An injectable black phosphorus nanosheet-loaded composite hydrogel as well as a preparation method and application thereof

Abstract

The application provides an injectable black phosphorus nanosheet-loaded composite hydrogel and a preparation method and application thereof. The composite hydrogel is prepared by mixing polydopamine-functionalized black phosphorus nanosheets, a 2-2.5% wt / vol sodium alginate suspension, a 1.5-2% wt / vol calcium alginate suspension and hyaluronic acid; wherein the volume ratio of the 2-2.5% wt / vol sodium alginate suspension, the 1.5-2% wt / vol calcium alginate suspension and the hyaluronic acid is 5:5:2, and the concentration of the polydopamine-functionalized black phosphorus nanosheets in the synthesized composite hydrogel is 100 μg / ml; and the application of the injectable black phosphorus nanosheet-loaded composite hydrogel in a myocardial infarction disease injection drug, through ROS scavenging to inhibit the NFκB signal pathway and reduce inflammatory reactions, the black phosphorus nanosheet-loaded composite hydrogel in the application can be used as a ROS scavenging, anti-inflammatory, anti-apoptosis, conductive and biodegradable platform for tissue engineering applications.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical hydrogel preparation, and specifically relates to an injectable black phosphorus nanosheet-loaded composite hydrogel for treating myocardial infarction and a preparation method and application thereof. BACKGROUND

[0002] Millions of people worldwide suffer from acute myocardial infarction every year, and the disease has become a major public health problem with high mortality and morbidity, and is one of the main causes of death in patients with coronary heart disease. The main cause of myocardial infarction is that atherosclerosis and thrombosis occur in the coronary artery, leading to coronary occlusion and reduced myocardial blood flow; coronary occlusion leads to large-area and irreversible death of myocardial cells, followed by fibrosis, affecting heart function, and pathological remodeling of surviving myocardium due to excessive pressure load, further exacerbating heart failure. After myocardial infarction, a series of pathological remodeling changes occur in the microenvironment of myocardial cells in the infarct area, such as excessive production of ROS and overexpression of hypoxia-inducible factor-1 alpha, which leads to the destruction of myocardial cell homeostasis, myocardial cell apoptosis, inflammatory cell infiltration, myocardial fibrosis, and further leads to reduced cardiac function. Early and timely rapid recovery of blood perfusion of ischemic myocardial tissue is still the main treatment method, and the current treatment method for acute myocardial infarction is mainly interventional surgery and drug therapy. Although these methods help to delay disease progression, they still cannot repair necrotic myocardial tissue.

[0003] Previous studies have shown that the development of acute myocardial infarction is closely related to ROS overproduction, calcium overload, inflammatory response, oxidative stress, etc. The imbalance between ROS overproduction and antioxidant system can lead to oxidative stress, further leading to cell apoptosis and inflammation. In myocardial cells, mitochondria account for 30%-40% of the volume, which is the main source of reactive oxygen species (ROS). Reactive oxygen species can also activate Toll-like receptor 4, thereby activating NF-κB, producing chemotactic factors and pro-inflammatory cytokines, and further inducing inflammatory response. Excessive inflammation can in turn induce ROS production, exacerbate myocardial injury and cardiac remodeling, thereby forming a vicious cycle between inflammation and ROS. A large number of studies have shown that the removal of ROS in myocardial cells can reduce inflammatory response and reduce myocardial cell apoptosis, thereby improving cardiac function. In addition, myocardial cell apoptosis is the final pathological result after myocardial ischemia, and oxidative stress-induced myocardial cell apoptosis is an important factor leading to irreversible myocardial loss and eventually heart failure. The Bcl-2 family is the main participant in cell apoptosis, throughout the whole process of cell apoptosis, Bax and Bcl-2 are important members of the family that regulate cell apoptosis. Bax, a protein of the Bcl-2 family, is a major control factor of the apoptosis model, and the activation of caspases is the central link in the apoptosis process. ROS overproduction, Ca2+ overload, etc. during myocardial ischemia-reperfusion period make mitochondria swell and rupture, activate caspase cascade, release a large amount of apoptosis-related proteins, and produce programmed cell death. Myocardial cell apoptosis is mainly related to ROS, inflammatory factors and neutrophil infiltration, etc. Therefore, inhibiting ROS production, rapidly removing ROS and reducing cardiac inflammation, and inhibiting apoptosis are considered effective methods to improve cardiac function after myocardial infarction.

[0004] More and more new materials have been applied to myocardial infarction treatment, such as conductive polymers, hydrogel patches, nanoparticles, etc. Injecting drug or cell-loaded hydrogel into the infarct area has been shown to have potential for treating acute myocardial infarction. Injectable hydrogel can replace infarct myocardium and provide sustained mechanical support for failing ventricular wall, and is widely used in myocardial repair process. Injectable hydrogel can also store drugs for long-term slow release of drugs, thereby regulating ROS and inflammation in myocardial infarction area. However, most new materials are not degradable and will remain as foreign bodies in the body, so developing a biodegradable biomaterial for tissue engineering is still a major challenge.

