CN119097719A - Molybdenum disulfide nano conveying system for co-carrying gambogic acid and cisplatin, and preparation method and application thereof - Google Patents
Molybdenum disulfide nano conveying system for co-carrying gambogic acid and cisplatin, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a molybdenum disulfide nano conveying system for co-carrying gambogic acid and cisplatin, which comprises the following components, 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 cisplatin prodrug Pt (IV) and 10-50 parts of polyethylene glycol. The invention also provides a preparation method of the nano-transport system. According to the MoS 2-MnO2 -PEG-Pt (IV)/GA nano drug delivery system constructed by the invention, through sequential release of GSH and near infrared light control drugs and combination of the characteristics of a carrier, tumor hypoxia drug resistance is reversed, so that 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, a new thought and research strategy is provided for clinical lung cancer treatment, and the application range of anticancer traditional Chinese medicines in tumor treatment is also expanded.
Description
Technical Field
The invention relates to a molybdenum disulfide nano-delivery system for co-carrying gambogic acid and cisplatin and a preparation method thereof, belonging to the field of pharmaceutical preparations.
Background
Lung cancer, whether in men or women, is one of the most common and fatal cancers worldwide, being the most frequently examined malignancy (11.6% of total cases) and the most important factor affecting cancer mortality (18.4% of total cancer mortality). Since the 30 s of the 20 th century, the incidence and mortality of lung cancer has steadily increased worldwide, and effective strategies are needed to prevent and treat lung cancer. Lung cancer can be classified, according to histopathology, into Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC being the most common, accounting for about 85% of the total lung cancer. The current treatment schemes for lung cancer mainly comprise surgery, drug treatment, external irradiation treatment, stereotactic radiosurgery, targeted therapy, immunotherapy and palliative therapy. Although surgical treatment is the first treatment for early stage lung cancer patients, the clinical application of surgery is limited to a certain extent due to the low tolerance of patients to surgical treatment, and most lung cancer patients are advanced in diagnosis due to certain limitations of early stage lung cancer screening technology, and the life span can be prolonged and the life quality can be improved only by chemotherapy or radiotherapy.
Chemotherapy is a common therapeutic strategy in cancer treatment, and several tens of anticancer drugs such as cisplatin, paclitaxel, docetaxel, doxorubicin (DOX), etc. have been developed by researchers in the field of lung cancer treatment for the past decades. Among them, cisplatin (CDDP) is one of the most widely known and effective chemotherapeutics, and plays a vital role as an antitumor drug in the treatment of various malignant tumors, and this platinum (II) -based molecule, due to its ability to form crosslinks with DNA, binds to DNA in tumor cells after entering cells, forms a platinum-DNA complex, causes changes in DNA structure, causes cytotoxic damage, induces apoptosis, and finally causes death of tumor cells. After FDA approval since 1978, it has been used to treat various solid human tumors, including bladder cancer, breast cancer, prostate cancer, ovarian cancer, testicular cancer, and neuroblastoma, etc., for first line treatment of malignant tumors along with the analogs carboplatin and oxaliplatin. The medicine is simple in administration form and good in curative effect, and becomes a gold standard chemotherapeutic medicine for treating various solid tumors clinically. However, cisplatin has a remarkable dose dependency in proportion to the dose, and most importantly, the tumor tissue lacks selectivity, and causes a plurality of adverse reactions such as vomiting, nausea, ototoxicity, neurotoxicity, nephrotoxicity and the like, so that the use of cisplatin is severely limited and the clinical treatment effect is weakened, and continuous chemotherapy and long-term administration can lead to drug resistance of tumors to drugs, greatly reduce the treatment effect, and cause serious damage to normal cells due to the fact that the drugs cannot specifically detect lung cancer cells, so that a plurality of patients have common gastrointestinal symptoms, bone marrow suppression and other toxic reactions. Although platinum drugs have better antitumor effect, the platinum drugs still face serious side effects and adverse reactions such as high toxicity caused by nonspecific binding with plasma essential proteins clinically, and the application of the platinum drugs is greatly limited. To overcome these limitations, a series of new cisplatin analogues have been synthesized.
Numerous studies have shown that Glutathione (GSH) and glutathione transferases (GSTs) that are overexpressed in most cancer cells are the primary causes of cisplatin adverse reactions and drug resistance. In order to promote the development of the platinum drugs, reduce the side effects of the cisplatin drugs and improve the application prospect of the cisplatin drugs in the tumor treatment field, a series of platinum compounds are synthesized and researched, wherein a scientific research staff develops cisplatin prodrug Pt (IV). Platinum (IV) prodrugs exhibit 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 fine tuning of pharmacological properties such as reduction potential, lipophilicity and kinetic stability. It is generally believed that platinum (IV) complexes with high coordination numbers are inert in motion and therefore have fewer side effects than the platinum (II) species. Pt (IV) complexes as prodrugs are first reduced in the extracellular or intracellular environment by bioreductive agents such as ascorbic acid (AsA) or Glutathione (GSH) releasing two axial ligands, followed by release of the cytotoxic Pt (II) drug. Cisplatin-based Pt (IV) complexes can be reduced by GSH to active Pt (II) species, with reduced levels of intracellular reducing species GSH throughout the reduction process, such that the toxic effects of Pt (II) analogs are mitigated.
The traditional Chinese medicine is the traditional medical treasure in China, and various traditional Chinese medicines are proved to have better anti-tumor, anti-inflammatory, immunoregulation and other effects, such as astragalus, curcuma zedoary, tripterygium wilfordii and the like, and have small toxic and side effects, and gamboge is one of the representatives of the anti-tumor traditional Chinese medicines. The traditional Chinese medicine gamboge is resin derived from gamboge (Garcinia hanburyi hook. F.), and the earliest record can be traced to five generations of "sea medicine materia Medica", mainly zhong tooth decay, and the "gamboge toxicity can attack toxicity" is recorded in Qing dynasty "book Jing Jiyuan", and "Chan mu Ji Yi" describes "treating carbuncle, stopping bleeding and resolving toxicity", treating carbuncle, swelling and sore, and the like. The main chemical components in resina Garciniae areKetones, benzophenones and the like, which are easy to functionalize in structure, have wide biological and pharmacological activities, especially anti-tumor activities, and are one of the hot spots of natural product research in recent years. In 1973, a compound with excellent anti-tumor activity, gambogic acid (gambogic acid, GA), was first extracted and separated from gamboge in China. A large number of researches prove that GA has better inhibition effect on stomach cancer, malignant tumor of blood system, liver cancer, malignant melanoma, lung cancer, leukemia, breast cancer and the like, wherein the treatment effect on the lung cancer is remarkable, and the GA is approved by Chinese food and drug administration to carry out phase II clinical test of lung cancer treatment. Currently, GA's antitumor mechanism mainly comprises induction to increase active oxygen accumulation, promotion of tumor cell apoptosis, induction of cell cycle arrest, inhibition of telomerase activity, inhibition of antitumor angiogenesis, inhibition of tumor metastasis, and the like. However, GA has the defects of poor solubility (the solubility in water is only 0.013mg/mL at room temperature), short half-life and the like as a small-molecule low-polarity substance, and is easy to cause side effects such as hepatotoxicity and the like, so that the clinical application of GA is severely limited. A large number of experimental researches show that the combination of the active ingredient GA and CDDP of the traditional Chinese medicine has the advantages of low administration dosage, good effect, small toxic and side effects and the like, and can avoid the occurrence of multi-drug resistance phenomenon and even reverse drug resistance phenomenon. Wang et al found that GA was able to modulate expression of pro-apoptotic proteins Fas (fatty acid synthase) and Bax (Bcl-2 related X gene) and anti-apoptotic protein Bcl-2 by inhibiting activation of the NF- κb and MAPK/HO1 pathways induced by CDDP, synergistically enhancing the ability of CDDP to inhibit proliferation of lung cancer cells, especially under sequential dosing regimens (CDDP first, GA later), very effectively inhibited cellular activity at the cellular level, promoting cell entry into the apoptotic phase, with far higher antitumor effects than either single or combination drug simultaneous dosing.
However, this strategy has not been successfully applied to clinic, and in vivo, CDDP and GA combined or alone have obvious inhibition effect on the growth of A549 tumors, but sequential administration of CDDP and GA cannot effectively inhibit the growth of tumors, and compared with other experimental groups, the method has no obvious difference, mainly because GA has strong hydrophobicity and low solubility and is easy to decompose under alkaline conditions,
The half-life is short, the in vivo distribution is wide, toxic and side effects (Zhao X,Ding S,Li S,et al.Construction of Gambogic Acid HPMACopolymer Coupling Drug System and Study on Anti-tumor Activity[J].Current Drug Delivery,2022,19(4):491-507.), can be generated on normal tissues while the activity of tumor cells is reduced, the bioavailability of the drug in the tumor tissues is insufficient due to the control of the absorption, distribution and metabolism and excretion of the drug by the pharmacokinetics of the traditional drug preparation, and in addition, different drug administration routes and different auxiliary materials related to each traditional drug form lead to different pharmacokinetic processes, so that the time of the absorption and distribution of the drug in the body is severely limited, and the synergistic effect among the drugs is avoided. Thus, there is an urgent need to design an appropriate drug carrier to solve the above-mentioned critical problems. In recent years, many scholars have developed various types of biological nanomaterials, and have further constructed more GA-or CDDP-loaded nano-delivery systems. Fang et al found that magnetic nanoparticles of Fe 3O4 could synergistically enhance GA induction of and inhibit proliferation of LOVO apoptosis in a dose-dependent and time-dependent manner. Du et al prepared a novel multi-environment sensitive gambogic acid polymeric prodrug micelle (GFCP) based on chitosan grafts, the nano micelle was stable and had good biocompatibility, and had pH, esterase and temperature dependent drug release modes, high loading capacity, targeted tumor accumulation and specific reactivity to tumor microenvironment, and showed potential for improving GA anticancer effect both in vivo and in vitro. Anees et al modified cisplatin by irradiated chitosan coating and successfully prepared cisplatin nanocomposite (Cis NC) with MgO nanoparticles, the nanocomposite can promote cisplatin release for a long time, reduce renal toxicity of cisplatin, and improve therapeutic effect. But no nano-delivery system co-supporting GA and CDDP has been seen so far.
Disclosure of Invention
The technical scheme of the invention is to construct a nano drug delivery system for carrying CDDP and GA together, control the sequential release of combined drugs and realize the drug synergistic treatment of lung cancer.
The invention provides a molybdenum disulfide nano conveying system for co-carrying gambogic acid and cisplatin, which comprises the following components, 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 cisplatin prodrug Pt (IV) and 10-50 parts of polyethylene glycol.
Further preferably, the composition comprises 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.
The nano-transport system is characterized in that molybdenum disulfide (MoS 2) nano-sheets are used as a substrate, manganese dioxide (MnO 2) nano-particles are modified, and meanwhile GA and CDDP are loaded, wherein MoS 2-MnO2 nano-sheets have a two-dimensional layered structure, and GA and CDDP are loaded through amide reaction and hydrophobic action.
The molybdenum disulfide (MoS 2) nanosheets are single-layer or few-layer molybdenum disulfide nanosheets prepared by a Morrison method, and comprise the following steps:
Putting MoS2 crystals into an n-butyllithium solution containing 15% of n-hexane under nitrogen, and stirring for 2 days under the protection of nitrogen;
after the reaction is finished, the mixture is washed by redistilled n-hexane to remove residues of n-butyllithium and other impurities;
Removing black powder after cleaning, immediately adding ddH2O, ultrasonically stripping for 2 hours, collecting solution, centrifuging at 8000rpm for 20 minutes to remove incompletely stripped crystals, transferring the upper layer solution into a dialysis bag with MWCO=100 kDa after centrifugation, and dialyzing for one week to remove residual lithium ions and other impurities to obtain solution containing MoS2 nanosheets;
The manganese dioxide (MnO 2) nanoparticle is prepared by adopting oxidation-reduction reaction, and the preparation method comprises the following steps:
Weighing KMnO4 and bovine serum albumin BSA, adding into ddH2O, stirring at room temperature for 5h, reducing KMnO4 by BSA to form MnO2 nanoparticles, centrifuging at 12000rpm for 20min after the reaction is finished to remove free KMnO4, BSA and MnO2 particles with larger particle size;
The preparation method of the Pt (IV) comprises the steps of adding CDDP and H 2O2 into ddH 2 O, stirring at 76 ℃ for 8 hours in a dark place, stopping heating and continuing stirring overnight after the reaction is finished, standing the mixture for crystallization at 4 ℃ for about 8 hours after overnight, then washing a crystallization product with cold water, ethanol and diethyl ether sequentially, centrifuging at 8000rpm for 10 minutes, collecting precipitates, drying in vacuum, obtaining bright yellow crystallization powder, namely cisplatin (Pt (II) oxide, after overnight, taking Pt (II) and succinic anhydride, adding into DMSO, stirring at 76 ℃ in a dark place for 48 hours, then freezing and drying to remove the DMSO to obtain yellow powder, adding pre-chilled glacial acetone into the powder, recrystallizing at-20 ℃, collecting the solution, washing 3 times with 10mL pre-chilled glacial acetone, centrifuging at 8000rpm for 10 minutes, collecting precipitates, and drying in vacuum, and obtaining white powder, namely Pt (IV).
