A nano material for combined treatment of tumors based on near-infrared light response and preparation and application thereof

Abstract

The application belongs to the technical field of tumor treatment, and relates to a kind of nano material for combined (photothermal, photodynamic, nitric oxide) treatment of tumor based on near-infrared light response and its preparation and application.A kind of nano material for combined treatment of tumor based on near-infrared light response, material is spherical structure, it is with the hollow mesoporous copper sulfide with photothermal performance as carrier, load nitric oxide donor, and wrap phase change material containing photosensitizer.The nano material of the application produces high temperature by the photothermal conversion of hollow mesoporous copper sulfide nanoparticles triggered by near-infrared light, melts phase change material, promotes the release of photosensitizer IR780 and L-Arg;Under the irradiation of near-infrared light, IR780 produces active oxygen, further triggers the release of L-Arg nitric oxide.This "linkage mechanism" of heat-controllable drug release closely combines photothermal, photodynamic and nitric oxide treatment, and the construction of the obtained nano material solves the problems that O2 and NO cannot be effectively supplied to tumor sites. 1 O2 and NO cannot be effectively supplied to tumor sites.

Description

Technical Field

[0001] This invention belongs to the field of tumor treatment technology, and relates to a nanomaterial for combined (photothermal, photodynamic, and nitric oxide) tumor treatment based on near-infrared light response, as well as its preparation and application. Background Technology

[0002] Cancer is one of the leading causes of death worldwide. According to the latest results released by the World Health Organization, approximately one-sixth of the global population dies from cancer each year, and the incidence rate is increasing annually, seriously threatening human health. To overcome the limitations of traditional treatments such as chemotherapy, photothermal therapy (PTT), photodynamic therapy (PDT), and gas therapy have been widely used in cancer treatment in recent years due to their advantages such as non-invasiveness, high accuracy, and safety. However, single treatment methods have many shortcomings, thus limiting their clinical application. For example, during PTT inducing tumor cell necrosis, the damage to cell membrane integrity leads to the release of intracellular components into the extracellular environment, triggering an inflammatory response and inducing side effects such as tissue damage, tumor regeneration, and secondary treatment resistance. Furthermore, the high temperatures generated during photothermal therapy induce the upregulation of heat shock proteins in cells, and a series of self-protective mechanisms induced by cells under stress can directly or indirectly weaken the therapeutic effect of PTT. For phototherapy-induced tumor irradiation (PDT), photosensitizers generate cytotoxic reactive oxygen species (ROS) under laser irradiation. However, commonly used photosensitizers have low water solubility and poor tumor targeting, thus affecting the practical application of PDT in tumor treatment. More importantly, insufficient oxygen supply to the tumor area and high intracellular glutathione consumption of ROS severely hinder the clinical application of PDT. 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, and inhibits thrombus formation and platelet aggregation for cardiovascular diseases. Furthermore, NO has shown exciting effects in tumor treatment; locally high concentrations (>400 nM) of NO have a significant inhibitory effect on tumors. However, NO has a short half-life and is gaseous, so it cannot be directly used clinically. To facilitate the transport and release of NO to the lesion site, numerous responsive NO donors have been researched and developed. However, the problem of ineffective NO supply to the tumor site severely hinders the application of NO therapy.

[0003] Compared to monotherapy, combining multiple methods can achieve better toxicity reduction and efficacy enhancement. Currently, combining PDT and PTT simultaneously or sequentially can fully utilize the advantages of each modality and offset their limitations. On the one hand, the photothermal effect increases the temperature of tumor tissue, making it more sensitive to reactive oxygen species, thereby enhancing the effect of PDT. On the other hand, the reactive oxygen free radicals generated by PDT damage the membrane structure of tumor cells, increasing the heat's killing effect on tumors, achieving a "1+1>2" therapeutic effect. However, the synergistic therapeutic effect of PTT and PDT is still challenged by the limitations of PDT and PDT. Typically, O2 concentration often limits the therapeutic effect of PDT due to the severe hypoxic environment of the tumor, thus necessitating the alleviation of tumor hypoxia. The high temperature (T>50℃) generated during PTT often inevitably causes unnecessary damage to nearby tissues, making the realization of mild PTT (mPTT, T<45℃) crucial. However, the lower temperature in mPTT also means a reduction in the cytotoxicity of the treatment, therefore, sensitization of mPTT is essential.

