Near-infrared light response-based nano material for combined treatment of tumors as well as preparation and application of nano material

Abstract

The invention belongs to the technical field of tumor treatment, and relates to a nanometer material for treating tumors based on near-infrared light response in combination with photo-thermal, photodynamic and nitric oxide as well as preparation and application of the nanometer material. The invention relates to a near-infrared light response-based nano material for combined treatment of tumors, which is of a sphere-like structure, takes hollow mesoporous copper sulphide with photo-thermal performance as a carrier, loads a nitric oxide donor, and wraps a phase change material containing a photosensitizer. According to the nano material, photo-thermal conversion of the hollow mesoporous copper sulfide nano particles is triggered through near-infrared light, high temperature is generated, the phase change material is molten, and release of the photosensitizer IR780 and L-Arg is promoted; under irradiation of near-infrared light, IR780 generates active oxygen, and L-Arg is further triggered to release nitric oxide. The linkage mechanism of thermal controllable drug release closely combines photo-thermal, photodynamic and nitric oxide treatment, and the construction of the obtained nano material solves the problem that < 1 > O2 and NO cannot be effectively supplied to tumor sites.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor treatment, and relates to a nano material for combined (photothermal, photodynamic, nitric oxide) tumor treatment based on near-infrared light response, and its preparation and application. Background Art

[0002] Cancer is one of the leading causes of death worldwide. According to the latest results released by the World Health Organization, about one-sixth of the world's population dies from cancer in just one year, and the incidence rate is increasing year by year, which has seriously threatened human health. In order to overcome the limitations of traditional treatments such as chemotherapy, in recent years, photothermal therapy (PTT), photodynamic therapy (PDT) and gas therapy have been widely used in the treatment of tumors due to their advantages such as non-invasiveness, high accuracy and safety. However, there are many shortcomings in a single treatment method, which limits the clinical application of the above treatment methods. For example, in the process of PTT causing tumor cell necrosis, due to the damage of cell membrane integrity, intracellular components are released into the extracellular environment, triggering an inflammatory response, thereby inducing tissue damage, tumor regeneration and secondary treatment resistance and other side effects. In addition, the high temperature generated during photothermal therapy will induce cells to upregulate the expression of heat shock proteins, and a series of self-protection mechanisms induced by cells under stress will also directly or indirectly weaken the therapeutic effect of PTT. For PDT, photosensitizers produce cytotoxic reactive oxygen species under laser irradiation. However, commonly used photosensitizers have low water solubility and poor tumor targeting, which affects the real application of PDT in tumor treatment. More importantly, the clinical application of PDT is seriously hindered by the shortcomings of insufficient oxygen supply in the tumor area and high intracellular glutathione consumption of reactive oxygen. In the past few decades, nitric oxide (NO) has been widely used as a physiological regulator and tumor inhibitor in various pathological processes. For example, it promotes wound healing through cell proliferation, angiogenesis and collagen deposition, inhibits thrombosis and platelet aggregation for cardiovascular diseases, etc. In addition, NO shows exciting effects in tumor treatment, and local high concentrations (>400nM) of NO have a significant inhibitory effect on tumors. However, NO has a short half-life and is in gaseous state, so it cannot be used directly in clinical practice. In order to facilitate the transportation and release of NO to the lesion site, a large number of responsive NO donors have been studied and developed. However, the problem that NO cannot be effectively supplied to the tumor site seriously hinders the application of NO therapy.

[0003] Compared with single therapy, the combination of multiple methods can achieve better toxicity reduction and efficacy. At present, combining PDT and PTT simultaneously or sequentially can make full use of the advantages of each mode and offset their limitations. On the one hand, the temperature of tumor tissue is increased by photothermal effect, making tumor tissue more sensitive to reactive oxygen, thereby enhancing the effect of PDT. On the other hand, the reactive oxygen free radicals generated by PDT destroy the membrane structure of tumor cells, improve the killing effect of heat on tumors, and achieve the therapeutic effect of "1+1>2". However, the therapeutic effect of the synergistic effect of PTT and PDT is still challenged by the bottleneck of PDT and PTT. Generally, the concentration of O2 often limits the therapeutic effect of PDT because of the severe tumor hypoxia environment, so it is urgent to relieve tumor hypoxia. The high temperature (T>50℃) generated during PTT often inevitably causes unnecessary damage to nearby tissues, so the realization of mild PTT (mPTT, T<45℃) is very important. However, the reduction of temperature in mPTT also means that the cytotoxicity of the treatment method is reduced, so it is necessary to sensitize mPTT.

[0004] Studies have shown that when NO concentration is high (>1 μM), it can exert anti-tumor effects by inducing mitochondrial and DNA damage, blocking DNA synthesis and repair, and inhibiting cellular respiration. In particular, NO-mediated vasodilation increases blood perfusion, thereby effectively alleviating tumor-related hypoxia. In addition, NO can react with reactive oxygen species to produce more harmful reactive nitrogen oxides, such as peroxynitrite (ONOO - ) molecules, which can aggravate DNA breakage and trigger cell apoptosis, ultimately enhancing the therapeutic effect of ROS on cancer cells. NO combined with PTT, on the one hand, can act on a wider range of tumor cells, reduce the expression of P-glycoprotein, and reverse the multidrug resistance of tumors. On the other hand, NO can prevent cell autophagy protection, downregulate the expression of heat shock proteins, and make cells more sensitive to temperature, which is expected to achieve mild PTT tumor treatment and achieve anti-tumor effects with reduced toxicity and increased efficacy. In summary, further combining it with other methods can be used as a new trend in cancer treatment. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of low therapeutic effect of existing therapies and to provide a nanomaterial for treating tumors based on near-infrared light response combined (photothermal, photodynamic, nitric oxide) for reducing toxicity and increasing efficacy of clinical cancer treatment.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A nanomaterial for combined tumor treatment based on near-infrared light response. The material has a spherical structure, uses hollow mesoporous copper sulfide with photothermal properties as a carrier, is loaded with nitric oxide donors, and is wrapped with a phase change material containing a photosensitizer.

[0008] The material has a diameter of 100-140 nm and a potential of -20.2 to -23.0 mV.

[0009] The nitric oxide donor is a water-soluble nitric oxide donor, which is L-arginine (L-Arg) or a metal-nitroso compound (M-NO); wherein the nitric oxide donor accounts for 18-24% of the material mass.

