CN111097070B

CN111097070B - Injectable bioactive hydrogel for inhibiting tumor and promoting repair

Info

Publication number: CN111097070B
Application number: CN202010022549.6A
Authority: CN
Inventors: 李海燕; 何丹
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-01-09
Filing date: 2020-01-09
Publication date: 2021-06-22
Anticipated expiration: 2040-01-09
Also published as: CN111097070A

Abstract

The invention discloses an injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair, a preparation method and application thereof, and relates to the fields of biomedical materials, tissue engineering and medicine. The injectable bioactive hydrogel contains high molecular material, multifunctional active peptide and water. The polymer material is grafted with the multifunctional active peptide through a grafting agent, and is subjected to photo-crosslinking and curing under ultraviolet light through a photoinitiator to form the injectable bioactive hydrogel. The injectable bioactive hydrogel disclosed by the invention can continuously and slowly release the multifunctional active peptide grafted to the hydrogel high polymer material in the whole stage from skin tumor excision to wound repair, is implemented by liquid and photo-crosslinking curing, can fill defect parts with different shapes, better realizes the combined treatment effect of inhibiting tumor recurrence and promoting tissue repair, is simple to manufacture, convenient to use, safe and durable in curative effect and has clinical application potential.

Description

Injectable bioactive hydrogel for inhibiting tumor and promoting repair

Technical Field

The invention relates to the fields of biomedical materials, tissue engineering and medicine, in particular to an injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair, and a preparation method and application thereof.

Background

The clinical treatment means aiming at the tumor comprises operation treatment, medicament treatment, radiation treatment and the like. For some tumors with larger volume and invasiveness, the operation is the most traditional treatment mode, and the growth and the metastasis of the tumors can be fundamentally inhibited. Tumor recurrence remains a major challenge for patient survival due to incomplete cancer cell eradication after surgical resection. Meanwhile, the wound surface after tumor resection is difficult to repair normally, and even a wound which is difficult to heal for a long time is caused.

Chemotherapy and radiation therapy are commonly used to prevent tumor recurrence. Many studies report nanoparticles loaded with small molecule drugs, such as doxorubicin, paclitaxel, and dexamethasone, for tumor chemotherapy. However, conventional chemoradiotherapy can destroy tumors and also cause toxicity to healthy cells, resulting in adverse side effects including severe immunosuppression, renal toxicity, cardiotoxicity, and the like. In addition, systemic administration of drugs by intravenous injection limits the ability of the drugs to reach and penetrate distant tumor cells, and the short half-life reduces their effectiveness while also causing damage to other normal tissues in the body. Removal of tumor tissue results in large areas of tissue defect and inevitably impaired healing of the incision wound in the chronic inflammatory microenvironment.

Many studies have focused on the treatment of tumors, and the repair of tissue defects after tumor removal has a great influence on whether normal tissue can then function. At present, biological materials for tumor treatment have single functions, medicines can only play an anti-tumor role generally, other repairing medicines can be selected generally for the problem of subsequent tissue defect repair, the problems of ordered release of multiple medicine molecules and the like exist, and the further development of tissue repair after tumor resection of tissue engineering is limited.

For local tissues after tumor resection, a great deal of medicine loss can be caused by directly using medicine powder or solution, high-dose medicine can generate great toxicity to surrounding normal tissues, and the medicine is required to play a role continuously and orderly in the whole tumor recurrence inhibiting and repair promoting stage. The use of hydrogels as carriers for prolonged drug release has been extensively studied and various polymeric materials have been reported to load biopharmaceuticals (e.g., proteins and polypeptides) to mitigate in vivo enzymatic degradation and the like to enhance the therapeutic efficacy of the drug, such as polyethylene glycol, sodium alginate, hyaluronic acid, and the like.

Hyaluronic acid is one of the main components of extracellular matrix, and is involved in the activation and transmission processes of various cell signaling pathways, such as the formation of inflammation, wound healing, the development and metastasis of tumors, and the like. The hyaluronic acid hydrogel has the characteristics of high hydrophilicity, biocompatibility and the like, so the hyaluronic acid hydrogel is a promising wound repair dressing in clinic. However, the traditional chemical crosslinking of hyaluronic acid requires a long time, and the hydrogel with a certain shape and size needs to be prepared in advance for experiments, so that the requirements of injection and filling of defects with any shape cannot be met. Meanwhile, the hydrogel encapsulated drugs are easy to cause drug burst release, which affects the treatment effect.

