Molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cis-platinum and preparation method and application of molybdenum disulfide nano-delivery system

Abstract

The invention discloses a gambogic acid and cis-platinum co-loaded molybdenum disulfide nano delivery system. The system comprises the following components in parts by weight: 1-5 parts of molybdenum sulfide, 5-50 parts of manganese dioxide, 0.1-5 parts of gambogic acid, 1-50 parts of a cis-platinum prodrug Pt (IV) and 10-50 parts of polyethylene glycol. The invention also provides a preparation method of the nano conveying system. According to the MoS2-MnO2-PEG-Pt (IV) / GA nano-drug delivery system constructed by the invention, the sequential release of the drug is controlled through GSH and near-infrared light, the tumor hypoxia drug resistance is reversed in combination with the characteristics of the carrier, the synergistic treatment effect of GA and CDDP is maximized, and finally, the combined drug synergistic treatment and photo-thermal combined treatment of lung cancer are realized, so that the application prospect is wide. A new thought and a research strategy are provided for clinical treatment of lung cancer, and the application range of anti-cancer traditional Chinese medicines in tumor treatment is expanded.

Description

Technical Field

[0001] The invention relates to a molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin and a preparation method thereof, belonging to the field of pharmaceutical preparations. Background Art

[0002] Lung cancer is the most commonly diagnosed malignancy (accounting for 11.6% of the total number of cases) and the most important factor affecting cancer mortality (accounting for 18.4% of the total number of cancer deaths) in both men and women. It is one of the most common and deadly cancers in the world. Since the 1930s, the incidence and mortality of lung cancer have steadily increased worldwide, so effective strategies are needed to prevent and treat lung cancer. According to histopathology, lung cancer can be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), of which NSCLC is the most common, accounting for about 85% of the total number of lung cancers. At present, the treatment options for lung cancer mainly include surgery, drug therapy, external beam radiation therapy, stereotactic radiosurgery, targeted therapy, immunotherapy and palliative care. Although surgical treatment is the first choice for patients with early lung cancer, the low tolerance of patients to surgical treatment limits the clinical application of surgery to a certain extent. In addition, due to certain limitations in early lung cancer screening technology, most lung cancer patients are already in the advanced stage when diagnosed, and can only prolong life and improve quality of life through chemotherapy or radiotherapy.

[0003] Chemotherapy is a commonly used treatment strategy in cancer treatment. In the past few decades, researchers have developed dozens of anticancer drugs for lung cancer treatment, such as cisplatin, paclitaxel, docetaxel, doxorubicin (DOX), etc. Among them, cisplatin (CDDP) is one of the most well-known and effective chemotherapy drugs. As an anti-tumor drug, it plays a vital role in the treatment of various malignant tumors. This platinum (II)-based molecule can form cross-links with DNA. After entering the cell, it binds to the DNA in the tumor cell to form a platinum-DNA complex, causing changes in the DNA structure, leading to cytotoxic damage, inducing cell apoptosis, and ultimately leading to tumor cell death. Since it was approved by the FDA in 1978, it has been used to treat various human solid tumors, including bladder cancer, breast cancer, prostate cancer, ovarian cancer, testicular cancer, and neuroblastoma, and is used together with analogs carboplatin and oxaliplatin in the first-line treatment of malignant tumors. Because of its simple administration form and good efficacy, it has become the gold standard chemotherapy drug for the treatment of various solid tumors in clinical practice. However, the efficacy of cisplatin is proportional to its dose and shows significant dose dependence. The most important of these is the lack of selectivity for tumor tissues. It can also cause many adverse reactions, such as vomiting, nausea, ototoxicity, neurotoxicity, and nephrotoxicity, which severely limit its use and weaken its clinical therapeutic effect. In addition, continuous chemotherapy and long-term medication can cause tumors to develop drug resistance, greatly reducing its therapeutic effect. Since these drugs cannot specifically detect lung cancer cells, they can cause serious damage to normal cells, leading to common gastrointestinal symptoms and bone marrow suppression in many patients. Although platinum drugs have achieved good anti-tumor effects, they still face serious side effects and high toxicity caused by nonspecific binding to essential plasma proteins in clinical practice, which greatly limits their application. In order to overcome these limitations, a series of new cisplatin analogs have been synthesized.

[0004] Numerous studies have shown that glutathione (GSH) and glutathione transferases (GSTs), which are overexpressed in most cancer cells, are the main causes of adverse reactions and resistance to cisplatin. In order to promote the development of platinum drugs, reduce the side effects of cisplatin drugs, and improve their application prospects in the field of tumor treatment, a series of platinum compounds have been synthesized and studied, among which researchers have developed cisplatin prodrugs Pt(IV). Platinum(IV) prodrugs show several advantages over traditional platinum(II) complexes. After chemical oxidation of the active square planar Pt(II) species, two "axial" ligands are added to the metal center, allowing pharmacological properties such as reduction potential, lipophilicity, and kinetic stability to be fine-tuned. It is generally believed that platinum(IV) complexes with high coordination numbers are inert in motion and therefore have fewer side effects than platinum(II) species. As a prodrug, the Pt(IV) complex is first reduced by a biological reducing agent (such as ascorbic acid (AsA) or glutathione (GSH)) in the extracellular or intracellular environment to release two axial ligands, and then release the cytotoxic Pt(II) drug. The cisplatin-based Pt(IV) complex can be reduced to an active Pt(II) substance by GSH. During the entire reduction process, the content of the reducing substance GSH in the cell is reduced, which alleviates the toxic effects of the Pt(II) analog.

[0005] Traditional Chinese medicine is a treasure of traditional Chinese medicine. Many Chinese medicines have been proven to have good anti-tumor, anti-inflammatory, and immunomodulatory effects, such as Astragalus, Curcuma, Tripterygium wilfordii, etc., and have little toxic side effects. Garcinia is also one of the representatives of anti-tumor Chinese medicine. The traditional Chinese medicine Garcinia is derived from the resin of Garcinia hanburyi Hook.f. The earliest record can be traced back to the Five Dynasties "Haiyao Bencao", which is mainly used to treat tooth decay; later in the Qing Dynasty "Ben Jing Feng Yuan" recorded that "Garcinia is poisonous and can attack poisons", and "Gangmu Shiyi" explained that "Garcinia can treat carbuncle, stop bleeding and detoxify", and treat carbuncle, swelling, stubborn tinea, and malignant ulcers. The main chemical components of Garcinia are Ketones and benzophenones, etc., are easy to functionalize and have a wide range of biological and pharmacological activities, especially anti-tumor activity, which is one of the hot spots in natural product research in recent years. In 1973, my country first extracted and isolated a compound with excellent anti-tumor activity from Garcinia - gambogic acid (GA). A large number of studies have confirmed that GA has a good inhibitory effect on gastric cancer, hematological malignancies, liver cancer, malignant melanoma, lung cancer, leukemia, breast cancer, etc., among which the therapeutic effect on lung cancer is particularly significant. It has been approved by the China Food and Drug Administration for Phase II clinical trials for lung cancer treatment. It is currently believed that the anti-tumor mechanism of GA mainly includes inducing increased accumulation of reactive oxygen species, promoting tumor cell apoptosis, inducing cell cycle arrest, inhibiting telomerase activity, inhibiting anti-tumor angiogenesis, inhibiting tumor metastasis, etc. However, as a small molecule and low-polar substance, GA has defects such as poor solubility (the solubility in water at room temperature is only 0.013 mg / mL) and short half-life, and is prone to cause side effects such as hepatotoxicity, which severely limits its clinical application. A large number of experimental studies have shown that the combination of GA, an active ingredient of traditional Chinese medicine, and CDDP has the advantages of low dosage, good effect, and low toxicity and side effects, and can avoid the occurrence of multidrug resistance and even reverse the drug resistance. Wang et al. found that GA can inhibit the activation of NF-κB and MAPK / HO1 pathways induced by CDDP, regulate the expression of pro-apoptotic proteins Fas (fatty acid synthase) and Bax (Bcl-2-related X gene) and anti-apoptotic protein Bcl-2, and synergistically enhance the ability of CDDP to inhibit the proliferation of lung cancer cells. Especially under the sequential administration scheme (CDDP first, GA later), it can effectively inhibit cell activity at the cellular level and promote cells to enter the apoptosis phase. Its anti-tumor effect is much higher than that of single or combined drugs administered at the same time.

[0006] However, this strategy cannot be successfully applied in clinical practice. At the in vivo level, CDDP combined with GA or used alone has a significant inhibitory effect on the growth of A549 tumors. However, the sequential administration of CDDP and GA cannot effectively inhibit tumor growth. There is no significant difference compared with other experimental groups. The main reason is that GA is highly hydrophobic, has low solubility, and is easily decomposed under alkaline conditions.

[0007] Moreover, it has a short half-life and is widely distributed in the body. While reducing the viability of tumor cells, it may have toxic side effects on normal tissues (Zhao X, Ding S, Li S, et al. Construction of Gambogic AcidHPMACopolymer Coupling Drug System and Study on Anti-tumor Activity[J]. Current Drug Delivery, 2022, 19(4): 491-507.), and the pharmacokinetics of traditional drug preparations control drug absorption, distribution, metabolism and excretion, resulting in insufficient bioavailability of drugs in tumor tissues. In addition, different routes of administration and different excipients associated with each traditional drug form lead to different pharmacokinetic processes, which severely limits the time for drug absorption and distribution in the body, thereby avoiding the synergistic effect between drugs. Therefore, it is urgent to design appropriate drug carriers to solve the above-mentioned key problems. In recent years, many scholars have developed various types of bio-nanomaterials and constructed a large number of GA or CDDP-loaded nano-delivery systems. Fang et al. found that Fe3O4 magnetic nanoparticles can synergistically enhance the induction of GA on LOVO cell apoptosis and inhibit its proliferation in a dose-dependent and time-dependent manner. Du et al. prepared a new multi-environmentally sensitive garcinia acid polymer prodrug micelle (GFCP) based on chitosan grafts. The nanomicelles are stable and have good biocompatibility. They have pH, esterase and temperature-dependent drug release, high loading capacity, targeted tumor accumulation and specific responsiveness to the tumor microenvironment. They have shown the potential to improve the anticancer effect of GA both in vivo and in vitro. Anees et al. modified cisplatin with irradiated chitosan coating and successfully formulated cisplatin nanocomposite (Cis NC) with MgO nanoparticles. The nanocomposite can promote the release of cisplatin in a long-term manner, reduce the nephrotoxicity of cisplatin, and improve the therapeutic effect. However, there is still no nanodelivery system that co-loads GA and CDDP. Summary of the invention

[0008] The technical solution of the present invention is to construct a nano drug delivery system co-loaded with CDDP and GA, control the sequential release of the combined drugs, and achieve drug synergistic treatment of lung cancer.

[0009] The invention provides a molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin, which comprises the following components in weight ratio: 1-5 parts of molybdenum sulfide, 5-50 parts of manganese dioxide, 0.1-5 parts of gambogic acid, 1-50 parts of cisplatin prodrug Pt(IV), and 10-50 parts of polyethylene glycol.

[0010] More preferably, it contains the following components in the following weight ratio: 1 part of molybdenum sulfide, 10 parts of manganese dioxide, 0.1 parts of gambogic acid, 1 part of cisplatin prodrug Pt(IV), and 20 parts of polyethylene glycol.

[0011] Among them, it is a nano-delivery system based on molybdenum disulfide (MoS2) nanosheets, modified with manganese dioxide (MnO2) nanoparticles, and loaded with GA and CDDP at the same time. Among them, the MoS2-MnO2 nanosheets have a two-dimensional layered structure; GA and CDDP are loaded through amide reaction and hydrophobic effect.

[0012] The molybdenum disulfide (MoS2) nanosheets are prepared by Morrison method with single or few layers of molybdenum disulfide nanosheets, which comprises the following steps:

[0013] Under nitrogen, MoS2 crystals were placed in a n-butyllithium solution containing 15% n-hexane and stirred for 2 days under the protection of nitrogen;

[0014] After the reaction is completed, the mixture is washed with redistilled n-hexane to remove the residue of n-butyl lithium and other impurities;

[0015] After washing, the black powder was removed and ddH2O was added immediately, and ultrasonic stripping was performed for 2 hours. Then, the solution was collected and centrifuged at 8000rpm for 20 minutes to remove the crystals that were not completely stripped. After centrifugation, the upper layer solution was transferred to a dialysis bag with MWCO=100kDa and dialyzed for one week to remove residual lithium ions and other impurities; thus, a solution containing MoS2 nanosheets was obtained;

[0016] The manganese dioxide (MnO2) nanoparticles are prepared by oxidation-reduction reaction, and the preparation method is as follows:

[0017] Weigh KMnO4 and bovine serum albumin (BSA) and add them to ddH2O, and stir at room temperature for 5 hours; KMnO4 is reduced by BSA to form MnO2 nanoparticles; after the reaction is completed, high-speed centrifugation is performed at 12000rpm for 20 minutes to remove free KMnO4, BSA and MnO2 particles with larger particle size; collect the upper layer solution to obtain manganese dioxide (MnO2) nanoparticles;

[0018] The preparation method of Pt(IV) is as follows: CDDP and H2O2 are added to ddH2O, stirred at 76°C in the dark for 8 hours, heating is stopped after the reaction is completed and stirring is continued overnight; after overnight, the mixture is placed at 4°C for crystallization for about 8 hours; then, the crystallized product is washed with cold water, ethanol and ether in turn, the precipitate is collected by centrifugation at 8000rpm for 10 minutes, and then vacuum dried to obtain a bright yellow crystalline powder, i.e., oxidized cisplatin (Pt(II)); Pt(II) and succinic anhydride are added to DMSO; stirred at 76°C in the dark for 48 hours, and then freeze-dried to remove DMSO to obtain a yellow powder, pre-cooled ice acetone is added to the powder, and recrystallized at -20°C; the collected solution is washed with 10 mL of pre-cooled ice acetone for 3 times, the precipitate is collected by centrifugation at 8000rpm for 10 minutes, and then vacuum dried to obtain a white powder, i.e., Pt(IV).

