Preparation and Application of a Copper Chalcogenide Nanohydrogel

Abstract

The present invention relates to the preparation and application of a copper chalcogenide nanohydrogel. In this study, a copper chalcogenide nanocomposite system with high stability was successfully synthesized, and experiments were carried out in vitro and in vivo using this nanosystem to explore its application in the field of bladder cancer treatment, providing a new strategy to address the current limitations in bladder cancer treatment. The Cu<subgt;2-x< / subgt;Se@PDA@BTZ nanomaterial was prepared by a one-pot method. Through the surface modification of Cu<subgt;2-x< / subgt>Se with PDA, Cu<subgt;2-x< / subgt>Se was encapsulated inside PDA. In addition, bortezomib (BTZ) can be connected to PDA through a phenylboronic ester bond. At the same time, the PDA in the coating layer of the composite material can crosslink with tetra-armed polyethylene glycol-thiol (4ARM-PEG-SH) to form a nanohydrogel, which can prolong the retention time of the copper chalcogenide nanocomposite nanomaterial in the bladder, enhance the contact time between the bladder mucosa and the drug, play a drug sustained-release role, and achieve the efficient utilization of the drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to an anti-tumor nano preparation, a preparation method and uses thereof. Background Art

[0002] In recent years, with the continuous development of nanotechnology, more and more technical systems have emerged. As Figure 1-2 shown, nanomaterials can load various nano-scale particles and drugs. Through the precise design and surface modification by researchers, nano-drugs can maintain better specificity and bioavailability, while reducing the toxicity to healthy tissues, effectively overcoming the limitations of traditional chemotherapy regimens, not only improving the treatment effect, but also bringing a higher quality of life to patients.

[0003] Compared with other tumor types, the pathogenesis environment of bladder cancer has unique physiological structures and characteristics. The bladder is a hollow-structured organ located in the lower abdomen. Its main functions are to store and excrete urine, and the smooth muscle structure of the bladder provides a relatively large capacity. This enclosed cavity can accommodate various therapeutic drugs. The therapeutic drugs enter the bladder through the urethra, directly contacting and even covering the bladder tumor, which can increase the local therapeutic drug concentration and avoid drug dilution caused by systemic circulation drug use. In addition, local drug administration can reduce the side effects of drugs, improve the tolerance of patients and the safety of treatment, thereby achieving precise treatment of bladder cancer. Therefore, the unique spatial structure of the bladder provides great convenience for the delivery of nano-drugs for the treatment of bladder cancer. However, due to the urination mechanism of the bladder, the urine continuously flushes and dilutes the therapeutic drugs, resulting in a reduction in the effective treatment time and concentration of the drugs. Moreover, the inner wall of the bladder is lined with urothelium, and its tight junctions make it difficult for conventional drug molecules to penetrate the bladder wall and infiltrate into the tumor tissue. This stratified epithelium has a unique flat "umbrella cell" that increases the surface area when stretched. In addition, a tight glycosaminoglycan barrier forms a mucin hydrophilic layer outside the urothelium, which has strong resistance to the passage of molecules and becomes an important obstacle to the success of drug delivery in the bladder. At the same time, there are many limitations in the traditional perfusion treatment regimens for bladder cancer, such as short drug retention time, large local drug reactions, high tumor drug resistance and poor drug treatment effects. Therefore, finding a treatment regimen with superior treatment effect, low drug toxicity and side effects, high bioavailability and long-lasting action time is the focus of current bladder cancer treatment. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present application provides a copper chalcogenide nanocomposite material, which can achieve efficient killing of bladder cancer tumor cells by combining photothermal therapy (PTT), chemodynamic therapy (CDT) and the copper-induced cell death mechanism. It can also induce immunogenic cell death (ICD), thereby enhancing the anti-cancer effect and stimulating the long-term defense ability of the immune system against tumors.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A tumor-targeted nanocomposite material, the nanocomposite material has the following structural formula: Cu 2-x Se@PDA@BTZ, the composite nanomaterial has a core-shell structure, including a core and a shell layer coated on the surface of the core, the core is Cu 2-x Se nanoparticles, and the shell layer is a polydopamine (PDA) coating layer coated on the Cu 2-x Se nanoparticles, and bortezomib (BTZ) material uniformly loaded on the surface of the PDA coating layer through chemical bonds.