The documents report that the residual tumor cells can be killed locally and the tissue defect repair can be promoted by using a proper scaffold material in tissue engineering, for example, tumor cells can be killed by using anti-tumor drugs, certain small molecules or metal elements through chemotherapy, photo-thermal or photodynamic therapy, and then repair factors in a composite scaffold promote the repair of defective tissues. However, it is more important for the invisible tumor cells to inhibit the malignant proliferation of the tumor cells, and the treatment means such as photothermal treatment can cause great damage to the surrounding normal tissues. Whether the anti-tumor drugs and the repair drugs can play their functions in a spatiotemporal sequence or not is limited in clinical application.

In the fields of biological materials and medicine, the problem of multi-stage treatment after tumor resection is not effectively solved, and a great need still exists for a method for simultaneously inhibiting tumors and promoting repair. In the field of tissue engineering, there is still a great need for multifunctional sustained release materials for tissue engineering scaffolds. Accordingly, those skilled in the art are devoted to the development of an injectable bioactive hydrogel that inhibits tumor recurrence after resection and promotes tissue repair, and methods of preparation and use thereof.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is how to realize a multi-stage treatment with one drug for the tissue after tumor resection.

In order to achieve the purpose, the injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair is characterized in that the bioactive hydrogel contains a high polymer material and a multifunctional active peptide, the high polymer material is grafted with the multifunctional active peptide through a grafting agent, and the high polymer material with the multifunctional active peptide is crosslinked and cured under ultraviolet light through a photoinitiator to form the bioactive hydrogel.

Further, the polymer material is hyaluronic acid.

Furthermore, the multifunctional active peptide is multifunctional active peptide JM2 with the functions of tumor resistance and tissue repair, and the amino acid sequence of the multifunctional active peptide JM2 is VFFKGVKDRVKGRSDC.

Further, the grafting agent is methacrylic anhydride, and the photoinitiator is phenyl-2, 4, 6-trimethylbenzoyllithium phosphonate.

Further, the interior of the bioactive hydrogel is a porous structure.

The invention also provides a preparation method of the injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair, which is characterized by comprising the following steps:

step 1, mixing a hyaluronic acid aqueous solution and a methacrylic anhydride solution, reacting to prepare a modified hyaluronic acid solution grafted with methacrylic anhydride, and freeze-drying the modified hyaluronic acid solution for subsequent use;

step 2, mixing the modified hyaluronic acid solution obtained in the step 1 with multifunctional active peptide JM2, reacting to prepare functionalized hyaluronic acid grafted with multifunctional active peptide JM2, and then freeze-drying the functionalized hyaluronic acid for subsequent use;

and 3, mixing the functionalized hyaluronic acid and the photoinitiator in the step 2, adding the mixture into a mold, and performing crosslinking curing under ultraviolet light to obtain the injectable bioactive hydrogel.

Further, in the step 1, the mass volume percentage of the hyaluronic acid aqueous solution is 1%, and the molar ratio of the hyaluronic acid to the methacrylic anhydride is 1: 10.

further, the molar ratio of the modified hyaluronic acid to the multifunctional active peptide JM2 in the step 2 is 1: 10.

further, the mass ratio of the functionalized hyaluronic acid to the photoinitiator in the step 3 is 9: 1, the power of ultraviolet light is 25mW/cm²。

The invention also provides application of the injectable bioactive hydrogel in preparing a scaffold material for repairing tissue defects after various tumors are excised in tissue engineering.

Technical effects

The injectable bioactive hydrogel material has the capabilities of inhibiting tumor proliferation, promoting tumor cell apoptosis, relieving inflammation and promoting tissue repair, and the hyaluronic acid-multifunctional active peptide JM2 hydrogel continuously releases drug molecules and plays different treatment effects in the whole stage. The prepared hydrogel material has the characteristic of injectability, and can be used for filling defects with different shapes. Can better exert the effect of the combined treatment of inhibiting the tumor recurrence and promoting the tissue repair.