[0005] Black phosphorus (BP) is a new two-dimensional nanomaterial, which has attracted much attention due to its excellent physicochemical properties. BP is composed of phosphorus, which is abundant in the human body, and can be degraded into phosphate in the presence of oxygen and water, making it have good biocompatibility. In addition, the conductivity of BP can improve the bioelectric conduction ability of local tissues, which is crucial for the repair of electrically active tissues such as the heart. Previous studies have shown that BP has the ability to regulate oxidative stress, which can directly capture ROS in the microenvironment or capture transition metal ions such as copper ions to play an oxidative stress regulation role and antioxidant capacity. The excellent physicochemical properties of BP lay a solid foundation for its treatment in MI, however, the inappropriate degradation rate of BP may affect its therapeutic effect. Dopamine (DA) is a biocompatible substance, and in alkaline environment, DA can undergo oxidative self-polymerization to form a polydopamine (PDA) layer on the surface of the material with super strong adhesion. By modifying BP with this property of DA, the contact of oxygen or water with black phosphorus nanosheets can be isolated to some extent, thereby improving its stability. Therefore, reducing myocardial inflammation and promoting heart repair is a promising option to improve heart function, but currently there is no research on this technology. SUMMARY

[0006] The present application provides an injectable black phosphorus nanosheet-loaded composite hydrogel, its preparation method and application, which has good biocompatibility, conductivity and biodegradability. The hydrogel is directly injected into the myocardium, and the BP in the hydrogel reacts with the ROS in the myocardium to generate non-cytotoxic phosphate through slow release, and the ROS is cleared to inhibit the NFκB signaling pathway and reduce inflammation.

[0007] In order to achieve the above technical purpose, the present application provides an injectable black phosphorus nanosheet-loaded composite hydrogel, which is prepared by mixing polydopamine-functionalized black phosphorus nanosheets, a sodium alginate suspension with a concentration of 2-2.5% wt / vol, a calcium alginate suspension with a concentration of 1.5-2% wt / vol, and hyaluronic acid; wherein the volume ratio of the sodium alginate suspension with a concentration of 2-2.5% wt / vol, the calcium alginate suspension with a concentration of 1.5-2% wt / vol, and the hyaluronic acid is 5:5:2, and the concentration of polydopamine-functionalized black phosphorus nanosheets in the synthesized composite hydrogel is 100 μg / ml.

[0008] The polydopamine functionalized black phosphorus nanosheet is prepared by dispersing black phosphorus nanosheet in anhydrous ethanol, then adding NaOH solution with pH of 8.5-9, and then slowly adding polydopamine solution with concentration of 50-100 mg / ml and mixing and stirring; the mass-volume ratio of the black phosphorus nanosheet and the anhydrous ethanol is 1:1; the volume ratio of the anhydrous ethanol, the NaOH solution and the polydopamine solution is 20:3:20.

[0009] The sodium alginate suspension is prepared by dissolving sodium alginate powder in sterile water; and the calcium alginate suspension is prepared by dissolving calcium alginate powder in sterile water.

[0010] The black phosphorus nanosheet is synthesized by electrochemically exfoliating a black phosphorus crystal block.

[0011] The application provides a preparation method of an injectable black phosphorus nanosheet-loaded composite hydrogel.

[0012] (1) The black phosphorus nanosheet is synthesized by electrochemically exfoliating a black phosphorus crystal block, the black phosphorus nanosheet is dispersed in anhydrous ethanol, then NaOH solution with pH of 8.5-9 is added, and then polydopamine solution with concentration of 50-100 mg / ml is slowly added, the mixture is stirred in a dark environment for 4-6 hours to form a BP@PDA solution, after stirring, the BP@PDA solution is centrifuged at 4 DEG C for 8-12 minutes, the supernatant is discarded, and the polydopamine functionalized black phosphorus nanosheet (BP@PDA) is formed after washing; wherein the mass-volume ratio of the black phosphorus nanosheet and the anhydrous ethanol is 1:1; the volume ratio of the anhydrous ethanol, the NaOH solution and the polydopamine solution is 20:3:20.

[0013] (2) At room temperature, sodium alginate powder is dissolved in sterile water to prepare a 2-2.5% wt / vol suspension, calcium alginate powder is dissolved in sterile water to prepare a 1.5-2% wt / vol suspension, the 2-2.5% wt / vol sodium alginate suspension and the 1.5-2% wt / vol calcium alginate suspension are heated and dissolved in a water bath at 45-55 DEG C for 10-15 minutes, then hyaluronic acid is added, and finally the polydopamine functionalized black phosphorus nanosheet prepared in step (1) is added to prepare the injectable black phosphorus nanosheet-loaded composite hydrogel, wherein the concentration of the polydopamine functionalized black phosphorus nanosheet in the composite hydrogel is 100 μg / ml; the volume ratio of the sodium alginate suspension, the calcium alginate suspension and the hyaluronic acid is 5:5:2.

[0014] The preferred technical scheme of the present application is that in step (1), the BP@PDA solution is centrifuged at 4 DEG C and 12000 rpm for 10 minutes, the supernatant is discarded, and the polydopamine functionalized black phosphorus nanosheet is formed after being washed twice with ultrapure water.

[0015] The present application also provides an application of the injectable black phosphorus nanosheet loaded composite hydrogel in a myocardial infarction disease injection drug.

