Preparation method and application of self-supply nano-drug system based on dopamine-platinum (II) auto-polymerization O2 and H2O2

Abstract

The invention relates to preparation and application of a self-supply nano-drug system based on dopamine-platinum (II) auto-polymerization O2 and H2O2. The preparation comprises the following steps: firstly, synthesizing dopamine-platinum (II) auto-polymerization spherical nanoparticles (PtPDs) with near-infrared light response, and under the combined action of reductive intracellular environment and illumination by utilizing auto-polymerization between platinum (II) and dopamine molecules, carrying out self-supply on the nanoparticles (PtPDs) to prepare the self-supply nano-drug system based on the dopamine-platinum (II) auto-polymerization O2 and H2O2. The PtPDs with high Pt loading efficiency are decomposed, Pt ions are released, and efficient chemotherapy is achieved. Then, the outer surface of the PtPDs is coated with a layer of manganese dioxide shell, and the manganese dioxide shell is modified with ultra-small gold nanoparticles with glucose oxidase activity by electrostatic adsorption, so that the cascaded nano drug-loading system PtPDs (at) MnO2 (at) Au NPs (PtPDMA) capable of automatically supplying O2 and H2O2 is obtained. Finally, combined treatment integrating nano-drug chemical treatment, photo-thermal treatment, chemical kinetics treatment and enzyme-like catalytic treatment is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and health, and particularly relates to a preparation method and application of a nano-drug system based on dopamine-platinum(II) self-polymerization with self-supplied O2 and H2O2. Background Art

[0002] Polydopamine (PDA) is a natural melanin derived from marine mussels, with good biocompatibility, biodegradability, and photothermal conversion ability. It is polymerized from dopamine and can not only be used as a drug carrier to deliver chemotherapeutic drugs, but also the surface of PDA nanoparticles contains a large number of phenyl, amino, and hydroxyl groups. Therefore, it can load various chemical drugs through p-p stacking or hydrogen bond interactions. PDA nanoparticles can also achieve targeted delivery to specific diseased tissues, which makes PDA-based nanomaterials a research hotspot in tumor treatment. Manganese dioxide (MnO2) has Fenton-like activity. MnO2 has been proven to have high catalytic activity in the decomposition of H2O2 in the tumor microenvironment, and the generated O2 by the catalytic reaction also has the effect of relieving hypoxia. MnO2 is decomposed by GSH to produce Mn 2+ , which can undergo an oxidation-reduction reaction with reduced GSH to generate Mn 2+ and oxidized glutathione (GSSG). The consumption of GSH will further induce the imbalance of the antioxidant defense system in cancer cells, thereby avoiding the scavenging of ×OH and enhancing the effect of CDT. Ultra-small gold nanoparticles (Au NPs) have excellent glucose oxidase mimicking activity. Glucose oxidase (GOx) mainly uses the nutrient glucose as a substrate in the body, oxidizes glucose to gluconic acid and H2O2 in the presence of oxygen, and can further oxidize to produce ATP to provide energy for the body.

[0003] Although the discovery and application of platinum-based drugs have milestone significance for the development of cancer chemotherapy, there are also many problems in the clinical use of platinum-based anti-tumor drugs, such as poor targeting, low bioavailability, easy inactivation in the body, etc. These reasons seriously limit the curative effect and long-term use, and are the biggest problems that need to be solved urgently at present. Therefore, how to prepare a nano-drug that can deliver the chemotherapeutic drug cisplatin to the tumor site and control the drug release rate is a technical problem that needs to be seriously solved. Based on this, the present application was developed. Summary of the Invention

[0004] The main object of the present invention is to overcome the defects of poor targeting, low bioavailability, and easy inactivation in the body of the single chemotherapeutic drug cisplatin, and provide a nano-drug system based on dopamine-platinum(II) self-polymerization with self-supplied O2 and H2O2, which can achieve combined treatment integrating chemotherapy, photothermal therapy, chemodynamic therapy, and enzyme-like catalytic therapy.

[0005] Another object of the present invention is to provide a preparation method and application of the above-mentioned polydopamine-platinum(II) self-polymerized O2 and H2O2 self-supplying nanodrug system. The nanodrug system prepared by this method has the advantages of high safety, small size, high biocompatibility, etc.