[0019] The synthesis method of MoS2-MnO2 is as follows: MoS2 nanosheet aqueous solution and MnO2 nanoparticle aqueous solution are mixed, MoS2 and MnO2 are combined together by sulfur chemical reaction, and then the mixed solution is transferred into a dialysis bag (MWCO=100kDa) after stirring for 24 hours, and dialyzed for 3 days to remove free MoS2 and MnO2, thereby obtaining a MoS2-MnO2 solution.

[0020] The present invention also provides a method for preparing the molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin, which comprises the following steps:

[0021] a. Preparation of molybdenum disulfide (MoS2) nanosheets;

[0022] b. preparing manganese dioxide (MnO2) nanoparticles;

[0023] c. Synthesis of MoS2-MnO2;

[0024] d. Synthesis of Pt(IV);

[0025] e. Synthesis of LA-PEG-NH2: coupling LA and PEG to obtain LA-PEG-NH2;

[0026] f. LA-PEG-NH2 was used as a connector to graft Pt(IV) onto MoS2-MnO2 to prepare a molybdenum disulfide nanodelivery system co-loaded with gambogic acid and cisplatin.

[0027] Wherein, the synthesis method of LA-PEG-NH2 described in step e is:

[0028] Weigh 6-arm PEG-NH2, LA, and DMAP and add them to CH2Cl2 solution. Stir until the solid is completely dissolved, then add EDC and NHS to activate LA, and stir at room temperature in the dark for 3 days. After the reaction, evaporate the solution until white crystals are obtained. Dissolve the crystals in ddH2O and centrifuge at 8000 rpm for 15 min. Transfer the supernatant to a dialysis bag (MWCO=3.5 kDa) and dialyze for 48 h to remove small molecule impurities. Finally, freeze-dry to obtain white solid LA-PEG-NH2.

[0029] In step f, LA-PEG-Pt(IV) is first synthesized, and the Pt(IV) complex is covalently attached to LA-PEG-NH2 via an amide bond to obtain LA-PEG-Pt(IV). The synthesis method is:

[0030] Weigh Pt(IV), EDC, and NHS and dissolve them in ddH2O, stir in the dark, then add LA-PEG-NH2 and stir in the dark for 24 hours; after the reaction, centrifuge at 8000 rpm for 10 minutes, transfer the supernatant to a dialysis bag (MWCO=3.5 kDa), dialyze for 48 hours to remove small molecule impurities, and obtain LA-PEG-Pt(IV);

[0031] LA-PEG-Pt(IV) was then grafted onto MoS2-MnO2 through sulfur chemistry, and then GA was loaded onto MoS2-MnO2-PEG-Pt(IV) through hydrophobic interaction to construct the MoS2-MnO2-PEG-Pt(IV) / GA nanodrug delivery system.

[0032] The preparation method of the MoS2-MnO2-PEG-Pt(IV) / GA nano drug delivery system comprises the following steps:

[0033] Pt(IV) loading:

[0034] 100 mg of LA-PEG-Pt(IV) was dissolved in 10 mL of ddH2O and stirred until the solid was completely dissolved. Then, 20 mL of MoS2-MnO2 solution (0.5 mg / mL) was added. MoS2-MnO2 and LA-PEG-Pt(IV) were combined by sulfur chemistry. After stirring for 24 h, the mixture was transferred to a dialysis bag (MWCO = 100 kDa) and dialyzed for 3 days to obtain the MoS2-MnO2-PEG-Pt(IV) solution;

[0035] GA Load:

[0036] GA solutions with concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL were prepared, and an equal volume of MoS2-MnO2-PEG-Pt(IV) solution was added to the above solution; GA was loaded onto MoS2-MnO2-PEG-Pt(IV) through hydrophobic interaction, and the mixture was stirred at room temperature in the dark for 24 h, and then centrifuged at 2000 rpm for 10 min to remove the precipitated solid drug, and the supernatant was collected and ultrafiltrated and centrifuged at 3000 rpm for 15 min, and washed repeatedly with ddH2O, and ultrafiltrated and centrifuged until the lower layer of the solution was colorless, thereby obtaining MoS2-MnO2-PEG-Pt(IV) / GA solution loaded with GA.

[0037] The present invention provides use of the molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin in preparing a medicine for treating lung cancer.

[0038] The MoS2-MnO2 nanosheet of the present invention has a two-dimensional layered structure, similar to graphene, has a large specific surface area, and is suitable for being a drug carrier. In addition, this nanosheet also has the characteristics of high physiological stability and blood compatibility, outstanding photothermal conversion ability, high oxygen production ability, and strong magnetic resonance weighted imaging, indicating that MoS2-MnO2 is a safe and functionalized drug carrier. The combined drug was successfully loaded through amide reaction and hydrophobic interaction, and the release of Pt (IV) and GA was achieved under the dual stimulation of GSH and near-infrared light. The results of the anti-tumor cell proliferation experiment showed that compared with single drugs and combined drugs, MoS2-MnO2-PEG-Pt (IV) / GA always showed stronger anti-tumor activity, especially under the irradiation of near-infrared light, it showed the strongest cell killing effect, only 9.1% of the cells were in a viable state, and the number of cells in the apoptotic stage was as high as 89.17%. MoS2-MnO2-PEG-Pt(IV) / GA delivers drugs to the tumor site through the EPR effect and passive targeting. Under the irradiation of near-infrared light, the temperature of the tumor site in nude mice rises rapidly to 56°C, which not only directly causes apoptosis of tumor cells, but also effectively controls the release of the combined drugs, enhancing their synergistic therapeutic effects and realizing the synergistic treatment of the combined drugs and the combined photothermal treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the synthesis of MOS2-MNO2 (Note: (I) MOS2 is synthesized by chemical stripping; (II) MNO2 is synthesized by redox reaction; (III) MNO2 is grafted onto MOS2 through sulfur chemistry);

[0040] Figure 2:(a) Infrared absorption spectrum of MoS2 before and after MnO2 grafting;(b) UV-visible-near infrared absorption spectrum of MoS2 before and after MnO2 grafting;

[0041] Figure 3 :(a) TEM image of MoS2 nanosheets;(b) TEM image of MnO2 nanoparticles;(c) TEM image of MoS2-MnO2 nanosheets;

[0042] Figure 4 :(a) TEM image and EDS element distribution map of MoS2-MnO2;(b) EDS element spectrum of MoS2-MnO2;

[0043] Figure 5 :(a) AFM image of MOS2 nanosheets;(b) AFM image of MOS2-MNO2 nanosheets;

[0044] Figure 6 :(a) Particle size of MOS2, MNO2 and MOS2-MNO2;(b) Potential of MOS2, MNO2 and MOS2-MNO2;

[0045] Figure 7 :(a) Optical photographs of MoS2 placed in water, PBS, and cell culture medium for 7 days; (b) Optical photographs of MoS2-MnO2 placed in water, PBS, and cell culture medium for 7 days;

[0046] Figure 8 :(a) at 1W / cm 2 (a) Photothermal heating curves of water and MoS2-MnO2 solutions with different concentrations under 808nm laser irradiation; (b) Photothermal heating curves of MoS2-MnO2 solution (300μg / mL) under 808nm laser irradiation with different power densities;

[0047] Fig. 9 The temperature change curve of MoS2-MnO2 solution in three cycles of irradiation-natural cooling;

[0048] Fig.10 :(a) MnO2 and MoS2-MnO2 at different Mn 2+ NMR T1-weighted images at different Mn concentrations and pH values. (b) MnO2 and MoS2-MnO2 at different Mn concentrations and pH values. 2+ Relaxation curves at different concentrations and pH;

[0049] Fig.11 :(a) O2 concentration change curve of MoS2-MnO2 under different pH conditions;(b) O2 concentration change curve of different concentrations of MoS2-MnO2 reacting with H2O2 at pH 5.5;

[0050] Fig.12 :(a) UV-visible absorption spectra of RBCs incubated with MoS2-MnO2 solutions of different concentrations for 2 h, with PBS and ddH2O as negative and positive controls, respectively;(b) Hemolysis percentage and hemolysis optical photos of RBCs incubated with MoS2-MnO2 solutions of different concentrations for 2 h;

[0051] Fig.13 For CDDP, PT(II) and PT(IV) 1 HNMR spectra;

[0052] Fig.14 : MS spectra of (a) PT(II) and (b) PT(IV);

[0053] Fig.15 FT-IR images of CDDP, PT(II), and PT(IV);

[0054] Fig.16 Schematic diagram of the synthesis of LA-PEG-PT(IV);

[0055] Fig.17 LA, PEG-NH2, LA-PEG-NH2 and LA-PEG-PT(IV) 1 HNMR spectra;

[0056] Fig.18 FTIR graphs of LA, PEG-NH2, LA-PEG-NH2 and LA-PEG-PT(IV);

[0057] Fig.19 Schematic diagram of the synthesis of MoS2-MnO2-PEG-Pt(IV) / GA;

[0058] Fig. 20 :(a) TEM image and EDS element distribution map of MoS2-MnO2-PEG-Pt(IV) / GA;(b) EDS element spectrum of MoS2-MnO2-PEG-Pt(IV) / GA;(c) UV-Vis images of GA, MoS2-MnO2-PEG-Pt(IV) and MoS2-MnO2-PEG-Pt(IV) / GA;

[0059] Fig.21 Schematic diagram of drug release from MoS2-MnO2-PEG-Pt(IV) / GA;

[0060] Fig. 22 :(a) PT standard curve;(b) GA standard curve;

[0061] Fig.23:(a) UV-Vis images of MoS2-MnO2-PEG-Pt(IV) / GA at different drug concentrations; (b) Relationship between the amount of GA loaded on MoS2-MnO2-PEG-Pt(IV) / GA and drug concentration, and optical photos after standing in PBS for 1 day;

[0062] Fig.24 :(a) Cumulative release curve of Pt in response to GSH; (b) Cumulative release curve of GA with / without near-infrared light irradiation;

[0063] Fig.25 : Cell viability and IC of A549 cells incubated with CDDP, GA, CDDP+GA, CDDP→GA and MoS2-MnO2-PEG-Pt(IV) / GA for 96 h under normoxic and hypoxic conditions 50 value;

[0064] Fig.26 Figure 2 shows the killing effects of CDDP, GA, CDDP+GA, CDDP→GA, MOS2-MNO2, and MOS2-MNO2-PEG-PT(IV) / GA on A549 cells under near-infrared light irradiation (a) normoxia (b) hypoxia (Note: * P﹤0.05,

[0065] ** P﹤0.01);

[0066] Fig. 27 Flow cytometry was used to quantitatively analyze the percentage of apoptosis of A549 cells treated in different ways under hypoxic environment;

[0067] Fig.28 (a) Thermal imaging of nude mice before and after near-infrared light irradiation and the corresponding (b) temperature change curve;

[0068] Fig.29 :(a) Tumor growth curves of nude mice in each group after treatment with different methods; (b) Tumor volume inhibition rate of nude mice in each group after treatment with different methods (Note: * P﹤0.05, ** P﹤0.01);

[0069] Fig.30 H&E staining images of nude mouse tumors in each group after 21 days of treatment;

[0070] Fig.31 The curves of body weight changes over time of nude mice in each group after 21 days of intervention in different ways;

[0071] Fig.32H&E staining images of the main organs of nude mice in the PBS group and MoS2-MnO2-PEG-Pt(IV) / GA+NIR group after 21 days of treatment. DETAILED DESCRIPTION

[0072] Example 1 Preparation of the MoS2 Nano-delivery System Co-loaded with Garcinia Acid and Cisplatin

[0073] 1. Preparation of related solutions and handling of consumables

[0074] 1.1 Preparation of phosphate buffered saline (PBS)

[0075] Weigh 4.0 g NaCl, 0.1 g KCl, 1.79 g NaHPO4·12H2O and 0.12 g KH2PO4 and dissolve them in 500 mL secondary water (ddH2O). Stir magnetically until dissolved, filter with a 0.22 μm filter membrane, sterilize with high-pressure steam for 1 h, and store at 4°C for later use.

[0076] 1.2 Dialysis bag pretreatment

[0077] Weigh 20g NaHCO3 and 373.2mg EDTA·2Na and dissolve them in 1L ddH2O. Place the dialysis bag in the solution and boil it. After 10 minutes, take it out and wash it with ddH2O. Place it in 1L ddH2O containing 373.2mg EDTA·2Na and continue boiling. After 10 minutes, take it out and wash it with ddH2O. Store it in 30% ethanol for later use.

