostasis time is 80 seconds, while BDO@20 hemostasis time is 38 seconds). In the nude mouse transplant tumor model, the BDO system effectively inhibits tumor growth compared with the conventional injection of the same dose of DOX (the tumor inhibition rate of the DOX group is 35.9%, while the tumor inhibition rate of the BDO@20 group is 78.2%). The BDO system can effectively load the anti-tumor drug DOX and maintain long-term release through multiple sustained-release principles after thrombus formation. In the biocompatibility experiment, it was shown that the DOX-loaded polypeptide hydrogel can effectively reduce the toxicity of the tumor treatment drug DOX and improve the overall safety of the material.

[0037] In one aspect, the present invention provides a polypeptide hydrogel, which is formed by self-assembly of a polypeptide and a doxorubicin active drug, wherein the amino acid sequence of the polypeptide is selected from any one of the following:

[0038] (1) the amino acid sequence shown in SEQ ID NO: 1;

[0039] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 1, and which retains the biological function of the amino acid sequence shown in SEQ ID NO: 1;

[0040] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1, and which retains the biological function of the amino acid sequence shown in SEQ ID NO: 1.

[0041] SEQ ID NO: 1:

[0042] Ac-CTVELMGTVAKQLVPC-NH2

[0043] In certain embodiments, the polypeptide is a cyclic peptide, which is formed by cyclization of two cysteine ​​residues in the amino acid sequence of the polypeptide.

[0044] In the present invention, the cyclic peptide is formed by the cysteine ​​residue at position 1 and the cysteine ​​residue at position 16 through a disulfide bond.

[0045] The polypeptides of the present invention can be prepared by standard peptide synthesis methods, for example, by standard solid phase or liquid phase methods, stepwise or by fragment assembly, and separation and purification of the final peptide compound product, or by any combination of recombinant and synthetic methods. Alternatively, the polypeptide compounds of the present invention are synthesized by solid phase or liquid phase peptide synthesis methods. Alternatively, the polypeptides of the present application are obtained by conventional commercial means.

[0046] In the present invention, the polypeptide (B-cL1) can self-assemble to form a nanofiber structure under neutral conditions.

[0047] In certain embodiments, the mass ratio of the polypeptide to the doxorubicin active drug is (10-30):(1-4), for example, 10:1, 10:2, 10:3, 10:4, 15:1, 15:2, 15:3, 15:4, 20:1, 20:2, 20:3, 20:4, 25:1, 25:2, 25:3, 25:4, 30:1, 30:2, 30:3, 30:4, etc.

[0048] In certain embodiments, the mass ratio of the polypeptide to the doxorubicin active drug is (15-25):(1-4).

[0049] In certain embodiments, the mass ratio of the polypeptide to the doxorubicin active drug is 20:(1-4).

[0050] In a specific and preferred embodiment, the mass ratio of the polypeptide to the doxorubicin active drug is 20:1.

[0051] In certain embodiments, the self-assembly is accomplished at neutral pH.

[0052] In certain embodiments, the self-assembly is accomplished in ultrapure water at a neutral pH.

[0053] In certain embodiments, NaOH is used to adjust the pH of the drug-loaded polypeptide hydrogel solution to neutral.

[0054] In certain embodiments, the doxorubicin active drug is doxorubicin or doxorubicin liposome nanoparticles.

[0055] In the present invention, the active drug of doxorubicin means doxorubicin or its analogs / derivatives, such as natural doxorubicin and its analogs. Doxorubicin is an anthracycline antibiotic first isolated from the fungus Streptomyces peucetius. The chemical structure of doxorubicin consists of four rings connected to the sugar daunosamine by a glycosidic bond. The hydrochloride of doxorubicin is one of the most common forms. It has various names, such as doxorubicin hydrochloride; 14-hydroxydaunomycin hydrochloride; 3-hydroxyacetyldaunomycin hydrochloride; and 5,12-tetraphenylene, 10-[(3-amino-2,3,6-trideoxy-α-L-lyso-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-, hydrochloride, (8S-cis)-(9Cl). Doxorubicin hydrochloride has the molecular formula C 27 H 29 NO 11 .HCl, molecular weight (MW) 580.0, CAS No. 25316-40-9. It is soluble in water and slightly soluble in methanol. Therefore, in the present invention, the doxorubicin can also be in the form of doxorubicin liposome nanoparticles or other preparations. In the present invention, the content of the doxorubicin active drug in the polypeptide hydrogel and the concentration in the polypeptide hydrogel are calculated as active pharmaceutical ingredients.