Mixing the MoS2 nanosheet aqueous solution and the MnO2 nanoparticle aqueous solution, combining MoS2 and MnO2 through the chemical action of sulfur, stirring for 24 hours, transferring the mixed solution into a dialysis bag (MWCO=100 kDa), and dialyzing for 3 days to remove free MoS2 and MnO2, thereby obtaining the MoS2-MnO2 solution.
The invention also provides a preparation method of the molybdenum disulfide nano-delivery system for co-carrying gambogic acid and cisplatin, which comprises the following steps:
a. Preparing molybdenum disulfide (MoS 2) nanosheets;
b. Preparing manganese dioxide (MnO 2) nanoparticles;
c. synthesizing MoS 2-MnO2;
d. Synthesizing Pt (IV);
e. Synthesizing LA-PEG-NH 2 by coupling LA and PEG to obtain LA-PEG-NH 2;
f. And (3) taking LA-PEG-NH 2 as a connector, grafting Pt (IV) on MoS 2-MnO2, and preparing the molybdenum disulfide nano-delivery system carrying gambogic acid and cisplatin together.
The synthesis method of the LA-PEG-NH 2 in the step e comprises the following steps:
Weighing 6-arm PEG-NH 2, LA and DMAP, adding into CH 2Cl2 solution, stirring until the solid is completely dissolved, adding EDC, NHS to activate LA, stirring at room temperature for 3 days in dark, after the reaction is finished, spin-evaporating the solution to white crystal, re-dissolving the crystal in ddH 2 O, centrifuging at 8000rpm for 15min, transferring the supernatant into a dialysis bag (MWCO=3.5 kDa), dialyzing for 48h, removing small molecular impurities, and finally freeze-drying to obtain white solid LA-PEG-NH 2.
In the step f, firstly synthesizing LA-PEG-Pt (IV), wherein the Pt (IV) complex is covalently attached to LA-PEG-NH 2 through an amide bond to obtain the LA-PEG-Pt (IV), and the synthesis method comprises the following steps:
Weighing Pt (IV), EDC and NHS, dissolving in ddH 2 O, stirring in a dark place, adding LA-PEG-NH 2, stirring in a dark place for 24 hours, centrifuging at 8000rpm for 10 minutes after the reaction is finished, transferring the supernatant into a dialysis bag (MWCO=3.5 kDa), dialyzing for 48 hours, and removing small molecular impurities to obtain LA-PEG-Pt (IV);
And then grafting LA-PEG-Pt (IV) on MoS 2-MnO2 through a sulfur chemical effect, and loading GA on MoS 2-MnO2 -PEG-Pt (IV) through a hydrophobic effect to construct a MoS 2-MnO2 -PEG-Pt (IV)/GA nano drug-carrying system.
The preparation method of the MoS 2-MnO2 -PEG-Pt (IV)/GA nano drug-loading system comprises the following steps:
pt (IV) loading:
100mg of LA-PEG-Pt (IV) is dissolved in 10mL of ddH 2 O, after stirring until the solid is completely dissolved, 20mL of MoS 2-MnO2 solution (0.5 mg/mL) is added, moS 2-MnO2 and LA-PEG-Pt (IV) are combined together through the chemical action of sulfur and stirred for 24 hours, the mixed solution is transferred into a dialysis bag (MWCO=100 kDa) and dialyzed for 3 days, and the obtained MoS 2-MnO2 -PEG-Pt (IV) solution is obtained;
loading of GA:
Preparing 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL of GA solution, adding equal volume of MoS 2-MnO2 -PEG-Pt (IV) solution into the above solution, loading GA onto MoS 2-MnO2 -PEG-Pt (IV) by hydrophobic effect, stirring at room temperature and in dark place for 24h, centrifuging at 2000rpm for 10min to remove precipitated solid medicine, collecting supernatant, ultrafiltering and centrifuging at 3000rpm for 15min, repeatedly washing with ddH 2 O, ultrafiltering and centrifuging until the lower solution is colorless, and finally obtaining MoS 2-MnO2 -PEG-Pt (IV)/GA solution loaded with GA.
The invention provides application of the co-carried gambogic acid and cisplatin molybdenum disulfide nano-delivery system in preparing a medicine for treating lung cancer.
The MoS 2-MnO2 nano-sheet has a two-dimensional layered structure, is similar to graphene, has a larger specific surface area, and is suitable for being used as a drug carrier. In addition, the nanosheets have the characteristics of high physiological stability and blood compatibility, outstanding photothermal conversion capability, high oxygen production capability, strong magnetic resonance weighted imaging and the like, and the MoS 2-MnO2 is a safe and functionalized drug carrier. The combined medicine is successfully loaded through amide reaction and hydrophobic effect, and the sequential release of Pt (IV) and GA is realized under the dual stimulus of GSH and near infrared light. Compared with the single medicine and the combined medicine, the anti-tumor cell proliferation experimental result shows that MoS 2-MnO2 -PEG-Pt (IV)/GA always shows stronger anti-tumor activity, particularly the strongest cell killing effect under the irradiation of near infrared light, only 9.1% of cells are in a survival state, and the cells in an apoptosis stage are as high as 89.17%. MoS 2-MnO2 -PEG-Pt (IV)/GA conveys the medicine to a tumor part through the EPR effect and the passive targeting effect, and the temperature of the tumor part of a nude mouse rises to 56 ℃ rapidly under the irradiation of near infrared light, so that the apoptosis of tumor cells can be directly caused, the release of the combined medicine can be effectively controlled, the synergistic treatment effect of the combined medicine is enhanced, and the synergistic treatment of the combined medicine and the photo-thermal combined treatment are realized.
Drawings
FIG. 1 is a schematic diagram of the synthesis of MOS 2-MNO2 (note that (I) chemical stripping to synthesize MOS 2, and (II) redox to synthesize MNO 2;(III)MNO2 grafted onto MOS 2 by sulfur chemistry);
FIG. 2 (a) shows the infrared absorption spectrum of MoS 2 before and after grafting of MnO 2 and (b) shows the ultraviolet-visible-near infrared absorption spectrum of MoS 2 before and after grafting of MnO 2;
FIG. 3 (a) TEM image of MoS 2 nanoplates, (b) TEM image of MnO 2 nanoparticles, (c) TEM image of MoS 2-MnO2 nanoplates;
FIG. 4 (a) TEM image and EDS elemental profile of MoS 2-MnO2 (b) EDS elemental profile of MoS 2-MnO2;
FIG. 5 (a) AFM image of MOS 2 nanoplatelets, (b) AFM image of MOS 2-MNO2 nanoplatelets;
FIG. 6 (a) particle diameters of MOS 2、MNO2 and MOS 2-MNO2, (b) potentials of MOS 2、MNO2 and MOS 2-MNO2;
FIG. 7 (a) is an optical photograph of MoS 2 in water, PBS, cell culture for 7 days, (b) is an optical photograph of MoS 2-MnO2 in water, PBS, cell culture for 7 days;
FIG. 8 (a) photothermal temperature rise curves of water and MoS 2-MnO2 solutions of different concentrations under 808nm laser irradiation of 1W/cm 2, (b) photothermal temperature rise curves of MoS 2-MnO2 solutions (300 μg/mL) under 808nm laser irradiation of different power densities;
FIG. 9 is a graph showing the temperature change of MoS 2-MnO2 solution over 3 cycles of irradiation-free cooling;
FIG. 10 (a) weighted images of nuclear magnetic T 1 at different Mn 2+ concentrations and different pH values for MnO 2 and MoS 2-MnO2. (b) Relaxation curves of MnO 2 and MoS 2-MnO2 at different Mn 2+ concentrations, different pH;
FIG. 11 (a) MoS 2-MnO2 shows the concentration profile of O 2 at different pH values and (b) MoS 2-MnO2 at different concentrations reacts with H 2O2 at pH 5.5 to give the concentration profile of O 2;
FIG. 12 (a) ultraviolet-visible light absorption spectrum after 2h incubation of RBCs with different concentrations of MoS 2 -MnO solution with PBS and ddH 2 O as negative and positive controls, respectively, (b) percent hemolysis and hemolysis optical photograph after 2h incubation of RBCs with different concentrations of MoS 2-MnO2 solution;
FIG. 13 is a 1 HNMR diagram of CDDP, PT (II) and PT (IV);
FIG. 14 is a MS diagram of (a) PT (II) and (b) PT (IV);
FIG. 15 is a FT-IR diagram of CDDP, PT (II), PT (IV);
FIG. 16 is a schematic diagram of the synthesis of LA-PEG-PT (IV);
FIG. 17 is a 1 HNMR diagram of LA, PEG-NH 2、LA-PEG-NH2, and LA-PEG-PT (IV);
FIG. 18 is a FTIR view of LA, PEG-NH 2、LA-PEG-NH2 and LA-PEG-PT (IV);
FIG. 19 is a schematic diagram of MoS 2-MnO2 -PEG-Pt (IV)/GA synthesis;
FIG. 20 (a) TEM image of MoS 2-MnO2 -PEG-Pt (IV)/GA and EDS element profile, (b) EDS element profile of MoS 2-MnO2 -PEG-Pt (IV)/GA, (c) UV-Vis profile of GA, moS 2-MnO2 -PEG-Pt (IV) and MoS 2-MnO2 -PEG-Pt (IV)/GA;
FIG. 21 is a schematic of MoS 2-MnO2 -PEG-Pt (IV)/GA drug release;
FIG. 22 (a) PT standard curve, (b) GA standard curve;
FIG. 23 (a) UV-Vis plot of MoS 2-MnO2 -PEG-Pt (IV)/GA at various drug loading concentrations, (b) optical photograph of MoS 2-MnO2 -PEG-Pt (IV)/GA loading GA as a function of drug loading concentration, standing in PBS for 1 day;
FIG. 24 (a) cumulative release profile of Pt in GSH response, (b) cumulative release profile of GA with/without near infrared light irradiation;
FIG. 25 shows cell viability and IC 50 values of A549 cells incubated with CDDP, GA, CDDP +GA, CDDP→GA, and MoS 2-MnO2 -PEG-Pt (IV)/GA, respectively, for 96h under normoxic and hypoxic conditions;
FIG. 26 shows the killing effect of (a) normoxic (b) hypoxic CDDP, GA, CDDP +GA, CDDP→GA, MOS 2-MNO2、MOS2-MNO2 -PEG-PT (IV)/GA on A549 cells under near infrared light irradiation (note: * P <0.05,
**P<0.01);
FIG. 27 is a graph showing the quantitative analysis of apoptosis percentage of A549 cells treated in various ways in a hypoxic environment by flow cytometry;
FIG. 28 is a thermal imaging of (a) nude mice before and after near infrared light irradiation and the corresponding (b) temperature profile;
FIG. 29 (a) tumor growth curves for groups of nude mice treated differently, (b) tumor volume inhibition rates for groups of nude mice treated differently (note: *P﹤0.05,** P < 0.01);
FIG. 30 is an H & E staining pattern of tumors of each group of nude mice after 21 days of treatment;
FIG. 31 is a graph showing the body weight of each group of nude mice after 21 days of different dry prognosis over time;
FIG. 32 is a graph of major organ H & E staining of PBS group and MoS 2-MnO2 -PEG-Pt (IV)/GA+NIR group nude mice after 21 days of treatment.
Detailed Description
Example 1 preparation of a molybdenum disulfide nanosupport System Co-carrying gambogic acid and cisplatin according to the present invention
1. Preparation of related solutions and consumable treatment
1.1 Preparation of phosphate buffer (Phosphate buffered saline, PBS)
4.0G NaCl, 0.1g KCl, 1.79gNaHPO 4·12H2 O and 0.12g KH 2PO4 are weighed and dissolved in 500mL secondary water (ddH 2 O), and after magnetic stirring until dissolved, the solution is filtered by a 0.22 mu m filter membrane, sterilized by high-pressure steam for 1h and stored at 4 ℃ for standby.
1.2 Pretreatment of dialysis bag
Weighing 20gNaHCO 3 and 373.2 mgEDTA.2Na, dissolving in 1L ddH 2 O, placing a dialysis bag in the solution, boiling, taking out the solution after 10min, cleaning with ddH 2 O, putting into 1L ddH 2 O containing 373.2 mgEDTA.2Na again, boiling again, taking out the solution after 10min, cleaning with ddH2O, and storing in 30% ethanol for later use.
2. Experimental method
2.1 Preparation of MoS 2 nanosheets
And preparing a single-layer or less-layer molybdenum disulfide nano sheet according to a Morrison method. The method is as follows, 500mgMoS 2 crystals are placed in 10mL of n-butyllithium solution containing 15% n-hexane in a nitrogen glove box and stirred under nitrogen for 2 days. After the reaction was completed, the reaction mixture was washed several times with redistilled n-hexane to remove residues of n-butyllithium and other impurities. After the washing was completed, the black powder was removed from the glove box and immediately added with 200mL of ddH 2 O, sonicated for 2h, then the solution was collected and centrifuged at 8000rpm for 20min to remove incompletely exfoliated crystals, the supernatant solution was transferred to a dialysis bag (mwco=100 kDa) after centrifugation was completed, dialyzed for one week to remove residual lithium ions and other impurities, and finally the solution containing MoS 2 nanoplatelets was kept in a 4 ℃ refrigerator for further use.
