Preparation method and application of bimetallic composite nano-drug formed by modifying rare metal Ru on surface of HMCuS

Abstract

According to the invention, the surface of HMCuS is modified with rare metal Ru to form a bimetallic composite nano-drug carrier, that is, the surface of HMCuS is ingeniously modified with the rare metal Ru through an electrochemical replacement method to form an HMCuS-Ru composite material, and the strategy utilizes the higher potential energy characteristic of the metal Ru relative to Cu to realize firm combination of Ru on the surface of CuS. The HMCuS is famous for excellent photothermal conversion efficiency, excellent stability and economical and efficient manufacturing cost, the HMCuS is further endowed with double-enzyme activity through combination with the metal Ru, and the functional range and application potential of the HMCuS are remarkably widened. Besides, the nano-drug HMCuS-Ru-ICG-GOX-9R-P is prepared on the basis of the preparation method disclosed by the invention, and due to the design, the nano-drug is endowed with high specific recognition capability on liver cancer cells, the treatment selectivity is greatly improved, and the potential damage to normal cells is effectively reduced. In a subsequent tumor-bearing mouse experiment, the composite nano material shows a surprising tumor inhibition effect, and the tumor inhibition rate is as high as 91.1%.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and health technology, and specifically relates to a preparation method and application of a bimetallic composite nanomedicine formed by modifying the surface of HMCuS with a rare metal Ru. Background Art

[0002] Ruthenium (Ru) complexes, as candidates for the next generation of metallodrugs, have shown significant antitumor activity. In particular, certain photoresponsive Ru complexes have attracted much attention in the field of anticancer phototherapy, and one study has even entered the clinical trial stage. These Ru complexes usually show low toxicity in a dark environment, but once exposed to light, they can generate toxic Ru ligands for photoactivated chemotherapy or generate reactive oxygen species (ROS), playing a key role in photodynamic therapy.

[0003] Although small molecule ruthenium (Ru) drugs have shown great potential in anticancer therapy, their nonspecific delivery, rapid blood clearance, and low accumulation in tumor sites limit their further application. To address these challenges, two main strategies have been proposed. The first strategy is to physically encapsulate Ru drugs in nanocarriers, aiming to precisely deliver Ru complexes to tumor sites. However, this approach has the risk of drug leakage during circulation, which may cause serious side effects. In addition, achieving a reproducible formulation of nanocarrier drugs is also challenging, resulting in differences between different batches. The second strategy is to combine Ru complexes with polymers through covalent coupling to form a multi-prodrug system. Although multi-prodrug systems have high drug loading capacity, synthetic polymer backbones usually have difficult-to-control polydispersity. Therefore, developing a simple and precise ruthenium (Ru) drug delivery technology remains a very challenging task.

[0004] Based on this, this application was developed. Summary of the invention

[0005] The present invention aims to overcome the defects of the prior art and provide a HMCuS surface modified with rare metal Ru to form a bimetallic composite nanomedicine. The composite nanomedicine is a new material structure, which combines the rare metal Ru with the inorganic metal Cu to form a composite bimetallic nanomaterial with dual enzyme activity modified by targeted peptides. The material has the absolute advantages of strong stability, high selectivity and multipolarized therapeutic effect.

[0006] The present invention also provides a preparation method and application of the bimetallic composite nano-medicine formed by modifying the surface of the HMCuS with the rare metal Ru.

[0007] Another object of the present invention is to provide a novel use of inorganic metal anti-tumor drug delivery.

[0008] To achieve the above object, the present invention proposes the following technical solutions: A method for preparing a bimetallic composite nano drug carrier by modifying the surface of HMCuS with a rare metal Ru (i.e., how to modify the rare metal ruthenium on the surface of hollow mesoporous copper sulfide), comprising the following steps: Step 1, dispersing ruthenium chloride trihydrate and hollow mesoporous copper sulfide nano drug carrier HMCuS in ethylene glycol to obtain a mixed solution A; Step 2, add polyvinyl pyrrolidone and methyl benzoate to the mixed solution A, mix well, and then heat in a constant temperature oil bath at 160-200° C. for 2-4 h; Step 3: After the reaction is completed, the mixture is cooled to room temperature, and solid-liquid separation is performed (centrifugation at 16000 rpm for 10-15 min), followed by washing (ethanol / acetone = 1:5-20, ultrasonic washing for 2-4 times), and freeze-drying for 20-30 hours under ultra-low temperature and high vacuum conditions to obtain a solid black powder product C (HMCuS-Ru), which is a nano drug carrier. The black powder can be dispersed in water at a solid-liquid ratio of 1:1000-1500 for later use.