[0056] In one aspect, the present invention provides a method for preparing the above-mentioned polypeptide hydrogel, comprising the following steps:

[0057] (1) preparing a polypeptide solution;

[0058] (2) The polypeptide solution of step (1) is mixed and dissolved with the active drug doxorubicin to obtain a polypeptide hydrogel solution, and the solution is allowed to stand to form a gel.

[0059] In certain embodiments, the step (1) specifically comprises: completely dissolving the polypeptide under a neutral pH condition to obtain the polypeptide solution.

[0060] In certain embodiments, the step (1) specifically comprises: dissolving the polypeptide using ultrapure water with a neutral pH to obtain the polypeptide solution.

[0061] In certain embodiments, the concentration of the polypeptide solution is 10-30 mg / mL, for example, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, etc.

[0062] In certain embodiments, the concentration of the polypeptide solution is 10-20 mg / mL.

[0063] In certain embodiments, the concentration of the doxorubicin active drug in the polypeptide hydrogel solution is 1-4 mg / mL, such as 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL. In certain embodiments, the concentration of the doxorubicin active drug in the polypeptide hydrogel solution is 1-3 mg / mL.

[0064] In certain embodiments, the standing temperature is 0-6°C, such as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, etc.

[0065] In a specific and preferred embodiment, the standing temperature is 4°C.

[0066] In certain embodiments, the standing time is 5-20 hours, for example 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, etc.

[0067] In certain embodiments, the standing time is 10-15 hours.

[0068] In a specific and preferred embodiment, the standing time is 12 hours.

[0069] In certain embodiments, the preparation method comprises the following steps: completely dissolving the polypeptide under a neutral pH condition to obtain the polypeptide solution; then adding doxorubicin active drug (adriamycin) to the polypeptide solution, and after complete dissolution, standing to obtain the polypeptide hydrogel.

[0070] In one aspect, the present invention provides use of the polypeptide hydrogel according to any one of the above items or the polypeptide hydrogel obtained according to any one of the above preparation methods in the preparation of anti-tumor drugs and / or hemostatic drugs.

[0071] In one aspect, the present invention provides use of the polypeptide hydrogel according to any one of the above items or the polypeptide hydrogel obtained according to any one of the above preparation methods in preparing therapeutic agents for use during and / or after tumor surgery.

[0072] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0073] Example 1

[0074] Preparation of drug-loaded peptide hydrogel BDO@10: First, take 10 mg of peptide powder B-cL1 and prepare a 10 mg / mL peptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin (DOX), mix with the peptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it in the peptide solution (control the pH to be neutral), and obtain a DOX-loaded peptide solution with a DOX concentration of 1 mg / mL. Next, the peptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a peptide hydrogel (BDO@10) with a drug loading of 1 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0075] Example 2

[0076] Preparation of drug-loaded peptide hydrogel BDO@20: First, take 20 mg of peptide powder B-cL1 and prepare a 20 mg / mL peptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin (DOX), mix with the peptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it in the peptide solution (control the pH to be neutral), and obtain a DOX-loaded peptide solution with a DOX concentration of 1 mg / mL. Next, the peptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a peptide hydrogel (BDO@20) with a drug loading of 1 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0077] Example 3

[0078] Preparation of drug-loaded polypeptide hydrogel BDO@30: First, take 30 mg of polypeptide powder B-cL1 and prepare a 30 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin (DOX), mix with the polypeptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a DOX-loaded polypeptide solution with a DOX concentration of 1 mg / mL. Next, the polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a polypeptide hydrogel (BDO@30) with a drug loading of 1 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0079] Example 4

[0080] Preparation of drug-loaded peptide hydrogel BDO2@10: First, take 10 mg of peptide powder B-cL1 and prepare a 10 mg / mL peptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin (DOX), mix with the peptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it in the peptide solution (control the pH to be neutral), and obtain a DOX-loaded peptide solution with a DOX concentration of 2 mg / mL. Next, the peptide solution was placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a peptide hydrogel (BDO2@10) with a drug loading of 2 mg / mL DOX was obtained. The prepared gel was stored in a 4°C refrigerator.

