A composite hydrogel loaded with silver ions and black phosphorus nanosheets and a preparation method thereof

Abstract

The present invention belongs to the technical field of medical biomaterials, and particularly relates to a composite hydrogel loaded with silver ions and black phosphorus nanosheets and a preparation method thereof. In order to overcome the problems of recurrence, metastasis and postoperative infection urgently needed to be solved in clinical tumor treatment, the present invention first modifies hyaluronic acid with dopamine and prepares Pluronic F127 modified with terminal mercapto groups, and then crosslinks the two by an addition reaction to obtain a hyaluronic acid / dopamine / Pluronic hydrogel. Finally, the black phosphorus nanosheets loaded with silver ions are encapsulated in the hydrogel to prepare a composite hydrogel loaded with silver ions and black phosphorus nanosheets. The composite hydrogel can not only effectively prevent tumor recurrence and metastasis and resist infection, but also has excellent characteristics such as tissue adhesion, biocompatibility, self-healing property and injectability. It can be used for the prevention and treatment of postoperative recurrence, metastasis and wound infection of malignant tumors, providing new ideas and strategies for the research and development of new implants.

Description

Technical Field

[0001] The invention belongs to the technical field of medical biomaterials, and particularly relates to a composite hydrogel loaded with silver ions and black phosphorus nanosheets and a preparation method thereof. Background Art

[0002] Malignant tumors pose a very serious threat to human health. In the current clinical treatment of tumors, surgical resection is the main treatment method, which faces the problem of incomplete removal of cancer cells due to large-scale tumor infiltration. In addition, the patient's immunity is low after surgery, and recurrence and metastasis are very likely to occur. The anti-cancer situation is not optimistic. At the same time, after surgical resection of the tumor, the wound is in direct contact with the external environment, which is likely to cause bacterial infection. Antibacterial treatment of the surgical wound is also a necessary link to promote the patient's early recovery. In the current clinical treatment of solid tumors, surgical resection is the main treatment method and faces two major problems: postoperative recurrence caused by incomplete tumor cell removal and bacterial infection caused by wound exposure. Traditional postoperative wounds of tumors are often treated with antibacterial drugs and dressings, but there are problems such as the shape of the dressing not matching the nature of the wound, poor antibacterial effect, short antibacterial time, easy secondary infection, and failure to promote tissue regeneration. Specifically, commonly used tumor resections include open surgery and minimally invasive surgery. In open surgery, the lesion site is completely exposed, the surgical field of view is clear, and the tumor tissue can be removed as much as possible, but the large wound left is very susceptible to infection and the patient recovers slowly. On the contrary, although minimally invasive surgery can shorten the postoperative healing time, it is difficult to completely remove cancerous tissue due to the limited surgical space, which can easily lead to tumor recurrence. Therefore, there is an urgent need for a method that can balance infection and recurrence and is applicable to both surgical methods.

[0003] As a commonly used implantable biomedical material, hydrogel is a natural or synthetic polymer that is physically or chemically cross-linked. It has a three-dimensional network structure, can swell in water but is insoluble in water, and has the properties of both solids and liquids. Due to its high water content and similarity to the structure of human tissue, it has very good biocompatibility, can provide a moist and hypoxic microenvironment at the surgical wound, reduce fluid loss, and accelerate wound healing. It is one of the ideal materials for tumor surgical wound implants. At the same time, after years of development, on the basis of traditional hydrogels, it has been given more and more functional properties that are compatible with biomedical uses, such as increasing the tissue adhesion, biodegradability, and self-healing properties of hydrogels, which has laid a very good foundation for its preparation, storage, and biomedical functions. Therefore, hydrogel dressings have become a research hotspot in this field.

[0004] However, implantable hydrogels need to have sufficient strength and toughness to cope with the complex in-vivo environment. Therefore, they often lack fluidity and are difficult to completely fill into irregular tumor resection cavities, which brings certain difficulties to the implantation surgery. Currently, surgical hydrogels such as surgical adhesives in clinical practice often require liquid precursors of multiple components for in-situ gelation. This not only makes the operation complex but also difficult for non-horizontal wound surfaces. Therefore, the development of "plug-and-play" hydrogels with good tissue adhesiveness and simplified pretreatment processes can greatly improve the convenience and efficiency of clinical surgeries.