[0009] Specifically, the mass ratio of ruthenium chloride trihydrate to HMCuS in step 1 can be: 1:0.3-0.7.

[0010] Specifically, the mass ratio of polyvinyl pyrrolidone to methyl benzoate in step 2 may be 1:0.1-0.3. The mass ratio of HMCuS to polyvinyl pyrrolidone may be 1:70-90.

[0011] Furthermore, the molecular weight of the polyvinyl pyrrolidone (PVP-K30) in step 2 is preferably 60,000-80,000 Da.

[0012] The invention provides a bimetallic composite nano drug carrier prepared by the above-mentioned preparation method, in which the surface of HMCuS is modified with the rare metal Ru.

[0013] The present invention also provides the application of the bimetallic composite nano drug carrier in drug loading, for example, the nano drug carrier is used to prepare drugs, especially anti-tumor drugs.

[0014] The present invention also provides a method for preparing a rare metal composite bimetallic nanomedicine modified with a liver cancer targeting peptide using the above-mentioned drug carrier, which comprises the following steps: Step a, dissolving product C and indocyanine green (ICG) in ultrapure water by ultrasonic treatment at room temperature, then stirring for 12-36 hours under light-proof conditions, centrifuging, washing, and freeze-drying for 12-24 hours to obtain product D; product D can be redissolved in ultrapure water for later use; Step b, under constant conditions of 2-8°C, glucose oxidase (Gox) is dissolved in a phosphate buffer with a pH value of 3-4, and then product D is added, and the mixture is allowed to stand in the dark for adsorption for 30-90 minutes, and then centrifuged, washed, and freeze-dried for 12-24 hours to obtain product E; product E can be redissolved in ultrapure water for later use; Step c, at room temperature, dissolve the liver cancer targeting peptide (9R-P peptide) in a phosphate buffer with a pH value of 7-9, add product E, stir in the dark for 12-48 h, centrifuge, wash, and freeze-dry for 20-30 h to obtain product F. Further preferably, product F can be re-dissolved in ultrapure water, centrifuged, washed, and freeze-dried for 20-30 h under low temperature and high vacuum conditions, and can be used for mild photothermal treatment of liver cancer targeting peptide-modified hollow mesoporous copper sulfide nanomedicine (HMCuS@HR-ICG-GOX-9R-P, abbreviated as HRIG9R).

[0015] Specifically, the mass ratio of product C to indocyanine green in step a can be 1:1-2; the mass ratio of glucose oxidase to product D in step b can be 1:1-2; the mass ratio of liver cancer targeting peptide to product E in step c can be 1-2:1, and the sequence of the liver cancer targeting peptide (9R-P peptide) is: RRRRRRRRRGSGSWHLDYPSMWYLD.

[0016] The present invention also provides a rare metal composite bimetallic nanomedicine modified with a liver cancer targeting peptide prepared by the above method.

[0017] The present invention provides a liver cancer targeting peptide modified composite bimetallic nanomedicine prepared by the above-mentioned innovative method, which not only has high selectivity, but also integrates a variety of treatment methods including photothermal therapy, chemodynamic therapy and photodynamic therapy. Specifically, the present invention provides a synergistic therapeutic drug integrating chemodynamics-photothermal-photodynamic therapy, the core of which is the composite nanomedicine carrier containing the rare metal Ru. The present invention cleverly introduces indocyanine green (ICG) and glucose oxidase (Gox) into the composite metal material, so that the nanomaterial can be applied to a variety of combined treatment strategies such as photodynamic therapy and mild photothermal therapy, showing a wide range of therapeutic potential and application prospects. The present invention cleverly introduces indocyanine green (ICG) with fluorescent properties as a fluorescent marker, which not only facilitates the precise positioning of the nanomaterial in subsequent biological experiments, but also enhances its advantages in visual operation. Glucose oxidase (Gox), as a vital oxidoreductase, plays a diversified function in the present invention: it can promote the oxidation of β-D-glucose into gluconic acid, which can block the generation of ATP and achieve the effect of starvation therapy. In addition, Gox can also efficiently degrade overexpressed heat shock proteins (HSPs), thereby enhancing the clearance effect of photothermal therapy (PTT) on tumor cells. By precisely controlling the pH value to make Gox positively charged, it is tightly coated on the surface of the HMCuS-Ru material using the principle of charge adsorption, thereby achieving further functional integration. Finally, the present invention wraps a layer of liver cancer targeting peptide (9R-P) on the surface of the material. This design gives the nanomaterial a highly specific recognition ability for liver cancer cells, greatly improves the selectivity of treatment, and effectively reduces potential damage to normal cells. In subsequent tumor-bearing mouse experiments, the composite nanomaterial showed an amazing tumor inhibition effect, with a tumor inhibition rate of up to 91.1%. In summary, the present invention successfully synthesized a composite nanomaterial that integrates efficient recognition and precise killing, bringing a new solution to the field of tumor treatment.