[0016] The preferred technical scheme of the present application is that the injectable black phosphorus nanosheet loaded composite hydrogel is a single substance of the myocardial infarction disease injection drug.

[0017] The preferred technical scheme of the present application is that the injection amount of the injectable black phosphorus nanosheet loaded composite hydrogel is 150 microliters.

[0018] The present application coats black phosphorus nanosheet (BPN) with polydopamine (PDA) by in-situ oxidation polymerization method, and prepares a biological hybrid hydrogel (GelHA-BP@PDA) that is a composite hydrogel loaded with black phosphorus nanosheet by cross-linking with GelHA molecules. Through comprehensive research, it is shown that the composite hydrogel in the present application has good biocompatibility, conductivity and biodegradability.

[0019] The hydrogel in the present application can be directly injected, and myocardial injection is adopted, three regions around the infarction area are selected for injection, after myocardial injection, black phosphorus (BP) in the hydrogel is slowly released to react with ROS in the myocardium to generate non-cytotoxic phosphate. The composite hydrogel inhibits the NFκB signaling pathway by eliminating ROS to reduce inflammatory response, and the black phosphorus nanosheet loaded composite hydrogel in the present application can be used as a ROS eliminating, anti-inflammatory, anti-apoptotic, conductive and biodegradable platform for tissue engineering application. The strategy of integrating biodegradable ROS eliminating BP nanomaterials into biocompatible hydrogel in the present application provides new insights for the design of tissue engineering biomaterials. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Fig. a is an electron microscope image of BPNs, and Fig. b is an electron microscope image of BP@PDA;

[0021] Figure 2 and Figure 3 Fig. is an atomic force microscope picture and thickness of BPNs in test one;

[0022] Figure 4 Fig. is a curve graph of XRD detection results of BPNs in test two;

[0023] Figure 5 Fig. is a comparison graph of Raman results of BPNs and BP@PDA in test three;

[0024] Figure 6 The zeta potential comparison chart of BPNs and BP@PDA in Test Four;

[0025] Figure 7 The surface element distribution chart of BPNs and BP@PDA in Test Five;

[0026] Figure 8 The scanning electron microscope image of ALGHA, ALGHA-BP and ALGHA-BP@PDA in Test Six;

[0027] Figure 9 The Raman characterization curve comparison chart of ALGHA-BP@PDA and ALGHA hydrogel matrix in Test Six;

[0028] Figure 10 The photograph of the injectability of ALGHA-BP@PDA hydrogel prepared in the embodiment;

[0029] Figure 11 The photograph of the adhesion of ALGHA-BP@PDA hydrogel prepared in the embodiment to plastic;

[0030] Figure 12 The comparison chart of the swelling property of ALGHA-BP@PDA and ALGHA-BP hydrogels prepared in the embodiment;

[0031] Figure 13 The comparison chart of the conductivity of ALGHA-BP@PDA and ALGHA-BP hydrogels prepared in the embodiment;

[0032] Figure 14 The comparison chart of the DPPH scavenging effect of ALGHA-BP@PDA hydrogels with different concentrations in Test Six;

[0033] Figure 15 The photograph of the reaction of DPPH solution and DPPH / ALGHA-BP@PDA hydrogel at 24 h;

[0034] Figure 16 The photograph of ABTS solution and ABTS / ALGHA-BP@PDA hydrogel;

[0035] Figure 17 The comparison chart of the ABTS scavenging effect of ALGHA-BP@PDA hydrogels with different concentrations in Test Six;

[0036] Figure 18 The comparison chart of the OH-radical scavenging effect of ALGHA-BP@PDA hydrogels with different concentrations in Test Six;

[0037] Figure 19 H9C2 cells were incubated on blank group, control group, ALGHA group and ALGHA-BP@PDA group for 24h, and CCK-8 method was used to detect the cell survival of different groups.

[0038] Figure 20 H9C2 cells were incubated in control group, ALGHA group and ALGHA-BP@PDA group for 24h, and Calcein-AM method was used to detect the representative pictures of cell activity;

[0039] Figure 21 and Figure 22 are the representative fluorescence images of intracellular superoxide anion radical activity (DHE) and intracellular total ROS (DCFH-DA) of H9C2 cells cultured on blank group, control group, ALGHA group and ALGHA-BP@PDA group for 24h, respectively;

[0040] Figure 23 and Figure 24 are the fluorescence intensity of DHE and DCFH-DA;

[0041] Figure 25 are the representative fluorescence images of mitochondrial membrane potential (JC-1) of H9C2 cells cultured on blank group, control group, ALGHA group and ALGHA-BP@PDA group for 24h;

[0042] Figure 26 is the animal experiment diagram of myocardial infarction rat model;

[0043] Figure 27 and Figure 28 are the comparison diagrams of TTC staining of rat heart to quantify myocardial infarction area of control group, postoperative MI, postoperative MI myocardial injection of ALGHA and postoperative MI myocardial injection of ALGHA-BP@PDA group;

[0044] Figure 29 and Figure 30 are the comparison diagrams of TUNEL staining detection results of rats in control group, postoperative MI, postoperative MI myocardial injection of ALGHA and postoperative MI myocardial injection of ALGHA-BP@PDA group at 28d after operation;

[0045] Figure 31 are the comparison diagrams of myocardial tissue apoptosis related protein Caspase-3 staining of rats in control group, postoperative MI, postoperative MI myocardial injection of ALGHA and postoperative MI myocardial injection of ALGHA-BP@PDA group at 28d after operation;

[0046] Figure 32Figure 8a-g: Figure 8a-g are the results of the Western blot analysis of the apoptosis-related proteins BCL-2, Bax and Caspase-3 of the rats in the control group, the post-MI group, the post-MI myocardial injection of ALGHA group and the post-MI myocardial injection of ALGHA-BP@PDA group at 28 days after the operation; Figure 8h-i are the quantitative analysis results of the Bax protein expression and the BCL-2 protein expression; Figure 8j is the quantitative analysis result of the Caspase-3 protein expression.