[0005] In addition, with the in-depth research on composite nano-therapy platforms in the field of biomedicine, the research on treatment methods alone can no longer fully meet the needs of precise tumor treatment. In contrast, using hydrogels as postoperative implantation treatment platforms for tumors can integrate multiple treatment methods, including chemotherapy, photothermal therapy, photodynamic therapy, immunotherapy, etc., to achieve the prevention and treatment of tumor recurrence, metastasis, and bacterial infection. Therefore, it is necessary to develop a composite hydrogel with good adhesion performance, fast coagulation, and anti-tumor and bactericidal functions. Summary of the Invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets, which encapsulates black phosphorus nanosheets loaded with silver ions (BP-Ag) in the hydrogel system for inhibiting tumor recurrence and metastasis and bacterial reproduction. As a strong photothermal agent, black phosphorus nanosheets (BP NSs) can achieve light-controlled local multiple photothermal treatments to kill residual tumor cells after resection, while silver ions, as a broad-spectrum antibacterial agent, can achieve long-term antibacterial effects without generating drug resistance. This hydrogel system can not only effectively prevent tumor recurrence, metastasis, and anti-infection but also has excellent characteristics such as tissue adhesiveness, biocompatibility, self-healing property, and injectability, providing new ideas and strategies for the research and development of new implants, overcoming the problems of recurrence, metastasis, and postoperative infection that urgently need to be solved in current clinical tumor treatment, and having good clinical application prospects.

[0007] In order to achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0008] The first aspect of the present invention provides a preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets, comprising the following steps:

[0009] S1. Dissolve hyaluronic acid in phosphate buffer solution, add an activator for activation, and then add dopamine. After reaction, dopamine-modified hyaluronic acid (HA-DA) is obtained;

[0010] S2. Add Pluronic F127 ( F127 was dissolved in an organic solvent, and then triethylamine and p-nitrophenylchloroformate (p-NPC) solvent were added for activation. Then, the activated Pluronic F127 was dissolved in an organic solvent again, and cysteamine was added for terminal thiol modification. After concentration and precipitation, Pluronic F127 modified with terminal thiol (Plu-SH) was obtained.

[0011] S3. First, a black phosphorus nanosheet solution was prepared by a liquid-phase exfoliation method, and then a silver salt solution was added to the black phosphorus nanosheet solution. After reaction, black phosphorus nanosheets loaded with silver ions (BP-Ag) were prepared.

[0012] S4. The dopamine-modified hyaluronic acid in step S1 and the Pluronic F127 modified with terminal thiol in step S2 were crosslinked in a phosphate buffer solution through a Michael addition reaction between the thiol group and the quinone structure of the oxidized catechol structure of dopamine. Then, the black phosphorus nanosheets loaded with silver ions in step S3 were added to continue the reaction to prepare a composite hydrogel loaded with silver ions and black phosphorus nanosheets (BP-Ag@HA-DA-Plu composite hydrogel).

[0013] Hyaluronic acid (HA) is a linear polyanionic polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine. It has good moisture retention, viscoelasticity, non-immunogenicity, degradability, and biocompatibility. F127 is a triblock copolymer of polyoxyethylene-polyoxypropylene ether. Because of its injectability and good biocompatibility, it has been approved by the FDA for use in humans. It has unique thermoresponsive properties, which can keep the hydrogel in a liquid state at room temperature and solidify into a semi-solid hydrogel at body temperature and physiological pH environment. By adjusting The addition ratio and crosslinking degree of F127 can control the critical gel temperature (CGT) between the body temperature and the operating room temperature, realizing the "plug and play" of the pre-crosslinked hydrogel during the operation. The lattice of black phosphorus (BP) is an interconnected six-membered ring, and each atom is connected to three other atoms. Due to the bond angle, each layer forms a corrugated and wrinkled plane. This makes the black phosphorus nanosheets prepared by liquid-phase ultrasonic exfoliation method have an extremely high specific surface area, which can be used for drug loading and has excellent drug loading capacity. At the same time, the black phosphorus nanosheets have good photothermal conversion efficiency and can generate photothermal effects by absorbing near-infrared light. Due to their good biocompatibility, degradability, and good photothermal conversion efficiency, the black phosphorus nanosheets can generate photothermal effects by absorbing near-infrared light and effectively kill tumor tissues. By being loaded into the long-lasting adhesive hydrogel for in-situ implantation after surgery, the black phosphorus nanosheets can repeatedly perform photothermal therapy on the affected area under the control of an external near-infrared light source, thereby more thoroughly killing residual tumor cells and achieving the purpose of inhibiting local recurrence. At the same time, silver ions can kill bacteria in various ways, such as dissolving cell membranes, changing cell permeability, inhibiting the activity of bacterial dehydrogenases, and binding to DNA to destroy its structure. And because silver ions can form a complexation reaction with sulfhydryl groups and have a charge adsorption effect with negatively charged black phosphorus nanosheets, they are very easy to be loaded into this hydrogel system to improve the overall antibacterial effect of the system.