[0007] Preferably, the Cu 2-x Se@PDA@BTZ nanocomposite material also contains tetra-arm polyethylene glycol-thiol (4ARM-PEG-SH), and 4ARM-PEG-SH crosslinks with the Cu 2-x Se@PDA@BTZ nanocomposite material to form a nanohydrogel, and the nanohydrogel is represented by the structural formula Cu 2-x Se@PDA@BTZ-Gel.

[0008] On the other hand, the present invention also provides a preparation method of the tumor-targeted nanocomposite nanomaterial, and the specific steps are as follows:

[0009] S1: First, prepare Cu 2-x Se nanoparticles;

[0010] S2: Synthesize Cu 2-x Se, dopamine hydrochloride and BTZ by a one-pot method to synthesize Cu 2-x Se@PDA@BTZ.

[0011] Cu 2-x Se@PDA@BTZ-Gel is carried out according to the following steps:

[0012] Weigh a certain amount of tetra-arm polyethylene glycol-thiol (4ARM-PEG-SH) and dissolve it in deionized water, mix the Cu 2-x Se@PDA@BTZ with the tetra-arm polyethylene glycol-thiol (4ARM-PEG-SH) solution, and a colloid can be quickly formed at room temperature, that is, Cu 2- x Se@PDA@BTZ-Gel.

[0013] Preferably, the preparation of Cu 2-x Se nanoparticles in S1 includes the following steps:

[0014] (a) Add a certain amount of poly(4-styrenesulfonic acid) solution to pure water and stir to obtain a mixed solution A;

[0015] (b) Weigh a certain amount of SeO2 and dissolve it in deionized water to obtain a mixed solution B. Weigh a certain amount of Vc and dissolve it in deionized water to obtain a mixed solution C. Weigh a certain amount of CuSO4·5H2O and dissolve it in deionized water to obtain a mixed solution D.

[0016] (c) Add solution A and solution C to the reaction vessel in sequence, stir. Wait until the mixed solution completely turns orange-red, then add a certain amount of the mixed solution of B and C to the orange-red solution, stir for a period of time until it completely turns orange-red;

[0017] (d) Continue to stir at room temperature for a period of time, observe that the solution color turns emerald green, then centrifuge and wash, and finally obtain Cu 2-x Se nanoparticles.

[0018] Preferably, in S2, the one-pot synthesis of Cu 2-x Se@PDA@BTZ includes the following steps:

[0019] (a) Prepare a Tris-HCl solution. Weigh a certain amount of Tris and dissolve it in deionized water. After calibrating the pH detector, use it and adjust the pH value of the Tris-HCl solution to 8 - 10 with an acid solution;

[0020] (b) Prepare a reaction vessel with a rotor. First, add a certain amount of Cu 2-x Se solution; then add a certain amount of Tris-HCl solution;

[0021] (c) Weigh a certain amount of dopamine hydrochloride and dissolve it in the Tris-HCl solution, and add a certain volume of the dopamine hydrochloride solution to the reaction vessel;

[0022] (d) Weigh a certain amount of BTZ and dissolve it in DMSO to obtain a mixed solution containing BTZ, and add the said mixed solution to the reaction vessel, place it on a stirrer and stir for 6 - 8 h until the solution color turns dark green, then centrifuge, filter, wash, and dry to obtain Cu 2-x Se@PDA@BTZ composite material.

[0023] Preferably, the stirring time in step c of step S1 is 5 - 30 min; the stirring time in step D is 15 - 24 h;

[0024] Preferably, the pH value in step a of step S2 is 8.5;

[0025] Preferably, the acid in step a of step S2 may be one or more of phosphoric acid, sulfuric acid, nitric acid, acetic acid, formic acid, and hydrochloric acid, and further preferably hydrochloric acid.

[0026] Preferably, the centrifugation conditions for centrifugation in step d of step S1 are: speed 10000 - 14000 rpm / min, time 10 - 15 min.

[0027] Preferably, the centrifugation conditions for centrifugation in step d of step S2 are: speed 10000 - 14000 rpm / min, time 10 - 15 min.

[0028] Use of the above tumor - targeting nanocomposite or the nanocomposite prepared by the above method in the preparation of anti - tumor drugs; the tumor is bladder cancer.