[0047] Figure 33 Figure 9a-d: Figure 9a-d are the echocardiograms of the rats in the control group, the post-MI group, the post-MI myocardial injection of ALGHA group and the post-MI myocardial injection of ALGHA-BP@PDA group at 24 hours, 7 days, 14 days and 28 days after the operation.

[0048] Figure 34 Figure 10a-c: Figure 10a-c are the HE, Sirius red and Masson trichrome staining results of the myocardial tissues of the rats in the control group, the post-MI group, the post-MI myocardial injection of ALGHA group and the post-MI myocardial injection of ALGHA-BP@PDA group at 28 days after the operation.

[0049] Figure 35 Figure 11: Figure 11 is the quantitative analysis result of the Masson staining of the myocardial tissues of the rats in the control group, the post-MI group, the post-MI myocardial injection of ALGHA group and the post-MI myocardial injection of ALGHA-BP@PDA group at 28 days after the operation.

[0050] Figure 36 Figure 12: Figure 12 is the quantitative analysis result of the Sirius red staining of the myocardial tissues of the rats in the control group, the post-MI group, the post-MI myocardial injection of ALGHA group and the post-MI myocardial injection of ALGHA-BP@PDA group at 28 days after the operation. DETAILED DESCRIPTION

[0051] The application will be further described below in conjunction with the examples. The technical solutions shown in the drawings below are specific solutions of the embodiments of the application, and are not intended to limit the scope of the claimed application. Based on the examples in the application, all other examples obtained by those of ordinary skill in the art without creative labor fall within the scope of the application.

[0052] The injectable black phosphorus nanosheet-loaded composite hydrogel provided by the example specifically comprises the following steps:

[0053] (1) Synthesis of black phosphorus nanosheets by electrochemical exfoliation of bulk BP crystals, 1 mg of BP nanosheets was dispersed in 1 ml of absolute ethanol, then 150 ul of NaOH solution was added, the pH of the NaOH solution was 8.5, then 1 ml of PDA solution with a concentration of 50-100 mg / ml was slowly added, a magnetic stirrer was used, and the mixture was stirred in the dark for 5 hours to prepare a BP@PDA solution; after stirring, the BP@PDA solution was centrifuged at 12000 rpm for 10 minutes at 4°C, the supernatant was discarded, and ultrapure water was added for washing twice to form polydopamine functionalized black phosphorus nanosheets (BP@PDA).

[0054] (2) At room temperature 26°C, sodium alginate powder was dissolved in sterile water to prepare a 2-2.5% wt / vol suspension, calcium alginate powder was dissolved in sterile water to prepare a 1.5-2% wt / vol suspension, the 2-2.5% wt / vol concentration of sodium alginate suspension and the 1.5-2% wt / vol concentration of calcium alginate suspension were mixed in a volume ratio of 1:1, then heated and dissolved in a 50°C water bath for 10-15 minutes, then added hyaluronic acid, the amount of hyaluronic acid added was 20%, and finally added BP@PDA nanosheets to make the concentration 100 μg / ml to prepare an injectable black phosphorus nanosheet-loaded composite hydrogel (i.e. ALGHA-BP@PDA).

[0055] The inventors of the present application conducted the following experimental studies on the performance of the black phosphorus nanosheets, polydopamine functionalized black phosphorus nanosheets and injectable black phosphorus nanosheet-loaded composite hydrogel in the above examples.

[0056] Experiment one: The black phosphorus nanosheets (BPNs) and polydopamine functionalized black phosphorus nanosheets (BP@PDA) in the examples were respectively characterized by transmission electron microscopy (TEM), as shown in Figure 1 a and Figure 1 b, it can be seen that the BP and BP@PDA nanosheets are two-dimensional multi-layer sheet structures with a diameter of about 300-500 nm; the morphology of the BP nanosheets was characterized by atomic force microscopy (AFM), as shown in Figure 2 and Figure 3 Atomic force microscopy shows that the thickness of the black phosphorus nanosheets is about 5-6 nm.

[0057] Experiment two: In order to study the crystal form and crystalline state of the prepared black phosphorus nanosheets, the inventors of the present application conducted X-ray powder diffraction (XRD) analysis. As shown in Figure 4The display shows that the black phosphorus nanosheet (BPNs) has high and sharp characteristic diffraction peaks, proving that it has excellent crystallinity, and the XRD diffraction peaks of the black phosphorus nanosheet can be substantially coincided with the standard black phosphorus XRD diffraction peaks (JCPDS No. 73-1358). The position of the characteristic diffraction peak of the black phosphorus nanosheet does not change, indicating that no new lattice is formed, and the original crystalline type of black phosphorus is maintained.