[0014] Therefore, aiming at the problems of recurrence, metastasis and postoperative infection that urgently need to be solved in current clinical tumor treatment, the present invention modifies hyaluronic acid with dopamine, then prepares Pluronic F127 with modified terminal mercapto group, and finally crosslinks through the Michael addition reaction between the mercapto group at the end of Pluronic F127 and the quinone group structure formed by the oxidation of dopamine catechol to obtain a hyaluronic acid / dopamine / Pluronic F127 hydrogel. And encapsulate black phosphorus nanosheets loaded with silver ions in the hydrogel to construct a multifunctional treatment system that is injectable, adhesive and has good biocompatibility for combating postoperative infection, recurrence and metastasis of tumors. After the hydrogel system is injected into the tumor resection wound surface, due to the presence of Pluronic F127, it rapidly solidifies at body temperature and adheres to tissues through the dopamine catechol structure therein, and is fixed at the wound surface for a long time. Irradiating with near-infrared light in vitro can cause primary killing of residual tumor cells, inducing necrotic and apoptotic tumor cells to release tumor antigens. In addition, repeated photothermal heating can cause the destruction of the hydrogel system structure, release black phosphorus nanosheets loaded with silver ions, strongly kill in-situ residual tumor cells through the photothermal effect, and use the broad-spectrum antibacterial effect of silver ions to completely eliminate the hidden danger of postoperative wound infection and promote the recovery of patients. The hydrogel system has thermal responsiveness and strong tissue adhesiveness, can well achieve "plug and play" without pretreatment, simplifies the surgical process, and shows excellent effects of preventing tumor recurrence and anti-infection both in vitro and in vivo. The hydrogel system is practical in operation, reliable in effect, low in cost, safe in composition, simple in preparation, and has good clinical application prospects.

[0015] Preferably, in step S1, the activator is carbodiimide hydrochloride and N-hydroxysuccinimide, and the mass ratio of carbodiimide hydrochloride to N-hydroxysuccinimide is 2:1. The carboxyl group in hyaluronic acid is activated by carbodiimide hydrochloride for forming an amide bond with the amino group of dopamine to obtain HA-DA.

[0016] Preferably, in step S1, the mass concentration of the hyaluronic acid in the phosphate buffer solution is 20-30 mg / mL, and the mass ratio of the hyaluronic acid to dopamine is 2:(0.5-2).

[0017] Preferably, in step S2, the mass ratio of Pluronic F127 to an organic solvent (such as dichloromethane, etc.) is 1:5-1:7, the mass ratio of Pluronic F127 to triethylamine is 1:15-25, and the mass ratio of Pluronic F127 to p-nitrophenyl chloroformate solvent is 10:1-3..

[0018] Preferably, in step S2, the mass ratio of the activated Pluronic F127 to cysteamine is (20-30):1.

[0019] Preferably, in step S2, the activation time is more than 48 hours, and the terminal thiol modification time is more than 24 hours.

[0020] Preferably, in step S3, the preparation method of the black phosphorus nanosheet solution is as follows: Add black phosphorus particles (BP) into an organic solvent, blow air to remove the dissolved oxygen in the solution, and then place the mixture in an ice bath for probe sonication for 24 - 48 h. After centrifugation of the obtained dispersion, collect the brown supernatant containing black phosphorus nanosheets to obtain it.

[0021] Preferably, in step S3, the silver salt is silver nitrate, and the volume ratio of the silver salt solution to the black phosphorus nanosheet solution is 1:1.

[0022] Preferably, in step S4, the mass ratio of the dopamine - modified hyaluronic acid to the terminal thiol - modified Pluronic F127 is 1:(2 - 3).

[0023] Preferably, in step S4, after the dopamine - modified hyaluronic acid and the terminal thiol - modified Pluronic F127 are respectively in a phosphate - buffered solution, then add the black phosphorus nanosheet solution loaded with silver ions. The mass concentrations of both the dopamine - modified hyaluronic acid and the terminal thiol - modified Pluronic F127 are 50 mg / 400 - 500 μL, the mass concentration of the black phosphorus nanosheet solution loaded with silver ions is 0.2 - 0.5 mg / mL, and the volume ratio of the total solution of the dopamine - modified hyaluronic acid and the terminal thiol - modified Pluronic F127 to the black phosphorus nanosheet solution loaded with silver ions is 8 - 10:1.

[0024] Preferably, in step S2, the p - nitrophenyl chloroformate solvent includes but is not limited to p - nitrophenyl chloroformate.

[0025] Preferably, in step S1, the pH of the phosphate - buffered solution used is controlled at 5.0. In step S4, the pH of the phosphate - buffered solution used is controlled at 7.4.

[0026] The second aspect of the present invention provides a composite hydrogel loaded with silver ions and black phosphorus nanosheets prepared by using the preparation method described in the first aspect.

[0027] The third aspect of the present invention provides the application of the composite hydrogel loaded with silver ions and black phosphorus nanosheets described in the second aspect in the preparation of anti - tumor and / or antibacterial implantable biomedical materials.