[0081] Example 5

[0082] Preparation of drug-loaded polypeptide hydrogel BDO2@20: First, take 20 mg of polypeptide powder B-cL1 and prepare a 20 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin (DOX), mix with the polypeptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it in the polypeptide solution to obtain a DOX-loaded polypeptide solution with a DOX concentration of 2 mg / mL. Next, the polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a polypeptide hydrogel (BDO2@20) with a drug loading of 2 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0083] Example 6

[0084] Preparation of drug-loaded polypeptide hydrogel BDO2@30: First, take 30 mg of polypeptide powder B-cL1 and prepare a 30 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin (DOX), mix with the polypeptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a DOX-loaded polypeptide solution with a DOX concentration of 2 mg / mL. Next, the polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a polypeptide hydrogel (BDO2@30) with a drug loading of 2 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0085] Example 7

[0086] Preparation of drug-loaded polypeptide hydrogel BDO3@10: First, take 10 mg of polypeptide powder B-cL1 and prepare a 10 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin (DOX), mix with the polypeptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a DOX-loaded polypeptide solution with a DOX concentration of 3 mg / mL. Next, the polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a polypeptide hydrogel (BDO3@10) with a drug loading of 3 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0087] Example 8

[0088] Preparation of drug-loaded polypeptide hydrogel BDO3@20: First, take 20 mg of polypeptide powder B-cL1 and prepare a 20 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin (DOX), mix with the polypeptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a DOX-loaded polypeptide solution with a DOX concentration of 3 mg / mL. Next, the polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a polypeptide hydrogel (BDO3@20) with a drug loading of 3 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0089] Example 9

[0090] Preparation of drug-loaded polypeptide hydrogel BDO3@30: First, take 30 mg of polypeptide powder B-cL1 and prepare a 30 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin (DOX), mix with the polypeptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a DOX-loaded polypeptide solution with a DOX concentration of 3 mg / mL. Next, the polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a polypeptide hydrogel (BDO3@30) with a drug loading of 3 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0091] Example 10

[0092] Preparation of drug-loaded polypeptide hydrogel BDO4@10: First, take 10 mg of polypeptide powder B-cL1 and prepare a 10 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin DOX, mix with the polypeptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve in the polypeptide solution (control the pH to be neutral), and obtain a DOX-loaded polypeptide solution with a DOX concentration of 4 mg / mL. Next, the polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a polypeptide hydrogel (BDO4@10) with a drug loading of 4 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0093] Embodiment 11

[0094] Preparation of drug-loaded polypeptide hydrogel BDO4@20: First, take 20 mg of polypeptide powder B-cL1 and prepare a 20 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin DOX, mix with the polypeptide solution, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve in the polypeptide solution (control the pH to be neutral), and obtain a DOX-loaded polypeptide solution with a DOX concentration of 4 mg / mL. Next, the polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a polypeptide hydrogel (BDO4@20) with a drug loading of 4 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0095] Example 12

[0096] Preparation of drug-loaded polypeptide hydrogel BDO4@30: First, take 30 mg of polypeptide powder B-cL1 and prepare a 30 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin DOX, mix with the polypeptide solution, vortex for 15 seconds, and then dissolve in the polypeptide solution by ultrasound for 10 minutes (control the pH to be neutral), and obtain a DOX-loaded polypeptide solution with a DOX concentration of 4 mg / mL. Next, the polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally a polypeptide hydrogel (BDO4@30) with a drug loading of 4 mg / mL DOX is obtained. The prepared gel is stored in a 4°C refrigerator.

[0097] Example 13

[0098] Preparation of drug-loaded polypeptide hydrogel BDI@10: First, take 10 mg of polypeptide powder B-cL1 and prepare a 10 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 1 mg / mL polypeptide solution of doxorubicin liposomes. Finally, the drug-loaded polypeptide solution was placed in a 4°C refrigerator for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI@10) was obtained. The prepared gel was stored in a 4°C refrigerator.

[0099] Embodiment 14

[0100] Preparation of drug-loaded polypeptide hydrogel BDI@20: First, take 20 mg of polypeptide powder B-cL1 and prepare a 20 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 1 mg / mL polypeptide solution of doxorubicin liposomes. Finally, the drug-loaded polypeptide solution was placed in a 4°C refrigerator for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI@20) was obtained. The prepared gel was stored in a 4°C refrigerator.

[0101] Embodiment 15

[0102] Preparation of drug-loaded polypeptide hydrogel BDI@30: First, take 30 mg of polypeptide powder B-cL1 and prepare a 30 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 1 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI@30) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0103] Example 16

[0104] Preparation of drug-loaded polypeptide hydrogel BDI2@10: First, take 10 mg of polypeptide powder B-cL1 and prepare a 10 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 2 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI2@10) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0105] Embodiment 17

[0106] Preparation of drug-loaded polypeptide hydrogel BDI2@20: First, take 20 mg of polypeptide powder B-cL1 and prepare a 20 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 2 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI2@20) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0107] Embodiment 18