[0058] Test three: in order to study the influence of PDA modification on the two-dimensional layered structure of black phosphorus nanosheet, the inventors of the present application respectively carried out Raman characterization on the black phosphorus nanosheet (BPNs) and the polydopamine functionalized black phosphorus nanosheet (BP@PDA) in the examples; Raman generally has A 1 g , B 2g , A g 2 three characteristic vibration peaks, and the Raman spectrum Figure 5 shows that the black phosphorus nanosheet (BPNs) and the PDA modified black phosphorus nanosheet (BP@PDA) have three significant characteristic vibration peaks at about 365, 440 and 465 cm -1 , which can be respectively attributed to A 1 g , B 2g , A g 2 three vibration modes. The shift of the peak basically does not move, which confirms that after the PDA modification, the two-dimensional structure of the black phosphorus nanosheet is not changed, and the PDA modified black phosphorus nanosheet still maintains the three characteristic peaks and basically does not change.

[0059] Test four: in order to further study the influence of PDA on the surface charge of the BP nanosheet, the inventors of the present application carried out experiments by using the zeta potential of two different samples. As Figure 6 shown, the zeta potential of the black phosphorus nanosheet (BPNs) without PDA modification is -31 mV, and the zeta potential of the polydopamine functionalized black phosphorus nanosheet (BP@PDA) after PDA modification is -44.9 mV. Since PDA is negatively charged, the potential after modification is lower, which further confirms the success of PDA modification.

[0060] Test five: in order to further study the element composition and distribution of the black phosphorus nanosheet, the inventors of the present application carried out element surface scanning by using a scanning transmission electron microscope. As Figure 7As shown, by comparing the element distribution of different colors, it is found that the main element of black phosphorus nanosheet is phosphorus, which further confirms that the prepared black phosphorus nanosheet has high purity. In addition, there is part of oxygen element in the black phosphorus nanosheet, which is mainly related to the oxidation of the black phosphorus nanosheet in the preparation process. Although the preparation process is carried out in a solvent, it is impossible to completely avoid oxidation, but the degree of oxidation has no obvious effect on the properties of the black phosphorus nanosheet. The increase of C and N elements on the surface of BP@PDA nanosheet proves that PDA is successfully modified. In summary, although there are nitrogen, carbon and other elements in the black phosphorus nanosheet, the main element is still phosphorus, which proves that the surface of the prepared black phosphorus nanosheet is relatively clean.

[0061] Test six: In order to study the concentration of BP@PDA in the injectable black phosphorus nanosheet composite hydrogel, the inventors of the present application carried out the following test, specifically: at room temperature 26℃, sodium alginate and calcium alginate powder were dissolved in sterile water to prepare sodium alginate suspension with concentration of 2-2.5%wt / vol and calcium alginate suspension with concentration of 1.5-2%wt / vol, respectively, after mixing the two in a volume ratio of 1:1, dissolving in a 50℃ water bath for 10 minutes, then adding hyaluronic acid, the amount of hyaluronic acid added is 20%, finally adding BP@PDA nanosheet. Adjust the amount of BP@PDA to prepare composite hydrogel containing 10ug, 20ug, 50ug, 100ug of BP@PDA nanosheet per ml of gel, respectively, and are marked as ALGHA-BP@PDA10, ALGHA-BP@PDA20, ALGHA-BP@PDA50, ALGHA-BP@PDA100; For the above composite hydrogels with different concentrations, the inventors of the present application carried out the following experimental study:

[0062] (1) First, the pore state of ALGHA (such as Figure 8 a), ALGHA-BP (such as Figure 8 b) and ALGHA-BP@PDA (such as Figure 8 c) three kinds of hydrogel were analyzed by scanning electron microscope (SEM), three kinds of hydrogel have clear sponge-like pore structure, which is conducive to cell adhesion; as shown in Figure 8 , ALGHA-BP@PDA hydrogel has smaller pore size, which may be caused by the introduction of PDA to promote additional crosslinking points in the hydrogel matrix. Raman characterization of BP@PDA and ALGHA hydrogel matrix Figure 9 , Raman spectrum shows that the combination of BP@PDA and ALGHA hydrogel matrix does not change the performance of BP@PDA nanosheet.

[0063] (2) To prove the ROS scavenging ability of ALGHA-BP@PDA hydrogel, the inventors of the present application used the antioxidant effect of ALGHA-BP@PDA hydrogel on 1,1-diphenyl-2-methyl hydroxyl radical (DPPH) and hydroxyl radical (OH - ) to characterize the ROS scavenging activity. DPPH is a stable RNS compound, and its ethanol solution is dark purple with a characteristic absorption peak at 517 nm. The inventors of the present application tested the scavenging of DPPH by the above-mentioned hydrogels with different concentrations of BP@PDA, and the results are shown in Figure 14 , which shows that ALGHA-BP@PDA hydrogel has a certain scavenging ability for DPPH. The DPPH scavenging ability of the hydrogel shows a concentration-dependent effect. With the increase of BP@PDA content, the ability of the hydrogel to scavenge DPPH gradually increases. When the content of BP@PDA is 100 ug / ml, the DPPH scavenging rate is as high as about 81%. At the same time, it was found through experiments that the DPPH reagent showed obvious decolorization after reacting with ALGHA-BP@PDA hydrogel, as shown in Figure 15 , after 24 hours of incubation, the dark purple Figure 15-1 of the DPPH solution turned into light purple Figure 15-2 and yellow Figure 15-3 , 4, 5, indicating that the DPPH free radical decolorized in the presence of the antioxidant. The macroscopic color change intuitively reflects the fact that the ROS scavenging activity is significantly enhanced when the content of BP@PDA is high.