In practical application, the injectable bioactive hydrogel material can inhibit tumor proliferation, promote tumor cell apoptosis, simultaneously relieve inflammation and promote tissue repair, and has good treatment effect on multi-stage treatment after tumor resection.

The preparation method of the injectable bioactive hydrogel for inhibiting the tumor recurrence after resection and promoting tissue repair is simple and easy to operate. When the injection is clinically used for treating after excision of skin tumor, the injection mode can be used due to the liquid application and the photo-crosslinking solidification, the operation is convenient, the pain of a patient in the treatment process is greatly relieved, the working strength of medical staff is reduced, the application is wide, and the practicability is high.

The injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair has good hydrophilicity, no cytotoxicity, stability, easy molding property and degradation performance, and has the capability of continuously releasing drug molecules, so that the defect that the drug molecules are easy to burst in tissue engineering is overcome, and the curative effect is safe and lasting.

The injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair has a simple system, can exert various treatment effects, can reduce inflammation and promote tissue repair while inhibiting tumor proliferation and promoting tumor cell apoptosis, and has a good treatment effect on multi-stage treatment after tumor resection.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a diagram illustrating the gelation process and appearance of injectable bioactive hydrogel of hyaluronic acid-bioactive peptide JM2 according to a preferred embodiment of the present invention;

FIG. 2 is a scanning electron microscope image of an injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 according to a preferred embodiment of the present invention;

FIG. 3 is a graph showing the release of the injectable bioactive hydrogel bioactive peptide JM2 molecule from hyaluronic acid-multifunctional bioactive peptide JM2 according to a preferred embodiment of the present invention over time.

FIG. 4 is a confocal laser microscope of an injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 encapsulating macrophages according to a preferred embodiment of the present invention;

FIG. 5A is a photograph showing the wound size of the wound on the back of C57BL/6 mouse treated with injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 as varied with time in accordance with the preferred embodiment of the present invention.

FIG. 5B is a graph showing the relative wound area as measured over time for dorsal wounds of C57BL/6 mice treated with an injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 in accordance with a preferred embodiment of the present invention.

FIG. 6 is a graph showing H & E staining of wound surface on back of C57BL/6 mouse treated with hyaluronic acid-multifunctional active peptide JM2 injectable bioactive hydrogel according to a preferred embodiment of the present invention;

FIG. 7 is a graph showing the infiltration of neutrophils and macrophages on the wound surface of the dorsal wound surface of a C57BL/6 mouse treated with the injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 according to a preferred embodiment of the present invention;

FIG. 8 is a graph showing the number of new blood vessels and mature blood vessels in the wound surface of the back wound surface of a C57BL/6 mouse treated with the injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 according to a preferred embodiment of the present invention;

FIG. 9 is a diagram of the recurrence of tumor in the wound surface after excision of the back tumor of the C57BL/6 mouse treated with the injectable bioactive hydrogel of hyaluronic acid-multiple-function active peptide JM2 in accordance with the preferred embodiment of the present invention;

FIG. 10 is a H & E staining chart of the wound surface of the hyaluronic acid-multifunctional active peptide JM2 subjected to injectable bioactive hydrogel after the resection of the tumor on the back of the C57BL/6 mouse in the preferred embodiment of the invention;

FIG. 11 is a diagram of the expression of wound surface malignant proliferation cell marker Ki67 of the wound surface after the resection of the back tumor of the C57BL/6 mouse treated by the hyaluronic acid-multifunctional active peptide JM2 injectable bioactive hydrogel in the preferred embodiment of the invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

The starting materials and equipment used in this example were known products and were obtained by purchasing commercially available products.