[0028] The composite hydrogel system prepared by the present invention, on the one hand, makes it rapidly solidify after injection through Pluronic, and uses the catechol structure of dopamine to make it have tissue adhesion behavior; on the other hand, it strongly kills tumor cells through black phosphorus nanosheets loaded with silver ions and eliminates the hidden danger of postoperative wound infection. It is a composite hydrogel with good biocompatibility, good adhesion performance, fast solidification, and can effectively prevent tumor recurrence, metastasis and anti-infection, and can be used for the prevention and treatment of postoperative recurrence, metastasis and wound infection of malignant tumors.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] The present invention discloses a preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets. First, hyaluronic acid is modified with dopamine, and Pluronic F127 modified with terminal thiol is prepared. Then, a hyaluronic acid / dopamine / Pluronic hydrogel is crosslinked through a Michael addition reaction between the thiol group and the quinone structure formed by the oxidation of the catechol structure of dopamine. Finally, black phosphorus nanosheets loaded with silver ions are encapsulated in the hydrogel to obtain a composite hydrogel loaded with silver ions and black phosphorus nanosheets. The hydrogel system of the present invention has the following advantages:

[0031] (1) By modifying hyaluronic acid with dopamine to construct a hydrogel substrate, good tissue adhesion properties can be generated on the premise of ensuring biocompatibility and biodegradability, which is convenient for continuous action at the surgical trauma site;

[0032] (2) The introduction of catechol groups can not only generate crosslinking through oxidative polymerization and Michael addition reaction with thiol groups, but also form reversible π-π stacking interactions and hydrogen bond interactions, endowing the hydrogel with the ability to resist external stress and self-healing properties, and being more adaptable to complex in vivo environments;

[0033] (3) Introducing Pluronic F127 into the system can endow the hydrogel with injectable properties, enabling the hydrogel to maintain a liquid state at room temperature and solidify into a semi-solid hydrogel at body temperature and physiological pH environment. By adjusting the addition ratio and crosslinking degree, the critical gel temperature (CGT) can be controlled between the body temperature and the operating room temperature, realizing the "plug and play" of the pre-crosslinked hydrogel during the operation;

[0034] (4) The high specific surface area characteristic of black phosphorus nanosheets enables them to efficiently load silver ions as a carrier. Through the high photothermal conversion rate of black phosphorus nanosheets, strong killing of in-situ residual tumor cells is achieved through photothermal effects, which can effectively solve the problems of postoperative tumor recurrence and metastasis at the same time;

[0035] (5) Silver ions can form a complexation reaction with sulfhydryl groups and have a charge adsorption effect on negatively charged black phosphorus nanosheets, making it easy for black phosphorus nanosheets to be loaded into the hydrogel system, thereby enhancing the overall antibacterial effect of the system. In addition, the broad-spectrum antibacterial effect of silver ions is utilized to completely eliminate the hidden danger of bacterial infection in the postoperative wound surface and promote the recovery of patients.

[0036] In summary, the composite hydrogel provided by the present invention can not only effectively prevent tumor recurrence and metastasis and resist infection, but also has excellent characteristics such as tissue adhesiveness, biocompatibility, self-healing property, and injectability. It can be used for the prevention and treatment of postoperative recurrence, metastasis, and wound infection of malignant tumors, providing new ideas and strategies for the research and development of new implants. Brief Description of the Drawings

[0037] Figure 1 It is the ultraviolet-visible absorption spectrum (UV-Vis) and nuclear magnetic resonance spectrum of hyaluronic acid-dopamine nanoparticles (HA-DA).

[0038] Figure 2 It is the nuclear magnetic resonance spectrum of Pluronic F-127 ( F127) and mercapto-substituted Pluronic F-127 (Plu-SH).

[0039] Figure 3 It is the particle size distribution diagram of black phosphorus nanosheets (BP) and black phosphorus nanosheets loaded with silver ions (BP-Ag).

[0040] Figure 4 It is the physical morphology characterization of BP-Ag nanoparticles and BP-Ag@HA-DA-Plu hydrogel platform. Among them, (A) is the scanning electron microscope image (a) and elemental mapping images (b-d) of BP-Ag, and the scale bar is 1:200 nm; (B) is the X-ray photoelectron spectroscopy of BP-Ag, (a) is the P2p spectrum, and (b) is the Ag3d spectrum; (C) is the scanning electron microscope images of BP-Ag@HA-DA-Plu hydrogel at different magnifications, and the scale bar of (a) is 1:50 μm and that of (b) is 1:10 μm.

[0041] Figure 5 It is the in vitro anti-tumor effect of the hydrogel. Among them, (A) is the cell viability of 4T1 tumor cells treated with different hydrogels. From left to right in A are G1, G2, G3, and G4. G1 is the HA-DA-Plu hydrogel group, G2 is the BP@HA-DA-Plu hydrogel group, G3 is the BP-Ag@HA-DA-Plu hydrogel group, and G4 is the BP-Ag@HA-DA-Plu hydrogel + near-infrared group. The data are expressed as mean ± SD (n = 6). ***P < 0.001; (B) shows the results of calcein / PI cell viability and cytotoxicity assays for G1 - G4 (a - d), scale bar: 200 μm; (C) shows the statistical results of the proportion of live and dead cells for G1 - G4, data are expressed as mean ± standard deviation (n = 3).