[0064] (3) ABTS is another stable RNS, and ABTS can be oxidized to blue-green ABTS + with a characteristic absorption peak at 405 nm. After adding the ALGHA-BP@PDA hydrogel prepared in the examples, the absorbance decreased significantly and the color faded obviously, as shown in Figure 16 , when the content of BP@PDA was 100 ug / ml, complete fading of the reaction system could be observed Figure 16-5 . In addition, the ABTS·+ scavenging ability of ALGHA-BP@PDA hydrogel is concentration-dependent Figure 17 .

[0065] (4) The scavenging effect of the composite hydrogel on hydroxyl radical (OH - ) also showed a similar trend. We used Fenton reaction to detect the inhibitory effect of ALGHA-BP@PDA hydrogel on OH - generation. As shown in Figure 18 , the OH - scavenging ability of the hydrogel shows a concentration-dependent effect. With the increase of BP@PDA content, the ability of the hydrogel to scavenge OH -The ability gradually increases; when the BP@PDA content is 100ug / ml, OH - The clearance rate is as high as approximately 89.6%.

[0066] The ALGHA-BP@PDA hydrogel prepared in the embodiments of this application is injectable, such as... Figure 10 The hydrogel injected through a needle, as shown, can be used to draw "HUST". Injectability is an important factor in hydrogel applications; good adhesion prevents the hydrogel from overflowing from the injection site, such as... Figure 11 As shown, the ALGHA-BP@PDA hydrogel also possesses certain adhesive properties, allowing it to adhere to object surfaces. The swelling property of the hydrogel is also an important characteristic, directly affecting its drug loading capacity. The ALGHA-BP@PDA hydrogel prepared in the examples exhibits good swelling properties, providing a good foundation for drug loading. Experiments demonstrate that the swelling property of the ALGHA-BP@PDA hydrogel is slightly lower than that of the ALGHA-BP hydrogel. Figure 12 This may be related to the fact that the addition of PDA changed the pore size of the hydrogel.

[0067] To investigate the conductivity of the ALGHA-BP@PDA hydrogel prepared in the examples, the inventors of this application used a four-probe resistivity meter to test the ALGHA-BP@PDA hydrogel. The results are as follows: Figure 13 As shown, the conductivity of ALGHA-BP@PDA hydrogel is approximately 0.085 S / m, which is close to the conductivity of natural heart tissue. Experiments have demonstrated that ALGHA-BP@PDA hydrogel can be used as a conductor to light LED tubes, indicating that ALGHA-BP@PDA hydrogel has good electrical conductivity.

[0068] The inventors of this application also tested the biocompatibility of the composite hydrogel. Studies showed that black phosphorus nanosheets had no significant toxicity to kidney cells, but their toxicity to cardiomyocytes is currently unknown. Good biocompatibility is crucial for maintaining cell viability and the application of hydrogels. After culturing H9C2 cells on the hydrogel for 24 hours, the biocompatibility of the hydrogel was tested using CCK-8 and Calcein-AM assays. The OD value detected by the CCK-8 kit is directly proportional to the number of cells; therefore, the OD value can be used to reflect cell survival in the hydrogel. Figure 19As shown, the OD value of H9C2 cells incubated on ALGHA-BP@PDA hydrogel for 24 hours had no obvious difference from the control group. Calcein is a calcium ion fluorescent indicator. Calcein-AM is enhanced in hydrophobicity on the basis of Calcein, so it can penetrate living cells, and has very low cytotoxicity, and can be used as a cell staining reagent for fluorescent labeling of living cells. Living cells can bind Calcein to show green fluorescence, such as Figure 20 As shown, the cell density and morphology of each group after Calcein-AM labeling had no obvious difference. The above studies show that ALGHA-BP@PDA hydrogel has low cytotoxicity and good biocompatibility. The reasons why ALGHA-BP@PDA hydrogel has good biocompatibility are as follows: (1) dopamine in the hydrogel has adhesion, and dopamine can enhance the water dispersibility of black phosphorus nanosheets, which can help cell adhesion and retention; (2) black phosphorus nanosheets have a wrinkled structure, which is also conducive to cell adhesion and retention; (3) phosphorus is a trace element in the human body, and phosphorus itself has no obvious toxicity to cells; (4) ALGHA hydrogel itself also has good biocompatibility and cell adhesion ability.