[0108] Preparation of drug-loaded polypeptide hydrogel BDI2@30: First, take 30 mg of polypeptide powder B-cL1 and prepare a 30 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 2 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI2@30) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0109] Embodiment 19

[0110] Preparation of drug-loaded polypeptide hydrogel BDI3@10: First, take 10 mg of polypeptide powder B-cL1 and prepare a 10 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 3 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI3@10) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0111] Embodiment 20

[0112] Preparation of drug-loaded polypeptide hydrogel BDI3@20: First, take 20 mg of polypeptide powder B-cL1 and prepare a 20 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 3 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI3@20) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0113] Embodiment 21

[0114] Preparation of drug-loaded polypeptide hydrogel BDI3@30: First, take 30 mg of polypeptide powder B-cL1 and prepare a 30 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 3 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI3@30) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0115] Embodiment 22

[0116] Preparation of drug-loaded polypeptide hydrogel BDI4@10: First, take 10 mg of polypeptide powder B-cL1 and prepare a 10 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 4 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI4@10) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0117] Embodiment 23

[0118] Preparation of drug-loaded polypeptide hydrogel BDI4@20: First, take 20 mg of polypeptide powder B-cL1 and prepare a 20 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 4 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI4@20) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0119] Embodiment 24

[0120] Preparation of drug-loaded polypeptide hydrogel BDI4@30: First, take 30 mg of polypeptide powder B-cL1 and prepare a 30 mg / mL polypeptide solution in ultrapure water with a neutral pH. Secondly, add doxorubicin liposomes, mix with the polypeptide solution, vortex for 15 seconds, and then ultrasonicate for 10 minutes to disperse and dissolve it in the polypeptide solution (control the pH to be neutral), and obtain a 4 mg / mL polypeptide solution of doxorubicin liposomes. Next, the drug-loaded polypeptide solution is placed in a 4°C refrigerator and allowed to stand for 12 hours to allow it to fully self-assemble, and finally the drug-loaded polypeptide hydrogel (BDI4@30) is obtained. The prepared gel is stored in a 4°C refrigerator.

[0121] Embodiment 25

[0122] Preparation of polypeptide hydrogel B-cL1@10: First, take 10 mg of polypeptide powder B-cL1, prepare 10 mg / mL polypeptide solution in ultrapure water with neutral pH, mix the polypeptide with ultrapure water, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it (control the pH to be neutral). Next, put the polypeptide solution in a 4°C refrigerator for 12 hours to allow it to fully self-assemble, and finally obtain the polypeptide hydrogel (B-cL1@10), and store the prepared gel in a 4°C refrigerator.

[0123] Embodiment 26

[0124] Preparation of polypeptide hydrogel B-cL1@20: First, take 20 mg of polypeptide powder B-cL1, prepare 20 mg / mL polypeptide solution in ultrapure water with neutral pH, mix the polypeptide with ultrapure water, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it (control the pH to be neutral). Next, put the polypeptide solution in a 4°C refrigerator and let it stand for 12 hours to allow it to fully self-assemble, and finally obtain the polypeptide hydrogel (B-cL1@20), and store the prepared gel in a 4°C refrigerator.

[0125] Embodiment 27

[0126] Preparation of peptide hydrogel RADA@20: First, take 20 mg of peptide powder RADA16 (Ac-RADARADARADARADA-NH2) and prepare a 20 mg / mL peptide solution in ultrapure water with a neutral pH. Mix the peptide and ultrapure water, vortex for 15 seconds, and then sonicate for 10 minutes to dissolve it. Next, put the peptide solution in a 4°C refrigerator and let it stand for 12 hours to allow it to fully self-assemble, and finally obtain the peptide hydrogel (RADA@20). The prepared gel is stored in a 4°C refrigerator.

[0127] Test Case

[0128] 1. SEM analysis of hydrogel

[0129] Objective: To observe the microscopic morphology of the doxorubicin polypeptide hydrogel products in Examples (Examples 1, 2, and 25-27)

[0130] Methods: In order to observe the surface morphology of peptide hydrogel, the surface of peptide hydrogel samples was gold-plated and then scanned using a scanning electron microscope (Hitachi S-4800, Japan). The microstructures of RADA@20 and different concentrations of B-cL1 and BDO were observed. The results are shown in the attached figure. Figure 1 shown.

[0131] Results: Attached Figure 1 The results showed that the fibers of the RADA@20 group were thinner and sparser. Compared with BDO@10, B-cL1@10 had a finer mesh structure. After adding DOX, its fiber density was denser and the mesh structure was more obvious. Compared with BDO@20, B-cL1@20 had a denser mesh structure and a higher fiber density. After adding DOX, the mesh structure was further denser and the fiber density was stacked layer by layer. The addition of DOX made the structure of B-cL1 denser, the fiber density was higher, and the nanofiber network was more obvious.