[0010] In the phase change material containing photosensitizer, the photosensitizer accounts for 12-16% of the mass of the phase change material; and the phase change material accounts for 30-40% of the mass of the material.

[0011] The photosensitizers are IR780, indocyanine green (ICG), and chlorin e6 (Ce6).

[0012] The phase change material (PCM) is a mixture of 1-hexadecanol and oleic acid in a mass ratio of 4:1.

[0013] The carrier is hollow mesoporous copper sulfide nanoparticles (HMCuS NPs). The HMCuS NPs are spherical in shape, with a diameter of 70-90nm, a potential of -22 to -25.9mV, and a mesopore diameter of 5-8nm.

[0014] The preparation method of the HMCuS NPs comprises:

[0015] Add 200-260mg PVP-K30 to 20-30mL deionized water to dissolve, add to 120-150μL 0.5mol / L CuCl2 solution, stir at room temperature for 5-10min. Add 20-30mL NaOH (pH=9.0) solution and stir for 2-5min, then add 3.2-6.4μL N2H4·H2O, stir for 5-10min to form a yellow Cu2O suspension. Add 150-200μL 160mg / mL Na2S aqueous solution to the suspension and stir for 5-10min, reflux at 75℃ for 2-4h. After the reaction is completed, cool in an ice bath, centrifuge at 10000rpm for 15min, wash twice with deionized water, and store at 4℃.

[0016] A method for preparing a nanomaterial for combined tumor treatment based on near-infrared light response uses a hollow mesoporous copper sulfide with photothermal properties as a carrier, loads a nitric oxide donor, and after loading, uses a phase change material containing a photosensitizer for wax sealing and packaging.

[0017] Further speaking

[0018] 1) Preparation method of HMCuS NPs: 200-260 mg PVP-K30 was dispersed in 20-30 mL deionized water, and 120-150 μL of 0.5 mol / L CuCl2 solution was added and mixed. After mixing, the system was adjusted to alkalinity with NaOH solution, and then 3.2-6.4 μL N2H4·H2O and 150-200 μL of Na2S aqueous solution were added to the system, and after mixing, the mixture was heated in a water bath at 75-80°C and refluxed for 2-3 h.

[0019] 2) adding nitric oxide donor and amphiphilic phospholipid to the above solution, stirring at room temperature for 24-48 hours to obtain solution 1;

[0020] 3) adding the photosensitizer to the phase change material and mixing them uniformly by ultrasonication to obtain solution 2;

[0021] 4) Solution 2 was quickly added to solution 1 preheated to 50-55° C., and ultrasonicated for 10-15 min. After ultrasonication, it was placed in an ice bath, and the dispersion was dialyzed overnight to remove impurities to obtain the material (CuS-Arg@PCM-IR780 NPs, referred to as CAPINPs).

[0022] In step 2), the mass ratio of the nitric oxide donor to the amphiphilic phospholipid is 4:1 to 5:1; in step 3), the concentration of the photosensitizer in the phase change material is 1.2-1.5 mg / mL; in step 4), solution 2 accounts for 4% of the volume of solution 1.

[0023] The amphiphilic phospholipids may be one or more of L-α-phosphatidylcholine, distearoyl phospholipids, and phosphatidylcholine.

[0024] An application of the nanomaterial for combined tumor treatment based on near-infrared light response, wherein the material is used for anti-tumor application by combining photothermal therapy (PTT), photodynamic therapy (PDT) and gas therapy (GT) under near-infrared light (NIR) response.

[0025] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0026] The present invention is based on a near-infrared light-responsive combined (photothermal, photodynamic and nitric oxide) therapeutic nanomaterial, which uses copper sulfide nanoparticles with hollow mesopores as carriers. Due to its good biocompatibility, excellent photothermal conversion performance, and excellent drug loading capacity, it provides favorable conditions for the construction of a multifunctional therapeutic nanoplatform based on photothermal therapy; a wax-sealed phase change material PCM is used to block the mesopores to solve the problem of premature release of L-Arg in the circulation. At the same time, the drug loading effect of PCM is utilized to disperse IR780 in the PCM in advance to achieve the loading of fat-soluble photosensitizers. Finally, the reactive oxygen species of water-soluble L-Arg responds to the nitric oxide release mechanism. Under near-infrared light, the reactive oxygen species generated by the photodynamic properties of the photosensitizer IR780 triggers the release of nitric oxide through a cascade reaction.

[0027] The nanomaterial of the present invention triggers the photothermal conversion of hollow mesoporous copper sulfide nanoparticles through near-infrared light, generates high temperature, melts the phase change material, and promotes the release of photosensitizer IR780 and L-Arg; under near-infrared light irradiation, IR780 produces reactive oxygen, which further triggers L-Arg to release nitric oxide. This "linkage mechanism" of thermally controllable drug release closely combines photothermal, photodynamic and nitric oxide therapy, and the construction of the resulting nanomaterial solves the problem of 1 It solves the problem that O2 and NO cannot be effectively supplied to the tumor site, and achieves effective treatment of the tumor at a mild temperature, making up for the poor effect of single and double treatment methods on tumor treatment.

[0028] The preparation method of the present invention is simple, has low requirements on equipment during the preparation process, is low in cost, is environmentally friendly, and has a high yield of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The present invention provides a flow chart for preparing hollow mesoporous copper sulfide nanoparticles (a); and a flow chart for preparing near-infrared light-responsive photothermal / photodynamic / nitric oxide therapeutic nanomaterials (b).

[0030] Figure 2 This is a transmission electron micrograph of HMCuS NPs provided by the present invention (a); a transmission electron micrograph of CAPI NPs (b); the Zeta potential of HMCuS NPs, CA NPs and CAPI NPs (c); the UV-visible-near infrared absorption spectrum of HMCuS NPs, CA NPs, CAPI NPs and IR780 (d); and the infrared absorption spectrum of HMCuS NPs, CA NPs, CAPI NPs and L-Arg (e).

[0031] Figure 3 Schematic diagram of the anti-tumor mechanism of near-infrared light-responsive photothermal / photodynamic / nitric oxide therapeutic nanomaterials.

[0032] Figure 4 The CAPI NPs of different concentrations provided by the present invention were exposed to near-infrared laser (808 nm, 1 W / cm 2 ) irradiation temperature change curve (a) and photothermal cycle curve (b); CAPI NPs under near-infrared laser (808nm, 1W / cm 2 ) 1 Continuous release behavior curve of O2 (c) and near-infrared controlled release behavior curve (d); CAPI NPs were exposed to near-infrared laser (808nm, 1W / cm 2 ) Cumulative release curve (e) and controlled release curve (f) of NO under irradiation.