2.2 Preparation of MnO 2 nanoparticles
According to the invention, mnO 2 nano particles are modified on the surface of the MoS 2 nano sheet to improve the physiological stability of the MoS 2 nano sheet.
MnO 2 nanoparticles are prepared according to the oxidation-reduction reaction. 20mg of KMnO 4 and 100mg of BSA were weighed into 50mL of ddH 2 O and stirred at room temperature for 5 hours. KMnO 4 can be reduced by BSA to form MnO 2 nanoparticles. After the reaction, the mixture was centrifuged at 12000rpm for 20min to remove free KMnO 4, BSA and MnO 2 particles with larger particle size, and the supernatant was collected and stored in a refrigerator at 4℃for further use. The BSA becomes a suitable candidate material for designing the nano-carrier due to good biocompatibility and low immunogenicity, and the formed nano-particles can carry wide therapeutic drugs, such as chemotherapeutic drugs and photosensitizers, can be stably conveyed into blood without easy degradation, enhance aggregation of the drugs in tumors, improve uptake rate of tumor cells and increase therapeutic activity.
2.3 Synthesis of MoS 2-MnO2
The synthesis of MoS 2-MnO2 is shown in fig. 1. 10mL of MoS 2 nanosheet aqueous solution (0.4 mg/mL) and 2mL of MnO 2 nanoparticle aqueous solution (4 mg/mL) are mixed, moS 2 and MnO 2 are combined together through chemical action of sulfur and stirred for 24 hours, the mixed solution is transferred into a dialysis bag (MWCO=100 kDa), free MoS 2 and MnO 2 are removed by dialysis for 3 days, and the obtained MoS 2-MnO2 solution is placed in a refrigerator at 4 ℃ for storage.
2.4 Characterization of MoS 2-MnO2
2.4.1 Fourier transform Infrared Spectroscopy (Fourier transforminfrared spectroscopy, FT-IR) analysis
The chemical structure of MoS 2、MnO2 and MoS 2-MnO2 was analyzed using FT-IR. Mixing a proper amount of freeze-dried samples of MoS 2、MnO2 and MoS 2-MnO2 with potassium bromide powder according to a mass ratio of 1:100 in an agate mortar, fully grinding to ensure that the samples are uniformly mixed with the potassium bromide powder and ground to be fine, placing a small amount of powder in an infrared mold, tabletting for about 1min to obtain a semitransparent sheet, placing the sheet in FT-IR, and detecting infrared spectrum information of different samples at 400-4000cm -1.
2.4.2 Ultraviolet-visible Spectrophotometer (Ultraviolet-visible spectroscopy, UV-vis) analysis
The UV-vis analysis of the change in UV absorbance of MoS 2-MnO2 was used. And respectively diluting a proper amount of MoS 2,MnO2,MoS2-MnO2 samples with ddH 2 O to a certain concentration, placing the diluted MoS 2,MnO2,MoS2-MnO2 samples in a quartz cuvette, and recording absorption peaks of different samples at 200-900 nm.
2.4.3 Field emission transmission electron microscope (Transmission electron microscope, TEM) analysis
The surface morphology and element distribution of MoS 2,MnO2,MoS2-MnO2 were observed using TEM. And respectively diluting a proper amount of MoS 2,MnO2,MoS2-MnO2 sample with ddH 2 O to a certain concentration, weighing 10 mu L of solution, dripping the solution on a carbon support film, and putting the carbon film into a TEM after the sample is dried overnight to observe the morphological characteristics of the carbon film. And simultaneously, performing elemental mapping analysis by using energy dispersive X-ray spectroscopy (EDS).
2.4.4 Atomic force microscope (Atomic force microscope, AFM) analysis
The dimensional thickness of MoS 2 before and after MnO 2 modification was analyzed using AFM. And respectively diluting a proper amount of MoS 2,MoS2-MnO2 sample with ddH 2 O to a certain concentration, weighing 20 mu L of solution, dripping the solution on a clean and smooth mica sheet, and putting the mica sheet into an AFM after the sample is dried overnight to observe the size and the thickness of the mica sheet.
2.4.5 Nanometer particle size distribution and potentiometric analysis
The particle size and Zeta potential of MoS 2,MnO2,MoS2-MnO2 were determined using a nanoparticle size and potential analyzer. Diluting a proper amount of MoS 2,MnO2,MoS2-MnO2 sample with ddH 2 O to a certain concentration, placing in a cuvette, measuring the particle size distribution of the sample, placing a proper amount of sample in a folded capillary sample cell, and measuring the Zeta potential of the sample.
2.4.6MoS 2-MnO2 Synthesis and characterization data
We first characterized the chemical structure of MoS 2-MnO2 using FTIR and UV-Vis spectroscopy. First, the chemical structure of MoS 2-MnO2 was identified by FTIR, and as shown in fig. 2 (a), we found from the infrared spectrum that a new absorption peak appears at about 1654cm -1 compared to MoS 2,MoS2-MnO2, which is attributed to the co-action of O-H bending vibration and Mn atoms, confirming that MnO 2 has been successfully grafted to MoS 2.
Meanwhile, as is clear from the UV-Vis of MoS 2-MnO2 in fig. 2 (b), the absorbance of MoS 2-MnO2 in the ultraviolet region is greater than that of MoS 2, especially in the range of 200nm to 280nm, because of the strong absorption of grafted MnO 2 in this region, and the successful combination of MoS 2 and MnO 2 has also been demonstrated.
The surface morphology and dimensional thickness of MoS 2-MnO2 were observed using TEM and AFM. As can be seen from FIG. 3, moS 2 has a two-dimensional layered structure, similar to graphene, a larger specific surface area, and is suitable for being used as a drug carrier, mnO 2 nanoparticles are highly dispersed and have a regular spherical morphology, and MnO 2 nanoparticles can be observed on MoS 2-MnO2 nanoparticles to be uniformly distributed on MoS 2 through chemical action of sulfur.
To further verify the above results, we performed elemental analysis of MoS 2-MnO2 using energy dispersive X-ray spectroscopy (EDS). From the mapping graph (fig. 4 a), it was found that Mn atoms were uniformly dispersed on the surface of MoS 2 nanoplatelets, while from the EDS graph (fig. 4 b) three peaks of Mo, mn, S were clearly seen, which further confirmed the successful preparation of MoS 2-MnO2.
The dimensions and thickness of MoS 2 before and after modification were determined by AFM. From fig. 5, it can be seen that the average thickness of MoS 2 nanoplatelets is 0.7nm, indicating that the MoS 2 nanoplatelets prepared are single-layered. After modification with MnO 2, the average thickness of MoS 2 nanoplatelets increased to 6nm, indirectly demonstrating the presence of MnO 2 on MoS 2 nanoplatelets.
The particle sizes of MoS 2 and MoS 2-MnO2 were determined using Zeta potential and a nanosize analyzer. As can be seen from FIG. 6, the average particle size of the prepared MoS 2 nano-sheets is 100nm, the average particle size of the MnO 2 nano-sheets is 10nm, the particle size of the MoS 2-MnO2 modified by MnO 2 is about 100nm, the nano-carrier has proper particle size and good dispersibility, and the nano-carrier can be effectively accumulated at tumor sites through high-permeability long-retention effect. The Zeta potential measurement results show that the potentials of MoS 2、MnO2 and MoS 2-MnO2 are-49.6 mV, -25.7mV and-35.1 mV respectively, and are negatively charged in the aqueous solution, wherein the Zeta potential absolute value of MoS 2-MnO2 is more than 30mV, which indicates that the surface of the particles is more charged and more prone to mutual repulsion, and the dispersion system of the particles is stable.
2.5 Physiological stability of MoS 2-MnO2
MoS 2,MoS2-MnO2 was resuspended in water, PBS solution and cell culture medium, respectively, and left at room temperature for one week to observe its stability in water, PBS solution and cell culture medium.
After confirming successful preparation of MoS 2-MnO2, we examined the physiological stability of MoS 2-MnO2 in different systems. MoS 2,MoS2-MnO2 is respectively resuspended in water, PBS solution and cell culture medium, and left at normal temperature for one week, and the stability of the MoS 2,MoS2-MnO2 in three systems is observed, and the result is shown in figure 7, wherein single MoS 2 is precipitated in PBS, and MoS 2-MnO2 nano-carrier is uniformly dispersed in water, PBS solution and cell culture medium for 7 days, which shows that the modification of MnO 2 improves the physiological stability of MoS 2, and provides precondition for the application of the MoS 2 in biomedicine.
2.6 Photothermal Properties of MoS 2-MnO2
2.6.1 Photo-thermal heating of MoS 2-MnO2
MoS 2-MnO2 solutions (0, 50, 100, 200 and 300. Mu.g/mL) of different concentrations were placed in a cuvette and the samples were irradiated with 808nm laser light at 1W/cm 2 for 10min. In the illumination process, the temperature value of the sample is recorded once every 1min by using an infrared thermal imager, and data are collected and a photo-thermal heating curve is drawn.
1MLMoS 2-MnO2 solution (300. Mu.g/mL) was placed in a cuvette and the sample was irradiated with 808nm laser light of different power densities (0.2W/cm 2、0.4W/cm2、0.6W/cm2、0.8W/cm2、1W/cm2) for 10min. And recording the temperature change value of the sample once every 1min by using an infrared thermal imager in the illumination process, collecting data and drawing a photo-thermal heating curve.
2.6.2 Photo-thermal stability of MoS 2-MnO2
1ML of MoS 2-MnO2 solution (300. Mu.g/mL) was placed in a cuvette and the sample was irradiated with 808nm laser light at 1W/cm 2 for 10min. And then turning off the laser, after the sample is naturally cooled to the initial temperature, irradiating the sample with laser light with the same power density for 10min again by using the laser, and circulating for 3 times, and monitoring a thermal imaging optical picture of the sample by using a thermal infrared imager and recording the change of the temperature of the sample in the whole experiment process.
2.6.3 Photothermal Properties of MoS 2-MnO2
Photothermal therapy is a therapeutic approach that uses heat generated by a photothermal agent under near infrared light irradiation to cause ablation of tumor cells, thereby killing the tumor cells. MoS 2 nano material is a popular material for photothermal treatment of tumors due to strong near infrared absorption. Considering the great potential of MoS 2 as a photothermal reagent, we studied the photothermal properties of MoS 2-MnO2 at different concentrations by near infrared laser irradiation. As can be seen in fig. 8, the presence of MnO 2 did not affect near infrared light absorption by MoS 2, indicating that MnO 2 retained the photo-thermal properties based on MoS 2. To further investigate the photothermal properties of MoS 2-MnO2, solutions of varying concentrations of MoS 2-MnO2 were prepared, followed by irradiation of the above samples with a 808nm laser at 1W/cm 2 for 10min. As a result, as shown in FIG. 8a, the rate of temperature rise of water was not significant, but increased by only 5℃within 10min of irradiation, whereas the higher the concentration of MoS 2-MnO2 sample, the greater the rate of temperature rise of the solution temperature, showing significant concentration dependence. The solution temperature rose from 29.3 ℃ to 61.4 ℃ in 10min when the sample concentration was 50 μg/mL, and from 29.6 ℃ to 87.6 ℃ in 10min when the sample concentration reached 300 μg/mL. Compared with the normal body temperature of 37 ℃, the irradiation of near-red light can raise the temperature of a tumor area, because MoS 2-MnO2 accumulated at a tumor part has better near-infrared light conversion efficiency, the near-infrared light is effectively converted into heat energy, and tumor cells are ablated and dead, so that the growth of the tumor is inhibited to a certain extent, and meanwhile, the relation between the laser power density and the photo-thermal effect is examined. MoS 2-MnO2 solution at a concentration of 300. Mu.g/mL was used followed by irradiation of the above samples with lasers of different power densities for 10min. The higher the laser power density, the greater the rate of rise of the solution temperature was found (fig. 8 b), showing a clear power density dependence. These results demonstrate that the photothermal effect of MoS 2-MnO2 has a close relationship with sample concentration, laser power density.
Photo-thermal stability is considered to be one of the important performance criteria for photo-thermal materials. Therefore, we studied the photo-thermal stability of MoS 2-MnO2 under near infrared laser irradiation. The MoS 2-MnO2 solution (300 μg/mL) was irradiated with 808nm laser at 1W/cm 2 for 10min, then the 808nm laser was turned off, the sample was naturally cooled to the initial temperature, the cycle was repeated 3 times, and the temperature change of the solution during the temperature increase and the temperature decrease was recorded, and as seen from fig. 9, the temperature increase rate and the temperature decrease rate were substantially maintained consistently in each temperature increase and decrease cycle, and there was no significant change, indicating that MoS 2-MnO2 had excellent photo-thermal stability.