Example 1: preparation of hyaluronic acid-multifunctional active peptide JM2 injectable bioactive hydrogel

Step 1, dissolving 1g of hyaluronic acid powder in 100mL of deionized water, and continuously stirring to prepare a 1% hyaluronic acid solution. The hyaluronic acid solution was placed at 4 ℃ and the pH was 8.0, and 7.4mL of methacrylic anhydride solution was slowly added dropwise to the hyaluronic acid solution and stirred uniformly. The solution was stirred at low speed overnight at pH 8.0 at 4 ℃. The solution was poured slowly into 1L of absolute ethanol, precipitated and left overnight. Collecting the precipitate, dialyzing with dialysis bag (molecular weight cut-off of 7kDa) for 7 days, collecting the dialyzed hyaluronic acid solution, and freeze-drying;

and 2, adding 100mg of the freeze-dried hyaluronic acid solution obtained in the step 1 into 50mL of triethanolamine buffer solution, and uniformly stirring. 37mg of multifunctional active peptide JM2 was added to the above solution and stirred well. The mixture is placed in an environment with the temperature of 37 ℃ for reaction for two hours. The above solution was collected and dialyzed through dialysis bag (cut-off molecular weight 7kDa) for 3 days. Collecting the dialyzed solution, and then freeze-drying the solution for subsequent use;

step 3, adding 20mg of the freeze-dried functionalized hyaluronic acid solution obtained in the step 2 into 2.2mg of lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate, and then adding2mL of phosphate buffered saline was added and stirred well. Adding about 100 μ L of the above mixed solution into a mold, and using power of 25mW/cm²Ultraviolet light irradiates for 30s to form hydrogel.

Example 2: appearance and microstructure analysis of injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2

As shown in fig. 1, the injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 prepared in example 1 has injectable properties and has a smooth appearance. The hyaluronic acid-multifunctional active peptide JM2 injectable bioactive hydrogel prepared in example 1 was freeze-dried, a dried sustained-release system sample was cut to obtain a relatively flat internal cross section, and the cross-sectional structure of the active substance sustained-release system was observed under an acceleration voltage of 10kV by an emission Scanning Electron Microscope (SEM) after surface gold spraying.

As shown in figure 2, the hyaluronic acid-multifunctional active peptide JM2 injectable bioactive hydrogel has a hydrogel sheet structure in the inner part under an emission scanning electron microscope, and a local enlarged view shows a porous structure.

Example 3: sustained release bioactive peptide JM2 molecular properties of injectable bioactive hydrogel of hyaluronic acid-multifunctional bioactive peptide JM2

Step a, taking 50mg of a sample of the lyophilized hyaluronic acid-multifunctional active peptide JM2 prepared in the step 2 in the example 1, adding 5.6mg of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate powder, adding 5mL of phosphate buffer solution to form a mixed solution, and uniformly stirring for later use;

step b, adding 1mL of the mixed solution obtained in the step a into a mold, wherein the use power is 25mW/cm²The hydrogel was formed by UV irradiation for 30s and incubated at 37 ℃ in 10mL phosphate buffered saline. 1mL of supernatant was collected at 8h, 16h, 24h, 32h, 48h, 72h, 168h, respectively, and 1mL of phosphate buffered saline was added. The amount of polypeptide concentration was measured for samples at different time points using BCA Protein quantification Kit (BCA Protein Assay Kit) and release curves were plotted.

As shown in figure 3, the hyaluronic acid-multifunctional peptide JM2 injectable bioactive hydrogel can continuously release polypeptide drugs, continuously release the active peptide JM2 molecules to inhibit malignant proliferation of residual tumor cells and promote apoptosis in the first few days after tumor resection, release the active peptide JM2 molecules to reduce inflammation and accelerate tissue repair process from 72h to 168h, and the whole time and space are consistent with the repair process after tumor resection, which shows that the hyaluronic acid-multifunctional peptide injectable bioactive hydrogel material has the dual functions of tumor regulation and repair.

Example 4: biological safety verification of hyaluronic acid-multifunctional active peptide JM2 injectable bioactive hydrogel on normal repair cells

Step i, taking 50mg of a sample of the lyophilized hyaluronic acid-multifunctional active peptide JM2 prepared in the step 2 of the example 1, adding 5.6mg of phenyl-2, 4, 6-trimethylbenzoyllithium phosphonate powder, and then adding 5mL of phosphate buffer solution to form a mixed solution, and uniformly stirring for later use;

step ii, macrophage RAW cells are treated at 1 × 10⁵Adding the mixture into 100 mu L of the mixed solution in the step i at a density of one/mL, gently blowing and uniformly mixing the mixture for later use, wherein the use power is 25mW/cm²Ultraviolet light irradiates for 30s to form hydrogel.