[0078] 2. Experimental methods

[0079] 2.1 Preparation of MoS2 nanosheets

[0080] Monolayer or few-layer molybdenum disulfide nanosheets are prepared according to the Morrison method. The method is as follows: 500 mg of MoS2 crystals are placed in 10 mL of n-butyl lithium solution containing 15% n-hexane in a nitrogen glove box and stirred for 2 days under the protection of nitrogen. After the reaction is completed, it is washed several times with redistilled n-hexane to remove the residues of n-butyl lithium and other impurities. After the washing is completed, the black powder is removed from the glove box and immediately added with 200 mL of ddH2O, ultrasonically stripped for 2 hours, and then the solution is collected and centrifuged at 8000 rpm for 20 minutes to remove the crystals that are not completely stripped. After the centrifugation, the upper layer solution is transferred to a dialysis bag (MWCO = 100 kDa) and dialyzed for one week to remove residual lithium ions and other impurities. Finally, the solution containing MoS2 nanosheets is placed in a 4°C refrigerator for storage.

[0081] 2.2 Preparation of MnO2 Nanoparticles

[0082] The present invention modifies the surface of MoS2 nanosheets with MnO2 nanoparticles to improve the physiological stability thereof.

[0083] MnO2 nanoparticles were prepared according to the redox reaction. Weigh 20mg KMnO4 and 100mg BSA into 50mL ddH2O and stir at room temperature for 5h. KMnO4 can be reduced by BSA to form MnO2 nanoparticles. After the reaction, high-speed centrifugation at 12000rpm for 20min was performed to remove free KMnO4, BSA and MnO2 particles with larger particle size. The upper layer solution was collected and stored in a refrigerator at 4℃ for later use. BSA is a suitable candidate material for designing nanocarriers due to its good biocompatibility and low immunogenicity. The formed nanoparticles can carry a wide range of therapeutic drugs, such as chemotherapeutic drugs and photosensitizers, and can be stably delivered to the blood without easy degradation, enhancing the aggregation of drugs in tumors, improving the uptake rate of tumor cells, and increasing therapeutic activity.

[0084] 2.3 Synthesis of MoS2-MnO2

[0085] The synthesis of MoS2-MnO2 is as follows Figure 1 As shown. 10 mL of MoS2 nanosheet aqueous solution (0.4 mg / mL) and 2 mL of MnO2 nanoparticle aqueous solution (4 mg / mL) were mixed, and MoS2 and MnO2 were combined by sulfur chemical reaction. After stirring for 24 hours, the mixture was transferred into a dialysis bag (MWCO = 100 kDa) and dialyzed for 3 days to remove free MoS2 and MnO2. The obtained MoS2-MnO2 solution was stored in a refrigerator at 4 ° C for later use.

[0086] 2.4 Characterization of MoS2-MnO2

[0087] 2.4.1 Fourier transform infrared spectroscopy (FT-IR) analysis

[0088] Use FT-IR to analyze the chemical structure of MoS2, MnO2 and MoS2-MnO2. Take appropriate amount of freeze-dried samples of MoS2, MnO2 and MoS2-MnO2, mix them with potassium bromide powder in an agate mortar at a mass ratio of 1:100, grind them thoroughly to make the sample and potassium bromide powder mix evenly and grind them to fineness, take a small amount of powder and place it in an infrared mold, press it for about 1 minute to obtain a translucent sheet, put the sheet into FT-IR, and detect different samples at 400-4000cm -1 Infrared spectrum information.

[0089] 2.4.2 Ultraviolet-visible spectroscopy (UV-vis) analysis

[0090] UV-vis was used to analyze the ultraviolet absorption changes of MoS2-MnO2. Appropriate amounts of MoS2, MnO2, and MoS2-MnO2 samples were diluted to a certain concentration with ddH2O, placed in a quartz cuvette, and the absorption peaks of different samples at 200-900nm were recorded.

[0091] 2.4.3 Field emission transmission electron microscope (TEM) analysis

[0092] TEM was used to observe the surface morphology and element distribution of MoS2, MnO2, and MoS2-MnO2. Appropriate amounts of MoS2, MnO2, and MoS2-MnO2 samples were diluted with ddH2O to a certain concentration, and 10 μL of the solution was dropped on the carbon support film. After the sample was dried overnight, the carbon film was placed in TEM to observe its morphological characteristics. At the same time, energy dispersive X-ray spectroscopy (EDS) was used for elemental analysis.

[0093] 2.4.4 Atomic force microscope (AFM) analysis

[0094] AFM was used to analyze the size and thickness of MoS2 before and after MnO2 modification. Appropriate amounts of MoS2 and MoS2-MnO2 samples were diluted with ddH2O to a certain concentration, and 20 μL of the solution was dropped on a clean and smooth mica sheet. After the sample was dried overnight, the mica sheet was placed in the AFM to observe its size and thickness.

[0095] 2.4.5 Nanoparticle size distribution and potential analysis

[0096] The particle size and Zeta potential of MoS2, MnO2, and MoS2-MnO2 were measured using a nanoparticle size and potential analyzer. Appropriate amounts of MoS2, MnO2, and MoS2-MnO2 samples were diluted to a certain concentration with ddH2O, placed in a cuvette, and the particle size distribution of the samples was measured; appropriate amounts of samples were placed in a folded capillary sample cell, and the Zeta potential of the samples was measured.

[0097] 2.4.6 Synthesis and characterization data of MoS2-MnO2

[0098] We first used FTIR and UV-Vis spectroscopy to characterize the chemical structure of MoS2-MnO2. First, FTIR was used to identify the chemical structure of MoS2-MnO2. The results are as follows: Figure 2As shown in (a), from the infrared spectrum we can find that compared with MoS2, MoS2-MnO2 has a -1 A new absorption peak appeared at , which was attributed to the combined effect of OH bending vibration and Mn atoms, confirming that MnO2 had been successfully grafted onto MoS2.

[0099] At the same time, the UV-Vis Figure 2 (b) It can be seen that the absorbance of MoS2-MnO2 in the ultraviolet region is greater than that of MoS2, especially in the range of 200nm-280nm, which shows an upward trend. This is because the grafted MnO2 has strong absorption in this region, which also proves that MoS2 and MnO2 are successfully combined.

[0100] TEM and AFM were used to observe the surface morphology and thickness of MoS2-MnO2. Figure 3 It can be seen that MoS2 has a two-dimensional layered structure, similar to graphene, with a large specific surface area, and is suitable as a drug carrier; MnO2 nanoparticles are highly dispersed and have a regular spherical morphology; MnO2 nanoparticles can be observed on the MoS2-MnO2 nanocarrier to be uniformly distributed on MoS2 through sulfur chemistry.

[0101] To further verify the above results, we used energy dispersive X-ray spectroscopy (EDS) to perform elemental analysis on MoS2-MnO2. Figure 4 a), it is found that Mn atoms are uniformly dispersed on the surface of MoS2 nanosheets. At the same time, from the EDS image ( Figure 4 b) The three peaks of Mo, Mn and S can be clearly seen. These results further confirm the successful preparation of MoS2-MnO2.

[0102] The size and thickness of MoS2 before and after modification were measured by AFM. Figure 5 It can be seen that the average thickness of the MoS2 nanosheets is 0.7nm, indicating that the prepared MoS2 nanosheets are single-layer. After modification with MnO2, the average thickness of the MoS2 nanosheets increases to 6nm, which indirectly proves that MnO2 exists on the MoS2 nanosheets.

[0103] The particle sizes of MoS2 and MoS2-MnO2 were measured using Zeta potential and nanoparticle size analyzer. Figure 6It can be seen that the average particle size of the prepared MoS2 nanosheets is 100nm, the average particle size of the MnO2 nanosheets is 10nm, and the particle size of MoS2-MnO2 after MnO2 modification is about 100nm. The particle size of the nanocarrier is appropriate and the dispersibility is good, and it can be effectively accumulated in the tumor site through the high permeability and long retention effect. The Zeta potential measurement results show that the potentials of MoS2, MnO2 and MoS2-MnO2 are -49.6mV, -25.7mV and -35.1mV respectively, and they are all negatively charged in aqueous solution. The absolute value of the Zeta potential of MoS2-MnO2 is >30mV, indicating that it has more charges on its surface, tends to repel each other, and the dispersed system of particles is more stable.

[0104] 2.5 Physiological stability of MoS2-MnO2

[0105] MoS2 and MoS2-MnO2 were resuspended in water, PBS solution and cell culture medium, respectively, and left at room temperature for one week to observe their stability in water, PBS solution and cell culture medium.

[0106] After confirming the successful preparation of MoS2-MnO2, we investigated the physiological stability of MoS2-MnO2 in different systems. MoS2 and MoS2-MnO2 were resuspended in water, PBS solution and cell culture medium respectively, and left at room temperature for one week to observe their stability in the three systems. The results are as follows: Figure 7 As shown, MoS2 alone precipitated in PBS, while MoS2-MnO2 nanocarriers were uniformly dispersed in water, PBS solution and cell culture medium for 7 days, indicating that the modification of MnO2 improved the physiological stability of MoS2 and provided a prerequisite guarantee for its application in biomedicine.

[0107] 2.6 Photothermal properties of MoS2-MnO2

[0108] 2.6.1 Photothermal heating of MoS2-MnO2

[0109] MoS2-MnO2 solutions of different concentrations (0, 50, 100, 200 and 300 μg / mL) were placed in a cuvette and heated at 1 W / cm 2 The sample was irradiated with 808nm laser for 10min. During the irradiation process, the sample temperature was recorded every 1min using an infrared thermal imager, and the data was collected and the photothermal temperature rise curve was drawn.

[0110] 1 mL of MoS2-MnO2 solution (300 μg / mL) was placed in a cuvette and the power density (0.2 W / cm 2 , 0.4W / cm 2 、0.6W / cm2 , 0.8W / cm 2 , 1W / cm 2 ) irradiated the sample with 808nm laser for 10min. During the irradiation process, an infrared thermal imager was used to record the temperature change of the sample every 1min, collect data and draw a photothermal temperature rise curve.

[0111] 2.6.2 Photothermal stability of MoS2-MnO2

[0112] Take 1 mL of MoS2-MnO2 solution (300 μg / mL) and put it into the cuvette. 2 The sample was irradiated with an 808nm laser for 10 minutes. The laser was then turned off, and after the sample naturally cooled to the initial temperature, the laser was used again to irradiate the sample for 10 minutes at the same power density. This cycle was repeated three times. During the entire experiment, an infrared thermal imager was used to monitor the thermal imaging optical picture of the sample and record the change in sample temperature.

[0113] 2.6.3 Photothermal properties of MoS2-MnO2

[0114] Photothermal therapy is a treatment method that uses the heat generated by photothermal agents under near-infrared light to cause ablation of tumor cells and kill them. MoS2 nanomaterials have become a popular material for tumor photothermal therapy due to their strong near-infrared absorption. Considering the great potential of MoS2 as a photothermal agent, we irradiated MoS2-MnO2 with different concentrations of near-infrared laser to study its photothermal properties. Figure 8 It can be seen that the presence of MnO2 does not affect the near-infrared light absorption of MoS2, indicating that MnO2 retains the photothermal properties based on MoS2. In order to further study the photothermal properties of MoS2-MnO2, MoS2-MnO2 solutions with different concentrations were prepared and then 2 The sample was irradiated with 808 nm laser for 10 min. Figure 8As shown in a, within 10 minutes of irradiation, the heating rate of water was not obvious, only increasing by 5°C. The higher the concentration of the MoS2-MnO2 sample, the greater the heating rate of the solution temperature, showing obvious concentration dependence. When the sample concentration was 50μg / mL, the solution temperature rose from 29.3℃ to 61.4℃ within 10 minutes. When the sample concentration reached 300μg / mL, the solution temperature rose from 29.6℃ to 87.6℃ within 10 minutes. Compared with the normal body temperature of 37℃, the irradiation of near-infrared light can increase the temperature of the tumor area. This is because the MoS2-MnO2 accumulated in the tumor site has a good near-infrared light conversion efficiency, which effectively converts near-infrared light into heat energy, leading to tumor cell ablation and death, thereby inhibiting tumor growth to a certain extent. At the same time, we also investigated the relationship between laser power density and photothermal effect. A MoS2-MnO2 solution with a concentration of 300μg / mL was used, and then the above samples were irradiated with lasers of different power densities for 10 minutes. It was found that the higher the laser power density, the greater the heating rate of the solution temperature ( Figure 8 b), showing obvious power density dependence. These results indicate that the photothermal effect of MoS2-MnO2 is closely related to sample concentration and laser power density.

[0115] Photothermal stability is considered to be one of the important performance criteria of photothermal materials. Therefore, we studied the photothermal stability of MoS2-MnO2 under near-infrared laser irradiation. 2 The MoS2-MnO2 solution (300 μg / mL) was irradiated with an 808 nm laser for 10 min, and then the 808 nm laser was turned off to allow the sample to cool naturally to the initial temperature. The cycle was repeated three times, and the temperature changes of the solution during the heating and cooling processes were recorded. Fig. 9 It can be seen that the heating rate and cooling rate in each heating and cooling cycle remain basically consistent and there is no obvious change, indicating that MoS2-MnO2 has excellent photothermal stability.