[0132] 2. Transmission electron microscopy analysis of hydrogel

[0133] Objective: To observe the microscopic morphology of the doxorubicin polypeptide hydrogel products of Examples (1, 2 and 25-27)

[0134] Methods: In order to observe the microstructure of hemostatic hydrogel, hydrogels RADA@20, B-cL1@10, BDO@10, B-cL1@10, and BDO@20 were prepared and allowed to stand for 12 hours at 4°C for full self-assembly. Then, 5 μL of each was dropped onto a 100-mesh carbon copper grid and dried at room temperature. After the sample was attached, it was washed twice with ammonium nitrate (30 seconds each time) and then stained with uranyl acetyloxide for 60 seconds. The images were taken using a 120 kV transmission electron microscope (JEM-1400 Flash). The results are shown in Figure 2. Figure 2 shown.

[0135] result: Figure 2 The results show that the fibers of the RADA@20 group are thinner and sparser. Compared with BDO@10, the fibers of B-cL1@10 present a finer mesh structure. After adding DOX, the fiber density of B-cL1@10 is denser and the mesh structure is more obvious. Compared with BDO@20, the mesh structure of B-cL1@20 is denser and the fiber density is higher. After adding DOX, the mesh structure is further dense and the fiber density is stacked layer by layer. The addition of DOX makes the structure of B-cL1 denser, the fiber density is higher, and the nanofiber network is more obvious.

[0136] 3. In vitro sustained release and degradation analysis of drug-loaded polypeptide hydrogel

[0137] Objective: To determine the in vitro sustained release and degradation properties of the drug-loaded polypeptide hydrogels of Examples (2 and 25)

[0138] Method: Hydrogel degradation curve drawing: Prepare three tubes of B-cL1@20 and BDO@20 drug-loaded gels, add 500μL of sterile PBS, add lysozyme to a concentration of (1.5μg / mL) and incubate at 37℃ for 1h. Centrifuge at 6000rpm for 3min, remove the supernatant, and record its initial weight as W0. Centrifuge and weigh on the 1st, 3rd, and 5th day to record its weight W1. The degradation rate is calculated as follows:

[0139] Degradation rate (%) = (W0-W1) / W0×100%

[0140] Results: In vitro degradation analysis of BDO@20 and B-cL1@20 peptide hydrogels was performed. Figure 3 shown. Figure 3 The results showed that the degradation curve of BDO@20 was gentler than that of B-cL1@20. BDO@20 degraded by 50% on the first day, and then the curve flattened and reached 70% on the fifth day, while B-cL@20 degraded by 50% on the third day and 80% on the fourth day. The in vitro degradation curve proved that the addition of DOX made the degradation curve of B-cL1 gentler, in which the encapsulated DOX was released as the hydrogel slowly degraded, which may play a key role in tumor inhibition.

[0141] 4. In vitro coagulation analysis

[0142] Objective: To study the coagulation and blood cell enrichment properties of the hemostatic polypeptide hydrogel products of Examples (1, 2, and 25-27) in vitro

[0143] Method: 100 μL of peptide hydrogel sample was added to a 96-well plate and allowed to stand at room temperature for one hour, and 100 μL of rabbit blood was added. Every 100 seconds, the plate was rinsed with saline and the coagulation time was recorded, and this process was repeated three times.

[0144] Take the coagulated thrombus (after 30 minutes) and wash it several times with PBS to wash away the blood cells adhering to the surface, add 0.5mL of 2% glutaraldehyde to fix the thrombus for 2 hours for subsequent experiments. Then take out the thrombus sample for dehydration, add ethanol to immerse the thrombus and dehydrate it in different gradients (20, 50, 70, 100% for 12 hours in each step, and store in a refrigerator at 4°C). After the dehydration treatment is completed, the sample is completely dried, and then the thrombus is cut into small square pieces and fixed on an electron microscope silicon wafer for gold spraying. The adsorption of blood cells by the sample is observed using a scanning electron microscope.