Step iii, transferring the hydrogel in the step 2 into a 24-pore plate, adding a culture medium, and culturing the bioactive hydrogel wrapped with RAW cells (macrophages) in an incubator at 37 ℃ for 3 days;

step iv, preparing live-dead (live-dead) cell stain, adding 5 mu L B reagent (ethidium homodimer-1) and 1.25 mu L A reagent (calcein AM) into 2.5mL of phosphate buffer solution, uniformly mixing, wrapping with aluminum foil, and placing in a dark place for later use. The medium in the culture plate is sucked away, the phosphate buffer solution is washed for three times, 500 mu L of staining solution is added into each hole, the culture plate is placed in an incubator for 15min and then taken out, the staining solution is sucked away, the phosphate buffer solution is used for washing cells for two times, the hydrogel is taken out and placed on a glass slide, and the cells are observed through a laser confocal microscope (CLSM).

As shown in FIG. 4, the observation result of the living and dead state of RAW cells under a laser confocal microscope shows that the injectable bioactive hydrogel of the hyaluronic acid-multifunctional active peptide JM2 has biological safety for repairing cells.

Example 5: verification of in vivo tissue repair promoting capability of hyaluronic acid-multifunctional active peptide JM2 injectable bioactive hydrogel

Step a, taking 27C 57BL/6 mice, shaving the back hairs of the mice by using a shaver, manufacturing two wound surfaces with the diameter of 1cm on the back of the mice, and sewing and fixing the wound surfaces by using a sewing ring;

step b, according to the method shown in example 1, the hyaluronic acid-multifunctional active peptide JM2 mixed solution is dripped on the wound surface, and the power of application is 25mW/cm²Irradiating with ultraviolet light for 30s to form hydrogel for filling wound defect, and setting blank group and hyaluronic acid control group without multifunctional medicine, wherein each group is set for 3 times;

step C, photographing and killing the back wound of the C57BL/6 mouse at 2 days, 6 days and 12 days, and collecting a wound sample for subsequent analysis;

step d, performing statistical analysis on the surface picture created in the step c, comparing the relative sizes of the wound surface areas, and analyzing the wound healing condition;

FIG. 5A is a photograph showing the change of wound size with time of a wound surface of a back wound surface of a C57BL/6 mouse treated with an injectable bioactive hydrogel of hyaluronic acid-multifunctional peptide JM2 (blank in the drawing; HA represents a hyaluronic acid control group without a multifunctional drug; HA-JM2 represents an injectable bioactive hydrogel group of hyaluronic acid-multifunctional peptide JM 2); fig. 5B is a graph showing the relative wound area as a function of time measured on the dorsal wound surface of C57BL/6 mice treated with the injectable bioactive hydrogel of hyaluronic acid-multifunctional peptide JM2 (blank in the figure; HA represents a hyaluronic acid control group without multifunctional drug; HA-JM2 represents an injectable bioactive hydrogel group of hyaluronic acid-multifunctional peptide JM 2; and x represents a significant difference between the results of the two groups). As shown by the results of fig. 5A and 5B, the injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 can promote wound closure.

E, fixing the wound surface sample collected in the step c with paraformaldehyde overnight, fixing with 70% alcohol overnight, dehydrating with 80% alcohol for 1h, dehydrating with 90% alcohol for 1h, dehydrating with 100% absolute ethyl alcohol for 30min, and dehydrating with xylene and absolute ethyl alcohol for 1: 1, 30min of xylene I, 30min of xylene II, 2H of paraffin I wax immersion, 2H of paraffin II wax immersion, 2H of paraffin III wax immersion, embedding and the like, cutting the embedded tissues into 5-micron sections by a slicer, and performing H & E staining, immunohistochemical neutrophil granulocyte and macrophage staining and immunofluorescence staining on the sections through a series of steps from dewaxing to water.