[0042] Figure 6 Results of in vitro safety tests of HA - Plu - DA, Ag@HA - Plu - DA, BP@HA - Plu - DA, BP - Ag@HA - Plu - DA hydrogels (from left to right: Control, Ag@HA - Plu - DA hydrogel, BP - Ag@HA - Plu - DA hydrogel, HA - Plu - DA hydrogel, BP@HA - Plu - DA hydrogel).

[0043] Figure 7 Results of the in vitro antibacterial effect of the hydrogels, where (A) are the photos of Staphylococcus aureus and Escherichia coli colonies after different treatments; (B) is the bar chart of quantitative colony density; (C) is the N01 / PI staining result of Escherichia coli after different treatments, scale bar is 10 μm. The specific different treatment schemes in the figure are: G1 is the blank group; G2 is the HA - DA - Plu hydrogel; G3 is the Ag@HA - Plu - DA hydrogel; G4 is the BP@HA - Plu - DA hydrogel; G5 is the BP - Ag@HA - Plu - DA hydrogel; G6 is the BP@HA - Plu - DA hydrogel + near - infrared light irradiation; G7 is the BP - Ag@HA - Plu - DA hydrogel + near - infrared light irradiation. Detailed implementation manners

[0044] The following further describes the detailed implementation manners of the present invention. It should be noted here that the description of these implementation manners is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various implementation manners of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0045] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional channels unless otherwise specified.

[0046] Example 1 Preparation of a hyaluronic acid - dopamine - pluronic F - 127 composite hydrogel loaded with silver ions and black phosphorus nanosheets

[0047] 1. The preparation method of the composite hydrogel specifically includes the following steps:

[0048] S1. Preparation of dopamine-modified hyaluronic acid: Dissolve 500 mg of hyaluronic acid (HA) in 20 mL of phosphate buffer solution with pH = 5.0, stir for 5 minutes, then add 242.6 mg of carbodiimide hydrochloride and 145.7 mg of hydroxysuccinimide as activating agents, and supplement 10 mL of phosphate buffer solution with pH = 5.0. Continue stirring for 20 minutes, then add 250 mg of dopamine (DA), and add 10 mL of phosphate buffer solution with pH = 5.0. Stir and react at room temperature for 12 hours. After that, pour the obtained mixed solution into a dialysis bag (MWCO: 12000 - 14000), dialyze in deionized water for 36 hours, and finally freeze-dry the product to obtain dopamine-modified hyaluronic acid nanoparticles (HA-DA).

[0049] The HA-DA obtained in step S1 was characterized by a proton nuclear magnetic resonance spectrometer (AVANCE 400, Bruke, USA) and an ultraviolet-visible spectrophotometer (LAMBDA365, Perkin Elmer, USA). The results are as Figure 1 shown, further verifying the successful synthesis of HA-DA. Among them, the structure of HA-DA is as follows (HA:DA is approximately 2:1, that is, x = 2, y = 1):

[0050]

[0051] S2. Preparation of thiolated Pluronic F127: Dissolve 10 g of Pluronic F127 ( F127) in 70 mL of dichloromethane, add 221.2 μL of triethylamine, and slowly drop the obtained solution into p-nitrophenyl chloroformate (959.8 mg of p-nitrophenyl chloroformate dissolved in 10 mL of dichloromethane) and stir evenly for 48 hours. Then, extract the activated Pluronic F127 with a saturated aqueous solution of sodium chloride in deionized water, further dry the dichloromethane phase with sodium sulfate for 30 min and filter. Precipitate the product by adding ice-cold ether and dry it under vacuum. Then dissolve 5 g of the activated Pluronic F127 in 50 mL of dichloromethane, add 423.4 mg of cysteamine, stir and react at room temperature evenly for 24 hours. Extract the final product with a saturated aqueous solution of sodium chloride in deionized water, dry the dichloromethane phase with sodium sulfate for 30 min and filter. Precipitate the product by adding ice-cold ether and dry it to obtain Plu-SH.

[0052] The Plu-SH obtained in step S2 was characterized by a proton nuclear magnetic resonance spectrometer (AVANCE 400, Bruke, USA). The results are as Figure 2 shown, further verifying the successful synthesis of Plu-SH. Among them, the structures of F127 and Plu-SH are as follows:

[0053]

[0054] S3. Preparation of silver ion-loaded black phosphorus nanosheets: Add 10 mg of black phosphorus particles into 20 mL of N-methylpyrrolidone, and introduce argon to remove the dissolved oxygen in the solution. Then, perform probe sonication on the mixture in an ice-water bath for 24 hours (power: 700 W, cycle: turn on for 3 seconds and turn off for 3 seconds). The obtained dispersion is centrifuged at 5000 rpm for 20 minutes to remove the incompletely exfoliated black phosphorus particles. Collect the brown supernatant containing the exfoliated black phosphorus nanosheets and store it at 4 °C for subsequent use. Before use, centrifuge the collected supernatant at 15000 rpm for 10 minutes to remove the N-methylpyrrolidone solvent, and repeat the water washing three times. Then, gradually add 1.57 mL of 0.8 mM AgNO3 solution dropwise into 1.57 mL of the previously obtained black phosphorus nanosheet solution, stir evenly for 3 hours. Centrifuge the obtained silver ion-loaded black phosphorus nanosheets (BP-Ag) at 15000 rpm for 10 minutes, wash them three times with water, and disperse them in 1 mL of phosphate buffer solution with pH = 7.4 for standby.