[0033] Figure 5 The figures are the cellular uptake of CAPI NPs provided in the embodiments of the present invention (a); the anti-tumor activity of 4T1 cells in different treatment groups (b); the Calcein AM / PI staining of live cells (green) and dead cells (red) of 4T1 cells in different treatment groups (c); the Annexin V-FITC / PI double staining of 4T1 cells in different treatment groups (d) (n=3, *p<0.05, **p<0.01, ***p<0.001).

[0034] Figure 6 The figures are the distribution diagram of IR780 and CAPI NPs in 4T1 tumor-bearing mice (a) and in vitro organ distribution diagram (b) provided by the embodiments of the present invention; the in vivo thermal imaging diagram of tumor-bearing mice under near-infrared light irradiation after intravenous injection of PBS and CAPI NPs (c).

[0035] Figure 7 Schematic diagram of the animal experiment scheme of the nanomaterial provided in the embodiment of the present invention and different experimental groups (a); tumor growth curves (b) and tumor photos (c) of mice after different treatments (n=5, *p<0.05, **p<0.01, ***p<0.001); representative immunofluorescence staining (d) and HE staining (e) pictures of 4T1 tumor proliferating cells (Ki-67) after treatment.

[0036] Figure 8 The graphs are a weight change curve of the nanomaterials provided in the embodiments of the present invention and animals in different experimental groups (a); the hemolysis rate of CAPI NPs at different concentrations (b); and the H&E staining images of the main organs of different treatment groups (c). DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the protection scope of the present invention.

[0038] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0039] Based on the limitations of single and two treatment methods in tumor treatment, the present invention proposes to use three treatment methods of photothermal, photodynamic and nitric oxide in combination, and endow the nano-delivery system with three functions of PTT, PDT and NO. By utilizing the enhanced permeability and retention effect (EPReffect) of the nano-delivery system, the nano-delivery system can utilize the high permeability of tumor blood vessels to cross the vascular barrier, directly enter the tumor microenvironment, and enrich in tumor tissue. Afterwards, by controlling the precise release of the drug, while reducing damage to normal tissues, the therapeutic effect is improved, and the purpose of tumor treatment with reduced toxicity and increased efficacy is achieved. Specifically, hollow mesoporous copper sulfide nanoparticles with photothermal properties are used as carriers, nitric oxide donors are loaded, and then a phase change material containing a photosensitizer is wrapped to construct a nano-material with photothermal, photodynamic and nitric oxide therapeutic functions based on near-infrared light response.

[0040] Hollow mesoporous copper sulfide (HMCuS NPs) can be loaded with chemotherapeutic drugs due to their mesopores, hollow interior and high surface area, and can be used as drug delivery carriers in tumor cancer treatment. In addition, HMCuS NPs have strong light absorption in the near-infrared band and have become an ideal medium for photothermal therapy. L-arginine (L-Arg) is an important nitric oxide (NO) donor molecule that can produce NO under the catalysis of inducible NO synthase or under the action of reactive oxygen species (ROS). Therefore, it is the best NO donor molecule for PDT / NO combination. Phase change materials (PCMs) prepared based on fatty acids, fatty alcohols and their derivatives respond precisely to temperature changes, providing a simple and effective platform for controlled drug release.

[0041] Example 1

[0042] Preparation of a nanomaterial for combined tumor treatment based on near-infrared light response, such as Figure 1 As shown:

[0043] S1. Preparation of hollow mesoporous copper sulfide nanoparticles (HMCuS NPs).

[0044] Specific method: Add 200-260mg PVP-K30 to 20-30mL deionized water to dissolve, add to 120-150μL of 0.5mol / L CuCl2 solution, and stir at room temperature for 5-10min. Add 20-30mL NaOH (pH=9.0) solution and stir for 2-5min, then add 6.4μL N2H4·H2O, and stir for 5-10min to form a yellow Cu2O suspension. Add 150-200μL of 160mg / mL Na2S aqueous solution to the suspension, stir for 5-10min, and reflux at 75℃ for 2-4h. After the reaction is completed, cool in an ice bath, centrifuge at 10000rpm for 15min, wash twice with pure water, and store at 4℃ (see Figure 1 a).

[0045] S2. Preparation of CuS-Arg@PCM-IR780 nanoparticles (CAPI NPs).

[0046] The specific preparation process of the phase change material (PCM) is as follows: 140-180 mg of 1-hexadecanol and 30-40 mg of oleic acid are dissolved in 10-15 mL of anhydrous ethanol, ultrasonically mixed at 60° C. for 10-20 min, and stored at 4° C.

[0047] To obtain CAPI NPs, first, 10 mL of the above HMCuS NPs and 100 mg of L-Arg were stirred at room temperature overnight to obtain a CuS-Arg solution. Then, 400 μL of the phase change material containing IR780 (1.5 mg / mL) was quickly added to the CuS-Arg mixed solution preheated to 50-55 ° C, and then ultrasonicated for 10-15 min and then ice-bathed. Under the condition of rapid cooling, the PCM solidified instantly and the drug was encapsulated. Finally, the prepared CuS-Arg@PCM-IR780 nanoparticles (CAPI NPs) were dialyzed overnight to remove impurities (see Figure 1 b).

[0048] from Figure 2 (a) shows that the prepared HMCuS NPs are spherical, with a rough surface and an obvious hollow structure. The size of the nanoparticles is relatively uniform, with an average particle size of 70-90nm. CAPI NPs are spherical in shape with a diameter of 100-140nm ( Figure 2 (b)). Figure 2(c) It can be seen that the potential of HMCuS NPs is -24.1±1.66mV. When L-Arg is loaded, the potential of CuS-Arg (CANPs) drops to -43.26±1.67mV due to the negative charge of L-Arg. After wrapping PCM-IR780, the CAPI potential shows -21.6±1.38mV, which is because IR780 is positively charged, proving that IR780 is successfully coated on the surface. By UV-visible-near infrared spectroscopy, it is calculated that when the concentration ratio of CA NPs and PCM-IR780 is 1:0.5, it has a good drug loading capacity (5.33±0.32)% and encapsulation efficiency (42.3±0.48)%. Figure 2 As shown in (d), the characteristic absorption peak of IR780 appeared in CAPI NPs and was slightly red-shifted, indicating the successful loading of IR780. Figure 2 As shown in (e), at 1739 cm -1 At , both CA NPs and CAPINPs showed the characteristic peak of C=N stretching vibration of guanidine nitrogen in L-Arg, as well as the double peak of primary amine, which proved the successful loading of L-Arg. Thermogravimetric analysis showed that the drug loading amount of L-Arg was about 20.2%.