[0018] The present invention introduces liver cancer targeting peptides into the surface of the composite bimetallic drug carrier, which can be applied to the specific targeting of liver cancer. Compared with the prior art, the composite bimetallic nano drug carrier containing rare metals modified with liver cancer targeting peptides of the present invention has the following beneficial effects: 1) The liver cancer targeting peptide-modified composite bimetallic nanomedicine prepared by the present invention not only integrates rare metals, but also has low-cost and easy-to-obtain raw materials, and also exhibits the remarkable characteristics of low toxicity and high safety.

[0019] 2) In the binding reaction of HMCuS and metal Ru in the present invention, an efficient one-pot method is used for direct synthesis, which is not only simple to operate but also has a considerable yield. In addition, by modifying the surface of the material with a liver cancer-targeting polypeptide, not only the hydrophilicity of the prepared drug carrier in aqueous solution is significantly improved, but also its targeting accuracy is significantly improved.

[0020] 3) The present invention combines glucose oxidase (Gox) with nanomaterials, and this design enables the material to implement mild photothermal therapy. Compared with traditional photothermal therapy, this method significantly reduces damage to normal tissues, thereby greatly improving the practicality of the material and the safety of treatment.

[0021] 4) The present invention cleverly combines the excellent stability and excellent photothermal therapy effect of hollow mesoporous copper sulfide material (HMCuS) with the dual enzyme activity of rare metal ruthenium, achieving functional complementarity between the two, greatly enhancing the overall functionality and application range of the material. The indocyanine green (ICG) used in the preparation is not only convenient for tracking as a fluorescent marker, but also can produce singlet oxygen under light conditions, thereby promoting the generation of more reactive oxygen species (ROS) and achieving effective killing of tumor cells. At the same time, the introduced glucose oxidase (Gox) can decompose the overexpressed heat shock proteins produced by tumor cells during photothermal therapy, while inhibiting the generation of ATP, achieving the starvation treatment effect on tumor cells. By modifying the liver cancer targeting peptide, the material of the present invention has extremely low toxicity to normal tissue cells, but shows strong toxicity to liver cancer cells, successfully integrating photothermal therapy, photodynamic therapy and chemotherapy into a combined tumor treatment strategy.

[0022] 5) The present invention has many advantages such as simple operation, excellent safety, mild and controllable reaction conditions and low cost. The developed composite nano drug carrier containing rare metals not only exhibits significant synergistic anti-tumor effects, but also has a small size and specific targeting, which is conducive to circulation and transmission in the body. In addition, the reagents used in the entire preparation process are of low toxicity, which further ensures the safety and reliability of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 TEM images of HMCuS (a), product C (HMCuS-Ru) (b) and product E (HRIG) (c), and a transmission electron microscope (TEM) image of product F (HRIG9R) (d) in Example 1; Figure 2 The UV absorption graphs of HMCuS, GOX, product C (HMCuS-Ru, abbreviated as HR), and product E (HRIG) in Example 1; Figure 3is a Zeta potential diagram of HMCuS, product C (HMCuS-Ru, abbreviated as HR), glucose oxidase (Gox), product E (HRIG) and product F (HRIG9R) in the present invention; Figure 4 IR spectra of HMCuS and product C (HMCuS-Ru) in Example 1; Figure 5 is the EDS diagram of product E (HRIG) in Example 1; Figure 6 The product F (HRIG9R) in Example 1 was irradiated with 808 nm laser (1 W / cm 2 ) for 5 min at different concentrations (a) and the product F (HRIG9R) 100 mg / mL was irradiated at 808 nm laser for 5 min at different powers (b); Figure 7 The product F (HRIG9R) in Example 1 was irradiated with 808 nm laser (1 W / cm 2 ) under irradiation of 100 μg / mL during 5 laser on / off cycles; Figure 8 The ·OH release of product F (HRIG9R) in Example 1 under different pH conditions (a) and the ·OH release of product F (HRIG9R) at different concentrations (b); Fig. 9 The cytotoxicity screening data of (a) product C (HR), (b) product D (HRI), (c) product E (HRIG), and (d) product F (HRIG9R) in Example 1; and (e) cytotoxicity screening data of HRIG and HRIG9R after near-infrared (NIR) irradiation. Fig.10 In vivo fluorescence imaging of H22 tumor-bearing mice. (a) In vivo fluorescence imaging at different time points after tail vein injection of ICG, (b) quantitative analysis of its in vivo fluorescence intensity, (c) fluorescence imaging of isolated organ tissues, (d) quantitative analysis of fluorescence intensity of isolated organ tissues, (e) in vivo fluorescence imaging at different time points after tail vein injection of HRIG9R, (f) quantitative analysis of its in vivo fluorescence intensity, (g) fluorescence imaging of isolated organ tissues, (h) quantitative analysis of fluorescence intensity of isolated organ tissues; Fig.11These are (a) digital images of tumor tissue, (b) tumor growth curves, (c) graphs of changes in mouse body weight, (d) graphs of changes in tumor weight, and (e) graphs of tumor inhibition rates at the end of treatment of H22 tumor-bearing mice with products C (HMCuS-Ru, HR), product E (HRIG), product E+NIR, ICG+NIR, product F (HRIG9R), and product F+NIR in Example 1. DETAILED DESCRIPTION