[0055] Figure 3 Through size analysis by dynamic light scattering (DLS), it can be determined that the obtained BP-Ag nanocomposite is mainly in the range of 300 - 500 nm. Such a particle size distribution not only ensures the uniform distribution of the nanocomposite in the hydrogel but also largely restricts the entry of the nanocomposite into the blood circulation of the tumor site, thereby reducing the risk of systemic adverse reactions.

[0056] S4. Dissolve 50 mg of HA-DA in 450 μL of phosphate buffer (pH 7.4) and dissolve 113 mg of Plu-SH in 450 μL of phosphate buffer solution (pH 7.4) respectively. Then, mix the obtained HA-DA and Plu-SH solutions with 100 μL of BP-Ag solution (BP concentration is 2 mg mL -1 ) and stir in an ice-water bath for 6 hours. Let the obtained mixture stand and age at 4 °C for 1 day to prepare a silver ion- and black phosphorus nanosheet-loaded hyaluronic acid-dopamine-Pluronic F-127 composite hydrogel (BP-Ag@HA-Plu-DA hydrogel).

[0057] Obtain the SEM image of the BP-Ag@HA-Plu-DA hydrogel in Example 1 by scanning electron microscopy, and analyze the XPS of the hydrogel using an X-ray photoelectron spectrometer (ESCALab250, Thermo, USA). The results are as Figure 4 shown, Figure 4 A shows that Ag nanoparticles are uniformly distributed on the surface of BPNSs, with a particle size <5 nm. Elemental mapping and X-ray photoelectron spectroscopy further confirm the successful preparation of the BP-Ag nanocomposite (Figure 4 A-(b-d) and Figure 4 B). A porous microstructure can be seen under a high-resolution scanning electron microscope, with a pore size of about 10-20 μm( Figure 4 C-(a)), and further magnification shows that irregular nanoscale flake-like appendages are distributed in the hydrogel( Figure 4 C-(b)), indicating successful loading of the BP-Ag nanocomposite.

[0058] Preparation of hyaluronic acid-dopamine-Pluronic F-127 composite hydrogel loaded with black phosphorus nanosheets in Comparative Example 1

[0059] The preparation steps are the same as those in Example 1, except that in step S3, the black phosphorus nanosheet solution was not reacted with the AgNO3 solution, and the black phosphorus nanosheets were directly dispersed in the buffer solution for step S4 to obtain a hyaluronic acid-dopamine-Pluronic F-127 composite hydrogel loaded with black phosphorus nanosheets (BP@HA-Plu-DA hydrogel).

[0060] Preparation of hyaluronic acid-dopamine-Pluronic F-127 composite hydrogel loaded with silver ions in Comparative Example 2

[0061] The preparation steps are the same as those in Example 1, except that: there is no step S3, and in step S4, 100 μL of 0.8 mM AgNO3 solution was used to replace 100 μL of the BP-Ag solution to obtain a hyaluronic acid-dopamine-Pluronic F-127 composite hydrogel loaded with silver ions (Ag@HA-Plu-DA hydrogel).

[0062] Preparation of hyaluronic acid-dopamine-Pluronic F-127 composite hydrogel in Comparative Example 3

[0063] The preparation steps are the same as those in Example 1, except that: there is no step S3, and the BP-Ag solution was not added in step S4, to obtain a hyaluronic acid-dopamine-Pluronic F-127 composite hydrogel (HA-DA-Plu or HA-Plu-DA hydrogel).

[0064] Experimental Example 1 In vitro anti-tumor effect and safety of BP-Ag@HA-Plu-DA composite hydrogel

[0065] The 4T1 tumor cells were cultured in a medium containing 10% (v / v) fetal bovine serum, antibiotics penicillin (100 U·mL -1 ) and streptomycin (100 μg·mL -1The 1640 medium was incubated in an incubator at 37 °C with 5% CO2. Experimental group settings: G1 was the HA-DA-Plu hydrogel group, G2 was the BP@HA-Plu-DA hydrogel group, G3 was the BP-Ag@HA-DA-Plu hydrogel group, and G4 was the BP-Ag@HA-DA-Plu hydrogel + near-infrared group. Among them, the groups that required near-infrared irradiation were given 808 nm NIR irradiation (0.4 W cm -2 , 5 min) after 12 h of incubation. Hydrogels with different concentrations (0, 25, 50, 100, 20 μL·mL -1 ) were placed in transwell culture chambers and incubated with 4T1 cells (cell concentration was 5×10 4 cells / mL) for 24 h.