[0006] In order to achieve the above-mentioned invention objects, the present invention provides the following technical solutions: A preparation method of a dopamine-platinum(II) self-polymerized O2 and H2O2 self-supplying nanodrug system, which comprises the following steps: Step 1: At room temperature, disperse cisplatin and silver nitrate solids in water (stir for 5 - 10 min to disperse evenly) to obtain a mixed solution A; adjust the pH value of solution A to 2 - 2.5 with HNO3, then stir the mixture in the dark at 65 - 90 °C for 12 - 18 h, cool to room temperature, and perform solid-liquid separation to obtain activated cisplatin; Step 2: Uniformly disperse the activated cisplatin and dopamine hydrochloride in water (ultrasound for 8 - 15 min to disperse evenly) to obtain a mixed solution B; adjust the pH value of solution B to 8 ± 0.1 with Tris buffer solution, and stir at room temperature for 12 - 18 h to obtain the product PtPDs; Step 3: At room temperature, subject the product PtPDs to solid-liquid separation, washing, and freeze-drying to obtain a black-brown solid; disperse the black-brown solid in ultrapure water, adjust the pH value to 7.0 - 8.5 with ammonia water, then add potassium permanganate with stirring to obtain solution C; stir and react at room temperature for 4 - 6 h to obtain the product PtPDs@MnO2 NPs (abbreviated as PtPDM); Step 4: At room temperature, subject the product PtPDM to solid-liquid separation, washing, and freeze-drying to obtain a black solid; dissolve the black solid and polyvinylpyrrolidone (PVP-K30) in ethylene glycol, then heat to 90 ± 2 °C and mix evenly, add NaBH4 and HAuCl4, and continue to stir and react at 90 ± 2 °C in the dark for 60 - 70 min to obtain the product PtPDs@MnO2@Au NPs (abbreviated as PtPDMA).

[0007] Specifically, in step 1, the mass ratio of the cisplatin and silver nitrate solids can be 1:1 - 1.5. Preferably, the concentration of cisplatin in solution A is 2.25 mg / mL, and the concentration of silver nitrate is 2.25 - 2.75 mg / mL.

[0008] Specifically, in step 2, the mass ratio of the activated cisplatin to dopamine hydrochloride can be 1:2.5 - 3.5.

[0009] Specifically, in step 3, the mass ratio of the black-brown solid to potassium permanganate can be 1:1.5 - 3.

[0010] Specifically, in step 4, the mass ratio of the black solid to polyvinylpyrrolidone can be 1:0.6 - 1.5, and the molecular weight of the polyvinylpyrrolidone is 8000 - 12000 Da.

[0011] Furthermore, in step 4, the mass ratio of NaBH4 to HAuCl4 can be 1:1.2 - 1.5. The mass ratio of the black solid to NaBH4 can be 1:0.6 - 0.8.

[0012] The present invention provides a dopamine-platinum(II) self-polymerized O2 and H2O2 self-supplying nano-drug system prepared by the above method, which can be used for chemotherapy, hyperthermia, chemodynamic therapy, and enzyme-like catalytic therapy.

[0013] The present invention also provides the application of the above dopamine-platinum(II) self-polymerized O2 and H2O2 self-supplying nano-drug system in the preparation of anti-tumor drugs.

[0014] The present invention first synthesizes a near-infrared light-responsive dopamine-platinum(II) self-polymerized spherical nanoparticle (PtPDs). By the self-polymerization between platinum(II) and dopamine molecules, under the combined action of a reducing intracellular environment and light, the PtPDs with high Pt loading efficiency are decomposed, releasing Pt ions to achieve efficient chemotherapy. Then, a manganese dioxide shell is coated on the outer surface of the PtPDs, and ultrasmall gold nanoparticles with glucose oxidase-like activity are modified on the manganese dioxide shell layer by electrostatic adsorption, obtaining a cascade nano-drug delivery system PtPDs@MnO2@Au NPs (PtPDMA) that self-supplies O2 and H2O2, ultimately realizing the combined therapy integrating nano-drug chemotherapy, photothermal therapy, chemodynamic therapy, and enzyme-like catalytic therapy.

[0015] Compared with the prior art, the dopamine-platinum(II) self-polymerized O2 and H2O2 self-supplying nano-drug system of the present invention has the following advantages and beneficial effects: 1) The poly-dopamine-platinum(II) nanoparticle spheres (PtPDs) connected by chemical bonds can effectively protect the chemotherapy drug cisplatin from being consumed before entering the tumor site, reduce drug resistance, and greatly improve the efficacy of chemotherapy drugs.