[0069] The inventors of the present application also studied the in vitro antioxidant performance of the injectable black phosphorus nanosheet-loaded composite hydrogel (ALGHA-BP@PDA) prepared in the examples: After myocardial infarction, a large amount of ROS is produced in the infarcted myocardial tissue, and excessive ROS can damage DNA, cause protein denaturation, and further cause oxidative stress damage to myocardial cells. Oxidative stress can cause inflammatory reactions, further exacerbating myocardial cell damage. The antioxidant effect of the hydrogel is crucial to reduce myocardial damage. Therefore, the inventors of the present application detected the ROS resistance of ALGHA-BP@PDA prepared in the examples at the cellular level, and DHE and DCFH-DA were used as probes to detect ROS in H9C2 cells, and the detection results are as follows: Figure 21 、 22 As shown, strong green fluorescence can be observed in the H2O2 stimulation group and the ALGHA hydrogel application group, indicating that after H2O2 treatment, H9C2 cells produce a large amount of ROS. Through comparison experiments, it was found that the DCFH-DA (green fluorescence) fluorescence intensity of H9C2 cells treated with ALGHA-BP@PDA hydrogel was significantly reduced. As shown in Figure 23 、 24 As shown, strong red fluorescence appeared in the H2O2 stimulation group and the ALGHA hydrogel application group, indicating a high level of ROS. The red fluorescence intensity of H9C2 cells treated with ALGHA-BP@PDA hydrogel was significantly reduced. The above studies show that BP@PDA can scavenge ROS, which is conducive to cell survival.

[0070] Removing ROS further restores membrane potential, which is beneficial for cell survival and normal function. Therefore, the inventors of this application also used an enhanced mitochondrial membrane potential detection kit to detect the mitochondrial membrane potential of H9C2 cells. JC-1 is a sensitive fluorescent sensor used to detect the mitochondrial membrane potential of cells and tissues. When the mitochondrial membrane potential is high, JC-1 forms a polymer in the mitochondrial mechanism, producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 exists in monomeric form, producing green fluorescence. Figure 25 As shown, the red fluorescence was significantly reduced in the H2O2 stimulation group and the ALGHA hydrogel treatment group, while the red fluorescence was significantly enhanced in the ALGHA-BP@PDA hydrogel treatment group. Exogenous BP@PDA can reduce intracellular ROS levels and restore mitochondrial function. Previous studies have indicated that mitochondria are the main organelles producing ROS; therefore, the application of ALGHA-BP@PDA hydrogel can inhibit and clear ROS from its main source, helping to reduce oxidative stress damage in H9C2 cells and restore mitochondrial function.

[0071] To demonstrate that the prepared ALGHA-BP@PDA hydrogel alleviates myocardial injury in rats with myocardial infarction through anti-apoptosis, the inventors of this application, after completing in vitro verification, further tested the hydrogel's ROS scavenging function in animals by conducting animal experiments using a rat model of myocardial infarction (e.g., Figure 26 First, TTC staining was used to compare the degree of cardiac injury 4 weeks after myocardial infarction among different groups. TTC staining was used to quantify the myocardial infarction area in rats (n=3 per group) from the control group, post-MI group, post-MI group with intramyocardial injection of ALGHA, and post-MI group with intramyocardial injection of ALGHA-BP@PDA. Results... Figure 27 and 28 The results showed that the infarct area of ​​rats treated with ALGHA-BP@PDA hydrogel was significantly reduced compared with other groups. Subsequently, the inventors of this application used TUNEL staining to study cardiomyocyte apoptosis after myocardial infarction; rats in the control group, after MI surgery, after MI surgery with intramyocardial injection of ALGHA, and after MI surgery with intramyocardial injection of ALGHA-BP@PDA were used. The number of apoptotic cardiomyocytes (scale bar = 100 μm) and the quantification of positive cell nuclei (3 rats per group) were detected by TUNEL staining 28 days after surgery; the results are as follows: Figure 29 and Figure 30As shown, compared with the control group, the myocardial cell apoptosis of the MI group was significantly increased, and the number of TUNEL positive myocardial cells was significantly reduced after treatment with ALGHA-BP@PDA hydrogel. The inventors of the present application also used rats in the control group, after MI, after intramyocardial injection of ALGHA after MI, and after intramyocardial injection of ALGHA-BP@PDA after MI, and stained the myocardial tissue apoptosis related protein Caspase-3 at 28d after operation, and used caspase-3 immunofluorescence image to show as Figure 31 As shown, the ALGHA-BP@PDA hydrogel treatment group showed significantly reduced myocardial tissue apoptosis. In addition, by Western blot analysis of the expression of apoptosis related proteins, the inventors of the present application respectively analyzed the Western blot of the apoptosis related proteins BCL-2, Bax and Caspase-3 of rats in the control group, after MI, after intramyocardial injection of ALGHA after MI, and after intramyocardial injection of ALGHA-BP@PDA after MI, at 28d after operation, and the analysis results are as shown in Figure 32 As shown, after treatment with ALGHA-BP@PDA hydrogel, the expression of anti-apoptotic molecule BCL-2 was up-regulated, while the expression of pro-apoptotic molecules Bax and Caspase 3 was down-regulated at the protein level.