[0066] After incubation, the cell counting kit-8 (CCK-8) method was used to verify the ability of the hydrogel to resist 4T1 tumor cells in vitro. At the same time, the CCK-8 method was used to detect cell viability, and the experimental results were observed by live / dead staining: Take 200 μL mL -1 hydrogel, add Calcein AM / PI dye to the incubated cells, and then observe under a fluorescence microscope (Eclipse Ti2-U, Nikon, Japan) after incubating for 30 min.

[0067] As Figure 5 shown in the photothermal effect test results of A, the killing ability of the BP-Ag@HA-DA-Plu hydrogel against tumor cells was significantly higher than that of other hydrogel groups and the hydrogel group without near-infrared irradiation. When the BP-Ag@HA-DA-Plu hydrogel exceeded 50 μL·mL -1 , a certain trend of decreasing activity began to appear under 808 nm near-infrared irradiation; when the concentration reached 200 μL·mL -1 , the survival rate of tumor cells was only 35%. At the same time, the Calcein / PI cell activity and cytotoxicity detection and their ratio statistical results could more intuitively obtain the same results ( Figure 5 B and Figure 5 C).

[0068] In addition, the same treatment method was used to further incubate 3T3 fibroblasts with the hydrogel to verify its safety. The experimental settings were a blank control group, an HA-Plu-DA hydrogel group, an Ag@HA-Plu-DA hydrogel group, a BP@HA-Plu-DA hydrogel group, and a BP-Ag@HA-Plu-DA hydrogel group. The concentration of each group of hydrogel was 200 μL·mL -1 , and the cell concentration was 5×10 4 cells / mL. The results were asFigure 6 As shown, after a total incubation of 72 h, the BP-Ag@HA-Plu-DA hydrogel had no significant effect on cell viability, indicating that the hydrogel had very reliable safety.

[0069] Experimental Example 2 Testing the in vitro antibacterial ability of the BP-Ag@HA-Plu-DA composite hydrogel

[0070] In this experimental example, the typical Staphylococcus aureus 186335 (purchased from BNCC, Gram-positive) and Escherichia coli 133264 (purchased from BNCC, Gram-negative) were selected to evaluate the broad-spectrum antibacterial ability of the BP-Ag@HA-DA-Plu hydrogel. The standard plate counting method was used for the antibacterial experiment, and the specific experimental method was as follows:

[0071] 1) Dilute the bacteria to 1×10 5 CFU / mL, take 200 μL and add it to a 96-well plate. Add 300 μL of the hydrogel to each group. Set G1 as the blank group in the experimental group; G2 as the HA-DA-Plu hydrogel; G3 as the Ag@HA-Plu-DA hydrogel; G4 as the BP@HA-Plu-DA hydrogel; G5 as the BP-Ag@HA-Plu-DA hydrogel; G6 as the BP@HA-Plu-DA hydrogel + near-infrared light irradiation; G7 as the BP-Ag@HA-Plu-DA hydrogel + near-infrared light irradiation. The near-infrared irradiation group was given 808 nm NIR irradiation (0.4 W·cm -2 , 5 min) after incubation for 12 h; place each group in an incubator at 37 °C for incubation for 6 h, and then use the plate dilution method to count the number of bacteria in each group.

[0072] 2) Live / dead bacteria staining method: After incubation, add the N01 / PI fluorescent nucleic acid dye and incubate for another 30 min. After resuspending the bacteria, take 8 μL of the bacterial solution and drop it on a glass slide, and observe it with a laser confocal microscope (LSN880, Zeiss, Germany).

[0073] Figure 7A and 7B are the antibacterial results of the standard plate count method. Compared with the HA-DA-Plu hydrogel, Ag@HA-DA-Plu hydrogel, and BP-Ag@HA-DA-Plu hydrogel treatment groups, the introduction of Ag nanoparticles has a greater inhibitory effect on the proliferation of both bacteria. In addition, by comparing the BP@HA-DA-Plu hydrogel and the BP@HA-DA-Plu hydrogel + NIR group, it is proved that the photothermal effect also has a certain killing effect on both bacteria. Therefore, through the synergistic broad-spectrum antibacterial ability of Ag nanoparticles and the photothermal effect of BP NSs, the BP-Ag@HA-DA-Plu hydrogel combined with 808 nm near-infrared laser irradiation has the best inhibitory effect on Staphylococcus aureus and Escherichia coli. In addition, similar results can also be obtained by observing the fluorescence images through N01 / PI staining ( Figure 7 C).