2.7 In vitro imaging of MoS 2-MnO2
And performing in-vitro MRI imaging and relaxation test on MnO 2 and MoS 2-MnO2 before and after pH response by using a magnetic resonance contrast agent imaging analyzer, and analyzing in-vitro imaging effect, relaxation time and relaxation efficiency. The magnetic field strength was 0.5t, and the t 1 imaging test used sequence was set mainly tr=1500 ms, te=18.2 ms. Relaxation tests tw=6000 ms using the sequence main parameter settings r 1 value test. 202 mL centrifuge tubes were separated into four groups of MnO 2 (pH 5.5 and pH 7.4) and MoS 2-MnO2 (pH 5.5 and pH 7.4), each group having 5 centrifuge tubes. 1mL of MnO 2 and MoS 2-MnO2 (the concentration is calculated by Mn 2+) solutions of different concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0mM are respectively prepared by phosphate buffer solutions of pH=7.4 and pH=5.5, 200 mu L of 1X 10 -2mmol/L H2O2 solution is added to each tube, 1mL of sample is placed in the corresponding sample tube after standing for 5min, a T 1 weighted image of each sample is detected by a 0.5T nuclear magnetic resonance contrast agent imaging analyzer, and the relaxation time (T 1) and the relaxation efficiency (r 1) of each sample are measured.
MoS 2-MnO2 nanometer system is uniformly dispersed in the pH5.5-7.4 environment, no precipitate is generated, when H 2O2 is singly added, gas (O 2) is generated in the solution, H 2O2 is added again after the pH is reduced, oxidation-reduction reaction occurs, nanometer particles are decomposed, and the color of the solution is gradually disappeared. The relaxation efficacy of the H 2O2 response of MoS 2-MnO2 was investigated using the property of MnO 2 to release Mn 2+ in slightly acidic conditions containing H 2O2. As shown in fig. 10, which is a T 1 weighted MRI plot of MnO 2 and MoS 2-MnO2 under different pH environments, it can be seen that the T 1 weighted MRI signal of MoS 2-MnO2 (pH 5.5) significantly increased with increasing concentration, showing a significant concentration dependence, indicating that MRI performance was enhanced upon degradation of MnO 2 to Mn 2+. Meanwhile, under the condition of the same MnO 2 concentration, the T 1 weighted image of MoS 2-MnO2 (pH 5.5) is brighter than that of MnO 2 (pH 5.5), which indicates that the MRI contrast capability of MoS 2-MnO2 is stronger. While the enhancement of MRI signals by MoS 2-MnO2 (ph 7.4) was not evident, further demonstrating the properties of MnO 2 with higher release of Mn 2+ in slightly acidic conditions containing H 2O2. To explore the reasons for this result, we determined the relaxation times (T 1) of MnO 2 and MoS 2-MnO2 at different Mn 2+ concentrations, followed by a relaxation curve with the inverse of the relaxation time (T 1) (1/T 1) as the ordinate and the slope of the straight line as the relaxation rate (r 1) with the Mn 2+ concentration as the abscissa. At pH5.5, r 1 of MnO 2 is 2.195mM -1S-1, in contrast to r 1 of MoS 2-MnO2 being 11.12mM - 1S-1, 5 times that of MnO 2, because conjugation of Mn-based paramagnetic metal complexes to nanocarriers can reduce the turnover rate of Mn, increase the molecular rotation-related time of the complexes, and thus increase r 1 thereof. these results fully prove that MoS 2-MnO2 has stronger MRI contrast capability, can be used as a good contrast agent, and lays a foundation for realizing the diagnosis and treatment integration of tumors.
2.8 Oxygen analysis by MoS 2-MnO2
The O 2 production capacity of MoS 2-MnO2 was measured using a dissolved oxygen meter. (1) 10mL of MoS 2-MnO2 (300. Mu.g/mL) solution with pH=5.5 and pH=7.4 were prepared in 50mL beakers, respectively, and 10mL of MoS 2-MnO2 (50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, 300. Mu.g/mL) solution with different concentrations with pH=5.5 was prepared in 50mL beakers, respectively. After the oxygen dissolving instrument probe is inserted below the liquid level, 10mL of 1X 10 -2mmol/L H2O2 solution is added into the solution, the change of the oxygen concentration with time is monitored by using the oxygen dissolving instrument, and after 10min, the probe is taken out to record the value.
Hypoxia is one of the features of the tumor microenvironment and can severely limit the efficacy of O 2 -dependent therapies. MnO 2 reacts with intracellular H 2O2 to generate O 2 in the tumor microenvironment, which lays a foundation for improving tumor hypoxia and increasing the generation of O 2 at the tumor part. Therefore, by introducing MoS 2-MnO2 nano-carriers, it is expected to realize degradation of MnO 2 by means of tumor microenvironment, and then O 2 is generated to relieve the hypoxic environment of tumors so as to enhance the treatment effect. We evaluated the dissolved oxygen levels of MoS 2-MnO2 in solution in different environments and at different concentrations. As shown in fig. 11 (a), the dissolved oxygen content in the MoS 2-MnO2 -containing solution rapidly increases under the action of H 2O2, and then tends to stabilize, and more dissolved oxygen is generated in an acidic environment. The degradation of MnO 2 under the combined action of H 2O2 under the acidic condition is more complete. At the same time, we also compare the dissolved oxygen levels of MoS 2-MnO2 solutions at different concentrations, as shown in fig. 11 (b), which shows that the dissolved oxygen content is somewhat concentration dependent, i.e., the dissolved oxygen level in the high concentration MoS 2-MnO2 solution is higher.
2.9 Blood compatibility of MoS 2-MnO2
2.9.1 Preparation of Red Blood Cells (RBCs) solution
0.5ML of blood was removed from the vein of the healthy nude tail and placed in a heparin tube. The serum is removed by centrifugation at 2000rpm for 3min, the obtained precipitate is redissolved in PBS solution, and is gently shaken to be uniformly dispersed in the PBS solution, and the solution is placed in a refrigerator at 4 ℃ for standby.
2.9.2 Hemolysis experiments
0.2ML of RBCs solution was added to the corresponding samples (0.8 mL) of (1) ddH 2 O, (2) PBS solution, (3) PBS solution containing different concentrations of MoS 2-MnO2 (50, 100, 300, 600, 900, 1200 and 1500. Mu.g/mL), mixed well, placed on a shaking table, and shaken at 100rpm for 2 hours at 37℃to take out all samples, centrifuged at 24000rpm for 15 minutes at high speed, after which the absorbance (A) of the supernatant of each sample at 541nm wavelength was measured, and the percent hemolysis (hemolysis) of the samples was calculated according to the formula (1-1).
The calculation formula of the percent hemolysis (hemolysis) is:
Hemolysis (%) = (A Sample of -APBS)/(AH2O-APBS) ×100% (1-1)
Wherein, A Sample of is the absorbance value of the sample solution, A PBS is the absorbance value of the negative control group, and A H2O is the absorbance value of the positive control group.
2.9.3 Results of blood compatibility test of MoS 2-MnO2
When nanomaterials are absorbed into the body, they are first exposed to erythrocytes, causing varying degrees of erythrocyte damage. If the ratio of hemolysis caused by the nanomaterial exceeds 5%, red blood cells are broken, thereby affecting the whole body. Therefore, it is important to examine the blood compatibility of MoS 2-MnO2. PBS solutions containing varying concentrations of MoS 2-MnO2 were added to dilutions containing RBCs and ddH 2 O and PBS solutions were used as positive and negative controls, respectively. After incubation at 37℃for 2h, high speed centrifugation at 24000rpm, the supernatant was removed, absorbance at 541nm wavelength was measured for each sample supernatant, and the percent hemolysis of the sample was calculated according to formula (1-1). As shown in fig. 12, the supernatant of the positive control group was red in color, and showed a remarkable hemolysis phenomenon, mainly due to the large difference in extracellular and intracellular osmotic pressure, the cells absorbed a large amount of water and burst, and hemoglobin in the cells penetrated the supernatant. In contrast, the supernatants of the experimental and negative control groups were relatively clear and most of the cells were centrifuged to the bottom of the centrifuge tube. At the same time, we have also found that the percent hemolysis of MoS 2-MnO2 on red blood cells is proportional to the sample concentration. However, even if the sample concentration reached a higher 1500 μg/mL, the percent hemolysis (2.9%) of the red blood cells by MoS 2-MnO2 was still less than 5%. These results are sufficient to demonstrate that MoS 2-MnO2 has better blood compatibility and can also provide a basis for intravenous administration.
2.10 Synthesis and characterization of MoS 2-MnO2 -PEG-Pt (IV)/GA
2.10.1 Synthesis of Pt (IV)
The synthesis of Pt (IV) is divided into two steps. In the first step, 500mg of CDDP and 2.84mL of H 2O2 were added to 22.5mL of ddH 2 O, stirred at 76℃in the dark for 8 hours, and after the reaction was completed, the heating was stopped and stirring was continued overnight. After overnight the mixture was left to crystallize at 4 ℃ for about 8h. Subsequently, the crystallized product was washed with 20mL of cold water, 20mL of ethanol and 10mL of diethyl ether in this order, centrifuged at 8000rpm for 10min to collect the precipitate, which was dried in vacuo, and the bright yellow crystallized powder, cisplatin oxide (Pt (II)), was obtained after overnight. In the second step, 200mg Pt (II) and 240mg succinic anhydride were weighed and added to 2mL DMSO. Stirring at 76℃for 48h in the absence of light, followed by lyophilization to remove DMSO, gives a yellow powder, to which 10mL of pre-chilled glacial acetone was added, and recrystallized at-20℃for about 8h. The collected solution was washed 3 times with 10mL of pre-chilled ice acetone, centrifuged at 8000rpm for 10min to collect the precipitate, which was dried in vacuo to give a white powder, pt (IV), after overnight, which was stored in a refrigerator at 4 ℃ for further use.
Characterization of 2.10.2Pt (IV)
2.10.2.1 Nuclear magnetic resonance Hydrogen Spectrometry (1Hnuclearmagneticresonance,1 HNMR) analysis
CDDP, pt (II), pt (IV) molecular structures were analyzed using 1 HNMR. And taking a proper amount of CDDP, pt (II) and Pt (IV) in a DMSO-d6 solution, carrying out ultrasonic treatment until the sample is completely dissolved, and transferring the prepared sample solution into a Nuclear Magnetic Resonance (NMR) measuring tube for carrying out 1 H-NMR analysis.
2.10.2.2 Ultra high performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-QqQ-MS/MS) analysis
Molecular weights of Pt (II) and Pt (IV) were determined using UPLC-QqQ-MS/MS. And dissolving Pt (II) and Pt (IV) powder into 50% mass spectrum grade methanol solution to prepare 700ng/mL solutions respectively, and detecting the molecular weight of Pt (II) and Pt (IV).
2.10.2.3 Fourier transform Infrared Spectroscopy (Fourier transforminfrared spectroscopy, FT-IR) analysis
The chemical structures of CDDP, pt (II), pt (IV) were analyzed using FT-IR. Mixing a proper amount of CDDP, pt (II) and Pt (IV) dry powder with potassium bromide powder according to a mass ratio of 1:100 in an agate mortar, fully grinding to uniformly mix a sample with the potassium bromide powder, grinding to be fine, placing a small amount of powder in an infrared mold, tabletting for about 1min to obtain a semitransparent sheet, placing the sheet in FT-IR, and detecting infrared spectrum information of different samples at 400-4000cm -1.
The synthesis of cisplatin prodrug includes first oxidizing cisplatin with 30% H 2O2 to produce hydroxylation reaction to obtain intermediate Pt (II), and then reacting the intermediate with succinic anhydride to obtain precursor Pt (IV) with high yield up to 75%. Compared with cis-platinum with a quadrilateral space structure, the hexacoordinated Pt (IV) has an octahedral structure in the space structure, is more stable, can reduce the drug resistance of tumor cells and the toxicity to normal cells, has high water solubility, and is easy to package and prepare into a dosage form with high drug loading.
Cisplatin (CDDP), oxidized cisplatin (Pt (II)), and cisplatin prodrugs (Pt (IV)) were structurally characterized using 1 HNMR. As shown in fig. 13, the-NH 2 proton peak in cisplatin appeared at δ=6.02 ppm on CDDP, the-NH 2 proton peak in cisplatin and the-OH proton peak in cisplatin oxide appeared at δ=6.54 ppm and δ=5.46 ppm, respectively, the-COOH proton peak generated after the reaction of cisplatin oxide with succinic anhydride appeared at δ=12.06 ppm on Pt (IV), and the-NH 2 proton peak in cisplatin appeared at δ=6.48 ppm, confirming that the synthesized compound was Pt (IV).
Molecular weights of Pt (II) and Pt (IV) were determined using UPLC-QqQ-MS/MS. FIG. 14 shows MS spectra of Pt (II) and Pt (IV), wherein the molecular weight of Pt (II) is 332.93, the molecular weight of Pt (IV) is 532.75, and the molecular weight is correct.