[0116] 2.7 In vitro imaging of MoS2-MnO2

[0117] The in vitro MRI imaging and relaxation tests of MnO2 and MoS2-MnO2 before and after pH response were performed using a magnetic resonance contrast agent imaging analyzer to analyze the in vitro imaging effect, relaxation time and relaxation efficiency. The magnetic field intensity was 0.5T, and the sequence used for T1 imaging test mainly set TR=1500ms and TE=18.2ms. The main parameter settings of the sequence used for relaxation test were: r1 value test Tw=6000ms. Take 20 2mL centrifuge tubes and divide them into four groups of MnO2 (pH5.5 and pH7.4) and MoS2-MnO2 (pH5.5 and pH7.4), with 5 tubes in each group. Use phosphate buffer at pH=7.4 and pH=5.5 to prepare different concentrations of MnO2 and MoS2-MnO2 (concentration is MnO2) of 0.2, 0.4, 0.6, 0.8, and 1.0mM, respectively. 2+ 1 mL of solution was added to each tube. -2 200 μL of mmol / L H2O2 solution was allowed to stand for 5 min, and then 1 mL of sample was placed in the corresponding sample tube. The T1-weighted image of each sample was detected using a 0.5 T nuclear magnetic resonance contrast agent imaging analyzer, and the relaxation time (T1) and relaxation efficiency (r1) of each sample were determined.

[0118] The MoS2-MnO2 nanosystem disperses evenly in a pH 5.5-7.4 environment without precipitation. When H2O2 is added alone, gas (O2) is generated in the solution. When the pH is lowered and H2O2 is added again, a redox reaction occurs, the nanoparticles decompose, and the color of the solution gradually disappears. 2+ The relaxation efficiency of MoS2-MnO2 in response to H2O2 was studied. Fig.10 The T1-weighted MRI images of MnO2 and MoS2-MnO2 under different pH conditions show that the T1-weighted MRI signal of MoS2-MnO2 (pH 5.5) is significantly enhanced with increasing concentration, showing obvious concentration dependence, indicating that MnO2 is degraded into Mn 2+ At the same time, under the condition of the same MnO2 concentration, the T1-weighted image of MoS2-MnO2 (pH 5.5) is brighter than that of MnO2 (pH 5.5), indicating that the MRI contrast imaging ability of MoS2-MnO2 is stronger. However, the MRI signal enhancement of MoS2-MnO2 (pH7.4) is not obvious, which further indicates that MnO2 has a higher release capacity of Mn under slightly acidic conditions containing H2O2. 2+ In order to explore the reasons for this result, we measured the properties of MnO2 and MoS2-MnO2 at different Mn 2+ The relaxation time (T1) at the concentration of Mn 2+The concentration is the horizontal axis, and the reciprocal of the relaxation time (T1) (1 / T1) is the vertical axis. Draw a relaxation curve, and the slope of the straight line is the relaxation rate (r1). At pH 5.5, the r1 of MnO2 is 2.195mM -1 S -1 In contrast, the r1 of MoS2-MnO2 is 11.12 mM - 1 S -1 , which is 5 times that of MnO2. This is because the conjugation of Mn-based paramagnetic metal complexes with nanocarriers can reduce the turnover rate of Mn, increase the molecular rotation correlation time of the complex, and thus increase its r1. These results fully prove that MoS2-MnO2 has strong MRI imaging ability and can be used as a good contrast agent, laying the foundation for the realization of integrated diagnosis and treatment of tumors.

[0119] 2.8 Analysis of oxygen evolution of MoS2-MnO2

[0120] The O2 production capacity of MoS2-MnO2 was tested using a dissolved oxygen meter. (1) 10 mL of MoS2-MnO2 (300 μg / mL) solution with pH = 5.5 and pH = 7.4 was prepared in a 50 mL beaker; (2) 10 mL of MoS2-MnO2 solution with different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL) with pH = 5.5 was prepared in a 50 mL beaker. After inserting the probe of the dissolved oxygen meter below the liquid surface, 1×10 -2 10mL of mmol / L H2O2 solution was added, and a dissolved oxygen meter was used to monitor the change of oxygen concentration over time. After 10 minutes, the probe was removed and the value was recorded.

[0121] Hypoxia, as one of the characteristics of the tumor microenvironment, will severely limit the efficacy of O2-dependent treatments. MnO2 reacts with intracellular H2O2 in the tumor microenvironment to produce O2, which lays the foundation for improving tumor hypoxia and increasing the production of O2 in the tumor site. Therefore, by introducing MoS2-MnO2 nanocarriers, we hope to use the tumor microenvironment to achieve the degradation of MnO2, and then produce O2 to alleviate the hypoxic environment of the tumor and enhance the therapeutic effect. We evaluated the dissolved oxygen levels of MoS2-MnO2 in solution in different environments and at different concentrations. Fig.11 As shown in (a), under the action of H2O2, the dissolved oxygen content in the solution containing MoS2-MnO2 rises rapidly, and then tends to be stable, and more dissolved oxygen is produced in an acidic environment. This shows that MnO2 is more completely degraded under acidic conditions and the combined action of H2O2. At the same time, we also compared the dissolved oxygen levels of MoS2-MnO2 solutions at different concentrations, such as Fig.11(b) It was found that the dissolved oxygen content showed a certain concentration dependence, that is, the dissolved oxygen level was higher in high concentration MoS2-MnO2 solution.

[0122] 2.9 Blood compatibility of MoS2-MnO2

[0123] 2.9.1 Preparation of Red Blood Cell (RBCs) Solution

[0124] 0.5 mL of blood was taken from the tail vein of healthy nude mice and placed in a heparin tube. The serum was removed by low-speed centrifugation at 2000 rpm for 3 min, and the obtained precipitate was redissolved in PBS solution, gently shaken to evenly disperse it in the PBS solution, and stored in a 4°C refrigerator for later use.

[0125] 2.9.2 Hemolytic test

[0126] Take 0.2 mL of RBCs solution and add it to the following corresponding samples (0.8 mL): (1) ddH2O; (2) PBS solution; (3) PBS solution containing different concentrations of MoS2-MnO2 (50, 100, 300, 600, 900, 1200 and 1500 μg / mL). After mixing evenly, place it on a shaker and oscillate at 100 rpm for 2 h at 37°C. Take out all samples and centrifuge them at 24000 rpm for 15 min. Then measure the absorbance (A) of the supernatant of each sample at a wavelength of 541 nm, and calculate the hemolysis percentage (hemolysis) of the sample according to formula (1-1).

[0127] The calculation formula for hemolysis percentage is:

[0128] Hemolysis (%) = (A 样品 -A PBS ) / (A H2O -A PBS ) × 100% (1-1)

[0129] Among them, A 样品 is the absorbance value of the sample solution, A PBS is the absorbance value of the negative control group, A H2O is the absorbance value of the positive control group.

[0130] 2.9.3 Blood compatibility test results of MoS2-MnO2

[0131] When nanomaterials are absorbed into the body, they are first exposed to red blood cells, causing varying degrees of red blood cell damage. If the hemolysis ratio caused by the nanomaterials exceeds 5%, it will cause red blood cells to rupture, which will in turn affect the entire body. Therefore, it is particularly important to examine the blood compatibility of MoS2-MnO2. Take PBS solutions containing different concentrations of MoS2-MnO2, add them to the dilution containing RBCs, and use ddH2O and PBS solutions as positive and negative controls, respectively. After incubation at 37°C for 2h, high-speed centrifugation at 24000rpm was performed, and the supernatant was taken out, and the absorbance of each sample supernatant at a wavelength of 541nm was measured, and the hemolysis percentage of the sample was calculated according to formula (1-1). The results are shown in Figure 1. Fig.12 As can be seen from the optical photographs, the supernatant of the positive control group is red, showing obvious hemolysis, mainly due to the large difference in osmotic pressure between the extracellular and intracellular regions. The cells absorbed a large amount of water and burst, and the hemoglobin in the cells infiltrated into the supernatant. In contrast, the supernatants of the experimental group and the negative control group were relatively clear, and most of the cells were centrifuged to the bottom of the centrifuge tube. At the same time, we also found that the hemolysis percentage of MoS2-MnO2 on red blood cells was proportional to the sample concentration. However, even when the sample concentration reached a higher 1500μg / mL, the hemolysis percentage of MoS2-MnO2 on red blood cells (2.9%) was still less than 5%. These results fully demonstrate that MoS2-MnO2 has good blood compatibility and can also provide a basis for intravenous administration.

[0132] 2.10 Synthesis and characterization of MoS2-MnO2-PEG-Pt(IV) / GA

[0133] 2.10.1 Synthesis of Pt(IV)

[0134] The synthesis of Pt(IV) is divided into two steps. Step 1: Add 500mg CDDP and 2.84mL H2O2 to 22.5mL ddH2O, stir at 76℃ in the dark for 8h, stop heating after the reaction is completed and continue stirring overnight. After overnight, place the mixture at 4℃ for crystallization for about 8h. Subsequently, wash the crystallized product with 20mL cold water, 20mL ethanol and 10mL ether in turn, collect the precipitate by centrifugation at 8000rpm for 10min and vacuum dry, and obtain a bright yellow crystalline powder overnight, i.e., oxidized cisplatin (Pt(II)). Step 2: Weigh 200mg Pt(II) and 240mg succinic anhydride and add them to 2mL DMSO. Stir at 76℃ in the dark for 48h, then freeze-dry to remove DMSO to obtain a yellow powder, add 10mL pre-cooled ice acetone to the powder, and recrystallize at -20℃ for about 8h. The collected solution was washed three times with 10 mL of pre-cooled ice acetone, centrifuged at 8000 rpm for 10 min, and the precipitate was collected and dried in vacuum to obtain a white powder, namely Pt(IV), which was stored in a 4°C refrigerator for future use.

[0135] 2.10.2 Characterization of Pt(IV)

[0136] 2.10.2.1 H NMR Spectrum ( 1 Hnuclearmagneticresonance, 1 HNMR) analysis

[0137] use 1 HNMR analysis of the molecular structure of CDDP, Pt(II), and Pt(IV). Take an appropriate amount of CDDP, Pt(II), and Pt(IV) in a DMSO-d6 solution, sonicate until the sample is completely dissolved, and transfer the prepared sample solution to a nuclear magnetic resonance measurement tube for 1 H-NMR analysis.

[0138] 2.10.2.2 Ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-QqQ-MS / MS) analysis

[0139] UPLC-QqQ-MS / MS was used to determine the molecular weight of Pt(II) and Pt(IV). Pt(II) and Pt(IV) powders were dissolved in 50% mass spectrometry grade methanol solution to prepare 700 ng / mL solutions, and the molecular weights of Pt(II) and Pt(IV) were determined.

[0140] 2.10.2.3 Fourier transform infrared spectroscopy (FT-IR) analysis

[0141] FT-IR was used to analyze the chemical structure of CDDP, Pt(II), and Pt(IV). Take appropriate amount of dry powder of CDDP, Pt(II), and Pt(IV), mix them with potassium bromide powder in an agate mortar at a mass ratio of 1:100, grind them thoroughly to make the sample and potassium bromide powder mix evenly and grind them to fineness, take a small amount of powder and place it in an infrared mold, press it for about 1 minute to obtain a translucent sheet, put the sheet into FT-IR, and detect different samples at 400-4000cm -1 Infrared spectrum information.

[0142] The synthesis of cisplatin prodrugs first involves oxidizing cisplatin with 30% H2O2 to produce a hydroxylation reaction to obtain an intermediate product, Pt(II); then the intermediate product reacts with succinic anhydride to obtain the prodrug Pt(IV) with a high yield of up to 75%. Compared with cisplatin, which has a quadrilateral spatial structure, the hexacoordinated Pt(IV) has an octahedral spatial structure, which is more stable and can reduce the drug resistance of tumor cells and the toxicity to normal cells. It also has high water solubility and is easy to encapsulate and prepare into a high-drug loading dosage form.

[0143] use 1 HNMR was used to identify the structures of cisplatin (CDDP), oxidized cisplatin (Pt(II)) and cisplatin prodrug (Pt(IV)). Fig.13 As shown, the -NH2 proton peak of cisplatin appeared at δ=6.02ppm on CDDP, the -NH2 proton peak of cisplatin and the -OH proton peak of oxidized cisplatin appeared at δ=6.54ppm and δ=5.46ppm on Pt(II), respectively, the -COOH proton peak generated by the reaction of oxidized cisplatin and succinic anhydride appeared at δ=12.06ppm on Pt(IV), and the -NH2 proton peak of cisplatin appeared at δ=6.48ppm, confirming that the synthesized compound was Pt(IV).

[0144] The molecular weights of Pt(II) and Pt(IV) were determined using UPLC-QqQ-MS / MS. Fig.14 The MS spectra of Pt(II) and Pt(IV) show that the molecular weight of Pt(II) is 332.93 and the molecular weight of Pt(IV) is 532.75, which are correct.

[0145] The structures of CDDP, Pt(II) and Pt(IV) were characterized by FT-IR. Fig.15 As shown in the figure, Pt(II) at 3515cm -1 is the stretching vibration of OH bond, 3261cm -1 is the stretching vibration of the NH bond in NH3, 1588, 1040m -1 All of them are stretching vibrations of Pt-O in the Pt-OH bond, indicating that the hydroxyl group has been successfully modified on cisplatin and Pt(II) has been successfully prepared. -1 is the stretching vibration of the NH bond in NH3, 3281 cm -1 is the stretching vibration of the OH bond in COOH, 1730 cm -1 is the stretching vibration of C=O in COO-Pt, 1659 cm -1 It is the stretching vibration of C=O in COOH, proving the existence of carboxyl group and indicating that Pt(IV) was successfully synthesized.