[0016] 2) After synthesizing the PtPDs nanoparticles, the present invention does not change the photothermal conversion structure of the poly-dopamine nanoparticles and still maintains a high photothermal conversion efficiency.

[0017] 3) The black-brown solid obtained by using silver nitrate solid, HNO3 solution, dopamine hydrochloride and the chemotherapeutic drug cisplatin as raw materials in the present invention is PtPDs nanoparticles; then PtPDMA is prepared by using ammonia water, potassium permanganate, polyvinylpyrrolidone (PVP-K30), sodium borohydride, chloroauric acid and PtPDs nanoparticles as raw materials. The raw materials used in the present invention have low toxicity, high safety, simple synthesis operation, mild and controllable reaction conditions, and low cost.

[0018] 4) The surface potential of PtPDMA in the present invention is negative, and the negative potential is conducive to the dispersion of the nano-drug system in the body. The size is 200-250 nm, the size distribution is uniform and the dispersion is uniform, which is conducive to circulation in the organism.

[0019] In summary, polydopamine, as a good near-infrared photothermal conversion material, plays a photothermal conversion role and can enhance water solubility and biocompatibility. When local laser irradiation is performed externally on the tumor, the nanoparticles aggregated at the tumor convert light energy into heat energy under laser irradiation to achieve the purpose of tumor thermal ablation. At the same time, through laser irradiation, the chemical bonds of the polydopamine-platinum (II) nanoparticles sphere are broken, and cisplatin is released to play a synergistic chemotherapy role. Manganese dioxide decomposes into Mn 2+ 、H2O2、O2 in the slightly acidic tumor environment, and Mn 2+ undergoes a Fenton-like reaction with H2O2 to generate toxic ×OH, in which O 2， is released to relieve the hypoxic microenvironment of the tumor. Ultra-small gold nanoparticles have glucose oxidase-like activity and can be used as a mimic of GOx. They can not only catalyze glucose to produce H2O2 for CDT and in-situ generate O2 to improve PDT, but also play a role in starvation therapy by consuming glucose. The entire nano-drug delivery system realizes a combined therapy integrating chemotherapy, photothermal therapy, chemodynamic therapy and enzyme-like catalytic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 are transmission electron microscopy (TEM) images of the products PtPDs (A), PtPDM (B) and PtPDMA (C) in Example 1; Figure 2 are the ultraviolet absorption spectra of the products PtPDs, PtPDM and PtPDMA in Example 1; Figure 3 are the hydrated particle size distribution diagram (A) of the product PtPDMA and the Zeta potential diagram (B) of the products PtPDs, PtPDM and PtPDMA in Example 1; Figure 4Infrared spectra of product PDA, product activated cisplatin (CDDP), product PtPDs, product PtPDM and product PtPDMA in Example 1; Figure 5 In vitro drug release curves of product PtPDMA in Example 1 under different pH values and with or without laser irradiation; Figure 6 Hemolysis photos (A) and hemolysis percentages (B) of different concentrations of product PtPDMA in Example 1, n = 3; Figure 7 Temperature change graphs of different concentrations of product PtPDMA irradiated with 808 nm laser (1.5 W / cm²) for 10 min in Example 1 (A) and temperature change graphs of different powers of product PtPDMA with a concentration of 200 mg / mL irradiated with 808 nm laser for 10 min (B); Figure 8 For product PtPDMA in Example 1 irradiated with 808 nm laser (1 W / cm 2 ) for 10 min, infrared thermal imaging graphs of different concentrations; Figure 9 For product PtPDMA in Example 1 irradiated with 808 nm laser (1 W / cm 2 ) for 5 min on H22 tumor-bearing mice, in vivo infrared thermal imaging graphs; Figure 10 Survival rate graphs of cells of product PtPDM on different cell lines without laser irradiation in Example 1 (A), survival rate graphs of product PtPDM, product PtPDMA and activated cisplatin (CDDP) on HepG2 cells before and after laser irradiation (B); Figure 11 Digital images of tumor tissues (A), graphs of changes in tumor weight (B), tumor growth curves (C) and graphs of changes in mouse body weight (D) at the end of treatment of H22 tumor-bearing mice with product PtPDM, product PtPDMA and activated cisplatin (CDDP) before and after laser irradiation in Example 1; Figure 12 HE staining graphs of tissue sections of heart, liver, spleen, lung and kidney after dissection of H22 tumor-bearing mice at the end of treatment with product PtPDM, product PtPDMA and activated cisplatin (CDDP) before and after laser irradiation in Example 1; Figure 13 HE staining and TUNEL staining graphs of tumor tissue sections after dissection of H22 tumor-bearing mice at the end of treatment with product PtPDM, product PtPDMA and activated cisplatin (CDDP) before and after laser irradiation in Example 1. Detailed implementation manners

[0021] To further understand the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. However, it should be understood that these descriptions are only for further explaining the features and advantages of the present invention rather than limiting the protection scope of the present invention.