The CDDP, pt (II) and Pt (IV) were structurally characterized by FT-IR, and the results are shown in FIG. 15. From the figure, it can be seen that Pt (II) is the stretching vibration of O-H bond at 3515cm -1, 3261cm -1 is the stretching vibration of N-H bond in NH 3, 1588 and 1040m -1 are the stretching vibration of Pt-O bond in Pt-OH bond, which indicates that the hydroxyl group is successfully modified to cis-platinum, pt (II) is successfully prepared, 3455cm -1 is the stretching vibration of N-H bond in NH 3 in infrared diagram of Pt (IV), 3281cm -1 is the stretching vibration of O-H bond in COOH, 1730cm -1 is the stretching vibration of C=O in COO-Pt, 1659cm -1 is the stretching vibration of C=O in COOH, which proves that the carboxyl group exists, which indicates that Pt (IV) is successfully synthesized.
2.10.3LA Synthesis of PEG-NH 2
Lipoic Acid (LA) is a naturally occurring antioxidant vitamin whose therapeutic application has been approved by the FDA, without cytotoxicity even at high concentrations. This experiment resulted in LA-PEG-NH 2 by coupling LA with PEG, and grafting Pt (IV) onto MoS 2-MnO2 using it as a linker.
200Mg of 6-arm PEG-NH 2, 60mg of LA and 3mg of DMAP are weighed and added into 30mL of CH 2Cl2 solution, after stirring until the solid is completely dissolved, 25mg EDC,20mg NHS is added to activate the LA, and stirring is carried out for 3 days at room temperature in a dark place. After the reaction is finished, the solution is steamed to white crystals, the crystals are redissolved in 10mL ddH 2 O, high-speed centrifugation is carried out at 8000rpm for 15min, the supernatant is transferred into a dialysis bag (MWCO=3.5 kDa), dialysis is carried out for 48h, small molecular impurities are removed, finally, the white solid LA-PEG-NH 2 is obtained by freeze drying, and the white solid LA-PEG-NH 2 is stored in a refrigerator at-20 ℃ for standby.
2.10.4LA Synthesis of PEG-Pt (IV)
The synthesis of LA-PEG-Pt (IV) is shown in FIG. 16. The Pt (IV) complex is an analogue of cisplatin, and contains an additional coordination site, and is covalently attached to LA-PEG-NH 2 through an amide bond to obtain LA-PEG-Pt (IV).
Preparation of LA-PEG-Pt (IV) solid complexes. 200mg Pt (IV), 50mg EDC, 25mg NHS were weighed in 10mL ddH 2 O and stirred for 2h in the dark, followed by 400mg LA-PEG-NH 2 and stirred for 24 h in the dark. After the reaction, centrifuging at 8000rpm for 10min, transferring the supernatant into a dialysis bag (MWCO=3.5 kDa), dialyzing for 48h, removing small molecule impurities, and storing in a refrigerator at 4 ℃ for later use.
2.10.5LA characterization of PEG-NH 2 and LA-PEG-Pt (IV)
2.10.5.1 Nuclear magnetic resonance Hydrogen Spectrometry (1Hnuclearmagneticresonance,1 HNMR) analysis
The molecular structure of LA, PEG-NH 2、LA-PEG-NH2 and LA-PEG-Pt (IV) was analyzed using 1 HNMR. Proper amounts of LA, PEG-NH 2、LA-PEG-NH2 and LA-PEG-Pt (IV) are taken in DMSO-d6 solution, ultrasonic treatment is carried out until the sample is completely dissolved, and the prepared sample solution is transferred into a nuclear magnetic resonance measuring tube for 1 H-NMR analysis.
2.10.5.2 Fourier transform Infrared Spectroscopy (Fourier transforminfrared spectroscopy, FT-IR) analysis
The chemical structures of LA, PEG-NH 2、LA-PEG-NH2 and LA-PEG-Pt (IV) were analyzed using FT-IR. Mixing proper amounts of LA, PEG-NH 2、LA-PEG-NH2 and LA-PEG-Pt (IV) dry powder with potassium bromide powder according to the mass ratio of 1:100 in an agate mortar, fully grinding to ensure that a sample and the potassium bromide powder are uniformly mixed and ground to be fine, placing a small amount of powder in an infrared mold, tabletting for about 1min to obtain a semitransparent sheet, placing the sheet in FT-IR, and detecting infrared spectrum information of different samples at 400-4000cm -1.
2.10.5.3LA characterization of PEG-NH 2 and LA-PEG-Pt (IV)
Molecular structures of LA, PEG-NH 2、LA-PEG-NH2 and LA-PEG-Pt (IV) were analyzed using 1 HNMR. As can be seen from fig. 17, the-COOH proton peak of LA at δ=12.02 in the 1 HNMR of LA, the-CH 2 proton peak of LA at δ=1.67 and at δ=1.87, the-NH 2 proton peak of PEG-NH 2 at δ=7.65 in PEG-NH 2, the new-CO-NH-proton peak of LA-PEG-NH 2 at δ=6.65, which fully demonstrated successful PEG-NH 2 and LA bonding by amide condensation, and the-NH 2 proton peak of Pt (IV) at δ=12.02 and the-NH 2 proton peak of δ=7.65 in LA-PEG-NH 2 disappeared after amide condensation, the new-CO-NH-proton peak at δ=6.65, and the-CH 2 proton peak still remained in LA at δ=1.83, were also observed in 1 HNMR of LA-PEG-Pt (IV), further demonstrating successful LA-PEG-Pt (IV) synthesis.
The chemical structures of LA, PEG-NH 2、LA-PEG-NH2 and LA-PEG-Pt (IV) were analyzed using FT-IR. As a result, as shown in FIG. 18, the IR spectrum of LA-PEG-NH 2 showed some IR characteristic peaks of LA and PEG-NH 2, such as the telescopic vibration peak of-C-H-, (2898 cm -1)、CH2 bending vibration (1467 cm -1)), the variable angle bending vibration peak of-C-O-, (1153 cm -1) and the telescopic vibration of C-O-C (1112 cm -1), while LA-PEG-Pt (IV) showed a vibration of c=o in-CONH at 1626cm -1, this was mainly combined with LA-PEG-NH 2 by amide reaction.
2.10.6PT (IV) and GA Loading
The synthesis of MoS 2-MnO2 -PEG-Pt (IV)/GA is shown in FIG. 19. Firstly, LA-PEG-Pt (IV) is grafted on MoS 2-MnO2 through a sulfur chemical action, and then GA is loaded on MoS 2-MnO2 -PEG-Pt (IV) through a hydrophobic action, so that a MoS 2-MnO2 -PEG-Pt (IV)/GA nano drug-carrying system is constructed.
Construction of 2.10.6.1 platinum Standard Curve
Precisely weighing 100 mug of platinum standard substance, placing in a10 mL brown volumetric flask, adding water to the position of a scale mark, mixing and shaking uniformly to obtain platinum stock solution with the concentration of 20 mug/mL, and storing in a dark place for standby. A proper amount of mother solution was precisely sucked and diluted with ddH 2 O to prepare a platinum standard solution having a concentration of 0. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 15. Mu.g/mL, and 20. Mu.g/mL, and the absorption strength of the standard solution was measured by ICP-MS, and a standard curve was established with the platinum concentration (X,. Mu.g/mL) as the abscissa and the absorption strength value (Y) as the ordinate.
2.10.6.2Pt (IV) load
100Mg of LA-PEG-Pt (IV) is weighed and dissolved in 10mL of ddH 2 O, after stirring until the solid is completely dissolved, 20mL of MoS 2-MnO2 solution (0.5 mg/mL) is added, moS 2-MnO2 and LA-PEG-Pt (IV) are combined together through chemical action of sulfur and stirred for 24 hours, the mixed solution is transferred into a dialysis bag (MWCO=100 kDa) and dialyzed for 3 days, and the obtained MoS 2-MnO2 -PEG-Pt (IV) solution is placed in a refrigerator at 4 ℃ for storage for standby.
10Mg of MoS 2-MnO2 -PEG-Pt (IV) is weighed, added into 2mL of concentrated nitric acid solution, digested for several hours at room temperature, protected from light during digestion, centrifuged at 6000rpm for 15min after digestion is finished, the supernatant is sucked into an EP tube, ddH 2 O is added for dilution, the diluted solution is filtered by a microporous filter membrane with the diameter of 0.22 mu m, the concentration of a sample is measured by ICP-MS, and the content of Pt (IV) in the MoS 2-MnO2 -PEG-Pt (IV) nano drug-loading system is calculated according to a standard curve of platinum. The drug loading of Pt (IV) in MoS 2-MnO2 -PEG-Pt (IV) was calculated according to equation (1-2).
The calculation formula of the drug loading is that the drug loading=drug loading nano-sheet drug mass/total administration amount is multiplied by 100% (1-2)
Construction of 2.10.6.3GA Standard Curve
Precisely weighing 1.0mg of GA, placing in a 10mL volumetric flask, adding DMSO to the scale mark, mixing, shaking to obtain GA mother solution with mother solution concentration of 0.1mg/mL, and storing for use. GA mother liquor was diluted with DMSO to a concentration of 0 μg/mL,10 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL,100 μg/mL of GA standard solution. The absorbance of the standard solution at a wavelength of 360nm was measured using UV-Vis, and a standard curve was established with the GA concentration (X, μg/mL) as the abscissa and the absorbance value (Y) as the ordinate.
2.10.6.4GA determination of load and drug loading
5ML of GA solution of 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL was prepared, and an equal volume of MoS 2-MnO2 -PEG-Pt (IV) solution (0.4 mg/mL) was added to the above solution. The GA is loaded on MoS 2-MnO2 -PEG-Pt (IV) through hydrophobic effect, after being stirred for 24 hours at room temperature and in a dark place, the GA is centrifuged at 2000rpm for 10min at a low speed to remove precipitated solid medicines, supernatant is collected, subjected to ultrafiltration centrifugation at 3000rpm for 15min, and repeatedly washed by ddH 2 O, the supernatant is subjected to ultrafiltration centrifugation until the lower solution is colorless, finally the MoS 2-MnO2 -PEG-Pt (IV)/GA solution carrying the GA is obtained, the absorbance of the MoS 2-MnO2 -PEG-Pt (IV)/GA at a wavelength of 360nm is measured according to UV-Vis, and the mass of the GA is calculated according to a standard curve of the GA. And calculating the medicine carrying amount of GA in MoS 2-MnO2 -PEG-Pt (IV)/GA according to the formula (1-2).
2.10.6.5GA usage investigation
The average particle size, PDI, physiological stability and drug loading are used as evaluation indexes, and the prepared MoS 2-MnO2 -PEG-Pt (IV)/GA solution is inspected to determine the dosage of GA.
2.11 Characterization of MoS 2-MnO2 -PEG-Pt (IV)/GA
2.11.1 Field emission transmission electron microscope (Transmission electron microscope, TEM) analysis
The elemental distribution of MoS 2-MnO2 -PEG-Pt (IV)/GA was observed using TEM. Diluting a proper amount of MoS 2-MnO2 -PEG-Pt (IV)/GA sample to a certain concentration by using ddH 2 O, weighing 10 mu L of solution, dripping the solution on a carbon support film, drying the sample overnight, putting the carbon film into a TEM, and analyzing element patterns and element distribution and element content by using energy dispersive X-ray spectroscopy (EDS).
2.11.2 Ultraviolet-visible Spectrophotometer (Ultraviolet-visible spectroscopy, UV-vis) analysis
UV-vis analysis was used for MoS 2-MnO2 -PEG-Pt (IV)/GA UV absorbance change. An appropriate amount of MoS 2-MnO2 -PEG-Pt (IV)/GA sample was diluted to a certain concentration with ddH 2 O, placed in a quartz cuvette, and the absorption peak of the sample at 360nm was recorded.
Characterization of MoS 2-MnO2 -PEG-Pt (IV)/GA
The synthesis of drug-loaded nanosheets MoS 2-MnO2 -PEG-Pt (IV)/GA was examined using TEM and UV-vis. As shown in fig. 20, it can be seen from the mapping graph in fig. 20 (a), the Pt element is uniformly distributed on MoS 2-MnO2, and four element peaks Mo, S, mn, pt in the MoS 2-MnO2 -PEG-Pt (IV)/GA nano-drug delivery system can be also observed in the EDS graph in fig. 20 (b), which indicates that the cisplatin prodrug was successfully loaded on the MoS 2-MnO2 nano-carrier. The characteristic peak of GA was found at 360nm for MoS 2-MnO2 -PEG-Pt (IV)/GA by ultraviolet ray diagram pattern FIG. 20 (c), indicating that GA loading was successful.
2.12MoS 2-MnO2 -PEG-Pt (IV)/GA in vitro Release Studies
The drug release process for MoS 2-MnO2 -PEG-Pt (IV)/GA is shown in FIG. 21. The pH of blood in normal tissue is 7.35-7.45, and PBS buffer solution with pH7.4 is selected to simulate the in vivo microenvironment. Because the Pt (IV) complex can undergo molecular conversion under the reduction condition and release the Pt (II) drug by eliminating the axial dihydroxyl, GSH is used as a reducing agent to test the controlled release behavior of the MoS 2-MnO2 -PEG-Pt (IV)/GA nano-drug-loaded system on the Pt (IV), and meanwhile, the accumulated release amount of GA in the MoS 2-MnO2 -PEG-Pt (IV)/GA nano-drug-loaded system is studied under the photo-thermal stimulation.