[0024] In order to have a deeper understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0025] Experimental instrument name and model: Japanese 200KV field emission transmission electron microscope JEOL JEM-F200; FreeZone 6L, a freeze-drying system from the United States; German electronic analytical balance BSA224S; Shanghai desktop centrifuge TGL-16C; NanoZS90 laser particle size and Zeta potential analyzer from the UK; American multifunctional microplate reader BioTek; American handheld thermal imager TiS75+.

[0026] Unless otherwise specified, room temperature refers to 25±5℃.

[0027] In the following examples, unless otherwise specified, the raw materials used are all common commercial products that can be directly purchased, or can be prepared by conventional methods in the art. For example, the hollow mesoporous copper sulfide nano drug carrier HMCuS can be prepared by referring to the method in the Chinese patent (patent number: ZL 202210695944.X, invention name: Preparation method and application of a hollow mesoporous copper sulfide nano drug carrier).

[0028] Example 1 A method for preparing a bimetallic composite nano drug carrier formed by modifying the surface of HMCuS with a rare metal Ru, comprising the following steps: Step 1, dispersing 50 mg of ruthenium chloride trihydrate and 25 mg of hollow mesoporous copper sulfide nanodrug carrier HMCuS in 50 mL of ethylene glycol to prepare a mixed solution A; Step 2: Subsequently, 0.424 g of methyl benzoate and 2 g of polyvinyl pyrrolidone were added to the mixed solution A to obtain a mixed solution B. The mixed solution B was subjected to ultrasonic treatment for 30 min until the solution became clear and free of obvious particles. Then, the mixture was heated in a constant temperature oil bath at 180°C for 3 h; the molecular weight of the polyvinyl pyrrolidone used was 60,000-80,000 Da; Step 3: After the reaction is completed, cool to room temperature and then centrifuge at 16000 rpm / min for 10 min. Use a solution prepared with ethanol and acetone in a volume ratio of 1:10 for three ultrasonic washings, and then freeze-dry for 24 h at an ultra-low temperature of -80°C and a high vacuum of 1 Pa to obtain a solid black powder product C (HMCuS-Ru, abbreviated as HR), which is a nano drug carrier. Redissolve with ultrapure water and set aside. This is a composite nano drug carrier containing rare metals.

[0029] The present invention also provides a method for preparing a composite bimetallic nanomedicine containing rare metals modified with a liver cancer targeting peptide using the above nanomedicine carrier, which comprises the following steps: Step a: Under room temperature, 35 mg of the above product C and 60 mg of indocyanine green (ICG) were ultrasonically dissolved in 80 mL of ultrapure water. Then, the mixture was magnetically stirred for 24 h under light-proof conditions, centrifuged at 1200 rpm for 10 min, washed three times with ultrapure water, and freeze-dried at low temperature (-80°C) and high vacuum (1 Pa) for 24 h to obtain product D (HMCuS-Ru-ICG, abbreviated as HRI). Step b, at a constant 4°C, 10 mg of glucose oxidase (Gox) was dissolved in 20 mL of phosphate buffered saline (PBS) at pH = 4 to obtain a mixed solution H. 10 mg of product D was dispersed in the mixed solution H, allowed to stand for adsorption for 1 h in the dark, centrifuged at 12000 rap for 10 min, washed three times with ultrapure water, and freeze-dried at low temperature (-80°C) and high vacuum (1 Pa) for 24 h to obtain product E (HRIG); Step c, at room temperature, dissolve 15 mg of 9R-P peptide (the sequence of the liver cancer targeting peptide 9R-P is: RRRRRRRRRGSGSWHLDYPSMWYLD) in 50 mL of phosphate buffer (PBS) with a pH value of 8, add 8 mg of product E, stir magnetically for 24 h in the dark, centrifuge at 12000 rpm for 15 min, wash three times with ultrapure water, freeze-dry for 24 h under low temperature (-80°C) and high vacuum (1Pa) conditions to obtain product F (HRIG9R), that is, a composite bimetallic nanodrug containing rare metals modified with a liver cancer targeting peptide.