[0146] 2.10.3 Synthesis of LA-PEG-NH2

[0147] Lipoic acid (LA) is a naturally occurring antioxidant vitamin that has been approved by the FDA for therapeutic applications and is non-cytotoxic even at high concentrations. In this experiment, LA and PEG were coupled to obtain LA-PEG-NH2, which was used as a linker to graft Pt(IV) onto MoS2-MnO2.

[0148] Weigh 200mg 6-arm PEG-NH2, 60mg LA, and 3mg DMAP and add them to 30mL CH2Cl2 solution. Stir until the solid is completely dissolved, then add 25mg EDC and 20mg NHS to activate LA. Stir for 3 days at room temperature and away from light. After the reaction is completed, the solution is evaporated to white crystals, and the crystals are redissolved in 10mL ddH2O, centrifuged at 8000rpm for 15min, and the supernatant is transferred to a dialysis bag (MWCO=3.5kDa) and dialyzed for 48h to remove small molecule impurities. Finally, freeze-dry to obtain white solid LA-PEG-NH2, which is stored in a -20℃ refrigerator for later use.

[0149] 2.10.4 Synthesis of LA-PEG-Pt(IV)

[0150] The synthesis of LA-PEG-Pt(IV) Fig.16 The Pt(IV) complex is an analog of cisplatin that contains an additional coordination site, which is covalently attached to LA-PEG-NH2 via an amide bond to obtain LA-PEG-Pt(IV).

[0151] Preparation of LA-PEG-Pt(IV) solid complex. Weigh 200 mg Pt(IV), 50 mg EDC, and 25 mg NHS and dissolve them in 10 mL ddH2O. Stir for 2 h in dark, then add 400 mg LA-PEG-NH2 and stir for 24 h in dark. After the reaction, centrifuge at 8000 rpm for 10 min, transfer the supernatant to a dialysis bag (MWCO = 3.5 kDa), dialyze for 48 h to remove small molecule impurities, and store in a 4 °C refrigerator for later use.

[0152] 2.10.5 Characterization of LA-PEG-NH2 and LA-PEG-Pt(IV)

[0153] 2.10.5.1 H NMR Spectrum ( 1 Hnuclearmagneticresonance, 1 HNMR) analysis

[0154] use 1 HNMR analysis of the molecular structure of LA, PEG-NH2, LA-PEG-NH2 and LA-PEG-Pt(IV). Take appropriate amounts of LA, PEG-NH2, LA-PEG-NH2 and LA-PEG-Pt(IV) in DMSO-d6 solution, sonicate until the sample is completely dissolved, and transfer the prepared sample solution to a nuclear magnetic resonance measurement tube for 1 H-NMR analysis.

[0155] 2.10.5.2 Fourier transform infrared spectroscopy (FT-IR) analysis

[0156] FT-IR was used to analyze the chemical structures of LA, PEG-NH2, LA-PEG-NH2 and LA-PEG-Pt(IV). Take appropriate amount of dry powder of LA, PEG-NH2, LA-PEG-NH2 and LA-PEG-Pt(IV), mix with potassium bromide powder in an agate mortar at a mass ratio of 1:100, grind thoroughly to make the sample and potassium bromide powder mix evenly and grind to fineness, take a small amount of powder and place it in an infrared mold, press for about 1 minute to obtain a translucent sheet, put the sheet into FT-IR, and detect different samples at 400-4000cm -1 Infrared spectrum information.

[0157] 2.10.5.3 Characterization results of LA-PEG-NH2 and LA-PEG-Pt(IV)

[0158] use 1 HNMR analysis of the molecular structures of LA, PEG-NH2, LA-PEG-NH2 and LA-PEG-Pt(IV). Fig.17 It is known that LA 1 In HNMR, δ=12.02 is the -COOH proton peak of LA, δ=1.67 and δ=1.87 are the -CH2 proton peaks of LA; δ=7.65 in PEG-NH2 is the -NH2 proton peak of PEG-NH2; a new -CO-NH- proton peak appears at δ=6.65 for LA-PEG-NH2, which fully proves that PEG-NH2 and LA are successfully combined through amide condensation; at the same time, in LA-PEG-Pt(IV) 1 It was observed in HNMR that the -COOH proton peak in Pt(IV) at δ=12.02 and the -NH2 proton peak in LA-PEG-NH2 at δ=7.65 disappeared after condensation through amide reaction, a new -CO-NH-proton peak appeared at δ=6.65, and the -CH2 proton peak in LA still existed at δ=1.83, further indicating that LA-PEG-Pt(IV) was successfully synthesized.

[0159] The chemical structures of LA, PEG-NH2, LA-PEG-NH2 and LA-PEG-Pt(IV) were analyzed by FT-IR. Fig.18 The infrared spectrum of LA-PEG-NH2 shows some infrared characteristic peaks of LA and PEG-NH2, such as the stretching vibration peak of -CH- (2898cm -1 ), bending vibration of CH2 (1467cm-1 ), -CO- angle bending vibration peak (1153cm -1 ) and the stretching vibration of COC (1112cm -1 ), while LA-PEG-Pt(IV) at 1626cm -1 The vibration of C=O in -CONH appeared, which was mainly due to the combination of Pt(IV) and LA-PEG-NH2 through amide reaction.

[0160] 2.10.6 Loading of PT(IV) and GA

[0161] The synthesis process of MoS2-MnO2-PEG-Pt(IV) / GA is as follows Fig.19 As shown. First, LA-PEG-Pt(IV) was grafted onto MoS2-MnO2 through sulfur chemistry, and then GA was loaded onto MoS2-MnO2-PEG-Pt(IV) through hydrophobic interaction to construct the MoS2-MnO2-PEG-Pt(IV) / GA nano-drug delivery system.

[0162] 2.10.6.1 Establishment of platinum standard curve

[0163] Accurately weigh 100 μg of platinum standard and place it in a 10 mL brown volumetric flask, add water to the scale line, mix and shake to obtain a platinum stock solution with a concentration of 20 μg / mL, and store it in the dark for later use. Accurately draw an appropriate amount of mother liquor and dilute it with ddH2O to prepare platinum standard solutions with concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL. Use ICP-MS to measure the absorption intensity of the above standard solutions, and use platinum concentration (X, μg / mL) as the horizontal coordinate and absorption intensity value (Y) as the vertical coordinate to establish a standard curve.

[0164] 2.10.6.2 Pt(IV) loading

[0165] Weigh 100 mg of LA-PEG-Pt(IV) and dissolve it in 10 mL of ddH2O. Stir until the solid is completely dissolved, then add 20 mL of MoS2-MnO2 solution (0.5 mg / mL). MoS2-MnO2 and LA-PEG-Pt(IV) are combined together through sulfur chemistry and stirred for 24 hours. Then, the mixture is transferred to a dialysis bag (MWCO=100 kDa) and dialyzed for 3 days. The obtained MoS2-MnO2-PEG-Pt(IV) solution is stored in a 4°C refrigerator for later use.

[0166] Weigh 10 mg of MoS2-MnO2-PEG-Pt(IV), add it to 2 mL of concentrated nitric acid solution, digest it at room temperature for several hours, protect it from light during the digestion process, centrifuge it at 6000 rpm for 15 minutes after the digestion, aspirate the supernatant into an EP tube, add ddH2O for dilution, filter the dilution with a 0.22 μm microporous filter membrane, and determine the sample concentration by ICP-MS. The content of Pt(IV) in the MoS2-MnO2-PEG-Pt(IV) nano drug delivery system is calculated based on the platinum standard curve. The drug loading amount of Pt(IV) in MoS2-MnO2-PEG-Pt(IV) is calculated according to formula (1-2).

[0167] The calculation formula of drug loading is: drug loading = drug mass of drug-loaded nanosheets / total drug dosage × 100% (1-2)

[0168] 2.10.6.3 Establishment of GA standard curve

[0169] Accurately weigh 1.0 mg of GA and place it in a 10 mL volumetric flask, add DMSO to the scale line, mix and shake to prepare a GA mother solution with a mother solution concentration of 0.1 mg / mL, and store for later use. Use DMSO to dilute the GA mother solution into GA standard solutions with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. Use UV-Vis to measure the absorbance of the above standard solution at a wavelength of 360 nm, and use GA concentration (X, μg / mL) as the horizontal coordinate and absorbance value (Y) as the vertical coordinate to establish a standard curve.

[0170] 2.10.6.4 Determination of GA loading and drug loading

[0171] Prepare 5 mL of GA solution with 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, take an equal volume of MoS2-MnO2-PEG-Pt(IV) solution (0.4 mg / mL) and add it to the above solution. GA is loaded onto MoS2-MnO2-PEG-Pt(IV) by hydrophobic interaction, stir at room temperature and avoid light for 24 hours, centrifuge at 2000 rpm for 10 minutes to remove the precipitated solid drug, collect the supernatant and ultrafiltrate at 3000 rpm for 15 minutes, and wash repeatedly with ddH2O, ultrafiltrate and centrifuge until the lower solution is colorless, and finally obtain MoS2-MnO2-PEG-Pt(IV) / GA solution loaded with GA, measure the absorbance of MoS2-MnO2-PEG-Pt(IV) / GA at a wavelength of 360 nm by UV-Vis, and calculate the mass of GA according to the standard curve of GA. The drug loading amount of GA in MoS2-MnO2-PEG-Pt(IV) / GA was calculated according to formula (1-2).

[0172] 2.10.6.5 Investigation of GA dosage

[0173] The prepared MoS2-MnO2-PEG-Pt(IV) / GA solution was investigated with the average particle size, PDI, physiological stability and drug loading as evaluation indicators to determine the dosage of GA.

[0174] 2.11 Characterization of MoS2-MnO2-PEG-Pt(IV) / GA

[0175] 2.11.1 Field emission transmission electron microscope (TEM) analysis

[0176] TEM was used to observe the elemental distribution of MoS2-MnO2-PEG-Pt(IV) / GA. An appropriate amount of MoS2-MnO2-PEG-Pt(IV) / GA sample was diluted to a certain concentration with ddH2O, and 10 μL of the solution was dropped on the carbon support film. After the sample was dried overnight, the carbon film was placed in TEM, and the elemental map was analyzed using energy dispersive X-ray spectroscopy (EDS) to analyze its elemental distribution and element content.

[0177] 2.11.2 Ultraviolet-visible spectroscopy (UV-vis) analysis

[0178] UV-vis was used to analyze the ultraviolet absorption changes of MoS2-MnO2-PEG-Pt(IV) / GA. An appropriate amount of MoS2-MnO2-PEG-Pt(IV) / GA sample was diluted to a certain concentration with ddH2O, placed in a quartz cuvette, and the absorption peak of the sample at 360nm was recorded.

[0179] 2.11.3 Characterization results of MoS2-MnO2-PEG-Pt(IV) / GA

[0180] The synthesis of drug-loaded nanosheets MoS2-MnO2-PEG-Pt(IV) / GA was investigated using TEM and UV-vis. Fig. 20 As shown, from Fig. 20 (a) As can be seen from the mapping diagram, the Pt element is evenly distributed on MoS2-MnO2. Fig. 20 (b) In the EDS image, it can also be observed that there are four element peaks of Mo, S, Mn, and Pt in the MoS2-MnO2-PEG-Pt(IV) / GA nano-drug delivery system, indicating that the cisplatin prodrug is successfully loaded on the MoS2-MnO2 nano-carrier. Fig. 20(c) It was found that MoS2-MnO2-PEG-Pt(IV) / GA showed a characteristic peak of GA at 360nm, indicating that GA was successfully loaded.

[0181] 2.12 In vitro release study of MoS2-MnO2-PEG-Pt(IV) / GA

[0182] Drug release process of MoS2-MnO2-PEG-Pt(IV) / GA Fig.21 As shown. The pH value of blood in normal tissues is 7.35-7.45, so a PBS buffer solution with a pH of 7.4 was selected to simulate the in vivo microenvironment. Since the Pt(IV) complex can undergo molecular transformation under reducing conditions and release the Pt(II) drug by eliminating its axial dihydroxyl group, GSH was used as a reducing agent to test the controlled release behavior of Pt(IV) by the MoS2-MnO2-PEG-Pt(IV) / GA nano-drug delivery system, and the cumulative release of GA in the MoS2-MnO2-PEG-Pt(IV) / GA nano-drug delivery system was studied under photothermal stimulation.