[0049] Further, if Figure 3 As shown in the figure, under the action of near-infrared light, the high temperature generated by HMCuS NPs first melts the phase change material, promoting the release of photosensitizer IR780 and L-Arg. Afterwards, the reactive oxygen species generated by photosensitizer IR780 are used for PDT on the one hand, and trigger L-Arg to release NO on the other hand. This "linkage mechanism" of heat-controlled drug release closely combines photothermal / photodynamic / nitric oxide therapy, solving the problem of 1 The problem of O2 and NO not being able to be effectively supplied to the tumor site can be solved, thereby achieving effective treatment of mild PTT.

[0050] Example 2

[0051] NO is a ligand with strong coordination ability with metal ions (such as Fe, Mn, Ru), so metal nitrosyl compounds (M-NO) can be used as potential NO donors. In addition, most M-NO are photosensitivity. Under ultraviolet light or near-infrared light irradiation, photoelectrons transition from the π orbit of metal ions to the π antibonding orbit of NO, resulting in the breaking of bonds of M-NO and rapid release of NO. The present invention can also use ruthenium nitrosyl compounds (Ru-NO) as NO donors. First, 10 mL of the above HMCuS NPs and 100-200 mg of Ru-NO are stirred at room temperature overnight to obtain a CuS-Ru-NO solution. Then, 400 μL of phase change material (1.5 mg / mL) containing IR780 is quickly added to the CuS-Ru-NO mixed solution preheated to 50-55°C, and an ice bath is performed after ultrasonication for 10-15 minutes. Under the condition of rapid cooling, the PCM solidifies instantly and the drug is encapsulated. Finally, the prepared CuS-Ru-NO@PCM-IR780 nanoparticles were dialyzed overnight to remove impurities.

[0052] Example 3

[0053] Indocyanine green (ICG) is composed of two hydrophobic polycyclic rings connected by a carbon chain in the middle, and each polycyclic ring is connected to a hydrophilic sulfate group. Therefore, it is an amphiphilic tricarbonyl cyanine dye with low toxicity to humans. Under near-infrared laser irradiation, ICG can stimulate the production of ROS to achieve PDT. The present invention can also use ICG as a photosensitizer. First, 10mL of the above HMCuS NPs and 100mg L-Arg are stirred at room temperature overnight to obtain a CuS-Arg solution. Then, 400μL of phase change material (1.5mg / mL) containing ICG is quickly added to the CuS-Arg mixed solution preheated to 50-55℃, and an ice bath is performed after ultrasonication for 10-15min. Under the condition of rapid cooling, the PCM solidifies instantly and the drug is encapsulated. Finally, the prepared CuS-Arg@PCM-ICG nanoparticles are dialyzed overnight to remove impurities.

[0054] Application Example 1

[0055] like Figure 4 As shown, the in vitro photothermal performance, photodynamic performance and NO release performance of the nanomaterial described in Example 1 were investigated.

[0056] S1. Investigation of in vitro photothermal performance.

[0057] In order to investigate the effect of concentration on the photothermal effect of CAPI NPs, pure water was used as the control group to measure the temperature rise of four different concentrations of CAPI NPs (0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, and 0.2 mg / mL). 1 mL of each sample was irradiated with 808 nm laser (1 W / cm 2 , 10min), record the temperature every 30s and draw a heating curve.

[0058] To investigate the photothermal stability of CAPI NPs, 1 mL of 0.2 mg / mL CAPI NPs solution was taken at 808 nm and 1.0 W / cm 2 The samples were irradiated with near-infrared laser for 10 min each time and then protected from light for 10 min. Four cycles of irradiation and protection from light were performed in total. The temperature was recorded every 30 s during the period to observe the temperature changes.

[0059] S2. Investigation of photodynamic performance.

[0060] 1 Determination of continuous O2 release: Prepare a 5 mM drug stock solution of singlet oxygen green fluorescent probe (SOSG) with methanol solution, shake well, and store at -20°C away from light. Thaw at 4°C before use, take an appropriate amount of stock solution and dilute it with deionized water to a working solution with a concentration of 1 μM. Mix 2 mL SOSG with 2 mL CAPI NPs and use a near-infrared laser (808 nm, 1 W / cm 2 ) for 10 min, take 200 μL of sample every 1 min in a 96-well plate, set up three replicate wells, use pure water containing SOSG as a blank control, and measure the fluorescence intensity (λE X :504nm,λE M :525nm).

[0061] 1 Determination of O2-responsive release: SOSG was prepared into a 5 mM drug stock solution with methanol solution, shaken, and stored at -20°C away from light. Thaw at 4°C before use, take an appropriate amount of the stock solution and dilute it with deionized water to a working solution with a concentration of 1 μM. Mix 2 mL of SOSG with 2 mL of CAPI NPs and use a near-infrared laser (808 nm, 1 W / cm 2 ) irradiation, one switch cycle was performed every 1 min, 200 μL of sample was taken at each time point in a 96-well plate, three replicate wells were set, pure water containing SOSG was used as a blank control, and the fluorescence intensity (λE X :504nm,λE M :525nm).

[0062] S3. Evaluation of NO release performance.

[0063] Draw NO standard curve: Take out Griess Reagent I and II and return them to room temperature. Dilute the standard with deionized water to the concentrations of 0μM, 2μM, 4μM, 6μM, 8μM, 10μM, 20μM, 40μM, 60μM, and 80μM. Add the standard to a 96-well plate at 50μL / well. Then add Griess Reagent I and Griess Reagent II at room temperature to each well at 50μL / well. Set up 3 replicate wells. Incubate at 37°C for 10 minutes and measure the absorbance at 540nm. Draw a standard curve based on the test results.

[0064] Evaluation of the NO continuous release performance of CAPI NPs: 0.2 mg / mL CAPI NPs solution was taken and the irradiation was performed at 808 nm and 1 W / cm 2 Laser irradiation. 100 μL of the sample was taken out at 0, 10, 15, 20, 25, and 30 minutes and placed in a 96-well plate. Three replicate wells were set for each sample, and then 50 μL of Griess Reagent I and Griess Reagent II were added to each well in turn. After incubation at 37°C for 10 minutes, the absorbance was measured at a wavelength of 540 nm, and the release of NO was calculated according to the NO standard curve. The CAPI NPs solution without NIR irradiation was used for comparison. The control group had the same conditions except that there was no NIR irradiation.