[0029] The beneficial effects of the present invention are as follows:

[0030] (1) In this study of the present invention, through the surface modification of Cu 2-x Se by PDA, Cu 2-x Se is wrapped inside PDA. In addition, bortezomib (BTZ) can be connected to PDA through phenylboronic acid ester bonds. The outermost layer PDA of the nanocomposite in the present invention can cross - link with tetra - arm - polyethylene glycol - thiol (4ARM - PEG - SH) to form a nano - hydrogel, which can make the chalcogenide nanocomposite hydrogel aim to extend the residence time of the drug in the bladder, enhance the contact time between the bladder mucosa and the drug, play a drug - slow - release role, and achieve the efficient utilization of the drug;

[0031] (2) Multi - mode tumor killing: By utilizing the photothermal effect (PTT), chemodynamic effect (CDT) of the copper chalcogenide nanohydrogel and the copper - induced cell death mechanism, a multi - functional tumor - killing method is provided to overcome the problem of insufficient efficacy of a single treatment method;

[0032] (3) Synergistic effect of chemotherapy drugs: Using the nanohydrogel to load bortezomib (BTZ), not only plays its chemotherapy effect, but also further enhances the anti - cancer effect by inducing immunogenic cell death (ICD), and solves the problem of insufficient ability of traditional chemotherapy drugs to stimulate the immune system.

[0033] (4) The copper chalcogenide nanohydrogel has high biocompatibility and biosafety. Its good bioavailability ensures the efficient action of the drug in the bladder, solves the problems of drug side effects and biosafety in traditional bladder cancer treatment. At the same time, through the long - term immune memory ability induced by the nanohydrogel, the recurrence risk of bladder cancer is effectively reduced, and the clinical problem of high recurrence rate of bladder cancer is solved. Description of the Drawings

[0034] Figure 1These are the TEM images of CS of the present invention, the TEM images of CSP, the TEM images of CSBP, and the EDS detection diagram of CSBP.

[0035] Figure 2 This is the SEM image of CSBP-Gel of the present invention.

[0036] Figure 3 These are the ultraviolet absorption spectra of CSBP of the present invention at different concentrations.

[0037] Figure 4 These are the changes in storage modulus (G’) and loss modulus (G”) during the gelation process of the nano-hydrogel.

[0038] Figure 5 This is the uptake behavior of MB49 cells of the present invention towards the nanomaterials.

[0039] Figure 6 These are the long-term retention effect and permeation effect of the nano-hydrogel. Detailed implementation manners

[0040] To make the technical solutions and advantages of the present invention clearer, the present invention and its beneficial effects will be further described in detail below in combination with the specific implementation manners and the accompanying drawings of the specification. However, the implementation manners of the present invention are not limited thereto.

[0041] In the present invention, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following items (pieces)" or its similar expressions refer to any combination of these items, including any combination of single item (piece) or plural items (pieces). For example, "at least one of a, b, or c", or, "at least one of a, b, and c" can all represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0042] The invention and its beneficial effects will be further described in detail. However, the implementation manners of the present invention are not limited thereto.

[0043] In the present invention, Cu 2-x Se is prepared by a one-pot method using Vc, SeO2, and CuSO4. The successfully prepared Cu 2-x Se nanoparticles are encapsulated in polydopamine (PDA), and the small molecule chemotherapeutic drug bortezomib (BTZ) is loaded on its surface. Subsequently, a hydrogel system is formed by reacting with four-arm polyethylene glycol thiol (4-ARM-PEG-SH). Based on the slow-release characteristics of the hydrogel, we can achieve the synergistic effect of the nano-system in the bladder cavity, and Cu 2-xSe@PDA@BTZ nanoparticles are slowly released into bladder cancer cells. Due to the weak acidity, high glutathione (GSH), and high hydrogen peroxide (H2O2) levels in the tumor microenvironment, it can trigger the release of Cu 2-x2- x Se and BTZ, enhancing the targeting ability of the nanoparticles. On the one hand, Cu 2-x Se reacts with GSH in the tumor to generate cuprous ions, inducing cuproptosis; on the other hand, the released BTZ synergistically kills tumors through chemotherapy and immunogenic cell death (ICD) effects. Through the combined action of multiple mechanisms, effective treatment of bladder cancer is achieved. In addition, our research also found that Cu 2-x Se exhibits local surface plasmon resonance (LSPR) characteristics in the near-infrared region and has a high photothermal conversion efficiency. Irradiation with a 1064nm laser can trigger a photothermal effect, further inhibiting tumor cell proliferation and acting synergistically with cuproptosis and ICD effects, thus effectively improving the treatment effect.