[0004] Studies have shown that when NO concentrations are 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-associated hypoxia. Furthermore, NO can react with reactive oxygen species to produce more harmful reactive nitrogen oxides, such as nitrosamine peroxide (ONO). - These molecules can exacerbate DNA breaks and trigger apoptosis, ultimately enhancing the therapeutic effect of ROS on cancer cells. Combining NO with PTT (proton pump inhibitory therapy) leverages NO's ability to act on a wider range of tumor cells, reducing P-glycoprotein expression and reversing multidrug resistance in tumors. Furthermore, NO can inhibit cellular autophagy protection and downregulate the expression of heat shock proteins, making cells more sensitive to temperature, potentially achieving a milder form of PTT for tumor treatment, thus reducing toxicity and increasing efficacy in anti-tumor therapy. In conclusion, further combining NO with other methods could represent a new trend in cancer treatment. Summary of the Invention

[0005] The purpose of this invention is to address the problem of low therapeutic efficacy of existing therapies and to provide a nanomaterial for the combined (photothermal, photodynamic, and nitric oxide) therapy of tumors based on near-infrared light response, which reduces toxicity and enhances efficacy in clinical cancer treatment.

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

[0007] A nanomaterial for combined tumor therapy based on near-infrared light response is described. The material has a spherical structure and uses hollow mesoporous copper sulfide with photothermal properties as a carrier to load a nitric oxide donor and encapsulate 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; the phase change material accounts for 30-40% of the mass of the material.

[0011] The photosensitizers are IR780, indocyanine green (ICG), and dihydroporphyrin e6 (Chlorine6, Ce6).

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

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

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

[0015] Dissolve 200-260 mg of PVP-K30 in 20-30 mL of deionized water, then add it to 120-150 μL of 0.5 mol / L CuCl2 solution and stir at room temperature for 5-10 min. Add 20-30 mL of NaOH (pH = 9.0) solution and stir for 2-5 min, followed by 3.2-6.4 μL of N2H4·H2O. Stir for 5-10 min to form a yellow Cu2O suspension. Add 150-200 μL of 160 mg / mL Na2S aqueous solution to the suspension and stir for 5-10 min. Reflux at 75 °C for 2-4 h. After the reaction is complete, cool in an ice bath, centrifuge at 10,000 rpm for 15 min, wash twice with deionized water, and store at 4 °C.

[0016] A method for preparing nanomaterials for combined tumor therapy based on near-infrared light response involves using hollow mesoporous copper sulfide with photothermal properties as a carrier, loading a nitric oxide donor, and then encapsulating it with a phase change material containing a photosensitizer using wax.

[0017] To elaborate

[0018] 1) Preparation method of HMCuS NPs: Disperse 200-260mg PVP-K30 in 20-30mL of deionized water, add 120-150μL of 0.5mol / L CuCl2 solution and mix. After mixing, adjust the system to alkalinity with NaOH solution. Then add 3.2-6.4μL of N2H4·H2O and 150-200μL of Na2S aqueous solution to the system. After mixing, heat in a water bath at 75-80℃ and reflux for 2-3h.

[0019] 2) Add a nitric oxide donor and an amphiphilic phospholipid to the above solution and stir at room temperature for 24-48 hours to obtain solution 1;

[0020] 3) Add the photosensitizer to the phase change material and mix it evenly by ultrasonication to obtain solution 2;

[0021] 4) Quickly add solution 2 to solution 1 preheated to 50-55℃, sonicate for 10-15 minutes, and after sonication, place it in an ice bath and dialyze the dispersion overnight to remove impurities, thus obtaining the material (CuS-Arg@PCM-IR780 NPs, abbreviated as CAPINPs).

[0022] In step 2), the mass ratio of nitric oxide donor to amphiphilic phospholipid is 4:1 to 5:1; in step 3), the concentration of photosensitizer in 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 phospholipid may be one or more of L-α-lecithin, distearate phospholipid, and phosphatidylcholine.

[0024] An application of the aforementioned nanomaterial for combined tumor therapy based on near-infrared light response, wherein the material is used for anti-tumor application through a combination of photothermal therapy (PTT), photodynamic therapy (PDT), and gas therapy (GT) in response to near-infrared light (NIR).

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

[0026] This invention relates to a combined (photothermal, photodynamic, and nitric oxide) therapeutic nanomaterial based on near-infrared light response. It utilizes hollow, mesoporous copper sulfide nanoparticles as a carrier. Due to their excellent biocompatibility, superior photothermal conversion performance, and excellent drug loading capacity, they provide favorable conditions for the construction of a multifunctional therapeutic nanoplatform based on photothermal therapy. A wax-sealed phase change material (PCM) is used to seal the mesopores, addressing the issue of premature L-Arg release during circulation. Simultaneously, the drug-carrying capacity of the PCM is utilized to pre-disperse IR780 within it, achieving the loading of a lipid-soluble photosensitizer. Finally, leveraging the reactive oxygen species (ROS) response mechanism of water-soluble L-Arg to nitric oxide release, under near-infrared light irradiation, the ROS generated by the photodynamic properties of the photosensitizer IR780 trigger the release of nitric oxide through a cascade reaction.