[0072] The inventors of the present application also carried out experiments to prove that ALGHA-BP@PDA hydrogel can reduce myocardial cell fibrosis and improve heart function in myocardial infarction rats, and the specific process is as follows: the heart function of rats was evaluated at 24h, 7d, 14d and 28d after treatment with ALGHA-BP@PDA hydrogel by echocardiography Figure 33 After 28d, compared with the control group, the left ventricular internal diameter of all myocardial infarction rats increased, and the left ventricular systolic function decreased, but compared with the MI group, ALGHA-BP@PDA hydrogel improved the left ventricular ejection fraction (LV-EF) and left ventricular fractional shortening (LV-FS) of rats. Therefore, it can be proved that ALGHA-BP@PDA hydrogel can improve the heart function of rats after myocardial infarction for 28d, and the reason may be that the hydrogel can be retained in the infarct myocardial area for a longer time, thereby better playing the roles of removing ROS, anti-inflammatory, anti-apoptosis, etc.

[0073] Myocardial infarction often leads to thinning of the ventricular wall and collagen deposition, and further leads to myocardial fibrosis. Therefore, 4 weeks after myocardial infarction, the inventors of the present application evaluated the therapeutic effect of the hydrogel on cardiac remodeling by HE, Sirius red and Masson's trichrome staining, respectively for the control group, the MI group, the MI group injected with ALGHA in myocardium and the MI group injected with ALGHA-BP@PDA in myocardium. The myocardial tissue was evaluated by HE, Sirius red and Masson's trichrome staining 28 days after operation, and the results are shown in Figure 34 Fig. 2. HE staining shows that Figure 34 c), compared with the MI group, the left ventricular wall of the ALGHA-BP@PDA hydrogel treatment group is significantly thickened; Masson group (such as Figure 34 d and Figure 35 ) shows that a large area of blue is present in the infarct area of the MI group, which is the deposition of cells and collagen, and the fibrosis is serious. However, the proportion of left ventricular fibrosis in the ALGHA-BP@PDA hydrogel treatment group is significantly reduced. Sirius red staining (such as Figure 34 e and Figure 36 ) also shows a similar trend, we observed that the infarct myocardium of the MI group and the ALGHA hydrogel group was stained red, while the proportion of red in the ALGHA-BP@PDA hydrogel treatment group was significantly reduced. These results show that the ALGHA-BP@PDA hydrogel can reduce myocardial fibrosis after myocardial infarction, inhibit scar formation, and inhibit left ventricular remodeling.

[0074] The above is only one embodiment of the present application, which is described in more detail and in detail, but it cannot be understood as a limitation on the scope of the present application. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. An injectable black phosphorus nanosheet-loaded composite hydrogel, characterized in that: The composite hydrogel is mixed and prepared from polydopamine functionalized black phosphorus nanosheets, a sodium alginate suspension with a concentration of 2-2.5% wt / vol, a calcium alginate suspension with a concentration of 1.5-2% wt / vol, and hyaluronic acid; wherein the volume ratio of the sodium alginate suspension with a concentration of 2-2.5% wt / vol, the calcium alginate suspension with a concentration of 1.5-2% wt / vol, and the hyaluronic acid is 5:5:2, and the concentration of the polydopamine functionalized black phosphorus nanosheets in the synthesized composite hydrogel is 100 μg / ml; the injectable black phosphorus nanosheet-loaded composite hydrogel is used as a single substance for preparing an injection medicament for myocardial infarction, and the injection medicament has a dosage of 150 μl. The preparation method of the injectable black phosphorus nanosheet-loaded composite hydrogel specifically includes the following steps: (1) Black phosphorus nanosheets are synthesized by electrochemically exfoliating black phosphorus crystal blocks, the black phosphorus nanosheets are dispersed in anhydrous ethanol, then a NaOH solution with a pH of 8.5-9 is added, and a polydopamine solution with a concentration of 50-100 mg / ml is slowly added dropwise, the mixture is stirred in a dark environment for 4-6 hours to form a BP@PDA solution, after the stirring is completed, the BP@PDA solution is centrifuged at 4°C for 8-12 minutes, the supernatant is discarded, and the polydopamine functionalized black phosphorus nanosheets are formed after washing; wherein the mass-volume ratio of the black phosphorus nanosheets to anhydrous ethanol is 1:1, and the volume ratio of the anhydrous ethanol, the NaOH solution, and the polydopamine solution is 20:3:20; (2) At room temperature, sodium alginate powder is dissolved in sterile water to prepare a suspension with a concentration of 2-2.5% wt / vol, calcium alginate powder is dissolved in sterile water to prepare a suspension with a concentration of 1.5-2% wt / vol, the sodium alginate suspension with a concentration of 2-2.5% wt / vol and the calcium alginate suspension with a concentration of 1.5-2% wt / vol are heated and dissolved in a water bath at 45-55°C for 10-15 minutes, then hyaluronic acid is added, and finally the polydopamine functionalized black phosphorus nanosheets prepared in step (1) are added to prepare the injectable black phosphorus nanosheet-loaded composite hydrogel, wherein the concentration of the polydopamine functionalized black phosphorus nanosheets in the composite hydrogel is 100 μg / ml, and the volume ratio of the sodium alginate suspension, the calcium alginate suspension, and the hyaluronic acid is 5:5:

2.

2. The injectable loadings of phosphorene nanosheets composite hydrogel according to claim 1, wherein: In step (1), the BP@PDA solution is centrifuged at 4°C at 12000 rpm for 10 minutes, the supernatant is discarded, and the polydopamine functionalized black phosphorus nanosheets are formed after being washed twice with ultrapure water.

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