[0030] The morphology, size and dispersion of the composites containing rare metals were characterized by transmission electron microscopy (TEM). Figure 1 shown. Figure 1 Part (a) clearly shows that HMCuS has a significant hollow mesoporous structure, the material size is uniform, the dispersion is good, and the particle size is approximately between 100-150 nm. In part (b), the product CHMCuS-Ru shows the morphology of the hollow copper sulfide surface successfully modified with the rare metal Ru. Compared with (a), there are obvious particles attached to the surface of HMCuS, which is preliminarily judged to be the successful loading of Ru metal. Further, the product E HRIG in part (c) is an image after a layer of glucose oxidase (Gox) film is modified on the surface of the complex. Compared with (b), the particles on the surface become less obvious, and it is preliminarily inferred that Gox has been coated on the surface of the material. Finally, the product F HRIG9R in part (d) is the morphology after the 9R-P peptide is connected to HRIG. It can be observed that there is a layer of transparent material coating around the material, which proves the successful modification of the 9R-P peptide.

[0031] The nanomaterials were scanned at full wavelength using UV absorption spectroscopy. Figure 2 shown. Figure 2 In the figure, it can be clearly observed that after modification with indocyanine green (ICG), the ultraviolet absorption wavelength of the material product EHRIG at 800 nm has been significantly enhanced. This change strongly proves that ICG has been successfully loaded into the material.

[0032] The Zeta potential of the nanoparticles in each synthesis step was analyzed using a Zeta potential instrument. Figure 3 shown. Figure 3 It is clearly shown that the Zeta potential value of HMCuS is -12.4 mV; after modification with Ru metal, the negative charge of the material is significantly enhanced to about -23.7 mV. Furthermore, when glucose oxidase (Gox) is modified to the HMCuS-Ru surface under pH=4, the overall charge of the material is weakened to -10.4 mV because the potential of Gox under this condition is +5.7 mV. Finally, since the 9R-P peptide itself has a strong positive charge, after it is modified to the HRIG surface, the Zeta potential of the material becomes +13.2 mV. This change once again verifies the successful modification of the 9R-P peptide.

[0033] HMCuS-Ru was characterized by infrared spectroscopy. Figure 4 shown. Figure 4 Clearly shows that metal Ru is between 3000 and 3500 cm -1Compared with the infrared spectrum of HMCuS, HMCuS-Ru has unique characteristic peaks in the range of 3000 cm -1 The characteristic peak appearing at clearly points to the changes brought about by the introduction of Ru, which further confirms that Ru has been successfully modified on the surface of HMCuS.

[0034] HRIG was analyzed by transmission electron microscopy. Figure 5 shown. Figure 5 It is clearly shown that the samples mainly contain copper (Cu), ruthenium (Ru), phosphorus (P) and other elements. Among them, phosphorus is a unique element of glucose oxidase (Gox), and this discovery provides strong evidence for the successful modification of Gox to the surface of the material.

[0035] The final nano drug product F was prepared into solutions of different concentrations with ultrapure water, and then irradiated with near-infrared light with a wavelength of 808 nm and a power density of 1 W / cm². The temperature change of the solution was monitored using a FLIR thermal imager, and a temperature change curve in the aqueous solution was plotted. The results are shown in Figure 6 .observe Figure 6 It can be seen that: Part (a) shows the effect of concentration change on temperature change. When the concentration is 100 μg / mL, the nano drug carrier can be heated to 42°C within 5 minutes under 808 nm laser (1 W / cm²), which meets the standard of photothermal therapy; Part (b) shows the temperature change of the solution irradiated with different powers at the same drug concentration (100 μg / mL). It can be seen from the figure that under 808 nm laser (1 W / cm 2 ) irradiated for 5 minutes to raise the temperature to about 42°C. Combined with starvation therapy, the nanomaterial can effectively kill tumor cells while reducing damage to normal tissues. Therefore, the nanomaterial designed by the present invention is suitable for mild photothermal tumor treatment.