[0044] This application also based on the multifunctional properties of copper chalcogenide nanohydrogels, effectively kills tumor cells through photothermal therapy (PTT), chemodynamic therapy (CDT), and copper-induced cell death mechanisms. At the same time, the nanohydrogel can load the chemotherapy drug bortezomib (BTZ), which not only has a chemotherapy effect but also can induce immunogenic cell death (ICD), further enhancing the anti-cancer effect. Copper chalcogenide nanohydrogels have high biocompatibility and safety, and their high bioavailability improves the treatment effect of bladder cancer. In addition, the nanohydrogel can also trigger long-term immune memory and reduce the risk of bladder cancer recurrence.

[0045] In the following examples, unless otherwise specified, the raw materials and instruments used are commercially available.

[0046] Example 1

[0047] First, Cu 2-x Se nanoparticles were synthesized.

[0048] (1) Add 0.16g of poly(4-styrenesulfonic acid) solution to 16ml of pure water, add it to a round-bottom flask, then add 55ml of deionized water to the round-bottom flask, and place a magnetic stirrer at the bottom of the flask for stirring.

[0049] (2) Weigh 22.192 mg of SeO2 and dissolve it in 1 ml of deionized water (0.2 M). Weigh 211.344 mg of Vc and dissolve it in 3 ml of deionized water (0.4 M). Weigh 99.876 mg of CuSO4·5H2O and dissolve it in 1 ml of deionized water (0.4 M). Weigh 282 mg of Vc and dissolve it in 4.7 ml of deionized water (0.4 M). The dissolution process can be accelerated using an ultrasonic instrument.

[0050] (3) Add SeO2 and 3 ml of Vc to the round-bottom flask in sequence, stir for 10 minutes. Wait until it completely turns orange-red, then add the mixed solution of CuSO4·5H2O and 4.7 ml of Vc, and the solution color turns brown.

[0051] (4) After stirring at room temperature for 17 h, observe that the solution color turns emerald green, transfer it to a small tube, centrifuge at 14,800 rpm for 10 minutes, discard the supernatant, and collect the precipitate. Wash the precipitate once with pure water, repeat centrifugation to discard the supernatant, and finally dissolve it in 5 ml of pure water, then Cu 2-x Se(CS) is successfully prepared. The prepared nanomaterial is placed in a refrigerator at 4 °C.

[0052] Example 2

[0053] We synthesize Cu 2-x Se, dopamine hydrochloride, and BTZ by a one-pot method to synthesize Cu 2-x Se@PDA@BTZ. The specific steps are as follows:

[0054] (1) Prepare Tris-Hcl solution. Weigh 0.363 g of Tris and dissolve it in 300 ml of deionized water. After calibrating the pH detector, use it. Adjust the pH value of the Tris-Hcl solution to 8.5 with HCl solution.

[0055] (2) Prepare a small bottle with a rotor. First, add 0.5 ml of Cu 2-x Se solution. Then add 8.5 ml of Tris-Hcl solution.

[0056] (3) Weigh 6 mg of dopamine hydrochloride and dissolve it in 1 ml of Tris-Hcl solution, and add 0.5 ml of dopamine hydrochloride solution to the bottle.

[0057] (4) Weigh 2 mg of BTZ and dissolve it in 800 μl of DMSO, and add it to the bottle. (If this step is not carried out, the finally obtained solution is Cu 2-x Se@PDA)

[0058] (5) Place the small bottle on a magnetic stirrer and stir for 6 h until the solution color turns dark green.

[0059] (6) Transfer the solution to a small tube, centrifuge at 14,800 rpm for 10 minutes, discard the supernatant, retain the precipitate, wash it once with pure water and then centrifuge in the above manner, discard the supernatant, and finally dissolve it in 1 ml of pure water. Place the prepared nanomaterials in a refrigerator at 4 °C.

[0060] Example 3

[0061] Cu 2-x Preparation of CuSe@PDA@BTZ-Gel:

[0062] Weigh 100 mg of 4ARM-PEG-Thiol and dissolve it in 1 ml of deionized water. Concentrate 1 ml of Cu 2-x Se@PDA@BTZ to 150 μl, mix it with the 4ARM-PEG-Thiol solution at a ratio of 1:1, and a colloid can be quickly formed at room temperature.