[0065] Evaluation of the NO responsive release performance of CAPI NPs: 0.2 mg / mL CAPI NPs solution was taken and the irradiation was performed at 808 nm and 1.0 W / cm 2 Laser irradiation, the laser was switched on and off every 5 minutes, and the "On, Off" cycle irradiation was performed. At each time point, 100 μL of the sample was taken out in a 96-well plate, and 3 replicate wells were set for each sample. Then 50 μL of Griess Reagent I and Griess Reagent II were added to each well in turn. After incubation at 37°C for 10 minutes, the absorbance was measured at a wavelength of 540 nm, and the release of NO was calculated according to the NO standard curve. The CAPI NPs solution without NIR laser irradiation was used for comparison. The control group had the same conditions except that there was no NIR laser irradiation.

[0066] Figure 4 (a) shows that the heat generated by CAPI NPs solution under near-infrared light irradiation is closely related to its concentration, showing a certain dose dependence, and the photothermal conversion efficiency is 40-45%. Figure 4(b) shows that after four cycles of light exposure and dark avoidance, the images of the heating and cooling curves are basically symmetrical, and the temperature change is not obvious, indicating that CAPI NPs have excellent photothermal stability and photothermal regeneration. Figure 4 (c) It can be seen that the CAPI NPs in the group without near-infrared light irradiation did not produce 1 O2. At 808nm, 1W / cm 2 Under laser irradiation, CAPI NPs can be detected 1 O2 fluorescence indicates the generation of ROS. More importantly, with the increase of irradiation time, CAPI NPs can continuously generate 1 O2, indicating that CAPI NPs are suitable as photosensitizers for photodynamic therapy. Figure 4 (d) It can be seen that in the absence of near-infrared light irradiation, almost no ROS is generated; after laser irradiation, ROS is immediately generated, and after the laser is turned off, no ROS is generated. The above rules are shown in several cycles, so the singlet oxygen produced by CAPI NPs is responsive to near-infrared light. Figure 4 (e) shows that the production of NO is almost undetectable in CAPI NPs without near-infrared light irradiation. 2 Under laser irradiation, the generation of NO can be detected in CAPI NPs. More importantly, as the irradiation time increases, CAPI NPs can continue to produce NO, which is consistent with the trend of CAPI NPs producing ROS. Figure 4 As shown in (f), in the absence of near-infrared light irradiation, almost no NO is produced; after laser irradiation, NO is produced immediately, and after the laser is turned off, the generation of NO stops. The above pattern is present in several cycles. Therefore, the production of NO by CAPI NPs is responsive to near-infrared light, which provides a basis for the precise control of NO administration dosage.

[0067] Application Example 2

[0068] like Figure 5 As shown, the cellular uptake and in vitro antitumor activity of the nanomaterial described in Example 1 were investigated.

[0069] The present invention uses mouse breast cancer 4T1 cell line as a model, utilizes the fluorescence characteristics of IR780 itself, and adopts flow cytometry to measure the cell uptake.

[0070] First, cells in the logarithmic growth phase were taken, washed twice with PBS, digested with trypsin, and digested with complete medium. After centrifugation, they were inoculated in a 6-well plate and cultured in an incubator. After the cells were completely attached to the wall, the old culture medium was discarded, washed twice with PBS, and 2 mL of CAPI NPs and IR780 (IR780 equivalent) solution prepared with complete medium was added to the wells respectively, and incubated with the cells. After 4 hours, the culture medium in the wells was aspirated and discarded, and washed several times with PBS. After digestion, the cell suspension was collected by centrifugation and tested on the machine.

[0071] The toxicity of the preparation to cells was determined by the MTT method to evaluate the in vitro antitumor effect of CAPI NPs. Cells in the logarithmic growth phase were washed twice with PBS, digested with trypsin, and digested with complete medium. After centrifugation, the cells were diluted with complete medium to form a cell suspension. 4 The cells were seeded at a density of 100 μL / well in a 96-well plate and placed in an incubator for incubation. After the cells were fully attached, the original culture medium was discarded, and 100 μL of HMCuS NPs, CPI NPs, and CAPI NPs (concentrations of 2.5, 5, 10, and 20 μg / mL, CuS equivalent) diluted with complete culture medium were added to each well. The incubation continued for 24 hours, and 4 replicate wells were set up in parallel for each group. The control group was treated with blank culture medium. After the preparation and cells were co-incubated for 24 hours, the irradiation group was irradiated with laser for 5 minutes (808 nm, 1 W / cm 2 ), and then continue to co-culture in a constant temperature incubator for 4 hours. Remove the residual culture medium and add 100 μL of complete culture medium containing 10% MTT (5 mg / mL) to each well. After incubation in the incubator for 4 hours, discard the culture medium and add 100 μL of DMSO to each well. Shake for 10 minutes in the dark to fully dissolve Formazan. Then use an ELISA reader to detect the absorbance value (OD) at a wavelength of 490 nm, and calculate the cell viability according to the following formula:

[0072]

[0073] Among them, OD sample is the absorbance of the drug-treated group; OD control is the absorbance of the control group; OD blank is the absorbance of the blank group.

[0074] Live and dead cell staining: In addition to detecting cell viability by MTT, this experiment also further explored the PDT / PTT / NO multifunctional synergistic effect of CAPI NPs by co-staining with Calcein AM and propidium iodide (PI). The experimental steps are as follows: 5×10 44T1 cells in the logarithmic growth phase were inoculated at a density of 1 / well and cultured overnight until the cells adhered to the wall. Then, 1 mL of HMCuS, CPI, and CAPI solution (CuS equivalent, 20 μg / mL) was added to the cells. Fresh culture medium was added to the blank control group and incubated for 24 h. The experimental group was given 1.0 W / cm 2 , 808nm NIR irradiation for 5min, after incubation for 4h, the cells were washed twice with PBS to ensure the removal of active esterase in the culture medium, and then sufficient working solution was added to ensure that the monolayer of cells was covered, and incubated in an incubator for 20min. After washing out the working solution, the incubation was terminated, and the cells were washed twice with PBS and then observed using an inverted fluorescence microscope.