[0022] Names and models of experimental instruments: Perkin-Elmer Lambda-850 ultraviolet spectrophotometer, USA; JEOL JEM-200CX transmission electron microscope, Japan; BioTek multi-functional microplate reader, USA; LEICA TCS SP8+STED laser confocal microscope, Germany; BD Facsverse flow cytometer, USA; LABCONCO-FreeZone 6L freeze-drying system, USA; Cytiva AI800flour ultrasensitive multi-functional imager, USA; Unless otherwise specified, room temperature refers to 25±5 °C.

[0023] Example 1 A dopamine-platinum(II) self-polymerizing O2 and H2O2 self-supplying nano-drug system, which comprises the following steps: Step 1: At room temperature, disperse 45 mg of cisplatin and 48 mg of silver nitrate solids in 20 mL of water (stir for 8 min to disperse evenly) to obtain a mixed solution A. The concentration of cisplatin in solution A is 2.25 mg / mL, and the concentration of silver nitrate is 2.4 mg / mL. Adjust the pH value of solution A to 2-2.5 with 0.1 M HNO3 solution, then stir the mixture in the dark at 80 °C overnight (12 h), and cool to room temperature. Then filter the precipitate using a 0.22 mm filter, repeat three times, and obtain activated cisplatin (CDDP).

[0024] Step 2: Disperse 100 mg of activated cisplatin and 300 mg of dopamine hydrochloride evenly in 25 mL of water (ultrasound for 10 min to disperse evenly) to obtain a mixed solution B. The mass ratio of activated cisplatin to dopamine hydrochloride in solution B is 1:3. Adjust the pH value of solution B to 8±0.1 with Tris buffer solution with a pH value of 7.5-9.5 and a concentration of 8-12 mM, and stir overnight (12 h) at room temperature to obtain the product PtPDs; Step 3: At room temperature, first centrifuge the product PtPDs at 12,000 rpm for 10 min, then wash it three times with ultrapure water, and freeze-dry it for 12 h under the conditions of low temperature (-80 °C) and high vacuum (1 Pa) to obtain 40 mg of black-brown solid. Disperse the 40 mg of black-brown solid in 200 mL of ultrapure water, adjust the pH value to 7.5 with 0.01 M ammonia water solution, add 500 mL of 120 mg / mL potassium permanganate solution under vigorous stirring, and stir vigorously for 15 min to fully dissolve it to obtain Solution C. The mass ratio of the black-brown solid to potassium permanganate in Solution C is 1:1.5. Stir and react at room temperature for 4 h to obtain the product PtPDs@MnO2 NPs (abbreviated as PtPDM); Step 4: At room temperature, first centrifuge the product PtPDM at 10,000 rpm for 10 min, then wash it three times with ultrapure water, and freeze-dry it for 12 h under the conditions of low temperature (-80 °C) and high vacuum (1 Pa) to obtain 20 mg of black solid. Add 20 mg of black solid and 13 mg of polyvinylpyrrolidone (PVP-K30, the molecular weight of polyvinylpyrrolidone is 8,000 - 12,000 Da) to 10 mL of ethylene glycol, ultrasonically treat for 15 min, and then stir vigorously at 90 °C for 10 min to fully mix. Then add 380 mL of 40 mg / mL NaBH4 solution and 380 mL of 60 mg / mL HAuCl4 solution to the above suspension in sequence. Among them, the mass ratio of NaBH4 to HAuCl4 is 1:1.5. Continue to stir in the dark at 90 °C for 60 min to obtain the product PtPDs@MnO2@Au NPs (abbreviated as PtPDMA).