[0063] Example 4

[0064] Cu 2-x Cell uptake verification of CuSe@PDA@BTZ and Cu 2-x Se@PDA:

[0065] (1) Sterilize the aseptic workbench. Prepare DMEM medium, DMSO, sterile PBS, FBS, double antibiotics (penicillin and streptomycin), sterile centrifuge tubes, and confocal culture dishes.

[0066] (2) Pretreat the confocal culture dish. Add 3 ml of culture medium to the confocal culture dish and take it out after placing it in the incubator for 15 minutes for use.

[0067] (3) Take out the culture dish containing MB49 cells and use it when the cell morphology and health are good. Wash with sterile PBS, digest with trypsin, terminate digestion, centrifuge, and resuspend the cells (the operation is the same as before).

[0068] (4) Remove the culture medium in the confocal dish, add 500 μl of complete medium with MB49 to the confocal culture dish, and the cell quantity is about 8×10^4. Place it in the incubator and incubate for 2 h to wait for the cells to adhere.

[0069] (5) Take out the confocal culture dish, add complete medium to 2 ml, and place it in the incubator for overnight incubation.

[0070] (6) Prepare the nanomaterials conjugated with CY5.5, 10 μl of Cy5.5-NHS (10 mg·ml-1), 1 ml of Cu 2-x Se@PDA@BTZ or Cu 2-xSe@PDA solution (concentration), EDC aqueous solution (100 mg / ml). Mix the three solutions and incubate overnight in the dark. The next day, centrifuge at 14,800 rpm, remove the supernatant, wash twice with deionized water, and use a UV spectrophotometer to detect that there is no Cy5.5 absorption peak in the supernatant. Finally, make the volume of the nanomaterial up to 1 ml.

[0071] (7) Observe the morphology and health of the cells in the confocal dish. If the state is good, aspirate the supernatant, add pure DMEM medium, and then add 20 μg of the nanomaterial loaded with Cy5.5 (calculated based on the copper content), a total of 2 ml with the medium, and place it in the incubator for 4 h. Add the lysosome green fluorescent probe (Lyso-Tracker Green) dye at a ratio of 1:20,000 to the medium, with a final concentration of 50 nM, and place it in the incubator for 1 h.

[0072] (8) After incubation, wash the confocal dish three times with PBS, 5 min each time, to remove the nanoparticles and lysosome dye that did not enter the cells.

[0073] (9) Fix the cells with 4% PFA and let it stand for 20 min.

[0074] (10) Prepare the DAPI stock solution by diluting 0.5 mg of DAPI dye with 5 ml of normal saline. The working solution is diluted 1000 times with normal saline before use.

[0075] (11) After fixation, continue to wash three times with PBS, 5 min each time, add 200 μl of the DAPI working solution, and stain in the dark for 10 min.

[0076] (12) After staining, wash three times with PBS, 5 min each time, to remove the free dye, and then it can be loaded onto the machine.

[0077] Example 5

[0078] Cu 2-x Se@PDA@BTZ and Cu 2-x Retention experiment of Se@PDA@BTZ-Gel:

[0079] (1) Label Cu 2-x Se@PDA@BTZ with Cy5.5, using the same method as before.

[0080] Cu 2-x Se@PDA@BTZ-Gel is formed by gelling 4arm-PEG-Thiol after Se@PDA@BTZ labeled with Cy5.5 by Cu 2-x Se@PDA@BTZ labeled with Cy5.5.

[0081] (2) Prepare anesthetic with normal saline, and the concentration of sodium pentobarbital is 1%.

[0082] (3) Select mice at 6 - 8 weeks old, with a body weight between 20 - 30 g, and divide them into two groups. One group is used for perfusion of Cu 2-x Se@PDA@BTZ - Gel, and the other group is used for Cu 2-x Se@PDA@BTZ.

[0083] (4) Prepare equipment: syringes, alcohol swabs, forceps, micro hemostatic clips, animal hair clippers, disposable intravenous indwelling needles (for perfusion), and weighing scales.

[0084] (5) Grasp the mice, weigh them, disinfect the abdomen with alcohol swabs, insert the needle obliquely into the lower abdomen of the mice, and inject anesthetic at a dose of 7.5 mg / kg.

[0085] (6) Aspirate the nanocomposite with a 29G fine needle and insert it into the disposable intravenous indwelling needle. Use forceps to pick up the urethra of the mice, slowly insert the intravenous indwelling needle into the bladder, inject an equal amount of nanocomposite, clamp the urethra with a hemostatic clip, slowly remove the intravenous indwelling needle, and remove the hemostatic clip after 1.5 h.