[0075] Cell apoptosis: Annexin V-FITC / PI double staining was used to detect cell apoptosis. Cells in the logarithmic growth phase were taken, washed twice with PBS, digested with trypsin, and digested with complete medium. After centrifugation, they were inoculated into 6-well plates and cultured in an incubator. When the cells grew to 80%, the old culture medium was discarded, washed twice with PBS, and 2 mL of CuS, CPI and CAPI solutions (CuS equivalent) were added respectively. The culture medium was used as a blank control and incubated in an incubator for 8 hours. The light group was given 808 nm (1 W / cm 2 ) laser irradiation for 5 minutes, and then continue to culture for 15 hours. Digest with EDTA-free trypsin, centrifuge after terminating digestion, wash twice with pre-cooled PBS, and then resuspend the cells in 100μL 1×Binding buffer. Add 5μL of two staining solutions to each group and incubate in the dark for 15 minutes, and finally add 400μL 1×Binding buffer and detect on the machine within 1 hour.

[0076] Three control groups were set up: (1) blank control: normal cells without dye and without any treatment; (2) dead cells and cells with only Annexin V-FITC dye; (3) dead cells and cells with only PI dye.

[0077] like Figure 5As shown in (a), after IR780 and CAPI NPs were co-incubated with cells for 4 hours, the fluorescence intensity was detected to be increased. However, compared with free IR780, the fluorescence intensity of the CAPI group was higher, indicating that the cells have a certain ability to take up free drugs, but have a stronger ability to take up CAPI NPs. The cellular uptake ability of nanoparticles is an important factor in determining their efficacy. Generally speaking, free IR780 enters cells by diffusion. Due to the polarity of the molecule, IR780 will be blocked by the lipid bilayer and the cellular uptake ability is low. However, after being loaded by a carrier to form nanoparticles, it can be internalized into the cells. Since the contact area between the nanoparticles and the cells is increased, the uptake efficiency will be higher. The toxicity of the preparation to cells was determined by the monothiazolyl tetrazolium (MTT) method to evaluate the in vitro anti-tumor effect of CAPI NPs. As shown in Figure 5 As shown in (b), when NIR irradiation was not applied, the anti-tumor ability of the HMCuS NPs group, the HMCuS@PCM-IR780 (CPI NPs) group, and the CAPI NPs group increased with the increase in concentration, among which the CAPI group had a better tumor inhibition effect. After NIR irradiation, the cell survival rate of the Control+NIR group remained almost unchanged, and the survival rates of the HMCuS+NIR group, the CPI+NIR group, and the CAPI+NIR group were all lower than those without irradiation, among which the cell survival rate of the CAPI+NIR group was the lowest. The above results show that NIR irradiation is an important factor in improving drug efficacy, and compared with single PTT and PTT / PDT synergy, the PTT / PDT / NO multiple combination of CAPI NPs has better anti-tumor efficacy. In addition to detecting cell survival rate by MTT, this experiment further explored the PDT / PTT / NO multifunctional synergistic effect of CAPI NPs by co-staining with calcein (Calcein AM)-propidium iodide (PI). As Figure 5 (c) shows that at 808nm, 1.0W / cm 2 After 5 minutes of near-infrared light irradiation, there were almost no dead cells in the Control+NIR group, a small amount of dead cells were observed in the HMCuS+NIR group and the CPI+NIR group, and most of the cells survived. However, in the CAPI+NIR group, most of the 4T1 cells could not survive, and almost no green living cells were observed in the entire field of view. All the results were consistent with the cytotoxicity test and apoptosis experiment, that is, the multiple synergistic effects of PTT / PDT / NO in the CAPI+NIR group induced the greatest degree of cell apoptosis and necrosis, achieving the best anti-tumor effect. The potential mechanism of cell death was studied using the Annexin V-FITC / PI apoptosis detection kit. Figure 5As shown in (d), the double-stained cell signals were tested by flow cytometry, and the treated cells were divided into four quadrants, namely Q1, Q2, Q3 and Q4, which corresponded to the mechanically damaged cells / debris group, late apoptosis / necrosis group, early apoptosis group and survival group. In the case of only near-infrared irradiation, there was almost no cell apoptosis, indicating that NIR was not a factor inducing cell apoptosis. After NIR irradiation, the apoptosis of the drug-treated group was aggravated. Compared with PBS+NIR and CPI+NIR, the CAPI+NIR group caused more cells to undergo late apoptosis or necrosis, and the proportion of induced apoptotic cells was nearly 50%, which was significantly higher than that of the PBS+NIR group and the CPI+NIR group, showing a strong apoptosis inducing ability. In summary, CAPI NPs can effectively kill tumor cells under near-infrared irradiation and achieve a good synergistic therapeutic effect. The experimental results are consistent with the results of the cytotoxicity experiment. Compared with the single PTT and PTT / PDT synergy, the multiple synergistic effects of PTT / PDT / NO produced by the CAPI+NIR group induced the greatest degree of cell apoptosis.

[0078] Application Example 3

[0079] The in vivo uptake, in vivo antitumor activity and in vivo safety of the nanomaterials described in Example 1 were investigated. Figure 6 , Figure 7 and Figure 8 shown.

[0080] In order to study the PTT / PDT / NO multi-synergistic anti-tumor effects of CAPI NPs on 4T1 tumor-bearing mouse models, the present invention established a breast cancer 4T1 tumor-bearing mouse model. When the tumor volume of the tumor-bearing mice reached 120 mm 3 Subsequent in vivo experiments were performed.

[0081] When the tumor volume of tumor-bearing mice reached 120 mm 3 At the same time, 6 tumor-bearing mice were selected, 3 mice in each group, and 200 μL IR780 and CAPI NPs (IR780 equivalent, dose of 10 mg / kg) were injected into the tail vein. At the set time points (3, 6, 12 and 24 h after injection), the mice were anesthetized and the fluorescence distribution of the preparations in vivo was observed using an in vivo imaging device.

[0082] The two groups of mice were injected with 200 μL IR780 and CAPI NPs (IR780 equivalent, dose of 10 mg / kg) through the tail vein, and the mice were anesthetized at 3, 6, 12 and 24 hours after administration, and the fluorescence distribution images were obtained using an optical small animal imaging system and IR780 as a fluorescent probe. In order to test the organ distribution of the drug in vivo, the mice were killed after 24 hours and the main organs (heart, liver, spleen, lung, kidney and tumor) were removed.