[0145] Results: Figure 4 As shown in the figure, after completing the performance test of the hydrogel, the in vitro coagulation ability of the polypeptide hydrogel product was evaluated first. 100 μL of rabbit blood was added to 100 μL of hydrogel and mixed evenly at room temperature to wait for coagulation. BDO@20 completely coagulated the blood within 100 seconds, demonstrating excellent coagulation ability (such as Figure 4 As shown in (a), there were significant differences between the RADA@20, B-cL1@10 and B-cL1@20 groups (***: P < 0.001) and the low-dose BDO@10 group (*: P < 0.1). Figure 4 As shown in b. BDO@10 completes coagulation in about 200s, RADA@20, B-cL1@10 and B-cL1@20 complete coagulation in about 300s. The blank control did not complete coagulation within 500s. The present invention found that the addition of DOX led to a significant improvement in the hemostatic performance of B-cL1, and the coagulation ability of BDO@10 far exceeded that of B-cL1@20 with twice the concentration. Rheological tests and dynamic light scattering experiments confirmed that the addition of DOX increased the nanofiber diameter and nanofiber network stacking efficiency of B-cL1, and this improvement greatly improved the hemostatic performance of the material. After the blood clot was processed and observed using a scanning electron microscope, it was found that a large number of blood cells and fibrinogen were attached to the stacked nanofiber networks, and this phenomenon was particularly evident in BDO@20. As Figure 4As shown in c, the drug-loaded gel BDO@20 separates blood cells from water in the blood through the fiber network on the surface, adsorbs larger blood cells on its surface, and promotes coagulation by continuously accumulating blood cells and fibrin that triggers coagulation to form blood clots. As a control, RADA@20 has only a small amount of blood cells attached to the surface and no obvious fibrin is observed. Fibrin is a necessary component for thrombosis during coagulation, which has a significant effect on the coagulation effect. B-cL1@10 and B-cL1@20 without DOX loading have a small layer of blood cells accumulated on the surface. A small amount of fibrin can be observed around the red blood cells instead of densely stacked blood cells like BDO@20. The overall number of blood cells is not enough to form a dense thrombus. BDO@10 in the low-dose group cannot effectively intercept blood cells due to the low density of the hydrogel fiber network, which has a certain impact on its coagulation ability.

[0146] 5. Analysis of the hemostasis model of SD rats with half tail amputation

[0147] Objective: To evaluate the coagulation performance of the polypeptide hydrogel products of Examples (2, 26-27) in the tail vein and artery models of SD rats with large blood flow and high blood pressure

[0148] Methods: Thirty female SD rats (220-230g) were fed for 3 days and then anesthetized with 50mg / kg of sildenafil. The rats were randomly divided into groups (blank control group, gauze control group, RADA@20 group, B-cL1@20 group, BDO@20 group), with 6 rats in each group. The rats were anesthetized, and the tails were cut open at the midpoint of the tails. The tails were immediately immersed in a 5mL EP tube containing 1mL of gel to stop bleeding. After the clot was formed, the suspension time was determined to be within 20s after no bleeding. The blood in the EP tube was weighed and the blood loss and coagulation time were recorded.

[0149] Results: After completing the in vitro coagulation evaluation experiment, the in vivo coagulation experiment was further conducted to evaluate the hemostatic effect of the hydrogel. Since there is an artery and multiple veins in the rat's tail, the amount of bleeding after tail amputation is very large, which is a great test of the hemostatic effect and adhesion of the hemostatic material.

[0150] As attached Figure 5 As shown in a, this test example sets up two experimental groups, a blank control group and two positive control groups (gauze control group and RADA@20). Figure 5As shown in b, in the blank control group, the tail of the rat did not achieve hemostasis within 300 seconds after amputation (the evaluation standard was that the wound stopped bleeding within 20 seconds). The gauze control group and B-cL@20 achieved hemostasis within 200 seconds, RADA@20 achieved hemostasis in about 122 seconds, and the BDO@20 group achieved hemostasis within 100 seconds. There was a very significant difference between the BDO@20 group and the blank control group (****: P<0.0001). Figure 5 As shown in c, compared with the blank control group, due to the long bleeding time, effective bleeding could not be stopped, with an average blood loss of 2.3 g. The blood loss in the gauze group was slightly less than that in the blank group, with an average blood loss of 1.46 g. The average blood loss in the RADA@20 group was 0.8 g, the blood loss in the B-cL1@20 group was 0.99 g, and the blood loss in the BDO@20 group was 0.27 g, which was significantly less than that in the blank control group (****: P<0.0001).

[0151] As a polypeptide hydrogel hemostatic agent, RADA has the following main disadvantages: low gel strength, slow hemostasis speed, and poor mechanical properties, which prevent it from being applied to cases of massive bleeding. B-cL1 makes up for this disadvantage after adding DOX. The gelation speed and mechanical strength of RADA@20 far exceed those of B-cL1 and RADA polypeptide hydrogels. This is due to its dense nanofiber network that can absorb a large number of blood cells and filter water, thus providing conditions for subsequent thrombosis and playing a key role in rapid hemostasis.