[0036] In order to evaluate the photothermal stability of nanodrugs, the present invention conducted multiple laser on / off cycle tests. In each cycle, the nanodrug solution (100 μg / mL) was subjected to 5 minutes of laser irradiation (808 nm laser, 1 W / cm 2 ), then turn off the laser and cool naturally to room temperature. This process was repeated 5 times. The temperature change in the cycle was recorded by plotting the temperature change over time. The results are shown in Figure 7 The results showed that HRIG9R nanoparticles (NPs) had excellent photothermal stability.

[0037] Figure 8The results of the ·OH release experiment are shown. In Figure (a), after setting different pH values ​​(5.5, 6.5, 7.5), it can be observed that the nanoparticles (NPs) exhibit certain acid response characteristics, specifically, when the pH value decreases, the release of OH gradually increases. This characteristic is expected to significantly reduce the potential damage to normal tissue cells. In Figure (b), by setting different drug concentrations (10, 20, 50, 100 μg / mL), it can be clearly seen that as the drug concentration increases, the amount of ·OH produced also shows a trend of gradually increasing.

[0038] In vitro cell inhibition The MTT experiment is used to detect the toxicity of the composite nano drug carrier containing rare metals to tumor cells.

[0039] The MTT method was used to detect the in vitro tumor (HL-7702, HeLa, Sun-739, HepG2) activity of the products C HMCuS-Ru (HR), D HMCuS-Ru-ICG (HRI), E HRIG and F HRIG9R nanoparticles (NPs). Taking HepG2 cells as an example, the experimental steps are as follows: First, HepG2 cells with good growth status were spread all over the bottom of the culture dish, then digested with trypsin and centrifuged by centrifuge. After the supernatant was removed, 1 mL of culture medium (DMEM culture medium, containing 1% penicillin-streptomycin and 10% fetal bovine serum, the same below) was added to blow the cells evenly, and then the cell suspension was diluted to an appropriate concentration to ensure that about 4500 cells were inoculated in each well of the 96-well plate and cultured for 24 h. Then, HR, HRI, HRIG and HRIG9R NPs were diluted to different concentrations with culture medium and placed in test tubes for later use. Four replicate wells were set for each concentration, and a blank control group without inoculated cells and a negative control group without drugs were set up for 48 h. For the laser control group experiment, it was divided into HRIG+NIR group and HRIG9R+NIR group. The laser group was irradiated with 808 nm laser (1 W / cm², 5 minutes) 12 hours after administration, and then continued to be cultured for 24 hours. The non-laser group was cultured for 24 hours after administration. After that, 50 µL of MTT solution with a concentration of 1 mg / mL was added to each well, and the liquid in the plate was aspirated after 4 hours of action. Then, 100 µL of DMSO was added to each well and shaken on a shaker for 10 minutes. Finally, the absorbance value A was measured at a wavelength of 570 nm using a multifunctional microplate reader. The cell inhibition rate was calculated by the formula: Cell inhibition rate × 100% = (average OD value of the negative group - average OD value of the experimental group) / (average OD value of the negative group - average OD value of the blank group). According to this formula, the inhibition rate of each concentration of samples on cells can be calculated, and the corresponding IC can be further calculated using the software. 50 value.

[0040] Fig. 9 The survival rate graphs (a), (b), (c), (d) of product C (HR), product D (HRI), product E (HRIG), and product F (HRIG9R) in Example 1 on HL-7702, HeLa, Sun-739, and HepG2 cells are given, as well as the toxicity test results (e) of product F (HRIG9R) and product E (HRIG) on four types of HepG2 cells under near-infrared (NIR) irradiation. Fig. 9 The proliferation rate results of HL-7702 cells were clearly displayed. Among them, the cell survival rate of the HRIG9R group remained at about 80% when the concentration was as high as 200 μg / mL. This data strongly proves that HRIG9R nanoparticles (NPs) have extremely low toxicity to normal tissue cells and are a nanomaterial with extremely high biosafety. At the same time, by comparing the inhibitory effects of different materials (HR, HRI, HRIG, HRIG9R) on tumor cells, it can be clearly seen that with the increase of material functions, its inhibitory effect on tumor tissue is becoming more and more significant. In particular, among three different tumor cells, HRIG9R showed obvious selective inhibition of HepG2 cells, which fully verified the targeting of 9R-P201. In addition, the present invention also investigated the toxicity of HRIG9R to HepG2 cells under laser irradiation. The results showed that the proliferation rate of cells decreased significantly under laser irradiation, which further proved that HRIG9R has a significant mild photothermal therapy effect.