[0183] Take four equal amounts of MoS2-MnO2-PEG-Pt(IV) / GA (0.5 mg / mL) sample (2 mL), disperse them in PBS solution (8 mL) with different GSH values ​​(10 mmol / L, 10 μmol / L), and seal them in dialysis bags (10 kDa). Immerse these dialysis bags in PBS solutions with corresponding GSH values, and all samples are shaken in a constant temperature shaker at 37°C and 100 rpm in the dark. Take out 1 mL of dialysate at fixed points at 0, 2, 4, 8, 12, 24, and 48 hours, and add an equal volume of the corresponding PBS buffer solution. After 48 hours, the samples in the light group were treated with 1 W / cm 2 The samples were irradiated with 808 nm laser for 10 min, and the control group was protected from light. Subsequently, 1 mL of dialysate was taken out at 50, 54, 60, 66, 72, 78, 84, 90, and 96 h, and an equal volume of PBS buffer solution was added. After dilution and filtration, the collected samples were measured for Pt concentration by ICP-MS and the absorbance at 360 nm by UV-Vis, and the cumulative drug release percentage of each sampling point was calculated, and the Pt (IV) and GA drug release curves were drawn respectively.

[0184] 2.13 Determination of drug loading of Pt(IV) and GA

[0185] 2.13.1 Establishment of platinum standard curve and determination of drug loading

[0186] The Pt content was determined by ICP-MS. Pt solutions of different concentrations were prepared and the linear equation was simulated according to the absorption intensity values ​​in ICP-MS. The results are shown in Fig. 22 As shown in (a), the regression equation of the Pt standard is Y=4154.51X, the correlation coefficient R is 1, and it has a good linear relationship between the concentration of 5-20μg / mL. The platinum element in MoS2-MnO2-PEG-Pt(IV) / GA was digested with concentrated nitric acid and then digested and centrifuged at room temperature. The supernatant was obtained and the platinum concentration was calculated by ICP-MS according to the standard curve. Finally, the drug loading of Pt(IV) in the MoS2-MnO2-PEG-Pt(IV) / GA nano drug delivery system was calculated to be 15.06% according to the formula.

[0187] 2.13.2 Establishment of GA standard curve

[0188] The content of GA was determined by UV-visible spectrophotometry. GA solutions of different concentrations were prepared and the linear equation was simulated according to the absorbance value at 360 nm. Fig. 22 (b) When the concentration was between 10 and 100 μg / mL, there was a good linear relationship between the concentration of GA and the absorbance value (R 2 =0.9996), the linear regression equation is Y=0.023X-0.007.

[0189] 2.13.3 GA dosage investigation and drug loading determination

[0190] To determine the dosage of GA, different concentrations of GA were mixed with MoS2-MnO2-PEG-Pt(IV), and free GA was removed by ultrafiltration and centrifugation to obtain MoS2-MnO2-PEG-Pt(IV) / GA solution. The particle size and PDI were measured and the drug loading was calculated to determine the dosage of GA. Fig.23 As shown in Table 1, the particle size and drug loading of MoS2-MnO2-PEG-Pt(IV) / GA gradually increase with the increase of GA dosage concentration. When the GA dosage concentration is 0.1-0.4 mg / mL, the MoS2-MnO2-PEG-Pt(IV) / GA system is relatively stable and the particle size is moderate. When the GA dosage concentration exceeds 0.6 mg / mL, the PDI of MoS2-MnO2-PEG-Pt(IV) / GA is as high as 0.337-0.674, and local coagulation occurs after being placed overnight, indicating that the drug loading has a great influence on the stability of the system. At the same time, in order to better load GA, 0.2 mg / mL is selected as the GA dosage concentration. Finally, according to the formula, the drug loading of GA in the MoS2-MnO2-PEG-Pt(IV) / GA nano drug delivery system is 14.79%.

[0191] Table 1 GA dosage investigation results (n=3)

[0192]

[0193] Most preferably, each gram of MoS2-MnO2-PEG-Pt(IV) / GA of the present invention contains 1 mg of molybdenum sulfide, 10 mg of manganese dioxide, 0.1 mg of gambogic acid, 1 mg of cisplatin prodrug Pt(IV), and 20 mg of polyethylene glycol.

[0194] 2.13.4 In vitro drug release study of MoS2-MnO2-PEG-Pt(IV) / GA

[0195] Fig.24 (a) is the release curve of Pt. It can be seen from the figure that the release rate of Pt(II) drug from MoS2-MnO2-PEG-Pt(IV) / GA depends on the concentration of GSH. When GSH is 10mM, which is similar to the concentration of glutathione in cancer cells, the cumulative release rate of Pt(II) drug in 96h reaches 84.73%, and the Pt release amount is close to the maximum at 12h. When GSH is 10μM, the cumulative release rate of Pt drug in 96h is only 29.42%, indicating that Pt(IV) can be better converted to Pt(II) in a strong reducing environment. This shows that the presence and concentration of GSH during the release of MoS2-MnO2-PEG-Pt(IV) / GA are highly sensitive to the controlled release of Pt(II) drug. This environmentally sensitive behavior of MoS2-MnO2-PEG-Pt(IV) / GA can effectively prevent the loss of drugs in the blood circulation process and ensure that the drugs are released in large quantities after reaching the tumor site. This GSH-sensitive behavior of MoS2-MnO2-PEG-Pt(IV) / GA can not only enhance its antitumor efficacy but also significantly reduce its drug toxicity during administration.

[0196] Fig.24 (b) is the release curve of GA. It can be seen from the figure that without near-infrared light irradiation, the release of GA within 48 hours is only 20.88%. The reason may be that the grafted MnO2 forms a polymer barrier on the MoS2 nanosheets, which limits the release of the drug. 2After 808 laser irradiation of the sample for 10 minutes, it was found that the cumulative release of GA in these 10 minutes was about 25%, and the release rate was 0.16ug / min, which was 5 times higher than that of the light-shielded group (0.032ug / min), indicating that near-infrared light can induce the release of GA. More importantly, after near-infrared light irradiation, the cumulative release within 48 hours was 37.6%, which was about 1.5 times that of the first 48 hours. This is mainly because MoS2 can efficiently convert the absorbed near-infrared light into heat energy, increase the temperature of the solution, accelerate the movement of GA molecules on the carrier surface, and then promote its detachment from the carrier as soon as possible. It shows that MoS2-MnO2-PEG-Pt(IV) / GA can achieve controlled release of Pt(IV) and GA under the stimulation of GSH and near-infrared light, which provides an important basis for subsequent in vitro and in vivo anti-tumor activity studies.

[0197] The beneficial effects of the present invention are demonstrated by the following efficacy test.

[0198] Experimental Example 1 In vitro antitumor activity of MoS2-MnO2-PEG-Pt(IV) / GA

[0199] 1. Synergistic treatment of MoS2-MnO2-PEG-Pt(IV) / GA at the cellular level

[0200] A549 cells with good growth status were obtained, digested with trypsin, and inoculated into 96-well cell culture plates (1×10 3 / well) and transferred to a 20% O2 incubator or a 1% O2 hypoxic culture bag. After the cells adhered to the wall, the initial culture medium was discarded and 200 μL of culture medium with different concentrations of CDDP, GA, CDDP+GA, CDDP→GA and MoS2-MnO2-PEG-Pt(IV) / GA was added (CDDP and GA were dissolved in DMSO, [CDDP] = 2, 4, 6, 8 and 10 μg / mL, [GA] = 0.2, 0.4, 0.6, 0.8 and 1.0 μg / mL), with 6 replicate wells in each group. (After incubation for 48 h in the CDDP, GA, CDDP+GA and MoS2-MnO2-PEG-Pt(IV) / GA groups, the old culture medium was aspirated and fresh culture medium was given for a further 48 h of incubation; CDDP→GA group: CDDP was incubated for 48 h and then GA was given for a further 48 h of incubation.) Finally, the cell activity was determined by the MTT method, and the relative cell survival rate R (%) was calculated according to formula (2-1).

[0201] 2. Synergistic treatment of MoS2-MnO2-PEG-Pt(IV) / GA at the cellular level

[0202] While verifying the hypoxia-induced drug resistance of tumors, the cytotoxicity of sequential administration was evaluated. In the clinical treatment of lung cancer, combination therapy is often an important treatment for lung cancer. Studies have shown that under the sequential administration scheme (CDDP first, GA later), the synergistic anti-tumor effect of GA and CDDP is very significant. In order to study whether the order of drug addition affects the synergistic effect, cells were treated with CDDP for 48 hours and then with GA for 48 hours. The results are as follows Fig.25 The drugs had a concentration-dependent inhibitory effect on cell growth, among which sequential administration had the strongest inhibitory effect on cell growth, indicating that sequential administration was the key factor in achieving synergistic treatment with CDDP and GA.

[0203] At the same time, it was found that the growth inhibitory effect of drugs on A549 cells after 96 hours in hypoxic environment was significantly lower than that in normoxic environment, showing higher cell activity, indicating that hypoxic environment can induce drug resistance in tumor cells. Under normoxic conditions, the cell survival rate of the free CDDP or GA group was more than 60%, while that of the free CDDP or GA group under hypoxic conditions was more than 80%. At the same time, the IC values ​​of CDDP and GA under hypoxic environment were calculated respectively. 50 The values ​​were 72.32μg / mL and 3.80μg / mL, which were 3.4 and 2.7 times those in the normoxic environment (20.67μg / mL and 1.369μg / mL), respectively, indicating that tumor cells have strong drug resistance to drugs in a hypoxic environment. This may be because under hypoxic conditions, A549 cells express more drug-resistant proteins on their surface, which excrete intracellular drugs to the outside of the cells, resulting in a decrease in the amount of drugs entering the cells and a decrease in anti-tumor efficiency. At the same time, it was found that the IC of the MoS2-MnO2-PEG-Pt(IV) / GA group 50 The value was 6.14 / 0.61 μg / mL (CDDP / GA), which was lower than that of other groups, but higher than that under normoxic conditions (5.26 / 0.53 μg / mL), indicating that MoS2-MnO2-PEG-Pt(IV) / GA could alleviate the external hypoxic state of tumor cells. This was because the oxygen production of MnO2 in the nanosystem improved tumor hypoxia and enhanced the toxicity of chemotherapeutic drugs to tumor-resistant cells.

[0204] 3. Cytotoxic effect of MoS2-MnO2-PEG-Pt(IV) / GA on lung cancer cells under near-infrared light irradiation

[0205] A549 cells in the logarithmic growth phase were obtained, digested with trypsin, and inoculated into 96-well cell culture plates (1×10 3 / well), placed in an incubator at 37°C and 20% O2 or a 1% O2 hypoxic culture bag. After the cells adhered, they were randomly divided into an illumination group and a non-illumination group, each of which was further divided into the following 7 subgroups: (1) cell culture medium, as a blank control group; (2) CDDP; (3) GA; (4) CDDP+GA; (5) CDDP→GA; (6) MoS2-MnO2; (7) MoS2-MnO2-PEG-Pt(IV) / GA. ([CDDP] = 2 μg / mL, [GA] = 0.2 μg / mL, [MoS2-MnO2] = 500 μg / mL), the initial culture medium was removed, 200 μL of the above materials were added to each well, and six replicates were set up for each group. After incubation for 6 hours, the drug-containing culture medium was aspirated, the cells were washed 3 times with PBS solution, and fresh culture medium was added to continue incubation for 42 hours (Note: after 48 hours, the group (5) was given a culture medium containing GA). Then, the cells in the illumination group were treated with a power density of 1 W / cm 2 The cells in the non-irradiated group were irradiated with 808 nm laser for 10 min, and the cells in the non-irradiated group were protected from light. After another 48 h of incubation (Note: After incubating in the GA medium for 6 h in group (5), the drug-containing medium was aspirated and fresh medium was added to continue incubation for 42 h), the drug-containing medium was aspirated and the cells were washed 3 times with sterile PBS, and then 10 μL MTT solution was added to each well and cultured in the incubator for another 4 h. The MTT solution was aspirated, 150 μL DMSO was added to each well, and the cell culture plate was placed in a shaker and shaken at 100 rpm for 15 min at 37 ° C. Finally, the absorbance (A) of each well at 490 nm was measured using an ELISA instrument, and the relative cell survival rate R (%) was calculated according to formula (2-1).

[0206] 4. Induction of apoptosis of lung cancer cells by MoS2-MnO2-PEG-Pt(IV) / GA under near-infrared light irradiation

[0207] A549 cells in the logarithmic growth phase were obtained, digested with trypsin, and inoculated into 6-well cell culture plates (2×10 5 / well). After the cells adhered, they were randomly divided into 9 groups: (1) cell culture medium, as a blank control group; (2) CDDP; (3) GA; (4) CDDP+GA; (5) CDDP→GA; (6) MoS2-MnO2; (7) MoS2-MnO2+NIR; (8) MoS2-MnO2-PEG-Pt(IV) / GA; (9) MoS2-MnO2-PEG-Pt(IV) / GA+NIR. ([CDDP]=2μg / mL, [GA]=0.2μg / mL, [MoS2-MnO2]=500μg / mL). The initial culture medium was removed and 1mL of the above materials was added to each well. After incubation for 6h, the drug-containing culture medium was aspirated, the cells were washed 3 times with PBS solution, and fresh culture medium was added to continue incubation for 42h (Note: after 48h, the group (5) was given GA-containing culture medium). Subsequently, the cells in the illumination group were treated with a power density of 1W / cm 2 The cells in the non-irradiated group were irradiated with 808 nm laser for 10 min, and the cells in the non-irradiated group were protected from light. After another 48 h of incubation (Note: the drug-containing medium in group (5) was aspirated after 6 h of incubation with GA medium, and fresh medium was added to continue incubation for 42 h), the Annexin V-FITC / PI cell apoptosis detection kit was used to examine cell apoptosis. The specific operation is as follows: after the incubation, the drug-containing medium was aspirated, and the cells were washed 3 times with PBS solution. During the washing, the cell culture plate was slightly shaken to remove the residual drug-containing medium on the cells. Then, the cells were digested with trypsin without EDTA. When the cells became round and fell off, the cells were collected into a 1.5 mL centrifuge tube, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded; the cells were resuspended with 1 mL of 4°C pre-cooled PBS solution, washed 3 times, centrifuged again, and the supernatant was discarded; 490 μL of 1× binding buffer was added to stain the resuspended cells (the binding buffer was diluted with deionized water), 5 μL of Annexin V-FITC was added to incubate at 4°C in the dark for 10 min, and then 5 μL of PI was added to incubate at 37°C in the dark for 10 min, and finally transferred to a flow tube. Flow cytometry was used for detection and data analysis was performed using FlowJo software.