[0152] 6. Analysis of the hemostasis model of liver injury in SD rats

[0153] Objective: To evaluate the coagulation performance of the polypeptide hydrogel products of Examples (2, 26-27) in the SD rat liver injury model

[0154] Methods: Thirty female SD rats (220-230g) were fed for 3 days and anesthetized with 50mg / kg of sildenafil. The rats were randomly divided into groups (blank control group, gauze group, RADA@20 group, B-cL1@20 group, BDO@20 group), with 6 rats in each group. After anesthesia, the abdominal hair of the rats was shaved clean for easy operation. The rat skin was cut open near the liver to expose the liver. The wound was fixed and clean gauze was laid flat under the liver. The excess liquid on the surface of the liver was wiped dry to avoid affecting the blood weighing. A cross wound (1cm×1cm×0.5cm) was cut in the left lobe of the rat liver with a sterile scalpel. After standing for a few seconds to wait for bleeding, 200μL of hydrogel was quickly given to stop bleeding. The bleeding time was recorded, the gauze was weighed to calculate the amount of bleeding, and the abdominal wound was sutured.

[0155] Results: The rat liver injury hemostasis model is one of the most commonly used models to evaluate the coagulation ability of coagulation materials. The amount of bleeding in the liver is less than that in the tail, but because heparin inhibits its own coagulation cascade reaction, it is also an important reference for hemostasis ability. Figure 6 As shown in Figure a, the rat liver was exposed, a 1 cm cross wound was made, and 200 μL of hydrogel was injected on the surface to stop bleeding. Figure 6 As shown in b, the average hemostasis time of the blank group was 140s, the average hemostasis time of the gauze group was 103s, the average hemostasis time of RADA@20 was 80s, the hemostasis time of B-cL1@20 was 107s, and the average hemostasis time of the drug-loaded gel BDO@20 group was 38s, which was significantly different from the gauze group, RADA group and B-cL1 (P<0.001). When the polypeptide hydrogel BDO@20 was mixed with blood, a preliminary clot was formed in 10s and coagulation was completed in about 30s. It was the hydrogel with the fastest hemostasis in each group. Figure 6 As shown in Figure c, in terms of blood loss, BDO@20 (0.29g) was significantly different from the gauze group (0.65g), RADA@20 (0.48g), and B-cL1@20 (0.60g) (P<0.01), and was extremely significantly different from the blank group (1.0g) (P<0.0001). In the non-arterial pressure bleeding model, the hemostasis speed of BDO compared with RADA was more obvious than that of the truncated model. When the blood flow rate was not fast, the hydrogel network could more quickly retain and adhere to aggregated blood cells and platelets to accelerate the coagulation cascade reaction process.

[0156] 7. Anti-tumor performance analysis

[0157] Objective: To evaluate the anti-tumor properties of the anti-tumor hemostatic polypeptide hydrogel products of Examples (1, 2 and 26)

[0158] Methods: Construction of nude mouse transplant tumor model: HCT116 colorectal cancer cells were cultured and inoculated (1×10 7 cell / 200μL / mouse, subcutaneously injected) into the left armpit of BALB / c nude mice, and the inoculated mice were randomly divided into different groups. 3 Each experimental group was injected with 100 μL of hydrogel around the tumor, the blank control group was injected with 100 μL of PBS, and the positive control group was injected with 100 μL of DOX at a concentration of 1 mg / mL into the tail vein. Each group was only given the drug once within three weeks. During the three weeks, the tumor diameter was weighed and recorded every three days. After three weeks, the nude mice were treated, the tumor was removed and weighed, and the tumor was cut into pieces for H&E staining and slice scanning.

[0159] Results: DOX is usually administered by intravenous drip as a tumor suppressor drug, but DOX itself is cytotoxic, and systemic intravenous drip has an impact on non-tumor tissues. It is urgent to develop a new drug delivery system to solve the above problems. By changing the administration method from intravenous injection to in situ injection, the content of DOX in the blood can be effectively reduced. At the same time, the use of gel-encapsulated DOX can reduce the toxicity to peritumoral tissues and prolong the drug action time due to the characteristics of multiple slow release. Therefore, in this experiment, DOX (equivalent dose of DOX polypeptide hydrogel) injected by tail vein was used as the positive control group, PBS was injected as the blank control group, and B-cL1 (BDO@10, BDO@20) loaded with 1 mg / mL DOX at different concentrations was used as the experimental group to evaluate the anti-tumor activity.