[0086] (7) Use the hair clipper to shave the hair on the abdomen of the mice clean. Image the mice with a dual - color infrared laser imaging system. The fluorescence signal of the material labeled with Cy5.5 is red, and imaging is performed at 2 h, 6 h, 24 h, 48 h, and 72 h respectively.

[0087] Preparation and detection of electron microscopy samples:

[0088] Take out the film from the carbon support film and place it on the filter paper. Carefully drop 2 μl of the nanomaterial on the surface of the film. After air - drying, sample for TEM and SEM electron microscopy shooting. For the sample preparation of the nanohydrogel, place the nanohydrogel at - 80 °C for 2 h, then place it in a freeze - dryer and freeze - dry for 24 h. After the sample preparation is completed, the morphology can be imaged by electron microscopy.

[0089] Cu 2-x Rheological properties of Cu

[0090] Use a rheological instrument to detect the rheological properties of the hydrogel. Place the concentrated solution of Cu 2-x Se@PDA@BTZ and tetra - arm thiol - terminated polyethylene glycol on the test platform of the rheometer for kinetic viscosity testing and modulus testing (storage modulus and loss modulus). The temperature condition is 37 °C, the angular velocity is set to 10 rad / s, and the stress is set to 0.1%.

[0091] We abbreviate the Cu 2-x Se nanoparticles as CS, and abbreviate the Cu 2-x Se@PDA nanoparticles as CSP, and abbreviate the Cu 2-x The abbreviation of Se@PDA@BTZ nanoparticles is CSBP, Cu 2-x The abbreviation of Se@PDA@BTZ-Gel is CSBP-Gel.

[0092] Figure 1 It is the TEM image of CS, scale bar: 50 nm; (b) The TEM image of CSP, scale bar: 50 nm; (c) The TEM image of CSBP, scale bar: 50 nm; (d) Element mapping of Cu, Se, C, N, B, and O in CSBP detected by EDS. The morphology of CS captured by TEM is spherical. Polydopamine is modified and wrapped on the surface of CS to form CSP, and the contrast of PDA is lower than that of Cu 2-x Se, and the dispersion is relatively uniform. Since BTZ is a small molecule substance, the morphologies of CSP and CSBP are similar. From the TEM images, the size range of the nanoparticles is between 50 - 100 nm. The element distribution can be observed from the EDS images (2 - 1d). We can observe that Cu, Se, N, C, O, and B are evenly distributed on each nanoparticle. Although the amount of B element is small, its morphology can still be observed, indicating that PDA has successfully encapsulated Cu 2-x Se and successfully loaded BTZ.

[0093] Figure 2 It is the SEM image of CSBP-Gel. We can see that the results show that under a field of view of 200 μm, the nano-hydrogel is a lamellar structure, while Figure 2-6 (b) The result is a field of view of 200 nm, and it can be observed that the nanomaterial has a 3D spherical structure with a diameter of less than 100 nm. The reason is that Cu 2-x Se stacked by PDA wrapping.

[0094] Figure 3 It is the UV absorption spectra of CSBP at different concentrations. It can be seen that the UV absorption peaks of the nanomaterial increase successively from low concentration to high concentration, and the whole curve is smooth without obvious broken line scatter plots, indicating that the concentration of the nanomaterial we measured is not exceeded, which proves the reliability of the data.

[0095] Figure 4are the changes in storage modulus (G’) and loss modulus (G”) during the gelation process of the nano-hydrogel; and the shear thinning ability of the nano-hydrogel. It can be observed that when the nanomaterial is mixed with 4arm-PEG-Thiol, the gel state appears within a very short time. The state where the storage modulus is greater than the loss modulus indicates the gelation state. The storage modulus increases rapidly within 500 s, indicating that the gelation time of the nano-hydrogel we prepared is very short, and the two can change from the liquid state to the gel state within seconds after mixing. In addition, we tested the shear thinning characteristics of the nanomaterial, with viscosity on the y-axis and shear rate on the x-axis. As the shear thinning rate increases, the viscosity also continuously decreases, indicating that the hydrogel has the ability of shear thinning, and also indicating that the nano-hydrogel can be injected through a syringe.