[0025] In addition, for comparison, the present invention provides the synthesis of polydopamine nanoparticles (PDA), which specifically includes the following steps: 300 mg of dopamine hydrochloride is uniformly dispersed in 25 mL of water. Adjust the pH value of the solution to 8 ± 0.1 with Tris buffer solution with a pH value of 7.5 - 9.5 and a concentration of 8 - 12 mM, and stir overnight (12 h) at room temperature to obtain the product PDA.

[0026] Characterize the sizes and dispersion degrees of the TEM images of PtPDs and the complexes PtPDM and PtPDMA by transmission electron microscopy (TEM), and the results are shown in Figure 1 . From Figure 1 it can be observed that: the prepared polydopamine-platinum(II) nanoparticles PtPDs are small in size and uniform in morphology, with a size of 100 - 150 nm (see Figure 1 A). Figure 1In B, the product after coating with a manganese dioxide shell is PtPDs@MnO2 NPs (PtPDM). It can be seen that a black shell is coated on the outer layer of PtPDs, inferring the successful coating of the manganese dioxide shell. Figure 1 In C, the product after loading ultrasmall gold nanoparticles is PtPDMA. It can be seen that there are particles with a diameter less than 10 nm around, proving the successful modification of ultrasmall gold nanoparticles. It proves the successful preparation of this nanoparticle, and it has good dispersibility and stability.

[0027] The nanoparticles were scanned at full wavelength using ultraviolet absorption spectroscopy, and the results are shown in Figure 2 . Figure 2 It can be seen that PtPDs has strong ultraviolet absorption in the near-infrared region, while PtPDs@MnO2 (PtPDM) after coating with a manganese dioxide shell shows an ultraviolet characteristic absorption peak at 420 nm, which means that manganese dioxide is successfully loaded onto the nanoparticle surface. For the product PtPDs@MnO2@Au (PtPDMA) after loading ultrasmall gold nanoparticles, an ultraviolet characteristic absorption peak appears at 560 nm, characterizing the successful loading of ultrasmall gold nanoparticles onto the nanoparticle surface and the successful preparation of this nanosystem.

[0028] The characterization results of the hydrodynamic diameter are as shown in Figure 3 (A). It can be seen from the figure that the hydrodynamic diameter of PtPDMA is: 290 nm, and it has good dispersibility (the PDI is less than 0.4), which is consistent with the particle diameter measured by TEM. The Zeta potential of PtPDMA nanoparticles and each component of the nanoparticles was studied using a Zeta potentiometer, and the results are shown in Figure 3 (B). Figure 3 It can be seen that the potential value of polydopamine nanoparticles (PDA) is -30.4 mV; the potential value of CDDP is +3.63 mV; the potential value becomes -13.35 mV after forming PtPDs; after loading the MnO2 shell, the potential value of PtPDs@MnO2 (PtPDM) becomes -38.10 mV; after modifying with ultrasmall gold nanoparticles, the potential value of PtPDs@MnO2@Au (PtPDMA) becomes -11.7 mV.

[0029] Through Figure 4 infrared absorption characterization, it can be seen that: in the figure, PDA shows three obvious characteristic bands at 1510 cm -1 , 1626 cm -1 and 1357 cm -1 , which are respectively attributed to the N-H bending vibration and stretching vibration of -OH, benzene ring and C-O bonds. Compared with pure PDA, the FTIR spectrum of PtPDs does not show the amine group at 1510 cm -1The stretching vibration at this position indicates that during the formation of PtPDs, the amine groups of PDA may have participated in the coordination or cross-linking reaction with platinum ions, resulting in the disappearance of the characteristic vibration of the amine groups. In addition, this may also mean that PDA formed nanoparticle spheres during the self-polymerization process, thus changing its infrared spectral characteristics. 548 cm -1 The corresponding position at this point is the characteristic peak of MnO2, indicating that the prepared material contains MnO2. 550 cm -1 and 557 cm -1 The corresponding position at this point is the characteristic peak of Au, indicating that the prepared material contains Au. This further illustrates the synthesis of the nanomaterials.