Four equal amounts of MoS 2-MnO2 -PEG-Pt (IV)/GA (0.5 mg/mL) samples 2mL were taken, dispersed in PBS solutions (8 mL) of different GSH values (10 mmol/L, 10. Mu. Mol/L), and sealed in dialysis bags (10 kDa). The dialysis bags were immersed in PBS solution corresponding to GSH value, and all samples were shaken in a constant temperature shaker at 37℃and 100rpm in the absence of light. 1mL of the dialysate was withdrawn at fixed points at 0, 2, 4, 8, 12, 24, 48h, respectively, and the corresponding PBS buffer was supplemented in equal volumes. After 48h, samples of the light group were irradiated with 808nm laser light at 1W/cm 2 for 10min and the control group was protected from light. Subsequently, 1mL of dialysate was withdrawn at fixed points at 50, 54, 60, 66, 72, 78, 84, 90, 96h, respectively, and an equal volume of the corresponding PBS buffer was replenished. After the collected samples are diluted and filtered, the concentration of Pt is measured by ICP-MS and the absorbance at the wavelength of 360nm is measured by UV-Vis, the cumulative drug release percentage of each sampling point is calculated, and the release curves of Pt (IV) and GA drugs are respectively drawn.
2.13 Determination of the drug loading of Pt (IV) and GA
Construction of 2.13.1 platinum standard curve and drug-loading capacity measurement
The content of Pt was measured by ICP-MS, pt solutions of different concentrations were prepared, and a linear equation was simulated according to the absorption intensity value in ICP-MS. As shown in fig. 22 (a), the regression equation of the Pt standard was y= 4154.51X, the correlation coefficient R was 1, and a good linear relationship was obtained between the concentrations of 5 to 20 μg/mL. And (3) digesting platinum element in MoS 2-MnO2 -PEG-Pt (IV)/GA by using concentrated nitric acid, then digesting and centrifuging at room temperature, calculating the platinum concentration of the obtained supernatant by ICP-MS according to a standard curve, and finally calculating according to a formula to obtain the drug loading rate of Pt (IV) in a MoS 2-MnO2 -PEG-Pt (IV)/GA nano drug loading system of 15.06%.
Construction of 2.13.2GA Standard Curve
And (3) measuring the content of GA by utilizing an ultraviolet-visible spectrophotometry, respectively preparing GA solutions with different concentrations, and simulating a linear equation according to the absorbance value at 360 nm. The results are shown in FIG. 22 (b), where GA concentration has a good linear relationship with absorbance (R 2 = 0.9996) at concentrations of 10-100 μg/mL, and the linear regression equation is Y = 0.023X-0.007.
2.13.3GA measurement of dose and drug loading
In order to determine the dosage of GA, different concentrations of GA are selected to be mixed with MoS 2-MnO2 -PEG-Pt (IV), free GA is removed by ultrafiltration and centrifugation, so that MoS 2-MnO2 -PEG-Pt (IV)/GA solution is obtained, the particle size, PDI and the calculated drug loading are measured, and the dosage of GA is determined. As shown in FIG. 23 and Table 1, the particle size and drug loading rate of MoS 2-MnO2 -PEG-Pt (IV)/GA gradually increased with increasing GA administration concentration, and when GA administration concentration was 0.1-0.4mg/mL, the MoS 2-MnO2 -PEG-Pt (IV)/GA system was stable, and the particle size was moderate, and when GA administration concentration exceeded 0.6mg/mL, the PDI of MoS 2-MnO2 -PEG-Pt (IV)/GA was as high as 0.337-0.674, and a phenomenon of local coagulation occurred after standing overnight, indicating that the drug loading rate had a great influence on the stability of the system. Meanwhile, in order to better load GA, 0.2mg/mL is selected as the administration concentration of GA, and finally the medicine loading amount of GA in a MoS 2-MnO2 -PEG-Pt (IV)/GA nano medicine loading system is 14.79% according to a formula.
Table 1GA dose investigation results (n=3)
Most preferably, each gMoS 2-MnO2 -PEG-Pt (IV)/GA of the invention contains 1mg of molybdenum sulfide, 10mg of manganese dioxide, 0.1mg of gambogic acid, 1mg of cisplatin prodrug Pt (IV) and 20mg of polyethylene glycol.
2.13.4MoS 2-MnO2 -PEG-Pt (IV)/GA in vitro drug Release Studies
FIG. 24 (a) is a graph showing the release profile of Pt, from which it can be seen that the rate of release of MoS 2-MnO2 -PEG-Pt (IV)/GA to Pt (II) drug is dependent on the concentration of GSH, and when GSH is 10mM, similar to the concentration of glutathione in cancer cells, the cumulative release rate of Pt (II) drug at 96h reaches 84.73%, and at 12h the Pt release rate is nearly the highest. While the cumulative release rate of Pt at 96h drug at GSH of 10 μm was only 29.42%, indicating that Pt (IV) can be better converted to Pt (II) in a strongly reducing environment, indicating that the presence and concentration of GSH at MoS 2-MnO2 -PEG-Pt (IV)/GA release is highly sensitive to the controlled release of Pt (II) drug. The environment-sensitive behavior of MoS 2-MnO2 -PEG-Pt (IV)/GA can effectively prevent the loss of the medicine in the blood circulation process and ensure that the medicine is released in a large amount after reaching the tumor part. The GSH sensitive behavior of MoS 2-MnO2 -PEG-Pt (IV)/GA can not only enhance the anti-tumor curative effect, but also obviously reduce the drug toxicity during the administration.
FIG. 24 (b) shows the GA release profile, which shows that the GA release amount is only 20.88% in 48 hours without irradiation with near infrared light, probably because grafted MnO 2 forms a polymer barrier on MoS 2 nm sheet, limiting drug release. After the sample is irradiated by 808 laser of 1W/cm 2 for 10min, the accumulated release amount of GA in the 10min is found to be about 25%, the release rate is 0.16ug/min, which is 5 times higher than that of a light shielding group (0.032 ug/min), which indicates that near infrared light can induce GA release, more importantly, the accumulated release amount in 48h is 37.6% after the near infrared light irradiation and is about 1.5 times that of the previous 48h, mainly because MoS 2 can efficiently convert the absorbed near infrared light into heat energy, the temperature of the solution is increased, the movement of GA molecules on the surface of a carrier is accelerated, and the GA molecules are further promoted to be separated from the carrier as soon as possible. The MoS 2-MnO2 -PEG-Pt (IV)/GA can realize the controlled release of Pt (IV) and GA under the stimulation of GSH and near infrared light, and provides an important basis for the subsequent in-vitro and in-vivo anti-tumor activity research.
The following proves the beneficial effects of the invention through the pharmacodynamic test.
Test example 1MoS2-MnO2-PEG-Pt (IV)/GA in vitro antitumor Activity
1. Co-therapy of MoS 2-MnO2 -PEG-Pt (IV)/GA at cellular level
A549 cells with good growth state are taken, digested by pancreatin, inoculated into 96-well cell culture plates (1×10 3/well) and transferred into an incubator with 20% O 2 or a 1% O 2 hypoxia culture bag. After cell attachment, the initial medium was discarded, 200. Mu.L of CDDP, GA, CDDP +GA, CDDP→GA, and MoS 2-MnO2 -PEG-Pt (IV)/GA medium were added (CDDP and GA were both dissolved in DMSO, [ CDDP ] = 2,4, 6, 8, and 10. Mu.g/mL, [ GA ] = 0.2, 0.4, 0.6, 0.8, and 1.0. Mu.g/mL), each set was plated with 6 multiplex wells. (CDDP, GA, CDDP +GA and MoS 2-MnO2 -PEG-Pt (IV)/GA groups are respectively incubated for 48 hours, then old culture medium is sucked out, fresh culture medium is added for further incubation for 48 hours, CDDP-GA groups are added for further incubation for 48 hours after CDDP incubation for 48 hours), finally the activity of cells is measured by an MTT method, and the relative cell survival rate R (%) is calculated according to a formula (2-1).
2. Co-therapy of MoS 2-MnO2 -PEG-Pt (IV)/GA at cellular level
Sequential administration was first evaluated for cytotoxicity while verifying hypoxia-induced resistance of tumors. In clinical treatment means of lung cancer, combined administration is often an important scheme for lung cancer treatment. Studies have shown that under sequential dosing regimens (CDDP followed by GA), the synergistic antitumor effect of GA and CDDP is very pronounced. To investigate whether the order of drug addition would affect the synergy produced, cells were treated with CDDP for 48h and then with GA for 48h, the results are shown in FIG. 25, where sequential administration has a concentration-dependent inhibition of cell growth, where sequential administration has the strongest inhibition of cell growth, indicating that sequential administration is a key factor in achieving synergy between CDDP and GA.
Meanwhile, the growth inhibition effect of the medicine on A549 cells for 96 hours in a hypoxia environment is obviously lower than that in a normoxic environment, and the medicine shows higher cell activity, which indicates that the hypoxia environment can induce tumor cells to generate drug resistance. Under normoxic conditions, the cell survival rate of the free CDDP or GA group reaches more than 60%, and under hypoxic conditions, the cell survival rate of the free CDDP or GA group reaches more than 80%, meanwhile, IC 50 values of the CDDP and the GA under the hypoxic conditions are respectively calculated to be 72.32 mug/mL and 3.80 mug/mL, which are respectively 3.4 times and 2.7 times (20.67 mug/mL and 1.369 mug/mL) of that under the normoxic conditions, the tumor cells have stronger drug resistance to drugs under the hypoxic conditions probably because the surfaces of the A549 cells express more drug resistant proteins, and the drug resistant proteins expel the intracellular drugs outside the cells, so that the drug quantity entering the cells is reduced, and the antitumor efficiency is reduced. Meanwhile, the IC 50 value of the MoS 2-MnO2 -PEG-Pt (IV)/GA group is 6.14/0.61 mug/mL (CDDP/GA), which is lower than that of other groups, but higher than that of the normal oxygen state (5.26/0.53 mug/mL), which shows that the MoS 2-MnO2 -PEG-Pt (IV)/GA can relieve the external hypoxia state of tumor cells, because the MnO 2 in the nano system generates oxygen to improve the tumor hypoxia, and the toxicity of the chemotherapeutic drugs to tumor drug-resistant cells is enhanced.
3. MoS 2-MnO2 -PEG-Pt (IV)/GA killing effect on lung cancer cells under near infrared light irradiation
A549 cells in logarithmic growth phase were inoculated into 96-well cell culture plates (1×10 3/well) after pancreatin digestion, and placed in an incubator of 37℃and 20% O 2 or 1% O 2 hypoxia bag. After cell attachment, the cells were randomly divided into illuminated and non-illuminated groups, each group being further divided into 7 groups of (1) cell culture medium, as a blank group ;(2)CDDP;(3)GA;(4)CDDP+GA;(5)CDDP→GA;(6)MoS2-MnO2;(7)MoS2-MnO2-PEG-Pt(IV)/GA.([CDDP]＝2μg/mL,[GA]＝0.2μg/mL,[MoS2-MnO2]＝500μg/mL),, the initial medium was removed, 200 μl of the above material was added per well, and six duplicate wells were set per group. After 6h of co-incubation, the drug-containing medium was aspirated, the cells were washed 3 times with PBS solution, fresh medium was added and incubation was continued for 42h (48 h later group (5) was given GA-containing medium). Then, the cells of the light group were irradiated with 808nm laser light having a power density of 1W/cm 2 for 10min, and the cells of the non-light group were protected from light. After further incubation for 48h (note: group (5) GA-containing medium was incubated for 6h, the drug-containing medium was aspirated, fresh medium was added for further incubation for 42 h), the drug-containing medium was aspirated, and cells were washed 3 times with sterile PBS, followed by addition of 10. Mu. LMTT solution per well for further incubation for 4h in an incubator. After aspiration of MTT solution and addition of 150. Mu.L DMSO per well, the cell culture plates were placed in a shaker and shaken at 100rpm for 15min at 37 ℃. Finally, absorbance (A) at 490nm was measured for each well using a microplate reader, and the relative cell viability R (%) was calculated according to formula (2-1).