[0083] To investigate the PTT effect of the preparation in vivo, the tumor volume reached 120 mm 3 Two mice were selected and divided into: (1) PBS group; (2) CAPI NPs group. 200 μL of PBS and CAPI NPs solution (dose of 10 mg / kg) were injected into the tail vein of each group of mice. After 24 h, the mice were anesthetized and stimulated with 808 nm, 1.0 W / cm 2 The laser was irradiated to the tumor site for 5 minutes. During the laser irradiation process, an infrared thermal imager was used to take images every 1 minute to record the temperature of the tumor surface.

[0084] An optical small animal imaging system was used to obtain fluorescence distribution images using IR780 as a fluorescent probe. Figure 6 (a) It can be seen that IR780 diffuses slowly in the body and is distributed throughout the body except for the tumor site. The fluorescence intensity at the tumor site reaches the highest after 12 hours, but the tail still retains a large amount of fluorescence. After 24 hours, the fluorescence gradually weakens. CAPI NPs diffuse faster in mice. Due to the EPR effect, the tumor site maintains strong fluorescence after 6 hours. At 12 hours, the fluorescence is almost only concentrated in the tumor area. After 24 hours, the fluorescence signal reaches the strongest. In order to test the distribution of drugs in organs in the body, mice were killed after 24 hours and the main organs (heart, liver, spleen, lungs, kidneys and tumors) were removed. Figure 6 As shown in (b), the fluorescence intensity of the free IR780 tumor site is weaker than that of the CAPI group. The above results show that CAPI NPs can effectively accumulate in the tumor site 24 hours after injection into the body. To explore the PTT effect of the preparation in vivo, the changes in the temperature of the tumor site after 5 minutes of NIR irradiation were compared after 24 hours of tail vein injection of PBS and CAPI NPs solution (dose of 10 mg / kg). Figure 6 (c) shows that the temperature at the tumor site in the PBS group only increased from 34.9°C to 37.2°C, an increase of 2.3°C. In contrast, within 5 minutes of NIR irradiation, the temperature at the tumor site of mice injected with CAPI NPs via the tail vein continued to rise, from 34.7°C to 45.9°C, a total of 11.2°C, showing a good photothermal warming effect. Therefore, its PTT effect can be used in anti-tumor treatment.

[0085] Figure 7 (a) is a schematic diagram of the animal experimental scheme of near-infrared light-responsive photothermal / photodynamic / nitric oxide therapeutic nanomaterials and other experimental groups. Specific scheme: When the tumor volume of tumor-bearing mice reaches ~120mm 3The mice were randomly divided into 4 groups, 5 in each group, including (1) PBS+NIR group; (2) HMCuS+NIR group; (3) CPI+NIR group; (4) CAPI+NIR group. After the mice were weighed, 200 μL of each solution was injected into the tail vein. The control group was injected with PBS intravenously. The remaining groups were treated with the same dose (10 mg / kg, CuS equivalent). 24 hours after the injection, the tumor site of each mouse was exposed to 808 nm, 1.0 W / cm 2 Laser irradiation for 5 min was performed, and drugs were administered on days 0, 3, 6, and 9, and NIR irradiation was performed on days 1, 4, 7, and 10. After that, the tumor volume and mouse body weight were measured, and all mice were killed on day 22. Figure 7 (b) It can be seen that at the end of the experiment, compared with the PBS+NIR, HMCuS+NIR, and CPI+NIR groups, the tumor volume of the CAPI+NIR group was the smallest, and the average tumor volume of the PBS+NIR group increased the most and fastest, about 8 times the original volume. The other groups all had a certain inhibitory effect, among which the tumor volume of the CAPI+NIR group was nearly 4.6 times and 3.3 times smaller than that of the HMCuS+NIR and CPI+NIR groups, respectively. This shows that the CAPI+NIR group has the best inhibitory effect on tumor growth. At the same time, the tumor peeled off after the end of treatment ( Figure 7 (c)), consistent with the above results, the tumor size in the CAPI+NIR group was the smallest and the treatment effect was the best, followed by the CPI+NIR group and the HMCuS+NIR group, and the largest was the tumor in the PBS+NIR group. This further proves the anti-tumor efficacy of CAPINPs in vivo.

[0086] After the treatment, the mice were killed, and the tumors in each group were collected and rinsed with PBS. The separated tumors were immersed in 4% paraformaldehyde fixative and sent to Wuhan Sewell Biotechnology Co., Ltd. for Ki67 immunohistochemistry and H&E staining to observe the proliferation and apoptosis of tumor cells.

[0087] Through frozen section experiments of tumor tissues, Ki67 immunohistochemical staining was used to examine the proliferation of tumor cells. Figure 7 (d) It can be seen that the proliferation level of tumor cells in mice treated with PBS+NIR group was significantly higher than that in other groups, which is why the tumors in this group of mice have been growing rapidly throughout the treatment cycle. The rate of tumor cell proliferation in mice in HMCuS+NIR and CPI+NIR groups has decreased. The proportion of Ki67-positive tumors in mice in CAPI+NIR group was significantly reduced, and the tumor proliferation ability of mice was the weakest, indicating that it effectively inhibited the proliferation of tumor cells. Hematoxylin-eosin reagent method, referred to as H&E staining, can be used to observe the apoptosis of tumor cells. From Figure 7(e) It can be seen that the above phenomenon can be observed in the HMCuS+NIR group, CPI+NIR group and CAPI+NIR group. The degree of cell apoptosis in each group increases successively. The CAPI+NIR group has the most serious light staining and the greatest degree of apoptosis.

[0088] like Figure 8 As shown in (a), during the treatment period, the body weight of mice in each group did not change significantly. Compared with before administration, the body weight generally increased and remained at a stable level. These results confirmed that the toxicity of the preparation was low and there was no obvious side effect on the growth of mice.

[0089] In order to investigate the biocompatibility of CAPI NPs, the hemolysis test was used as an indicator to evaluate the toxicity of CAPI NPs to blood. 1 mL of fresh blood from mice was taken into an anticoagulant tube, 3 mL of PBS was added and mixed, centrifuged at 3000 rpm for 15 min, the supernatant was aspirated and the supernatant was repeatedly centrifuged and washed until the supernatant was clear and transparent without blood color, and then it was prepared into a 2% red blood cell suspension with PBS for use. The negative control group took an equal volume of 2% red blood cell suspension and mixed it with PBS, the positive control group took an equal volume of 2% red blood cell suspension and mixed it with deionized water, and the experimental group used sterile PBS to prepare the preparation suspension with concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, and added an equal volume of 2% red blood cell suspension. After incubating the prepared mixed solution at 37 ° C for 2 h, centrifuged at 10000 rpm for 10 min, the supernatant was taken and the sample was measured at 541 nm using an enzyme marker. Each group was tested three times in parallel. The absorbance of the positive control tube was regarded as 100% hemolysis, and the hemolysis rate was calculated by substituting the following formula:

[0090]

[0091] Among them, OD sample is the OD value of the preparation group, OD control is the OD value of the negative control group, OD positive is the OD value of the positive control group.