[0030] Through inductively coupled plasma optical emission spectrometry (ICP-OES) technology, we successfully determined the standard curve of platinum element and obtained the drug loading efficiency of cisplatin as 17.13% based on this. An in-depth study was conducted on the in vitro drug release characteristics of the nano-drug PtPDMA, as Figure 5 shown. In the 48-hour drug release experiment, when there was no 808 nm laser irradiation, the drug release rate of the nano-drug PtPDMA in phosphate buffer at pH 7.4 was 8.24% ± 0.69%, while under the condition of pH 7.4 with laser irradiation, the drug release rate was significantly increased to 24.71% ± 3.62%, an increase of 16.47% compared with the former. This result indicates that when 808 nm laser irradiation was introduced, the drug release rate of the laser irradiation group was improved compared with the non-laser group. In particular, in phosphate buffer at pH 5.5, the drug release rate was as high as 46.40% ± 2.77%, and under the condition of pH 5.5 with laser irradiation, the drug release rate reached 69.34% ± 3.74%. These results reveal that in a slightly acidic environment, the nano-drug exhibits "acid-responsive" drug release characteristics. The MnO2 in the nano-drug undergoes acid-responsive decomposition, and at the same time, laser irradiation causes the chemical bonds in PtPDs to break, thereby promoting the release of divalent cisplatin and achieving dual-stimulus-responsive drug release. In addition, the heat generation effect of PDA under near-infrared laser irradiation reduces the viscosity of the surrounding medium, further promoting the diffusion of the drug and making the drug release rate more rapid under laser irradiation. This dual-stimulus-responsive drug release characteristic is expected to significantly enhance the anti-tumor effect of the drug at the tumor site.

[0031] Good biosafety is a prerequisite for in vivo experiments. Deionized water and normal saline were used as positive control and negative control respectively to observe the hemolysis phenomenon of the nano-drug PtPDMA, and the results are shown in Figure 6 As Figure 6 shown, even when the concentration of the nano-drug PtPDMA NPs was as high as 400 mg / mL -1 , the hemolysis rate was still less than 5%, indicating that the biocompatibility of the whole system was good and the safety was relatively high.

[0032] Figure 7 It shows that after the nano-drug PtPDMA NPs solution with different concentrations (0, 50, 100, 200, and 400 mg / mL) was irradiated with a laser of 1.5 W / cm² for 10 min, the temperature increased sharply. The temperatures rose to 41.9 °C, 50.1 °C, 58.1 °C, 64.5 °C respectively, with temperature increases of 16.0 °C, 24.7 °C, 33.6 °C, 39.6 °C respectively. While the PBS solution without adding nano-drug PtPDMA NPs only rose to 33.8 °C, with a temperature increase of 8.9 °C. In particular, Figure (B) depicts the temperature rise curve of the PtPDMA NPs solution with a concentration of 200 mg / mL after being irradiated with lasers of different powers (0.5, 0.8, 1.0, 1.25, 1.5 W / cm²) for 10 min. The figure shows that as the laser power increases, the solution temperature shows a gradually increasing trend, and the temperatures rise to 38.1 °C, 45.8 °C, 52.1 °C, 57.9 °C, 60.4 °C respectively, with temperature increases of 14.7 °C, 22.6 °C, 28.7 °C, 32.3 °C, 36.6 °C respectively. These results strongly prove that this nano-drug has excellent photothermal conversion performance, indicating that it can be used as an effective medium for photothermal therapy and shows broad application prospects in the field of anti-tumor therapy.

[0033] The photothermal properties of PtPDMA were systematically evaluated using near-infrared imaging technology. Figure 8 The infrared thermal imaging diagrams of different concentrations of the product PtPDMA irradiated with an 808 nm laser (1 W / cm²) for 10 min are given. As Figure 8 shown, the observed results are consistent with the expected photothermal effect curve. It gradually changes from the initial low-temperature blue or green area to the high-temperature red or yellow area. The higher-concentration PtPDMA samples show a faster heating rate and a higher equilibrium temperature under laser irradiation, which is mainly attributed to the higher absorption and conversion efficiency of light energy by the material at high concentrations, thus being able to convert light energy into heat more effectively.

[0034] Furthermore, the photothermal conversion efficiency of PtPDMA in living mice was explored. Figure 9 The infrared thermal imaging diagram of the product PtPDMA of the present invention on H22 tumor-bearing mice irradiated with an 808 nm laser (1 W / cm²) for 5 min in the mouse body is given. Figure 9The experimental results showed that when saline was injected into mice as a control group, there was no significant fluctuation in their body temperature. However, in the mice injected with PtPDs@MnO2@Au (PtPDMA), the temperature in the tumor area was significantly higher than that in the corresponding area of the mice injected with saline. This phenomenon was attributed to the enhanced permeability and retention effect (EPR) of the nanoparticles and their high selective enrichment ability for tumor tissues, enabling PtPDMA to accumulate efficiently at the tumor site. The effectiveness of PtPDMA in achieving photothermal conversion in mice was verified.