FIG. 6 shows the H & E staining results of the wound surface of the back wound of C57BL/6 mice treated with the injectable bioactive hydrogel of hyaluronic acid-multifunctional peptide JM2 (blank in the figure; HA represents hyaluronic acid control group without multifunctional drug; HA-JM2 represents injectable bioactive hydrogel group of hyaluronic acid-multifunctional peptide JM 2).

FIG. 7 shows the infiltration of neutrophils and macrophages due to immunohistochemical staining of the wound surface of the back of a C57BL/6 mouse treated by the injectable bioactive hydrogel of hyaluronic acid-multifunctional peptide JM2 (blank in the figure; HA represents hyaluronic acid control group without multifunctional drug; HA-JM2 represents hyaluronic acid-multifunctional peptide JM2 injectable bioactive hydrogel group).

FIG. 8 shows the number of new blood vessels and mature blood vessels in the wound surface of the back wound of C57BL/6 mice treated with the injectable bioactive hydrogel of hyaluronic acid-multifunctional peptide JM2 (blank in the figure; HA represents hyaluronic acid control group without multifunctional drug; HA-JM2 represents hyaluronic acid-multifunctional peptide JM2 injectable bioactive hydrogel group).

As shown in fig. 6 to 8, the injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 can promote wound closure, reduce inflammatory cell infiltration, and accelerate tissue repair process.

Example 6: verification of capability of hyaluronic acid-multifunctional active peptide JM2 in inhibiting recurrence after tumor resection and promoting tissue repair in vivo of injectable bioactive hydrogel

Step 1, 12C 57BL/6 mice were harvested and their back hair shaved using a shaver, whereuponRight dorsal injection 2X 10⁵Individual melanoma cells, after 7 days, developed tumors on the back, which were excised, leaving a small amount of tumor tissue. The wound surface after the tumor is removed is fixed by suturing with a suturing ring;

step 2, according to the method shown in example 1, the hyaluronic acid-multifunctional active peptide JM2 mixed solution is dripped on the wound surface, and the using power is 25mW/cm²Irradiating with ultraviolet light for 30s to form hydrogel for filling wound defect, and setting tumor-free normal wound group, blank group and hyaluronic acid control group without multifunctional medicine, wherein each group is set for 3 times;

step 3, photographing the back wound of the C57BL/6 mouse at 3 days, 7 days and 14 days, killing the mouse at 14 days, and collecting a wound sample for subsequent analysis;

step 4, fixing the wound surface sample collected in the step 3 with paraformaldehyde overnight, fixing with 70% alcohol overnight, dehydrating with 80% alcohol for 1h, dehydrating with 90% alcohol for 1h, dehydrating with 100% absolute ethyl alcohol for 30min, and dehydrating with xylene and absolute ethyl alcohol for 1: 1, carrying out transparency for 1H, carrying out transparency for 30min on xylene I, carrying out transparency for 30min on xylene II, carrying out paraffin I waxing for 2H, carrying out paraffin II waxing for 2H, carrying out paraffin III waxing for 2H, embedding and the like, cutting the embedded tissue into 5-micrometer sections through a slicing machine, carrying out H & E staining and immunohistochemical Ki67 proliferation index staining on the sections through a series of steps from dewaxing to water.

FIG. 9 is a diagram showing the recurrence of tumor in the wound surface after resection of the tumor on the back of the C57BL/6 mice treated with the injectable bioactive hydrogel of hyaluronic acid-multifunctional peptide JM2 (in the figure, "normal wound" represents the tumor-free normal wound group; blank "represents the blank group; HA" represents the hyaluronic acid control group without multifunctional drug; and "HA-JM 2" represents the injectable bioactive hydrogel group of hyaluronic acid-multifunctional peptide JM 2).

FIG. 10 shows the H & E staining results of the wound surface after resection of the tumor on the back of the C57BL/6 mouse treated by the bioactive hydrogel of hyaluronic acid-multifunctional peptide JM2 (in the figure, "normal wound" represents the tumor-free normal wound group; blank "represents the blank group; HA" represents the hyaluronic acid control group without multifunctional drug; and "HA-JM 2" represents the injectable bioactive hydrogel group of hyaluronic acid-multifunctional peptide JM 2).