[0208] 5. Statistical analysis

[0209] Unless otherwise specified, the experimental results were statistically analyzed using GraphPad Prism 8.0 software. The experimental data were expressed as mean ± standard deviation (Mean ± SD). The t test was used to analyze the differences between two experimental groups; the one-way analysis of variance (ANOVA) was used to analyze the differences between more than two experimental groups. * P﹤0.05 indicated a significant difference. ** P﹤0.01 indicates a very significant difference.

[0210] MoS2 nanomaterials have become a popular material for tumor photothermal therapy due to their strong near-infrared absorption. Therefore, we further verified whether the cell killing ability of MoS2-MnO2-PEG-Pt(IV) / GA is enhanced under near-infrared light irradiation. After incubating the cells according to method 3.4, the cell activity was calculated according to the MTT method. Fig.26 It can be seen that in a normoxic environment, near-infrared light irradiation alone does not increase the cytotoxicity of free drugs or combined drugs. However, after the tumor cells treated with MoS2 materials were irradiated with near-infrared light, their cell activity decreased significantly. In a normoxic environment, after the tumor cells treated with MoS2-MnO2 were irradiated with near-infrared light, the cell activity dropped from 96.3% to 57.01%. This is mainly because MoS2 nanomaterials can convert the absorbed near-infrared light into thermal energy, causing the ablation of tumor cells, inhibiting their proliferation, and then inducing tumor cell death. Importantly, the MoS2-MnO2 material loaded with CDDP and GA showed the strongest cell killing effect under near-infrared irradiation, with only 11.47% of cells in a viable state, which was much lower than that of other experimental groups. The reasons for this result are: first, near-infrared light effectively induced the release of GA, which not only significantly increased the concentration of free GA in cells, but also achieved the sequential administration of CDDP and GA to tumor cells, greatly improving the synergistic effect between the two drugs; second, the combined treatment of photothermal and synergistic chemotherapy. In a hypoxic environment, photothermal therapy alone is not affected by the lack of oxygen, while the drug group is restricted to varying degrees. MoS2-MnO2-PEG-Pt(IV) / GA binds to intracellular H + It reacts with H2O2 to produce oxygen, relieves the tumor hypoxia, weakens the tumor hypoxia resistance, and shows a strong ability to inhibit tumor cell proliferation, especially under near-infrared light irradiation. However, the drugs in other drug groups have a significant inhibitory effect on their ability to inhibit tumor cell proliferation regardless of whether they are exposed to light or near-infrared light.

[0211] 6. Induction of apoptosis of lung cancer cells by MoS2-MnO2-PEG-Pt(IV) / GA under near-infrared light irradiation

[0212] Apoptosis is a form of cell death, and many anticancer drugs, including CDDP and GA, can kill tumor cells by inducing apoptosis. Photothermal therapy can also effectively induce apoptosis by stimulating tumor cells to initiate the apoptotic program. To further prove that MoS2-MnO2-PEG-Pt(IV) / GA has a strong cell killing effect under near-infrared light irradiation, we investigated the ability of MoS2-MnO2-PEG-Pt(IV) / GA to induce cell apoptosis under near-infrared light irradiation. Flow cytometry is a detection method for quantitative analysis of cell apoptosis rate, in which the four quadrants of the cell density map represent cells in different states, Q1 represents necrotic or mechanically damaged cells, Q2 represents late apoptotic cells, Q3 represents early apoptotic cells, and Q4 represents living cells. This experiment uses the Annexin V-FITC and PI double staining method to detect cell apoptosis. The principle is as follows: during the apoptosis process, the phosphatidylserine (PS) on the inner surface of the cell membrane is destroyed due to the asymmetry of its distribution, resulting in PS being exposed from the inner surface of the cell membrane to the outer surface of the cell membrane. Annexin V has a high affinity for PS and can specifically bind to PS. Annexin V can detect PS through fluorescent FITC labeling. However, in addition to early and late apoptotic cells, the PS of necrotic cells will also be transferred to the outside of the cell membrane, so propidium iodide (PI) is used to distinguish them. PI cannot penetrate the intact cell membrane, and the integrity of the cell membrane of late apoptotic and necrotic cells has been destroyed, so they can be distinguished by PI detection.

[0213] Fig. 27 The density distribution picture of the flow cytometry results of A549 cells in a hypoxic environment. It can be found from the figure that after A549 cells were treated with different drugs, the cell apoptosis rate of sequential administration was stronger than that of single drugs or combination drugs, indicating that under the sequential administration scheme (CDDP first, GA later), the synergistic anti-tumor effect of CDDP and GA was very significant, which was consistent with the cell proliferation experiment. At the same time, it was observed that the MoS2-MnO2 carrier material had no obvious apoptosis-inducing effect on cells, and its apoptosis percentage was 4.26%, indicating that MoS2-MnO2 is relatively safe, but compared with the MoS2-MnO2+NIR (39.54%) group, it was found that the cell apoptosis rates of the two groups were significantly different, indicating that the photothermal warming effect caused by MoS2 may promote cell apoptosis. In addition, the percentage of apoptotic cells in the MoS2-MnO2-PEG-Pt(IV) / GA+NIR group was 83.6%, which was much higher than that in the other experimental groups, indicating that MoS2-MnO2-PEG-Pt(IV) / GA effectively induced tumor cell apoptosis under the irradiation of near-infrared light, which was consistent with the results of the tumor cell proliferation experiment.

[0214] Experimental Example 2 In vivo antitumor activity of MoS2-MnO2-PEG-Pt(IV) / GA

[0215] 1. Experimental methods

[0216] 1.1 Establishment of in vivo A549 subcutaneous transplant tumor model

[0217] After nude mice were adaptively raised in the animal experimental center for 1 week, tumor cells were subcutaneously inoculated. A549 cells in the logarithmic growth phase were obtained, digested with trypsin, collected and centrifuged. The cell pellet was then resuspended in PBS and the cell concentration was determined using a cell counter. To ensure cell viability, cell collection and cell inoculation should be completed in a short time. A disposable sterile syringe was used to draw 100 μL of a concentration of 5×10 7 / mL of the A549 cells were injected subcutaneously into the left forelimb of nude mice, and then the nude mice were returned to the cages for breeding. The general characteristics of the mice, such as their activities and mental state, were observed, and any swelling or infection caused by the injection was observed.

[0218] 1.2 In vivo antitumor activity of MoS2-MnO2-PEG-Pt(IV) / GA under near-infrared light irradiation

[0219] 1.2.1 Experimental Grouping

[0220] When the tumor volume grows to 60 mm 3 At about 3 4 h, the nude mice were randomly divided into 8 groups, with 5 mice in each group: (1) PBS, as a blank control group; (2) CDDP; (3) GA; (4) CDDP+GA; (5) MoS2-MnO2; (6) MoS2-MnO2+NIR; (7) MoS2-MnO2-PEG-Pt(IV) / GA; (8) MoS2-MnO2-PEG-Pt(IV) / GA+NIR; ([CDDP]=1.0 mg / kg, [GA]=0.1 mg / kg, [MoS2-MnO2-PEG]=5 mg / kg).

[0221] 1.2.2 Tail vein administration

[0222] Each group of nude mice was injected with the above-mentioned corresponding materials through the tail vein according to their body weight, once every 3 days, for a total of 7 times. Before administration, wipe the tail of the nude mouse with an alcohol cotton ball to dilate the tail vein. The drug was injected intravenously at 1 / 3 of the tail of the nude mouse. The drug was injected when there was no resistance when the needle was inserted and blood was sucked back. After the injection, sterile cotton balls were used to stop bleeding. In order to ensure that Pt(IV) in MoS2-MnO2-PEG-Pt(IV) / GA has enough time to increase the sensitivity of tumor cells to GA, the nude mice in the light group were treated with 1.0W / cm 2The tumor site was irradiated with 808nm laser for 10 minutes. During the irradiation process, the temperature value was recorded every 60 seconds with an infrared thermal imager, and the data was collected and the photothermal temperature rise curve was drawn.

[0223] 1.2.3 Evaluation indicators

[0224] (1) Body weight: During the 21 days of drug treatment, the nude mice were weighed every 3 days to observe their weight changes and investigate the toxicity of the drug to the nude mice.

[0225] (2) Tumor volume: The length and width of the nude mouse tumor were measured every 3 days and recorded in time. The tumor volume (V) of the nude mouse was calculated according to formula (3-1), and the tumor growth curve was drawn.

[0226] The calculation formula of tumor volume is: tumor volume (V) = (a × b 2 ) / 2(3-1)

[0227] Where a and b are the long and short diameters of the tumor, respectively.

[0228] (3) Tumor volume inhibition rate: The length and width of the nude mouse tumor were measured every 3 days and recorded in time. The tumor volume inhibition rate (TGI) of the nude mouse was calculated according to formula (3-2) and statistical analysis was performed.

[0229] The calculation formula of tumor volume inhibition rate TGI (%) is: TGI (%) = 1-[(V treated -V0) / V PBS -V0)]×100%(3-2)where V treated is the average tumor volume of the drug-treated group, V PBS is the average tumor volume of the control group, and V0 is the initial tumor volume.

[0230] 1.2.4 Hematoxylin-eosin (H&E) staining analysis

[0231] After 21 days of administration, all nude mice were killed, and the heart, liver, spleen, lung, kidney and tumor tissues were removed and fixed in 4% paraformaldehyde solution for 1 day. The tissues were then placed in 70% ethanol solution and finally stored in a 4°C refrigerator for later use. The tumor tissues of nude mice in each group and the heart, liver, spleen, lung and kidney tissues of the PBS group and the MoS2-MnO2-PEG-Pt(IV) / GA+NIR group were taken for H&E staining analysis. The operation steps are as follows:

[0232] (1) Preparation of tissue sections: The preserved heart, liver, spleen, lung and kidney tissues were cut into uniform small pieces and placed in an ethanol / water solution for dehydration. The tissues were permeabilized with xylene, embedded in paraffin, and sliced ​​with a microtome. The obtained tissue slices (5 μm) were then placed on glass slides and baked in a baking oven at 70°C for 10 min.

[0233] (2) Dewaxing: After the sections are dried, they are transferred to xylene solution and immersed twice, each time for 10 minutes. Then, the sections are immersed in 100% ethanol, 95% ethanol, 80% ethanol, and 75% ethanol solutions in sequence for 3 minutes each for dewaxing treatment, and finally placed in distilled water.

[0234] (3) Staining: Place the sections in hematoxylin stain for 5 min and rinse with distilled water three times, each time for 3 min.

[0235] (4) Differentiation: The sections were immersed in 1% hydrochloric acid ethanol solution and incubated for 10 min, followed by rinsing with distilled water for 15 min.

[0236] (5) Restaining: Immerse the sections in 0.5% eosin solution for 6-10 seconds, rinse the sections in 95% ethanol for 10 seconds, and rinse the sections in anhydrous ethanol for 10 seconds.

[0237] (6) Sealing: Add an appropriate amount of neutral gum diluted with xylene onto the tissue and cover with a coverslip.

[0238] (7) Taking photos: Observe the tissue under an upright microscope and take photos to save the images.

[0239] 1.3 Statistical analysis

[0240] Unless otherwise specified, the experimental results were statistically analyzed using GraphPad Prism 8.0 software. The experimental data were expressed as mean ± standard deviation (Mean ± SD). The t test was used to analyze the differences between two experimental groups; the one-way analysis of variance (ANOVA) was used to analyze the differences between more than two experimental groups. * P﹤0.05 indicated a significant difference. ** P﹤0.01 indicates a very significant difference.

[0241] 2. Results and Discussion

[0242] 2.1 In vivo antitumor activity of MoS2-MnO2-PEG-Pt(IV) / GA under near-infrared light irradiation

[0243] Based on the experimental results of MoS2-MnO2-PEG-Pt(IV) / GA inhibiting tumor cell proliferation and inducing tumor cell apoptosis in vitro, we investigated its anti-tumor effect in vivo. 3At about 24 hours, the nude mice were randomly divided into 8 groups: (1) PBS group, as the control group; (2) CDDP; (3) GA; (4) CDDP+GA; (5) MoS2-MnO2; (6) MoS2-MnO2+NIR; (7) MoS2-MnO2-PEG-Pt(IV) / GA; (8) MoS2-MnO2-PEG-Pt(IV) / GA+NIR. After each intravenous administration for 24 hours, 1W / cm 2 The tumor was irradiated with 808 nm laser for 10 min. Fig.28 Before and after laser irradiation, the tumor temperature of PBS, CDDP, GA, and CDDP+GA groups did not change much and was always below 40°C, while the tumor temperature of MoS2-MnO2+NIR group and MoS2-MnO2-PEG-Pt(IV) / GA+NIR group changed significantly, and the tumor temperature rose rapidly to 56°C within 10 minutes. This high temperature can not only cause thermal death of tumor cells, but also help release loaded drugs, enhance the chemotherapy effect of MoS2-MnO2-PEG-Pt(IV) / GA, and achieve the synergistic effect of chemotherapy and photothermal therapy.