[0035] In vitro cell inhibitory effect The cell viability was detected by the standard MTT method. First, the anti-tumor effect of PtPDMA was preliminarily evaluated through in vitro cell experiments. PtPDMA at different concentrations (0, 25, 50, 75, 100, and 150 mg / mL) was co-cultured with four cell lines, namely HL-7702, HeLa, 4T1, and HepG2. The results are shown in Figure 10 . As Figure 10 shown in A, the activity of tumor cells gradually decreased with the increase in the concentration of PtPDMA, showing an obvious downward trend. At the same concentration, the nanodrug PtPDMA had the least cytotoxicity to normal hepatocytes HL-7702 and was particularly significant to HepG2 tumor cells. Figure 10 B in shows the cytotoxicity results of the nanodrug PtPDMA under laser irradiation. PtPDMA can exert the effect of photothermal therapy (PTT) under the irradiation of 808 nm laser, further enhancing the killing ability of tumor cells. When the concentration of PtPDMA reached 150 mg / mL and combined with laser irradiation, the cell survival rate decreased to less than 30%, which fully confirmed that the prepared PtPDMA could successfully achieve the combined treatment effect.

[0036] In vivo tumor treatment For the in-depth exploration of the anti-tumor experiment in mice, we used H22 cells to construct a subcutaneous transplanted tumor model in BALB / c mice, and studied the anti-tumor effect of the nanodrug through tail vein injection combined with laser control. The experimental design was as follows: Mice with a tumor volume of about 100 mm³ were selected and randomly divided into six experimental groups: (1) PBS group; (2) CDDP group; (3) PADMA group; (4) PADMA+L group; (5) PtPDMA group; (6) PtPDMA+L group. Drugs were administered every other day (dosage: containing 5 mg / kg of CDDP), seven times in total, and drug injection and necessary laser irradiation treatment were carried out on the mice. The body weight and tumor volume of the mice were measured during drug administration, and 808 nm laser (1 W / cm 2, irradiate the tumor area (5 min). On the 14th day, all mice were sacrificed, and their tumors and major organs were collected and sectioned and stained to observe tissue changes. The results are shown in Figure 11 .

[0037] Figure 11 The data in (A), (B), and (C) in the figure show that compared with the PBS control group, the tumor volume of the mice treated only with the chemotherapeutic drug cisplatin (Pt) decreased slightly, but it indicated that the therapeutic effect of using chemotherapeutic drugs alone was not good. Compared with the PBS control group, the tumor volume of the PADMA nanocomposite treatment group decreased, which confirmed that the combined application of chemodynamic therapy (CDT) and enzyme activity could exhibit certain anti-tumor effects. Further, the tumor volume of the PADMA+L group irradiated treatment group decreased more significantly compared with the non-irradiated group, demonstrating the obvious curative effect of photothermal therapy (PTT). Particularly prominent is that the tumor volume of the mice in the PtPDMA+L group decreased relatively significantly, which strongly proved that under laser irradiation, the CDT / PTT / chemotherapy / enzyme activity quadruple therapy strategy mediated by the PtPDMA nanocomposite could efficiently exert anti-tumor effects and showed superior tumor treatment effects. In addition, Figure (D) shows the body weight changes of the mice during the 14-day experimental period, and there were no significant differences among the groups, indicating that the studied nano-drug delivery system had good biosafety and had no adverse effects on the overall health status of the mice.

[0038] The tumor tissue and tissue sections of the heart, liver, spleen, lung, and kidney were stained with hematoxylin-eosin (HE), and a fluorescence microscope was used to observe the tissue damage. In this staining process, the basic dye hematoxylin is mainly responsible for staining the chromatin in the cell nucleus and ribosomes in the cytoplasm purple-blue, while the acidic dye eosin mainly renders the components in the cytoplasm and extracellular matrix red, mainly making the components in the cytoplasm and extracellular matrix red. The results are shown in Figure 12 . Figure 12 The experimental results show that compared with the organs of the PBS control group, there were no obvious changes in the organs treated with the nano-drug PADMA, the nano-drug PADMA+L, the nano-material PtPDMA after loading drugs, and the nano-material PtPDMA+L after laser irradiation, indicating the biosafety of the nano-drugs and nano-materials. This result further confirmed that the synthesized multifunctional nano-drugs had good biocompatibility in vivo.