FIG. 11 shows the expression of the wound malignant proliferation cell marker Ki67 of the wound after the resection of the tumor on the back of the C57BL/6 mice treated with the injectable bioactive hydrogel of hyaluronic acid-multifunctional peptide JM2 (in the figure, "normal wound" represents the tumor-free normal wound group; blank "represents the blank group; HA" represents the hyaluronic acid control group without multifunctional drug; and "HA-JM 2" represents the injectable bioactive hydrogel group of hyaluronic acid-multifunctional peptide JM 2).

As shown in fig. 9 to 11, the injectable bioactive hydrogel of hyaluronic acid-multifunctional active peptide JM2 can inhibit malignant proliferation of tumor cells and promote tissue repair.

The injectable bioactive hydrogel has the capabilities of inhibiting tumor proliferation, promoting tumor cell apoptosis, relieving inflammation and promoting tissue repair, continuously and slowly releases the multifunctional active peptide JM2 grafted to a hyaluronic acid hydrogel material in the whole stage from skin tumor excision to wound repair through the hyaluronic acid-multifunctional active peptide JM2 injectable bioactive hydrogel, inhibits extracellular ATP release, reduces inflammatory cell infiltration and accelerates tissue repair process while inhibiting malignant proliferation and promoting apoptosis of tumor cells, and plays different treatment effects in the whole stage. Meanwhile, the prepared hydrogel material has the characteristic of injectability and can fill defects with different shapes. Thereby better exerting the effect of the combined treatment of simultaneously inhibiting the tumor recurrence and promoting the tissue repair.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. An injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair, wherein the bioactive hydrogel comprises a polymer material and a multifunctional active peptide, the polymer material is grafted with the multifunctional active peptide through a grafting agent, and the polymer material with the multifunctional active peptide is crosslinked and cured under ultraviolet light through a photoinitiator to form the injectable bioactive hydrogel; the polymer material is hyaluronic acid, the grafting agent is methacrylic anhydride, the multifunctional active peptide is multifunctional active peptide JM2 with anti-tumor and tissue repair effects, and the amino acid sequence of the multifunctional active peptide is VFFKGVKDRVKGRSDC.

2. The injectable bioactive hydrogel for inhibiting postoperative tumor recurrence and promoting tissue repair of claim 1 wherein the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate.

3. The injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair of any of claims 1 to 2, wherein the injectable bioactive hydrogel has a porous structure inside.

4. A method of preparing an injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair according to any of claims 1 to 3, comprising the steps of:

step 1, mixing a hyaluronic acid aqueous solution and the methacrylic anhydride solution, reacting to prepare a modified hyaluronic acid solution grafted with the methacrylic anhydride, and then freeze-drying the modified hyaluronic acid solution for subsequent use;

step 2, mixing the modified hyaluronic acid solution obtained in the step 1 and a multifunctional active peptide JM2 for reaction to prepare functionalized hyaluronic acid grafted with the multifunctional active peptide JM2, and then freeze-drying the functionalized hyaluronic acid for subsequent use;

and 3, mixing the functionalized hyaluronic acid and the photoinitiator in the step 2, adding the mixture into a mold, and performing crosslinking curing under the ultraviolet light to obtain the bioactive hydrogel.

5. The method for preparing an injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair according to claim 4, wherein the mass volume percentage of the hyaluronic acid aqueous solution in step 1 is 1%, and the molar ratio of the hyaluronic acid to the methacrylic anhydride is 1: 10.

6. the method for preparing injectable bioactive hydrogel for inhibiting postoperative tumor recurrence and promoting tissue repair of claim 4 wherein the molar ratio of the modified hyaluronic acid to the multifunctional active peptide JM2 in step 2 is 1: 10.

7. the method for preparing an injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair according to claim 4, wherein the mass ratio of the functionalized hyaluronic acid to the photoinitiator in step 3 is 9: 1, the power of the ultraviolet light is 25mW/cm²。

8. Use of an injectable bioactive hydrogel according to any one of claims 1 to 3 for the preparation of a scaffold material for tissue engineering of tissue defect repair after resection of various tumors.