[0096] Figure 5 are the confocal images after CSP-Cy5.5 and CSBP-Cy5.5 are incubated with MB49 for 4 h and lysosome staining for 1 h. The nucleus is stained with DAPI (blue), the lysosome is stained with Lyso-Tracker Green (green), and the cytoplasm is stained inside the cell after the nanomaterial enters (red). The uptake of CSBP and CSP by MB49 cells is measured by confocal laser microscopy, and the distribution of the nanomaterial after entering the cell is evaluated by labeling the fluorescent lysosome. Strong Cy-5.5 signals can be seen in both CSP and CSBP from the confocal images. It can be observed from the co-localization images of the lysosome staining and the nanomaterial that the lysosome can assist in transporting the nanomaterial and enhance the cell delivery ability of the nanomaterial. In short, the nanomaterial can be efficiently taken up by MB49 mouse bladder cancer cells.

[0097] Figure 6CSBP-Gel-Cy5.5 and CSBP-Cy5.5 were perfused into the bladders of mice, and imaging was performed at 2 h, 6 h, 24 h, 48 h, and 72 h respectively. (b) Frozen sections of mouse bladders were stained with DAPI for nuclei (blue), and Cy5.5 was used to localize CSBP-Gel and CSBP (red). Scale bar: 25 μm. Equal amounts of CSBP nanomaterials and nanohydrogels were perfused into the bladders of mice, and imaging was performed at 2 h, 6 h, 24 h, 48 h, and 72 h. CSBP was almost completely emptied within 2 h, while the red fluorescence signal emitted by Cy5.5 could still be detected in CSBP-Gel until 72 h, demonstrating the long retention effect of the nanohydrogel. During the whole process, the mice did not show any problems such as poor urination, weakness, and emaciation, indicating good biocompatibility of CSBP-Gel. Subsequently, the mice at the end of the observation were dissected, and mouse bladder tissue samples were prepared by frozen sectioning. After nuclear staining, it was observed that there was still hydrogel remaining on the bladder wall in the CSBP-Gel group, and some hydrogels had disintegrated. The nanoparticles loaded with Cy5.5 had entered the cytoplasm, indicating that the hydrogel could not only remain in the bladder for a long time but also further deliver CSBP into bladder cells.

[0098] From the above experiments, it can be seen that PDA of this application was successfully surface-modified on Cu 2-x Se. BTZ is a small molecule chemotherapeutic drug that is poorly soluble in water. By dissolving it in DMSO and connecting it to DA through a borate ester bond, it was stably loaded on CSBP, which not only solved the toxic side effects of chemotherapeutic drugs but also achieved targeted drug delivery. EDS proved that the nanocomposite was composed of Cu, Se, N, C, O, and B. The excellent photothermal conversion ability of CSBP and CSBP-Gel demonstrated the diversity of the functions of the nanomaterials. CSBP-Gel had the ability of rapid gelation and shear thinning. By perfusing CSBP-Gel into the bladder, due to the special structure and surface properties of the hydrogel, it could adhere to the bladder wall for a long time, verifying the excellent long-term retention ability and biocompatibility of the hydrogel. When the nanomaterials entered the cells through the lysosomal pathway, due to the weak acid environment, high GSH, and high H2O2 in the tumor microenvironment of bladder cancer, they could react with CSBP and cause complete decomposition of CSBP, further enhancing the bioavailability of the chemotherapeutic drug BTZ and realizing the chemodynamic effect of copper ions. The synthesized CSBP was a nanohydrogel with good biocompatibility and biosafety. As a carrier for delivering nanodrugs and chemotherapeutic drugs, it could realize multifunctional nanomaterials for synergistic treatment in multiple ways such as photothermal therapy, chemodynamic therapy, and chemotherapy, and had broad application prospects.

[0099] Based on the disclosure and teachings of the above specification, those skilled in the art to which the present invention pertains are also able to make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the above specific embodiments, and any obvious improvements, substitutions, or variations made by those skilled in the art based on the present invention fall within the protection scope of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. A tumor-targeting nanocomposite, the nanocomposite having the following structural formula: Cu 2-x Se@PDA@BTZ, characterized in that The composite nanomaterial has a core-shell structure, including a core and a shell layer coated on the surface of the core. The core is Cu 2-x Se nanoparticles, and the shell layer is a polydopamine (PDA) coating layer coated on the surface of the Cu 2-x Se nanoparticles, and bortezomib (BTZ) material uniformly loaded on the surface of the PDA coating layer through chemical bonds; The described Cu 2-x Se@PDA@BTZ nanocomposite also contains tetra-armed polyethylene glycol-thiol (4ARM-PEG-SH), and 4ARM-PEG-SH crosslinks with the Cu 2-x Se@PDA@BTZ nanocomposite to form a nano-hydrogel, and the nano-hydrogel is represented by the structural formula Cu 2-x Se@PDA@BTZ-Gel; Cu 2-x The preparation of Se@PDA@BTZ-Gel is carried out according to the following steps: Weigh a certain amount of tetra-armed polyethylene glycol-thiol (4ARM-PEG-SH) and dissolve it in deionized water. Mix Cu 2-x Se@PDA@BTZ with the tetra-armed polyethylene glycol-thiol solution (4ARM-PEG-SH), and a colloid can be quickly formed at room temperature, namely Cu 2-x Se@PDA@BTZ-Gel.