[0039] HE staining, Ki67 immunohistochemical analysis, and Tunel staining of tumor tissue sections were performed to detect the effects of different groups on the proliferation and apoptosis of HepG2 tumor cells. The results are shown in Figure 13 . As Figure 13As shown, in terms of the proliferation level, cell proliferation in the PBS group was the most obvious. The proliferation level in the PADMA group was similar to that in the PBS group. The proliferation level of tumor cells in the PtPDMA group was lower than that in the PBS group but higher than that in the PADMA+L group treated with laser irradiation. In terms of apoptosis, apoptosis of tumor cells in the PBS, PADMA, and PtPDMA groups was less, and the apoptosis phenomenon in the PADMA+L group was the most obvious. The results showed that the proliferation level of PtPDMA+L was the weakest while the apoptosis phenomenon was the most obvious. This experimental result once again proved that the combined treatment of PTT, CDT, enzyme activity treatment, and chemotherapy had good anti-tumor effects.

Claims

1. A preparation method of a dopamine-platinum (II) self-polymerized O2 and H2O2 self-supplying nanopharmaceutical system, characterized in that, It includes the following steps: Step 1: At room temperature, disperse cisplatin and silver nitrate solids in water to obtain a mixed solution A; adjust the pH value of solution A to 2 - 2.5, then stir the mixture in the dark at 65 - 90 °C for 12 - 18 h, cool to room temperature, and perform solid-liquid separation to obtain activated cisplatin; Step 2: Uniformly disperse the activated cisplatin and dopamine hydrochloride in water to obtain a mixed solution B; adjust the pH value of solution B to 8 ± 0.1 and stir at room temperature for 12 - 18 h to obtain the product PtPDs; Step 3: At room temperature, subject the product PtPDs to solid-liquid separation, washing, and freeze-drying to obtain a black-brown solid; disperse the black-brown solid in ultrapure water, adjust the pH value to 7.0 - 8.5, then add potassium permanganate with stirring to obtain solution C; stir and react at room temperature for 4 - 6 h to obtain the product PtPDM; Step 4: At room temperature, subject the product PtPDM to solid-liquid separation, washing, and freeze-drying to obtain a black solid; dissolve the black solid and polyvinylpyrrolidone in ethylene glycol, then heat to 90 ± 2 °C and mix evenly, then add NaBH4 and HAuCl4, and continue to stir and react at 90 ± 2 °C in the dark for 60 - 70 min to obtain the product.

2. The preparation method of the dopamine-platinum(II) self-polymerized O2 and H2O2 self-supplying nanomedicine system according to claim 1, wherein, In Step 1, the mass ratio of the cisplatin and silver nitrate solids is 1:1 - 1.

5.

3. The preparation method of the dopamine-platinum (II) self-polymerized O2 and H2O2 self-supplying nanopharmaceutical system according to claim 1, wherein, In Step 2, the mass ratio of the activated cisplatin to dopamine hydrochloride is 1:2.5 - 3.

5.

4. The preparation method of the dopamine-platinum (II) self-polymerized O2 and H2O2 self-supplying nanodrug system according to claim 1, characterized in that, In Step 3, the mass ratio of the black-brown solid to potassium permanganate is 1:1.5 - 3.

5. The preparation method of the dopamine-platinum (II) self-polymerized O2 and H2O2 self-supplying nano-drug system according to claim 1, wherein, In Step 4, the mass ratio of the black solid to polyvinylpyrrolidone is 1:0.6 - 1.5, and the molecular weight of the polyvinylpyrrolidone is 8000 - 12000 Da.

6. The preparation method of the dopamine-platinum (II) self-polymerized O2 and H2O2 self-supplying nano-drug system according to claim 1, characterized in that, In Step 4, the mass ratio of NaBH4 to HAuCl4 is 1:1.2 - 1.

5.

7. The preparation method of the dopamine-platinum(II) self-polymerized O2 and H2O2 self-supplying nanodrug system according to claim 1, wherein, In Step 4, the mass ratio of the black solid to NaBH4 is 1:0.6 - 0.

8.

8. A dopamine-platinum(II) self-polymerized O2 and H2O2 self-supplying nanodrug system prepared by the method according to any one of claims 1 to 7.

9. Use of the dopamine-platinum(II) self-polymerized O2 and H2O2 self-supplying nanodrug system according to claim 8 in the preparation of antitumor drugs.

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