4. Induction of apoptosis of lung cancer cells by MoS 2-MnO2 -PEG-Pt (IV)/GA under near infrared light irradiation
A549 cells in logarithmic growth phase were inoculated into 6-well cell culture plates (2X 10 5/well) after pancreatin digestion. After cell attachment, cells were randomly divided into 9 groups (1) cell culture medium, as a blank ;(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 medium was removed and 1mL of the above material was added per well. After 6h of co-incubation, the drug-containing medium was aspirated, the cells were washed 3 times with PBS solution, fresh medium was added and incubation was continued for 42h (48 h later group (5) was given GA-containing medium). Then, the cells of the light group were irradiated with 808nm laser light having a power density of 1W/cm 2 for 10min, and the cells of the non-light group were protected from light. After 48h incubation (note: group (5) GA-containing medium was incubated for 6h, then drug-containing medium was aspirated, fresh medium was added for further incubation for 42 h), and apoptosis was examined using the annexin V-FITC/PI apoptosis detection kit. The preparation method comprises the following steps of sucking out a drug-containing culture medium after incubation is finished, washing cells for 3 times by using a PBS solution, slightly shaking a cell culture plate during washing to clean the residual drug-containing culture medium on the cells, then digesting the cells by using pancreatin without EDTA, collecting the cells into a 1.5mL centrifuge tube when the cells are round and fall off, centrifuging at 1500rpm for 5min, discarding supernatant, resuspending the cells by using 1mL4 ℃ precooled PBS solution, washing the cells for 3 times, centrifuging again, discarding supernatant, adding 490 mu L of 1 Xbinding buffer for staining the resuspension cells (the binding buffer is diluted by deionized water), adding 5 mu L of Annexin V-FITC 4 ℃ for Wen Biguang min for incubation at 37 ℃ for 10min, then adding 5 mu L of PI for light-shielding incubation at 37 ℃ and finally transferring the cells into a flow tube. Data analysis was performed using flow cytometer detection and FlowJo software.
5. Statistical analysis
Unless otherwise indicated, the experimental results were all statistically analyzed using GRAPHPAD PRISM 8.0.0 software. Experimental data are expressed as Mean ± standard deviation (Mean ± SD). Differences between two experimental groups were analyzed using t-test, and differences between more than two experimental groups were analyzed using one-way analysis of variance (ANOVA). * P <0.05 indicates a significant difference and ** P <0.01 indicates a very significant difference.
The MoS 2 nano material becomes a hot material for tumor photothermal treatment due to strong near infrared absorption, so that we further verify whether the killing power of MoS 2-MnO2 -PEG-Pt (IV)/GA on cells is enhanced under the irradiation of near infrared light. After incubating the cells according to 3.4, the cell activity was calculated according to the MTT method. As can be seen from fig. 26, in normoxic environments, near infrared light irradiation alone does not increase cytotoxicity of free drug or combination drug. However, the activity of the tumor cells treated with the MoS 2 -containing material was significantly reduced after irradiation with near infrared light. In a normoxic environment, after the tumor cells treated by MoS 2-MnO2 are irradiated by near infrared light, the activity of the cells is reduced from 96.3% to 57.01%, mainly because the MoS 2 nano material can convert the absorbed near infrared light into heat energy to cause ablation of the tumor cells, inhibit proliferation of the tumor cells and further induce death of the tumor cells. Importantly, the MoS 2-MnO2 material loaded with CDDP and GA showed the strongest cell killing effect under near infrared irradiation, with only 11.47% of the cells in the viable state, far lower than the other experimental groups. The results are presented in that, firstly, near infrared light effectively induces the release of GA, not only remarkably improves the concentration of free GA in cells, but also realizes sequential administration of CDDP and GA to tumor cells, greatly improves the synergistic effect between the two drugs, and secondly, the combined treatment of photo-thermal and synergistic chemotherapy. In a hypoxic environment, photothermal therapy alone is not affected by the lack of oxygen content, while the drug group is limited to varying degrees. MoS 2-MnO2 -PEG-Pt (IV)/GA reacts with intracellular H + and H 2O2 through MnO 2 to generate oxygen, so that the tumor hypoxia state is relieved, the tumor hypoxia drug resistance is weakened, the strong tumor cell proliferation inhibition capability is shown, particularly under near infrared light irradiation, but the tumor cell proliferation inhibition capability of other drugs is obviously inhibited under the conditions of light shielding and near infrared light irradiation.
6. Induction of apoptosis of lung cancer cells by MoS 2-MnO2 -PEG-Pt (IV)/GA under near infrared light irradiation
Apoptosis is a form of cell death and many anticancer drugs, including CDDP and GA, can kill tumor cells by inducing apoptosis. Photothermal therapy is also effective in inducing apoptosis by stimulating tumor cells to initiate an apoptosis program. To further demonstrate that MoS 2-MnO2 -PEG-Pt (IV)/GA has a strong cell killing effect under near infrared light irradiation, we explored the ability of MoS 2-MnO2 -PEG-Pt (IV)/GA to induce apoptosis under near infrared light irradiation. Flow cytometry is used as a method for quantitatively analyzing apoptosis rate, wherein four quadrants of a cell density map represent cells in different states, respectively, Q1 represents necrotic or mechanically damaged cells, Q2 represents late apoptotic cells, Q3 represents early apoptotic cells, and Q4 represents living cells. The experiment adopts an Annexin V-FITC and PI double-staining method to detect apoptosis, the principle is approximately as follows, and phosphatidylserine (Phosphatidylserine, PS) on the inner surface of a cell membrane is destroyed due to the asymmetry of distribution in the apoptosis process of the cell, so that the PS is exposed on the outer surface of the cell membrane from the inner surface of the cell membrane. Annexin V has high affinity to PS, can be specifically combined with PS, and can detect PS through fluorescent FITC labeling. However, in addition to early and late apoptotic cells, the PS of necrotic cells also migrates to the cell membrane, so propidium iodide (Propidium Iodide, PI) is used to distinguish between the cells, PI cannot permeate the intact cell membrane, and late apoptotic and necrotic cells have their membrane integrity destroyed and can be distinguished by PI detection.
FIG. 27 is a graph showing the density distribution of A549 cells in a hypoxic environment. From the figure, it can be found that after the A549 cells are treated by different administration, the apoptosis rate of the sequential administration is stronger than that of single medicine or combination administration, which indicates that under the sequential administration scheme (CDDP is firstly, GA is secondly), the synergistic anti-tumor effect of the CDDP and the GA is very obvious, and is consistent with the cell proliferation experiment. Meanwhile, no obvious apoptosis induction effect of MoS 2-MnO2 carrier material on cells is observed, the apoptosis percentage is 4.26%, which shows that MoS 2-MnO2 is relatively safe, but compared with MoS 2-MnO2 +NIR (39.54%) groups, the apoptosis rate of MoS 2-MnO2 carrier material and MoS NIR (39.54%) group is found to be significantly different, which shows that the photo-thermal heating effect caused by MoS 2 can have a promotion effect on apoptosis. In addition, the percentage of apoptotic cells in the MoS 2-MnO2 -PEG-Pt (IV)/GA+NIR group was 83.6%, which is far higher than that in other experimental groups, indicating that MoS 2-MnO2 -PEG-Pt (IV)/GA effectively induces apoptosis of tumor cells under irradiation of near infrared light, and is consistent with the experimental results of proliferation of tumor cells.
Test example 2MoS 2-MnO2 -PEG-Pt (IV)/GA in vivo antitumor Activity
1. Experimental method
1.1 Establishing an in vivo A549 subcutaneous graft tumor model
Tumor cells were inoculated subcutaneously after 1 week of adaptive feeding of nude mice in animal experimental center. A549 cells in logarithmic growth phase were taken, digested with pancreatin, collected and centrifuged. The cell pellet was then resuspended in PBS and the cell concentration was determined using a cytometer. To ensure cell viability, the collection of cells and cell seeding should be completed in a short period of time. A549 cells with the concentration of 5 multiplied by 10 7/mL are sucked by a disposable sterile injector, injected into the subcutaneous part of the left forelimb of a nude mouse, then the nude mouse is put back into a mouse cage for feeding, the general characteristics of the mice such as activity, mental state and the like are observed, and the conditions of red swelling, infection and the like are observed.
1.2 In vivo anti-tumor Activity of MoS 2-MnO2 -PEG-Pt (IV)/GA under near-infrared light irradiation
1.2.1 Experimental groups
When the tumor volume increased to about 60mm 3, the nude mice were randomly divided into 8 groups of 5 (1) PBS as a blank 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]＝1.0mg/kg,[GA]＝0.1mg/kg,[MoS2-MnO2-PEG]＝5mg/kg).
1.2.2 Tail vein administration
Each group of nude mice was given 1 time every 3 days by injecting the above corresponding materials via the tail vein according to body weight, and total 7 times. Before administration, the tail of the nude mice is rubbed by an alcohol cotton ball to dilate tail vein, intravenous injection is carried out at 1/3 position of the tail of the nude mice, no resistance is caused by pricking a needle head, blood is sucked back and pumped out, and then the administration can be carried out, and after the injection is finished, the hemostasis is carried out by a sterile cotton ball. To ensure that Pt (IV) in MoS 2-MnO2 -PEG-Pt (IV)/GA had enough time to increase tumor cell sensitivity to GA, the nude mice in the light group were irradiated with 808nm laser light at 1.0W/cm 2 for 10min after tail vein injection of the corresponding drug for 24 h. In the illumination process, an infrared thermal imager is used for recording the temperature value according to one time every 60 seconds, collecting data and drawing a photo-thermal heating curve.
1.2.3 Evaluation index
(1) Body weight the body weight of the nude mice was weighed every 3 days within 21 days of the administration treatment of the nude mice, and the body weight change was observed to examine the toxicity of the drug to the nude mice.
(2) Tumor volume-the length and width of a nude mouse tumor was measured every 3 days and recorded in time, the tumor volume (V) of the nude mouse was calculated according to formula (3-1), and a tumor growth curve was drawn.
The calculation formula of the tumor volume is that the tumor volume (V) = (a multiplied by b 2)/2 (3-1)
Wherein a and b are the major and minor diameters of the tumor, respectively.
(3) Tumor volume inhibition ratio (TGI) of nude mice is calculated according to formula (3-2) and statistical analysis is performed.
The calculation formula of the tumor volume inhibition rate TGI (%) is TGI (%) =1- [ (V treated－V0)/VPBS－V0) ]. Times.100% (3-2) wherein V treated is the average tumor volume of the administration group, V PBS is the average tumor volume of the control group, and V 0 is the initial tumor volume.
1.2.4 Hematoxylin-eosin (H & E) staining analysis
After 21 days of administration, all nude mice were sacrificed, and tissues such as heart, liver, spleen, lung, kidney and tumor were removed and fixed in 4% paraformaldehyde solution for 1 day. The tissue was then placed in 70% ethanol solution and finally kept in a 4 ℃ refrigerator for further use. Tumor tissues and heart, liver, spleen, lung and kidney tissues of PBS group and MoS 2-MnO2 -PEG-Pt (IV)/GA+NIR group of each group of nude mice were taken for H & E staining analysis. The operation steps are as follows:
(1) Tissue section is prepared by cutting preserved heart, liver, spleen, lung and kidney into uniform small pieces, and dehydrating in ethanol/water solution. Tissues were permeabilized with xylene, paraffin embedded, microtomed, and the resulting tissue slices (5 μm) were then placed on slides and baked in a slide oven at 70 ℃ for 10min.
(2) Dewaxing, namely drying the slices, and transferring the slices into a xylene solution for soaking twice for 10min each time. Sequentially immersing the slices in 100% ethanol, 95% ethanol, 80% ethanol and 75% ethanol solutions for 3min for dewaxing, and finally putting into distilled water.
(3) Staining, namely placing the slice into hematoxylin dye for staining for 5min, and flushing with distilled water for 3 times each for 3min.
(4) Differentiation the sections were incubated in 1% ethanol hydrochloride for 10min followed by washing with distilled water for 15min.
(5) Counterstaining, namely immersing the slices into 0.5% eosin solution for 6-10s, rinsing the slices with 95% ethanol for 10s, and rinsing the slices with absolute ethanol for 10s.
(6) And (3) sealing, namely dripping a proper amount of neutral gum diluted by using dimethylbenzene onto the tissues, and covering with a cover glass.
(7) Photographing, namely observing tissues under a normal microscope and photographing and storing images.
1.3 Statistical analysis
Unless otherwise indicated, the experimental results were all statistically analyzed using GRAPHPAD PRISM 8.0.0 software. Experimental data are expressed as Mean ± standard deviation (Mean ± SD). Differences between two experimental groups were analyzed using t-test, and differences between more than two experimental groups were analyzed using one-way analysis of variance (ANOVA). * P <0.05 indicates a significant difference and ** P <0.01 indicates a very significant difference.
2. Results and discussion
2.1 In vivo anti-tumor Activity of MoS 2-MnO2 -PEG-Pt (IV)/GA under near-infrared light irradiation
Based on the experimental result that MoS 2-MnO2 -PEG-Pt (IV)/GA inhibits proliferation of tumor cells and induces apoptosis of the tumor cells in vitro, we examined the anti-tumor effect in vivo. When the tumor volume reached about 60mm 3, nude mice were randomly divided into 8 groups (1) PBS group, and after 24 hours of each intravenous administration as 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., tumors were irradiated with 808nm laser light of 1W/cm 2 for 10 minutes. As a result, as shown in fig. 28, the tumor temperature of PBS, CDDP, GA before and after laser irradiation and cddp+ga group was not greatly changed, and was always lower than 40 ℃, whereas the tumor temperature of MoS 2-MnO2 +nir group and MoS 2-MnO2 -PEG-Pt (IV)/ga+nir group were significantly changed, and the tumor temperature was rapidly increased to 56 ℃ within 10min. The high temperature not only can cause the thermal death of tumor cells, but also is beneficial to the release of loaded medicines, enhances the chemotherapeutic effect of MoS 2-MnO2 -PEG-Pt (IV)/GA, and realizes the synergistic effect of chemotherapy and photo-heat.