[0074] In summary, the present invention provides a BP-Ag@HA-Plu-DA composite hydrogel with good biocompatibility, adhesion performance, and fast coagulation. On the one hand, dopamine is used to modify hyaluronic acid to construct a hydrogel substrate, and the introduction of Pluronic F127 into the system can endow the hydrogel with injectable properties, enabling the "plug-and-play" of the pre-crosslinked hydrogel during surgery. On the other hand, black phosphorus nanosheets are used as carriers to efficiently load silver ions, which can produce a synergistic effect. The in-situ residual tumor cells can be strongly killed through the photothermal effect, effectively solving the problems of postoperative tumor recurrence and metastasis. The broad-spectrum antibacterial effect of silver ions is used to effectively solve the problem of bacterial infection, comprehensively eliminate the hidden danger of postoperative wound infection, and effectively accelerate wound healing. In addition, by introducing catechol groups, not only can crosslinking be generated through oxidative polymerization and Michael addition reaction with thiols, but also reversible π-π stacking and hydrogen bonding can be formed, endowing the hydrogel with the ability to resist external stress and self-healing properties, and being more adaptable to the complex in-vivo environment. It can be seen that the present invention designs and prepares a dynamic HA-DA-Plu hydrogel platform for long-term continuous treatment after tumor surgery. This platform simultaneously has excellent characteristics such as tissue adhesion, biocompatibility, self-healing property, and injectability, providing new ideas and strategies for the research and development of new implants and having important potential application value.

[0075] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations to these embodiments still fall within the protection scope of the present invention.

Claims

1. A preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets, characterized in that, It includes the following steps: S1. Dissolve hyaluronic acid in a phosphate buffer solution, add an activator for activation, and then add dopamine. After reaction, dopamine-modified hyaluronic acid is obtained; S2. Dissolve Pluronic F127 in an organic solvent, add triethylamine and p-nitrophenyl chloroformate solvent for activation, then dissolve the activated Pluronic F127 in the organic solvent again, add cysteamine for terminal thiol modification, and obtain terminal thiol-modified Pluronic F127 after concentration and precipitation; S3. First, prepare a black phosphorus nanosheet solution by liquid-phase exfoliation method, and then add a silver salt solution to the black phosphorus nanosheet solution. After reaction, black phosphorus nanosheets loaded with silver ions are prepared; S4. Crosslink the dopamine-modified hyaluronic acid in step S1 and the terminal thiol-modified Pluronic F127 in step S2 in a phosphate buffer solution through the Michael addition reaction between the thiol group and the quinone structure formed by the oxidation of the dopamine catechol structure, and then add the black phosphorus nanosheets loaded with silver ions in step S3 to continue the reaction to prepare a composite hydrogel loaded with silver ions and black phosphorus nanosheets.

2. The preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets according to claim 1, characterized in that, In step S1, the activator is carbodiimide hydrochloride and N-hydroxysuccinimide, and the mass ratio of carbodiimide hydrochloride to N-hydroxysuccinimide is 2:

1.

3. The preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets according to claim 1, characterized in that, In step S1, the mass concentration of hyaluronic acid in the phosphate buffer solution is 20-30 mg / mL, and the mass ratio of hyaluronic acid to dopamine is 2:(0.5-2).

4. The preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets according to claim 1, characterized in that, In step S2, the mass ratio of Pluronic F127 to the organic solvent is 1:5-1:7, the mass ratio of Pluronic F127 to triethylamine is 1:15-25, and the mass ratio of Pluronic F127 to p-nitrophenyl chloroformate solvent is 10:1-3.

5. The preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets according to claim 1, characterized in that, In step S2, the mass ratio of the activated Pluronic F127 to cysteamine is (20-30):1; the activation time is more than 48 hours, and the terminal thiol modification time is more than 24 hours.

6. The preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets according to claim 1, characterized in that, In step S3, the preparation method of the black phosphorus nanosheet solution is as follows: add black phosphorus particles to an organic solvent, blow air to remove the dissolved oxygen in the solution, and then place the mixture in an ice bath for probe sonication for 24-48 h. The obtained dispersion is centrifuged, and the brown supernatant containing black phosphorus nanosheets is collected to obtain the black phosphorus nanosheet solution.

7. The preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets according to claim 1, characterized in that, In step S3, the silver salt is silver nitrate, and the volume ratio of the silver salt solution to the black phosphorus nanosheet solution is 1:

1.

8. The preparation method of a composite hydrogel loaded with silver ions and black phosphorus nanosheets according to claim 1, characterized in that, In step S4, the mass ratio of the dopamine-modified hyaluronic acid to the terminal thiol-modified Pluronic F127 is 1:(2-3).

9. A composite hydrogel loaded with silver ions and black phosphorus nanosheets prepared by the preparation method according to any one of claims 1-8.

10. Use of the composite hydrogel loaded with silver ions and black phosphorus nanosheets according to claim 9 in the preparation of anti-tumor and / or antibacterial implantable biomedical materials.

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