[0160] As shown in Figure 7a, after the 21-day experimental period, the tumor size and tumor weight of nude mice in the BDO@10 and BDO@20 groups were significantly smaller than those in the blank group (PBS), the negative control group B-cL@20, and the positive control group (DOX). The tumor volume of nude mice in each group did not increase significantly within 21 days ( Figure 7 In (b), the tumor in the DOX group began to grow significantly around the 9th day, while the experimental groups BDO@10 and BDO@20 maintained a sustained inhibitory effect on the tumor within 21 days. Figure 7 As shown in Figure c, after the experiment, the tumors were weighed and it was found that the tumors in the BDO@10 and BDO@20 groups were significantly smaller than those in the PBS, B-cL1@20 and DOX groups. The tumor volume inhibition rate (TGI) was calculated and the results were: B-cL1@20 (32.33%), DOX (35.92%), BDO@10 (80.34%), and BDO@20 (78.21%). Figure 7 As shown in Figure d, the weight of mice in each group remained stable during the 21-day experiment, and no deaths occurred. Due to the cytotoxicity of DOX and the fact that it was only given once, the weight of the DOX group showed a slight downward trend in the first 9 days, and then increased significantly after 9 days. HE examination of tumor tissue showed ( Figure 7 Middle e) The tumor cells in the B-cL1@20 group and the PBS group had intact morphology, necrotic tumor cells were found in the DOX group, and more obviously necrotic tumor cells could be observed in the BDO@10 group and the BDO@20 group.

[0161] The present invention uses polypeptide B-cL1 and doxorubicin as raw materials to prepare a degradable drug-loaded hemostatic hydrogel, which is intended to stop bleeding quickly during tumor resection surgery and to act as a "warehouse" for anti-tumor drugs after surgery to play a role of long-acting, sustained-release anti-tumor drugs. The present invention utilizes the high fiber density and strong cell adhesion of the polypeptide hydrogel to stop bleeding quickly during tumor resection surgery, and utilizes the swollen gel to accurately administer drugs in situ and release anti-tumor drugs after surgery, thereby stopping bleeding quickly during tumor resection surgery and locally releasing anti-tumor drugs after surgery to inhibit tumor recurrence and metastasis.

[0162] 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 drug-loaded polypeptide hydrogel, characterized in that: It is formed by self-assembly of a polypeptide and a doxorubicin active drug, wherein the amino acid sequence of the polypeptide is selected from any one of the following: (1) the amino acid sequence shown in SEQ ID NO: 1; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 1, and which retains the biological function of the amino acid sequence shown in SEQ ID NO: 1; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1, and which retains the biological function of the amino acid sequence shown in SEQ ID NO:

1.

2. The polypeptide hydrogel according to claim 1, characterized in that The mass ratio of the polypeptide to the doxorubicin active drug is (10-30):(1-4), preferably (15-25):(1-4), more preferably 20:(1-4), and most preferably 20:

1.

3. The polypeptide hydrogel according to claim 1, characterized in that The polypeptide is a cyclic peptide, which is formed by cyclization of two cysteine ​​residues in the amino acid sequence of the polypeptide.

4. The polypeptide hydrogel according to any one of claims 1 to 3, characterized in that The self-assembly is completed under a neutral pH condition; Preferably, the doxorubicin active drug is doxorubicin or doxorubicin liposome nanoparticles.

5. The method for preparing the polypeptide hydrogel according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) preparing a polypeptide solution; (2) The polypeptide solution of step (1) is mixed and dissolved with the active drug doxorubicin to obtain a polypeptide hydrogel solution, and the solution is allowed to stand to form a gel.

6. The preparation method according to claim 5, characterized in that: The step (1) specifically comprises: completely dissolving the polypeptide under a neutral pH condition to obtain the polypeptide solution.

7. The preparation method according to claim 5, characterized in that: The concentration of the polypeptide solution is 10-30 mg / mL, preferably 10-20 mg / mL.

8. The preparation method according to any one of claims 5 to 7, characterized in that: The concentration of the doxorubicin active drug in the polypeptide hydrogel solution is 1-4 mg / mL, preferably 1-3 mg / mL.

9. Use of the polypeptide hydrogel according to any one of claims 1 to 4 or the polypeptide hydrogel obtained according to the preparation method according to any one of claims 5 to 8 in the preparation of anti-tumor drugs and / or hemostatic drugs.

10. Use of the polypeptide hydrogel according to any one of claims 1 to 4 or the polypeptide hydrogel obtained according to the preparation method according to any one of claims 5 to 8 in preparing therapeutic agents for use during and / or after tumor surgery.

Images