[0027] This invention utilizes near-infrared light to induce photothermal conversion in hollow mesoporous copper sulfide nanoparticles, generating high temperatures that melt the phase change material and promote the release of photosensitizers IR780 and L-Arg. Under near-infrared light irradiation, IR780 generates reactive oxygen species, further triggering the release of nitric oxide from L-Arg. This thermally controlled drug release "linkage mechanism" tightly integrates photothermal, photodynamic, and nitric oxide therapy, and the construction of the resulting nanomaterial solves the problem of... 1 This addresses the problem of O2 and NO not being effectively supplied to the tumor site, and enables effective tumor treatment at a gentle temperature, thus overcoming the shortcomings of single or dual treatment methods for poor tumor treatment efficacy.

[0028] The preparation method in this invention is simple, requires little equipment, is low-cost, environmentally friendly, and yields a high product. Attached Figure Description

[0029] Figure 1 The flowcharts (a) and (b) of the preparation of the hollow mesoporous copper sulfide nanoparticles provided by this invention are as follows:

[0030] Figure 2 The images shown are: (a) Transmission electron micrograph of HMCuS NPs provided by this invention; (b) Transmission electron micrograph of CAPI NPs; (c) Zeta potential of HMCuS NPs, CA NPs, and CAPI NPs; (d) Ultraviolet-visible-near-infrared absorption spectra of HMCuS NPs, CA NPs, CAPI NPs, and IR780; and (e) Infrared absorption spectra of HMCuS NPs, CA NPs, CAPI NPs, and L-Arg.

[0031] Figure 3 A schematic diagram of the antitumor mechanism of near-infrared light-responsive photothermal / photodynamic / nitric oxide therapy nanomaterials.

[0032] Figure 4 The different concentrations of CAPI NPs provided by this invention are used in near-infrared laser (808nm, 1W / cm²) 2 Temperature change curve (a) and photothermal cycling curve (b) under near-infrared laser irradiation; CAPI NPs under near-infrared laser (808nm, 1W / cm²) irradiation. 2 ) under irradiation 1 The continuous release behavior curve (c) and near-infrared controlled release behavior curve (d) of O2; CAPI NPs in near-infrared laser (808nm, 1W / cm²) 2 The cumulative NO release curve (e) and the controlled release curve (f) under irradiation.

[0033] Figure 5 The following are examples of CAPI NPs cell uptake (a); antitumor activity of 4T1 cells in different treatment groups (b); Calcein AM / PI staining images of live (green) and dead (red) 4T1 cells in different treatment groups (c); Annexin V-FITC / PI double staining images of 4T1 cells in different treatment groups (d) (n=3, *p<0.05, **p<0.01, ***p<0.001).

[0034] Figure 6 The images show the distribution of IR780 and CAPI NPs in 4T1 tumor-bearing mice (a) and in ex vivo organs (b) as provided in this embodiment of the invention; and the in vivo thermal imaging of tumor-bearing mice under near-infrared light irradiation after intravenous injection of PBS and CAPI NPs (c).

[0035] Figure 7 The following are schematic diagrams of the animal experimental protocols for the nanomaterials provided in the embodiments of the present invention (a); tumor growth curves (b) and tumor photographs (c) of mice after different treatments (n=5, *p<0.05, **p<0.01, ***p<0.001); and representative immunofluorescence staining (d) and HE staining (e) images of 4T1 tumor proliferating cells (Ki-67) after treatment.

[0036] Figure 8 The figures provided in this embodiment of the invention are: (a) a graph showing the change in body weight of animals in different experimental groups; (b) a graph showing the hemolysis rate of different concentrations of CAPI NPs; and (c) H&E staining images of the major organs in different treatment groups. Detailed Implementation

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

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

[0039] Addressing the limitations of single or dual treatment methods in tumor therapy, this invention proposes a combined approach using photothermal, photodynamic, and nitric oxide therapies. A nanodelivery system is endowed with PTT, PDT, and NO functions. Utilizing the enhanced permeability and retention effect (EPReffect) of the nanodelivery system, it can cross the vascular barrier through the high permeability of tumor blood vessels, directly enter the tumor microenvironment, and accumulate within the tumor tissue. Subsequently, by precisely controlling drug release, damage to normal tissues is reduced while improving therapeutic efficacy, achieving the goal of reduced toxicity and enhanced efficacy in tumor treatment. Specifically, hollow mesoporous copper sulfide nanoparticles with photothermal properties are used as carriers, loaded with nitric oxide donors, and then encapsulated with a phase change material containing a photosensitizer, constructing a nanomaterial with photothermal, photodynamic, and nitric oxide therapeutic functions based on near-infrared light response.