[0041] In vivo tumor therapy In order to comprehensively evaluate the biodistribution of HRIG9R nanoparticles (NPs) in vivo, the present invention adopts the following experimental method: the nanomaterials were injected (5 mg / kg) into mice bearing H22 tumors through the tail vein, and the mice were dissected at 0, 1, 2, 4, 8, 12, 24, and 36 h after injection, and their heart, liver, spleen, lung, kidney, and tumor tissues were collected for subsequent fluorescence imaging analysis. Fig.10As shown in the figure, the results of fluorescence imaging clearly reveal the dynamic distribution characteristics of HRIG9R NPs in vivo: after injection, the fluorescence signal at the tumor site gradually increased, reaching a peak 12 hours after injection, and then began to weaken at 24 hours. This time-dependent fluorescence signal enhancement phenomenon is likely due to the combined effect of HRIG9R NPs through enhanced permeability and retention effect (EPR) and active and specific targeting of liver cancer cells. At the same time, fluorescence signals were also observed in liver and kidney tissues, which may be related to the metabolic process of nanoparticles in these organs. However, no obvious fluorescence signals were observed in organs such as the heart, spleen, and lungs, indicating that HRIG9RNPs, as a multifunctional nanodrug carrier, specifically delivers nanodrugs while having low toxicity to other tissues throughout the body.

[0042] In addition, the present invention randomly divided the mice into 7 groups, each group containing 6 mice. In order to construct a subcutaneous transplant tumor model in BALB / c mice, H22 cells were used. When the tumor volume reached about 100 mm 3 At this time, each group of mice began to be treated with different functional material groups, and each group of mice was given 5 mg / kg of the corresponding material. The 7 groups of mice received the following treatments: ① control group (normal saline, Saline), ② HR group, ③ HRIG group, ④ HRIG+NIR group, ⑤ ICG+NIR group, ⑥ HRIG9P nanoparticles (NPs) group, ⑦ HRIG9R nanoparticles (NPs) + NIR group. The treatment method was to administer the drug once every other day, for a total of three times. At the same time as the drug was administered, the weight and tumor volume of the mice were measured. In addition, every other day after each dose, an 808 nm laser (1 W / cm 2 The tumor area was irradiated for 5 min. The tumor volume of the mouse was calculated by the formula V=W 2 ×L / 2, where V represents the tumor volume, W represents the shortest diameter of the tumor, and L represents the longest diameter of the tumor. This experimental design aims to comprehensively evaluate the anti-tumor effects of different nanomedicines and the potential impact of laser irradiation on the therapeutic effect.

[0043] After three doses, Fig.11 As shown, the weight gain of mice in each material group was similar to that in the control group, indicating that the material itself had little effect on the growth of mice and did not cause obvious growth damage. Compared with the control group, the HRIG9R nanoparticle (NPs) group and the HRIG9R NPs+laser group showed significant inhibitory effects on tumor growth. Among them, the inhibitory effect of the HRIG9RNPs+laser group was the most prominent, with the tumor weight of mice in this group being only 0.18 g, much lower than the 1.1 g in the blank control group. In addition, the tumor volume of mice in the HRIG9R NPs+laser group was also significantly reduced to only 131.28 mm3 , which is much lower than the 748.83 mm in the control group. 3 The tumor inhibition rate was as high as 91.1%. This result fully proves that under laser irradiation, nanomedicine HRIG9R NPs can more effectively inhibit tumor growth.