[0244] After the treatment started, the length and width of the nude mouse tumor were measured with a vernier caliper every 3 days to calculate the real-time volume of the tumor, draw a curve of the change in tumor volume, and calculate the tumor volume inhibition rate. Fig.29 It can be seen that the nude mice injected with MoS2-MnO2 alone had a faster tumor growth rate. After 21 days, the average volume of the tumor in this group reached 729.06 mm 3 , close to 770 mm in the PBS group 3, and the tumor volume inhibition rate was only 5.31%, indicating that MoS2-MnO2 at this dose is low toxic and has no significant inhibitory effect on tumors. Compared with the PBS group, the tumor growth rate of CDDP or GA alone was slightly slowed down, indicating that the single drug cannot effectively inhibit tumor growth. Compared with single drugs, the anti-tumor ability of combined administration is only slightly higher, but this is inconsistent with the results of cell proliferation experiments. The reasons are: (1) GA and CDDP are both small molecules with inherent defects such as poor water solubility, short half-life, and no targeting, which seriously limit their therapeutic effects in vivo. (2) The two have different administration routes and pharmacokinetic processes, making it impossible for the two to act on the tumor in a set order, eliminating strict time restrictions and eliminating the synergistic effect between them. When the combined drugs are co-loaded on MoS2-MnO2, this can improve the inherent defects of CDDP and GA such as poor water solubility and short half-life, and achieve a stronger tumor inhibition ability. More importantly, under the irradiation of near-infrared light, the release of drugs is controlled, and the two drugs act on the tumor one after another, which greatly improves the synergistic effect between the two. Combined with the photothermal effect of the carrier itself, the average volume of the tumor is only 57.68mm 3 The tumor inhibition rate was as high as 92.09%, showing the strongest anti-tumor ability.

[0245] The tumor tissue was stained with H&E and the tumor tissue sections were observed ( Fig.30 ) found that compared with other groups, after treatment with MoS2-MnO2-PEG-Pt(IV) / GA+NIR, the tumor cell density decreased, most tumor cells were destroyed, and the cell nucleus staining became lighter, showing isomerization, indicating that MoS2-MnO2-PEG-Pt(IV) / GA+NIR has a stronger therapeutic effect, further illustrating that the nanodrug delivery system can promote tumor cell apoptosis and necrosis and inhibit tumor growth under near-infrared light irradiation.

[0246] 2.2 In vivo safety evaluation

[0247] The safety of the drug was investigated by recording the body weight changes of nude mice and sections of major organs. Fig.31 The figure is the weight change curve of nude mice. The results show that the weight of nude mice increases slowly over time, but there is no significant change. This shows that MoS2-MnO2-PEG-Pt(IV) / GA has no obvious toxic side effects on nude mice. At the same time, we also used the H&E staining method to examine the toxic effects of MoS2-MnO2-PEG-Pt(IV) / GA on major organs in the body. The main tissue and organ sections of the two groups were observed, and the results are as follows Fig.32The results showed that there was no significant difference in the main organ sections between the PBS group and the MoS2-MnO2-PEG-Pt(IV) / GA+NIR group, indicating that MoS2-MnO2-PEG-Pt(IV) / GA did not cause obvious damage to the main organs of nude mice, nor did it cause pathological changes such as tissue necrosis and fibrosis, and no organ damage occurred. This further confirmed that MoS2-MnO2-PEG-Pt(IV) / GA had no obvious toxic side effects on the main tissues and organs of nude mice, indicating that MoS2-MnO2-PEG-Pt(IV) / GA is a safe and effective nanodrug preparation.

[0248] The antitumor activity of MoS2-MnO2-PEG-Pt(IV) / GA was investigated at the animal level. First, a nude mouse lung cancer A549 cell subcutaneous transplant tumor model was established, and MoS2-MnO2-PEG-Pt(IV) / GA was injected into the tail vein. This nano drug delivery system can be enriched in the tumor site by passive targeting and EPR effect. Under the irradiation of near-infrared light, the average temperature of the tumor site of nude mice rose rapidly to 56°C. This high temperature can not only cause ablation of tumor cells and inhibit tumor growth, but also effectively promote the release of combined drugs, enhance the therapeutic effect of the nano drug delivery system, and realize the synergistic chemotherapy and photothermal combined treatment of combined drugs for tumors. In terms of safety, the weight changes of nude mice and H&E staining results showed that MoS2-MnO2-PEG-Pt(IV) / GA had no obvious toxic side effects in the body and did not cause visible damage to major organs. It is a safe and effective nano drug preparation.

[0249] The MoS2-MnO2-PEG-Pt(IV) / GA nano drug delivery system constructed by the present invention controls the sequential release of drugs through GSH and near-infrared light, combines the characteristics of the carrier itself, reverses tumor hypoxia and drug resistance, maximizes the synergistic therapeutic effect of GA and CDDP, and ultimately achieves combined drug synergistic treatment and photothermal combined treatment of lung cancer, providing a new idea and research strategy for the clinical treatment of lung cancer, and also expanding the application scope of anti-cancer Chinese medicine in tumor treatment.

Claims

1. A molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin, characterized in that: The invention contains the following components in weight ratio: 1-5 parts of molybdenum sulfide, 5-50 parts of manganese dioxide, 0.1-5 parts of gambogic acid, 1-50 parts of cisplatin prodrug Pt(IV), and 10-50 parts of polyethylene glycol.

2. The molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin according to claim 1, characterized in that: The invention contains the following components in weight ratio: 1 part of molybdenum sulfide, 10 parts of manganese dioxide, 0.1 part of gambogic acid, 1 part of cisplatin prodrug Pt(IV), and 20 parts of polyethylene glycol.

3. The molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin according to claim 1 or 2, characterized in that: It is a nano-delivery system based on molybdenum disulfide (MoS2) nanosheets, modified with manganese dioxide (MnO2) nanoparticles, and loaded with GA and CDDP. The MoS2-MnO2 nanosheets have a two-dimensional layered structure; GA and CDDP are loaded through amide reaction and hydrophobic effect.

4. The molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin according to claim 3, characterized in that: The molybdenum disulfide (MoS2) nanosheets are prepared by Morrison method to form a single layer or a few layers of molybdenum disulfide nanosheets, which comprises the following steps: Under nitrogen, MoS2 crystals were placed in a n-butyllithium solution containing 15% n-hexane and stirred for 2 days under the protection of nitrogen; After the reaction is completed, the mixture is washed with redistilled n-hexane to remove the residue of n-butyl lithium and other impurities; After washing, the black powder was removed and ddH2O was added immediately, and ultrasonic stripping was performed for 2 hours. Then, the solution was collected and centrifuged at 8000rpm for 20 minutes to remove the crystals that were not completely stripped. After centrifugation, the upper layer solution was transferred to a dialysis bag with MWCO=100kDa and dialyzed for one week to remove residual lithium ions and other impurities; thus, a solution containing MoS2 nanosheets was obtained; The manganese dioxide (MnO2) nanoparticles are prepared by oxidation-reduction reaction, and the preparation method is as follows: Weigh KMnO4 and bovine serum albumin (BSA) and add them to ddH2O, and stir at room temperature for 5 hours; KMnO4 is reduced by BSA to form MnO2 nanoparticles; after the reaction is completed, high-speed centrifugation is performed at 12000rpm for 20 minutes to remove free KMnO4, BSA and MnO2 particles with larger particle size; collect the upper layer solution to obtain manganese dioxide (MnO2) nanoparticles; The preparation method of Pt(IV) is as follows: CDDP and H2O2 are added to ddH2O, stirred at 76°C in the dark for 8 hours, heating is stopped after the reaction is completed, and stirring is continued overnight; after overnight, the mixture is placed at 4°C for about 8 hours for crystallization; Subsequently, the crystalline product was washed with cold water, ethanol and ether in turn, the precipitate was collected by centrifugation at 8000 rpm for 10 min and then vacuum dried to obtain a bright yellow crystalline powder, i.e., oxidized cisplatin (Pt(II)); Pt(II) and succinic anhydride were added to DMSO; the mixture was stirred at 76°C in the dark for 48 h, and then freeze-dried to remove DMSO to obtain a yellow powder, pre-cooled ice acetone was added to the powder, and recrystallized at -20°C; the solution was collected and washed three times with 10 mL of pre-cooled ice acetone, the precipitate was collected by centrifugation at 8000 rpm for 10 min, and then vacuum dried to obtain a white powder, i.e., Pt(IV).

5. The molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin according to claim 3, characterized in that: The synthesis method of MoS2-MnO2 is as follows: MoS2 nanosheet aqueous solution and MnO2 nanoparticle aqueous solution are mixed, MoS2 and MnO2 are combined together by sulfur chemical reaction, and then the mixed solution is transferred into a dialysis bag (MWCO=100kDa) after stirring for 24 hours, and dialyzed for 3 days to remove free MoS2 and MnO2, thereby obtaining a MoS2-MnO2 solution.

6. A method for preparing the molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin according to any one of claims 1 to 5, characterized in that: It includes the following steps: a. Preparation of molybdenum disulfide (MoS2) nanosheets; b. preparing manganese dioxide (MnO2) nanoparticles; c. Synthesis of MoS2-MnO2; d. Synthesis of Pt(IV); e. Synthesis of LA-PEG-NH2: coupling LA and PEG to obtain LA-PEG-NH2; f. LA-PEG-NH2 was used as a connector to graft Pt(IV) onto MoS2-MnO2 to prepare a molybdenum disulfide nanodelivery system co-loaded with gambogic acid and cisplatin.

7. The method for preparing the molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin according to claim 6, characterized in that: The synthesis method of LA-PEG-NH2 described in step e is: Weigh 6-arm PEG-NH2, LA, and DMAP and add them to CH2Cl2 solution. Stir until the solid is completely dissolved, then add EDC and NHS to activate LA, and stir at room temperature in the dark for 3 days. After the reaction, evaporate the solution until white crystals are obtained. Dissolve the crystals in ddH2O and centrifuge at 8000 rpm for 15 min. Transfer the supernatant to a dialysis bag (MWCO=3.5 kDa) and dialyze for 48 h to remove small molecule impurities. Finally, freeze-dry to obtain white solid LA-PEG-NH2.

8. The method for preparing the molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin according to claim 8, characterized in that: In step f, LA-PEG-Pt(IV) is first synthesized, and the Pt(IV) complex is covalently attached to LA-PEG-NH2 via an amide bond to obtain LA-PEG-Pt(IV). The synthesis method is: Weigh Pt(IV), EDC, and NHS and dissolve them in ddH2O, stir in the dark, then add LA-PEG-NH2 and stir in the dark for 24 hours; after the reaction, centrifuge at 8000 rpm for 10 minutes, transfer the supernatant to a dialysis bag (MWCO=3.5 kDa), dialyze for 48 hours to remove small molecule impurities, and obtain LA-PEG-Pt(IV); LA-PEG-Pt(IV) was then grafted onto MoS2-MnO2 through sulfur chemistry, and then GA was loaded onto MoS2-MnO2-PEG-Pt(IV) through hydrophobic interaction to construct the MoS2-MnO2-PEG-Pt(IV) / GA nanodrug delivery system.

9. The method for preparing the molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin according to claim 8, characterized in that: The preparation method of the MoS2-MnO2-PEG-Pt(IV) / GA nano drug delivery system comprises the following steps: Pt(IV) loading: 100 mg of LA-PEG-Pt(IV) was dissolved in 10 mL of ddH2O and stirred until the solid was completely dissolved. Then, 20 mL of MoS2-MnO2 solution (0.5 mg / mL) was added. MoS2-MnO2 and LA-PEG-Pt(IV) were combined by sulfur chemistry. After stirring for 24 h, the mixture was transferred to a dialysis bag (MWCO = 100 kDa) and dialyzed for 3 days to obtain the MoS2-MnO2-PEG-Pt(IV) solution; GA Load: GA solutions with concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL were prepared, and an equal volume of MoS2-MnO2-PEG-Pt(IV) solution was added to the above solution; GA was loaded onto MoS2-MnO2-PEG-Pt(IV) through hydrophobic interaction, and the mixture was stirred at room temperature in the dark for 24 h, and then centrifuged at 2000 rpm for 10 min to remove the precipitated solid drug, and the supernatant was collected and ultrafiltrated and centrifuged at 3000 rpm for 15 min, and washed repeatedly with ddH2O, and ultrafiltrated and centrifuged until the lower layer of the solution was colorless, thereby obtaining MoS2-MnO2-PEG-Pt(IV) / GA solution loaded with GA.

10. Use of the molybdenum disulfide nano-delivery system co-loaded with gambogic acid and cisplatin as claimed in any one of claims 1 to 5 in the preparation of a drug for treating lung cancer.

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