2. A preparation method of the tumor-targeting nanocomposite nanomaterial as described in claim 1, characterized in that: Cu 2-x The preparation of Se@PDA@BTZ is carried out according to the following steps: S1: First, prepare Cu 2-x Se nanoparticles; S2: Synthesize the prepared Cu 2-x Se, dopamine hydrochloride and BTZ to synthesize Cu 2-x Se@PDA@BTZ via a one-pot method; Cu 2-x The preparation of Se@PDA@BTZ-Gel is carried out according to the following steps: Weigh a certain amount of four-armed polyethylene glycol thiol (4ARM-PEG-SH) and dissolve it in deionized water. Mix Cu 2-x Se@PDA@BTZ with the four-armed polyethylene glycol thiol solution (4ARM-PEG-SH). A colloid can be rapidly formed at room temperature, namely Cu 2-x Se@PDA@BTZ-Gel.

3. The preparation method according to claim 2, characterized in that, Prepare Cu 2-x The steps for preparing CuSe nanoparticles are as follows: (a) Add a certain amount of poly(4-styrenesulfonic acid) solution to pure water and stir to obtain a mixed solution A; (b) Weigh a certain amount of SeO2 and dissolve it in deionized water to obtain a mixed solution B, weigh a certain amount of Vc and dissolve it in deionized water to obtain a mixed solution C, and weigh a certain amount of CuSO4·5H2O and dissolve it in deionized water to obtain a mixed solution D; (c) Add solution A and solution C to the reaction vessel in sequence, stir, wait until the mixed solution completely turns orange-red, then add a certain amount of the mixed solution of B and C to the orange-red solution, stir for a period of time until it completely turns orange-red; (d)After stirring at room temperature for a period of time, observe that the solution color turns emerald green, and then obtain Cu 2-x Se nanoparticles by centrifugation and washing.

4. The preparation method according to claim 2, characterized in that, One-pot synthesis of Cu 2-x Se@PDA@BTZ, comprising the following steps: (a) Prepare a Tris-HCl solution, weigh a certain amount of Tris and dissolve it in deionized water. After calibrating the pH detector, use it, and adjust the pH value of the Tris-HCl solution to 8-10 with an acid solution; (b) Prepare a reaction vessel with a rotor. First, add a certain amount of Cu 2-x Se solution; then add a certain amount of Tris-HCl solution; (c) Weigh a certain amount of dopamine hydrochloride and dissolve it in the Tris-HCl solution, and add a certain volume of the dopamine hydrochloride solution to the reaction vessel; (d)Weigh a certain amount of BTZ and dissolve it in DMSO to obtain a mixed solution containing BTZ. Then add the said mixed solution to a reaction vessel, place it on a stirrer and stir for 6 - 8 h until the solution color turns dark green. Then centrifuge, filter and wash to obtain Cu 2-x Se@PDA@BTZ composite material.

5. The preparation method according to claim 3, characterized in that, The stirring time in step C is 5-30 min; the stirring time in step D is 15-24 h.

6. The preparation method according to claim 4, wherein The pH value in step a is 8.

5.

7. The preparation method according to claim 4, characterized in that, The acid in step a is one or more of phosphoric acid, sulfuric acid, nitric acid, acetic acid, formic acid, and hydrochloric acid.

8. The preparation method according to any one of claims 2-3, characterized in that, The centrifugation conditions are: speed 10000-14000 rpm / min, time 10-15 min.

9. Use of the nanocomposite material as described in claim 1 or the nanocomposite material prepared by the method as described in claims 2-8 in the preparation of anti-tumor drugs; the tumor is bladder cancer.

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