[0044] In summary, the present invention successfully prepared a composite nano drug carrier containing rare metals, which exhibits excellent hydrophilicity, high safety, small size and specific targeting ability for liver cancer. First, the present invention innovatively synthesized a near-infrared light-responsive nanomaterial-rare metal ruthenium-modified copper sulfide nanoparticles (HMCuS-RuNPs), whose unique cage structure enables the rare metal ruthenium to be firmly modified on the surface, thereby greatly improving the functionality and application range of the material. Subsequently, the present invention modified the outer surface of HMCuS-RuNPs with Gox, so that it has the ability to consume ATP and overexpress heat shock proteins, which is highly consistent with mild photothermal therapy. Furthermore, we modified the liver cancer targeting peptide 9R-P on the outer surface of HRIG NPs, thereby obtaining a nano drug HMCuS-Ru-ICG-GOX-9R-P (abbreviated as HRIG9R) with liver cancer targeting. The nano drug achieves the comprehensive therapeutic effect of chemodynamic therapy, mild photothermal therapy and photodynamic therapy. In in vitro and in vivo studies, HRIG9R nanomedicines can actively target and enter HepG2 cells through specific receptor-mediated endocytosis on the surface of liver cancer cells. And trigger drug release in the tumor microenvironment (TME). Under the irradiation of near-infrared (NIR) light, HRIG9R can not only effectively convert near-infrared light into the heat required for photothermal therapy, but also digest heat shock proteins, thereby reducing the tolerance of tumor tissue to photothermal therapy. At the same time, it can also generate high concentrations of reactive oxygen species (ROS) for photodynamic therapy. In the tumor-bearing mouse experiment, tumor-bearing mice treated with the HRIG9RNPs+laser group irradiated with near-infrared light (808 nm, 1 W / cm², 5min) showed a tumor inhibition rate of up to about 91.1%. Therefore, the HRIG9R nanomedicine developed in the present invention has great potential and can be used as an efficient and precise nanotherapeutic agent to induce chemokine therapy, mild photothermal therapy and photodynamic therapy, showing excellent anti-tumor effects and low side effects.

Claims

1. A method for preparing a bimetallic composite nano drug carrier by modifying the surface of HMCuS with a rare metal Ru, characterized in that: The steps include: Step 1, dispersing ruthenium chloride trihydrate and hollow mesoporous copper sulfide nano drug carrier HMCuS in ethylene glycol to obtain a mixed solution A; Step 2, adding polyvinyl pyrrolidone and methyl benzoate to the mixed solution A, mixing well, and then heating at 160-200° C. for reaction for 2-4 h; Step 3: After the reaction is completed, the solid and liquid are separated, and the product C is obtained after washing and drying, which is the nano drug carrier.

2. The method for preparing a bimetallic composite nano drug carrier by modifying the surface of HMCuS with a rare metal Ru as claimed in claim 1, characterized in that: The mass ratio of ruthenium chloride trihydrate to HMCuS in step 1 is: 1:0.3-0.

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3. The method for preparing a bimetallic composite nano drug carrier by modifying the surface of HMCuS with a rare metal Ru as claimed in claim 1, characterized in that: The mass ratio of polyvinyl pyrrolidone to methyl benzoate in step 2 is 1:0.1-0.

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4. The method for preparing a bimetallic composite nano drug carrier by modifying the surface of HMCuS with a rare metal Ru as claimed in claim 1, characterized in that: The mass ratio of the HMCuS to polyvinyl pyrrolidone is 1:70-90.

5. The method for preparing a bimetallic composite nano drug carrier by modifying the surface of HMCuS with a rare metal Ru as claimed in claim 1, characterized in that: The molecular weight of the polyvinyl pyrrolidone in step 2 is 60000-80000 Da.

6. A bimetallic composite nano drug carrier prepared by the preparation method of any one of claims 1 to 5, wherein the surface of HMCuS is modified with the rare metal Ru.

7. Use of the bimetallic composite nano drug carrier according to claim 6 in drug delivery.

8. A method for preparing a rare metal composite bimetallic nanomedicine modified with a liver cancer targeting peptide using the drug carrier according to claim 6, characterized in that: The steps include: Step a, dissolving product C and indocyanine green in ultrapure water at room temperature, stirring for 12-36 hours in a dark environment, centrifuging, washing, and freeze-drying to obtain product D; Step b, at 2-8°C, glucose oxidase is dissolved in a phosphate buffer with a pH value of 3-4, and then product D is added, and the mixture is allowed to stand in the dark for adsorption for 30-90 minutes, and then centrifuged, washed, and freeze-dried to obtain product E; Step c: at room temperature, dissolve the liver cancer targeting peptide in a phosphate buffer with a pH value of 7-9, add product E, stir in the dark for 12-48 h, centrifuge, wash, and freeze-dry to obtain product F.

9. The method according to claim 8, characterized in that In step a, the mass ratio of product C to indocyanine green is 1:1-2; in step b, the mass ratio of glucose oxidase to product D is 1:1-2; in step c, the mass ratio of liver cancer targeting peptide to product E is 1-2:1, and the sequence of the liver cancer targeting peptide is: RRRRRRRRRGSGSWHLDYPSMWYLD.

10. A rare metal composite bimetallic nanomedicine modified with a liver cancer targeting peptide prepared by the method of claim 8 or 9.

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