After the treatment is started, the length and the width of the tumor of the nude mice are measured every 3 days by using a vernier caliper, the real-time volume of the tumor is calculated, a change curve of the tumor volume is drawn, and the tumor volume inhibition rate is calculated. As can be seen from fig. 29, when the nude mice were injected with MoS 2-MnO2 alone, the tumor growth rate was relatively fast, the average tumor volume of the group reached 729.06mm 3 after 21 days, which is close to 770mm 3 of the PBS group, and the tumor volume inhibition rate was only 5.31%, indicating that MoS 2-MnO2 at this dose was low toxic and had no significant inhibition effect on tumors. The tumor growth rate was slightly slowed by CDDP or GA alone compared to the PBS group, indicating that single drug was not effective in inhibiting tumor growth. Compared with single drug, the combined drug has slightly higher anti-tumor capability, but the anti-tumor capability is not the same as the experimental result of cell proliferation, because (1) GA and CDDP are both small molecular substances, have the defects of poor water solubility, short half-life, no targeting and the like, and severely limit the therapeutic effect in vivo. (2) The two have different administration routes and pharmacokinetics processes, so that the two can not act on tumors according to a set sequence, and strict time limitation is eliminated, so that the synergistic effect between the two is eliminated. When the combined medicines are all co-carried on MoS 2-MnO2, the defects of poor water solubility, short half-life and the like of CDDP and GA can be overcome, and a stronger tumor inhibiting capability is obtained. More importantly, under the irradiation of near infrared light, the release of the medicine is controlled, the double medicines are sequentially acted on the tumor, the synergistic effect between the two medicines is greatly improved, and the average volume of the tumor is only 57.68mm 3 by combining the photo-thermal energy of the carrier, so that the tumor inhibition rate is up to 92.09%, and the strongest anti-tumor capability is shown.
H & E staining of tumor tissues is carried out, and a tumor tissue section (figure 30) is observed, so that compared with other groups, after MoS 2-MnO2 -PEG-Pt (IV)/GA+NIR treatment, the tumor cell density is reduced, most of tumor cells are destroyed, and the cell nucleus staining is shallow, isomerization is shown, the treatment effect of MoS 2-MnO2 -PEG-Pt (IV)/GA+NIR is stronger, and further, the nano drug-carrying system can promote apoptosis and necrosis of tumor cells and inhibit tumor growth under near infrared light irradiation.
2.2 In vivo safety assessment
The safety of the drug was investigated by recording the body weight change of nude mice and the sections of the major organs. Fig. 31 is a graph of body weight change in nude mice, which shows that the body weight of nude mice increases slowly with time, but without significant change. The MoS 2-MnO2 -PEG-Pt (IV)/GA has no obvious toxic or side effect on nude mice. At the same time, we also used the H & E staining method to examine the toxic effect of MoS 2-MnO2 -PEG-Pt (IV)/GA on major organs in vivo. The two groups of main tissue organ sections are observed, and the results are shown in fig. 32, and the PBS group and the main organ sections of the MoS 2-MnO2 -PEG-Pt (IV)/GA+NIR group are not obviously different, so that the MoS 2-MnO2 -PEG-Pt (IV)/GA does not cause obvious damage to the main organs of the nude mice, and pathological changes such as tissue necrosis, fibrosis and the like do not occur, and the phenomenon of organ damage does not occur, so that the MoS 2-MnO2 -PEG-Pt (IV)/GA has no obvious toxic or side effect on the main tissue organs of the nude mice, so that the MoS 2-MnO2 -PEG-Pt (IV)/GA is a safe and effective nano pharmaceutical preparation.
The antitumor activity of MoS2-MnO2-PEG-Pt (IV)/GA was examined at the animal level. Firstly, establishing a subcutaneous transplantation tumor model of a lung cancer A549 cell of a nude mouse, and injecting MoS 2-MnO2 -PEG-Pt (IV)/GA into a tail vein. The nano-drug delivery system can be enriched in tumor sites by virtue of passive targeting and EPR effect. Under the irradiation of near infrared light, the average temperature of the tumor part of the nude mice rises to 56 ℃, and the high temperature not only can cause the ablation of tumor cells and inhibit the growth of tumors, but also can effectively promote the release of combined medicines, enhance the treatment effect of a nano medicine carrying system and realize the cooperative chemotherapy and photo-thermal combined treatment of the combined medicines of the tumors. In the aspect of safety, the MoS 2-MnO2 -PEG-Pt (IV)/GA is found to have no obvious toxic or side effect in vivo and no visible damage to main organs through the weight change of a nude mouse and the H & E staining result, so that the preparation is a safe and effective nano-drug preparation.
According to the MoS 2-MnO2 -PEG-Pt (IV)/GA nano drug delivery system constructed by the invention, through sequential release of GSH and near infrared light control drugs and combination of the characteristics of a carrier, tumor hypoxia drug resistance is reversed, so that 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, a new thought and research strategy is provided for clinical lung cancer treatment, and the application range of anticancer traditional Chinese medicines in tumor treatment is also expanded.
Claims (10)
1. A molybdenum disulfide nano-delivery system for co-carrying gambogic acid and cisplatin is characterized by comprising the following components, 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 cisplatin prodrug Pt (IV) and 10-50 parts of polyethylene glycol.
2. The molybdenum disulfide nano-delivery system for co-carrying gambogic acid and cisplatin according to claim 1, which is characterized by comprising the following components in parts by weight.
3. The nano conveying system for co-supporting gambogic acid and cisplatin is characterized in that molybdenum disulfide nano conveying system takes molybdenum disulfide (MoS 2) nano sheets as a substrate, manganese dioxide (MnO 2) nano particles are modified, and meanwhile, GA and CDDP are loaded, wherein the MoS 2-MnO2 nano sheets have a two-dimensional lamellar structure, and GA and CDDP are loaded through amide reaction and hydrophobic effect.
4. The molybdenum disulfide nano-delivery system for co-carrying gambogic acid and cisplatin as claimed in claim 3, wherein the molybdenum disulfide (MoS 2) nano-sheet is a single-layer or less-layer molybdenum disulfide nano-sheet prepared by a Morrison method, and comprises the following steps:
Putting MoS2 crystals into an n-butyllithium solution containing 15% of n-hexane under nitrogen, and stirring for 2 days under the protection of nitrogen;
after the reaction is finished, the mixture is washed by redistilled n-hexane to remove residues of n-butyllithium and other impurities;
Removing black powder after cleaning, immediately adding ddH2O, ultrasonically stripping for 2 hours, collecting solution, centrifuging at 8000rpm for 20 minutes to remove incompletely stripped crystals, transferring the upper layer solution into a dialysis bag with MWCO=100 kDa after centrifugation, and dialyzing for one week to remove residual lithium ions and other impurities to obtain solution containing MoS2 nanosheets;
The manganese dioxide (MnO 2) nanoparticle is prepared by adopting oxidation-reduction reaction, and the preparation method comprises the following steps:
Weighing KMnO4 and bovine serum albumin BSA, adding into ddH2O, stirring at room temperature for 5h, reducing KMnO4 by BSA to form MnO2 nanoparticles, centrifuging at 12000rpm for 20min after the reaction is finished to remove free KMnO4, BSA and MnO2 particles with larger particle size;
The preparation method of the Pt (IV) comprises the steps of adding CDDP and H 2O2 into ddH 2 O, stirring at 76 ℃ for 8 hours in a dark place, stopping heating and continuing stirring overnight after the reaction is finished, standing the mixture for crystallization at 4 ℃ for about 8 hours after overnight, then washing a crystallization product with cold water, ethanol and diethyl ether sequentially, centrifuging at 8000rpm for 10 minutes, collecting precipitates, drying in vacuum, obtaining bright yellow crystallization powder, namely cisplatin (Pt (II) oxide, after overnight, taking Pt (II) and succinic anhydride, adding into DMSO, stirring at 76 ℃ in a dark place for 48 hours, then freezing and drying to remove the DMSO to obtain yellow powder, adding pre-chilled glacial acetone into the powder, recrystallizing at-20 ℃, collecting the solution, washing 3 times with 10mL pre-chilled glacial acetone, centrifuging at 8000rpm for 10 minutes, collecting precipitates, and drying in vacuum, and obtaining white powder, namely Pt (IV).
5. The molybdenum disulfide nano-delivery system for co-supporting gambogic acid and cisplatin according to claim 3, wherein the synthesis method of MoS2-MnO2 is characterized in that MoS2 nano-sheet aqueous solution and MnO2 nano-particle aqueous solution are mixed, moS2 and MnO2 are combined together through chemical action of sulfur and stirred for 24 hours, then the mixed solution is transferred into a dialysis bag (MWCO=100 kDa), and free MoS2 and MnO2 are removed after dialysis for 3 days, so that MoS2-MnO2 solution is obtained.
6. A method for preparing a molybdenum disulfide nano-delivery system for co-carrying gambogic acid and cisplatin according to any one of claims 1-5, which is characterized by comprising the following steps:
a. Preparing molybdenum disulfide (MoS 2) nanosheets;
b. Preparing manganese dioxide (MnO 2) nanoparticles;
c. synthesizing MoS 2-MnO2;
d. Synthesizing Pt (IV);
e. Synthesizing LA-PEG-NH 2 by coupling LA and PEG to obtain LA-PEG-NH 2;
f. And (3) taking LA-PEG-NH 2 as a connector, grafting Pt (IV) on MoS 2-MnO2, and preparing the molybdenum disulfide nano-delivery system carrying gambogic acid and cisplatin together.
7. The method for preparing the nano molybdenum disulfide delivery system for co-carrying gambogic acid and cisplatin as claimed in claim 6, wherein the synthetic method of LA-PEG-NH 2 in step e is as follows:
Weighing 6-arm PEG-NH 2, LA and DMAP, adding into CH 2Cl2 solution, stirring until the solid is completely dissolved, adding EDC, NHS to activate LA, stirring at room temperature for 3 days in dark, after the reaction is finished, spin-evaporating the solution to white crystal, re-dissolving the crystal in ddH 2 O, centrifuging at 8000rpm for 15min, transferring the supernatant into a dialysis bag (MWCO=3.5 kDa), dialyzing for 48h, removing small molecular impurities, and finally freeze-drying to obtain white solid LA-PEG-NH 2.
8. The method for preparing the molybdenum disulfide nano-delivery system for co-supporting gambogic acid and cisplatin as claimed in claim 8, wherein in the step f, firstly, LA-PEG-Pt (IV) is synthesized, and the Pt (IV) complex is covalently attached to LA-PEG-NH 2 through an amide bond to obtain the LA-PEG-Pt (IV), and the synthesis method comprises the following steps:
Weighing Pt (IV), EDC and NHS, dissolving in ddH 2 O, stirring in a dark place, adding LA-PEG-NH 2, stirring in a dark place for 24 hours, centrifuging at 8000rpm for 10 minutes after the reaction is finished, transferring the supernatant into a dialysis bag (MWCO=3.5 kDa), dialyzing for 48 hours, and removing small molecular impurities to obtain LA-PEG-Pt (IV);
And then grafting LA-PEG-Pt (IV) on MoS 2-MnO2 through a sulfur chemical effect, and loading GA on MoS 2-MnO2 -PEG-Pt (IV) through a hydrophobic effect to construct a MoS 2-MnO2 -PEG-Pt (IV)/GA nano drug-carrying system.
9. The method for preparing the molybdenum disulfide nano-delivery system for co-carrying gambogic acid and cisplatin as claimed in claim 8, wherein the method for preparing the MoS 2-MnO2 -PEG-Pt (IV)/GA nano-drug-carrying system comprises the following steps:
pt (IV) loading:
100mg of LA-PEG-Pt (IV) is dissolved in 10mL of ddH 2 O, after stirring until the solid is completely dissolved, 20mL of MoS 2-MnO2 solution (0.5 mg/mL) is added, moS 2-MnO2 and LA-PEG-Pt (IV) are combined together through the chemical action of sulfur and stirred for 24 hours, the mixed solution is transferred into a dialysis bag (MWCO=100 kDa) and dialyzed for 3 days, and the obtained MoS 2-MnO2 -PEG-Pt (IV) solution is obtained;
loading of GA:
Preparing 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL of GA solution, adding equal volume of MoS 2-MnO2 -PEG-Pt (IV) solution into the above solution, loading GA onto MoS 2-MnO2 -PEG-Pt (IV) by hydrophobic effect, stirring at room temperature and in dark place for 24h, centrifuging at 2000rpm for 10min to remove precipitated solid medicine, collecting supernatant, ultrafiltering and centrifuging at 3000rpm for 15min, repeatedly washing with ddH 2 O, ultrafiltering and centrifuging until the lower solution is colorless, and finally obtaining MoS 2-MnO2 -PEG-Pt (IV)/GA solution loaded with GA.
10. Use of the co-supported gambogic acid and cisplatin molybdenum disulfide nano-delivery system as defined in any of claims 1-5 in the manufacture of a medicament for treating lung cancer.
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