[0092] Pictures of different concentrations of CAPI NPs incubated with fresh blood and the hemolysis rate after centrifugation Figure 8 As shown in (b), CAPINPs did not show obvious hemolysis in the concentration range of 12.5-200μg / mL, and the hemolysis rate of each group was less than 4%, indicating that the preparation has good blood compatibility. This provides safety guarantee for the application of CAPI NPs in intravenous administration and further clinical application. After the treatment, the main organs (heart, liver, spleen, lung, kidney) of mice in each group were removed and H&E staining was performed, as shown in Figure 2. Figure 8As shown in (c), no obvious damage was observed in the main organs of the two groups of mice. The experimental results show that CAPI NPs is an effective PTT / PDT / NO synergistic therapeutic agent with good biocompatibility.

[0093] In summary, the novel composite nanoparticles (CAPI) with a "linkage mechanism" provided by the present invention are used for combined tumor treatment with near-infrared light (NIR)-responsive photothermal therapy (PTT), photodynamic therapy (PDT) and gas therapy (GT). Compared with traditional radiotherapy and chemotherapy, photothermal therapy has the advantages of high tumor ablation efficiency, less trauma, high specificity, and fewer side effects, and has broad application prospects in medicine and clinics. Hollow mesoporous copper sulfide nanoparticles (HMCuS NPs) have strong light absorption in the near-infrared band and have become an ideal medium for photothermal therapy. Utilizing the high absorbance of HMCuS NPs in the near-infrared band, the high temperature generated by the photothermal effect induced by HMCuS NPs will melt the phase change material (PCM), thereby accurately releasing L-arginine (L-Arg) and iodinated IR780 (IR780) at the tumor site. At the same time, near-infrared light can excite IR780 to produce a large amount of singlet oxygen ( 1 O2), which can not only play a photodynamic therapy role to kill cancer cells, but also oxidize L-Arg to generate nitric oxide (NO) for gas therapy. On the other hand, NO can prevent the cell's autophagy protection, downregulate the expression of heat shock proteins, make cells more sensitive to temperature, and increase the sensitivity of tumor cells to reactive oxygen species (ROS), destroying the antioxidant defense of intracellular glutathione, and ultimately achieving mild tumor photothermal therapy and efficient photodynamic anti-tumor therapy.

Claims

1. A nanomaterial for combined tumor treatment based on near-infrared light response, characterized in that: The material is a spherical structure, which uses hollow mesoporous copper sulfide with photothermal properties as a carrier, loaded with nitric oxide donors, and encapsulated with a phase change material containing a photosensitizer.

2. The nanomaterial for combined tumor treatment based on near-infrared light response according to claim 1, characterized in that: The material has a diameter of 100-140 nm and a potential of -20.2 to -23.0 mV.

3. The nanomaterial for combined tumor treatment based on near-infrared light response according to claim 1, characterized in that: The nitric oxide donor is a water-soluble nitric oxide donor, which is L-arginine (L-Arg) and a metal-nitrosyl compound (M-NO); wherein the nitric oxide donor accounts for 18-24% of the material mass.

4. The nanomaterial for combined tumor treatment based on near-infrared light response according to claim 1, characterized in that: In the phase change material containing photosensitizer, the photosensitizer accounts for 12-16% of the mass of the phase change material; and the phase change material accounts for 30-40% of the mass of the material.

5. The nanomaterial for combined tumor treatment based on near-infrared light response according to claim 4, characterized in that: The photosensitizers are IR780, indocyanine green (ICG), and chlorin e6 (Ce6).

6. The nanomaterial for combined tumor treatment based on near-infrared light response according to claim 4, characterized in that: The phase change material (PCM) is a mixture of 1-hexadecanol and oleic acid in a mass ratio of 4:

1.

7. A method for preparing a nanomaterial for combined tumor treatment based on near-infrared light response according to claim 1, characterized in that: Hollow mesoporous copper sulfide with photothermal properties is used as a carrier to load the nitric oxide donor, and after loading, a phase change material containing a photosensitizer is used for wax sealing and wrapping.

8. The method for preparing a nanomaterial for combined tumor treatment based on near-infrared light response according to claim 7, characterized in that: 1) Preparation method of hollow mesoporous copper sulfide nanoparticles (HMCuS NP): 200-260 mg of polyvinyl pyrrolidone (PVP-K30) was dispersed in 20-30 mL of deionized water, and 120-150 μL of 0.5 mol / L copper chloride (CuCl2) solution was added and mixed, and the system was adjusted to alkalinity with sodium hydroxide (NaOH) solution after mixing, and then 3.2-6.4 μL of hydrazine hydrate (N2H4·H2O) and 150-200 μL of sodium sulfide (Na2S) aqueous solution were added to the system, and after mixing, the mixture was heated in a water bath at 75-80°C and refluxed for 2-3 hours; 2) adding nitric oxide donor and amphiphilic phospholipid to the above solution, stirring at room temperature for 24-48 hours to obtain solution 1; 3) adding the photosensitizer to the phase change material and mixing them uniformly by ultrasonication to obtain solution 2; 4) Solution 2 is quickly added to solution 1 preheated to 50-55° C., and ultrasonicated for 10-15 min. After ultrasonication, it is placed in an ice bath, and the dispersion is dialyzed overnight to remove impurities, thereby obtaining the material.

9. The method for preparing a nanomaterial for combined tumor treatment based on near-infrared light response according to claim 8, characterized in that: In step 2), the mass ratio of the nitric oxide donor to the amphiphilic phospholipid is 4:1 to 5:1; in step 3), the concentration of the photosensitizer in the phase change material is 1.2-1.5 mg / mL; in step 4), solution 2 accounts for 4% of the volume of solution 1.

10. An application of the nanomaterial for combined tumor treatment based on near-infrared light response according to claim 1, characterized in that: The material is used for anti-tumor applications by combining photothermal therapy (PTT), photodynamic therapy (PDT) and gas therapy (GT) under near-infrared light (NIR) response.

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