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

[0041] Example 1

[0042] Preparation of a nanomaterial for combined tumor therapy 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: Dissolve 200-260 mg PVP-K30 in 20-30 mL of deionized water, then add 120-150 μL of 0.5 mol / L CuCl2 solution and stir at room temperature for 5-10 min. Add 20-30 mL of NaOH (pH=9.0) solution and stir for 2-5 min, followed by 6.4 μL of N2H4·H2O. Stir for 5-10 min to form a yellow Cu2O suspension. Add 150-200 μL of 160 mg / mL Na2S aqueous solution to the suspension and stir for 5-10 min. Reflux at 75 °C for 2-4 h. After the reaction is complete, cool in an ice bath, centrifuge at 10000 rpm for 15 min, wash twice with pure water, and store at 4 °C (see [reference]). Figure 1 a).

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

[0046] The specific preparation process of phase change material (PCM): 140-180 mg of 1-hexadecyl alcohol 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, firstly, 10 mL of the above HMCuS NPs was mixed with 100 mg L-Arg and stirred overnight at room temperature to obtain a CuS-Arg solution. Then, 400 μL of phase change material containing IR780 (1.5 mg / mL) was rapidly added to the CuS-Arg mixed solution preheated to 50-55 °C. After sonication for 10-15 min, the mixture was placed in an ice bath. Under rapid cooling, the PCM solidified instantaneously, encapsulating the drug. Finally, the prepared CuS-Arg@PCM-IR780 nanoparticles (CAPI NPs) were dialyzed overnight to remove impurities (see [link to relevant documentation]). Figure 1 b).

[0048] from Figure 2 As shown in (a), the prepared HMCuS NPs are spherical with a rough surface and a distinct hollow structure. The nanoparticles are relatively uniform in size, with an average particle size of 70-90 nm. CAPI NPs have a quasi-spherical structure with a diameter of 100-140 nm. Figure 2 (b)). From Figure 2(c) It can be seen that the potential of HMCuS NPs is -24.1±1.66mV. After 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 further coating with PCM-IR780, the CAPI potential is -21.6±1.38mV, which is due to the positive charge of IR780, proving that IR780 is successfully coated on the surface. By using UV-Vis-NIR spectroscopy, it was calculated that when the concentration ratio of CA NPs to PCM-IR780 is 1:0.5, it has a good drug loading of (5.33±0.32)% and an encapsulation efficiency of (42.3±0.48)%. Figure 2 As shown in (d), the characteristic absorption peak of IR780 appears in the CAPI NPs with a slight redshift, indicating successful loading of IR780. Figure 2 As shown in (e), at 1739cm -1 At the location, both CA NPs and CAPINPs showed characteristic peaks of C=N stretching vibration of guanidino nitrogen in L-Arg, as well as double peaks of primary amine, proving the successful loading of L-Arg. Thermogravimetric analysis showed that the drug loading of L-Arg was approximately 20.2%.

[0049] Furthermore, such as Figure 3 As shown, under near-infrared light, the high temperature generated by HMCuS NPs first melts the phase change material, promoting the release of photosensitizers IR780 and L-Arg. Subsequently, the reactive oxygen species generated by the photosensitizer IR780 are used for photothermal therapy (PDT) on one hand, and trigger the release of NO from L-Arg on the other. This thermally controlled drug release "linkage mechanism" tightly integrates photothermal / photodynamic / nitric oxide therapy, solving the problem of... 1 The inability of O2 and NO to effectively supply the tumor site can thus enable effective treatment with a mild PTT.

[0050] Example 2

[0051] NO is a ligand with strong coordination ability with metal ions (such as Fe, Mn, Ru), therefore metal nitrosyl compounds (M-NO) can serve as potential NO donors. Furthermore, most M-NOs are photosensitizing; under ultraviolet or near-infrared irradiation, photoelectrons jump from the π orbitals of the metal ion to the π antibonding orbitals of NO, leading to bond breaking in M-NO and rapid release of NO. This invention can also use ruthenium nitrosyl compounds (Ru-NO) as NO donors. First, 10 mL of the above-mentioned HMCuS NPs and 100-200 mg of Ru-NO are stirred overnight at room temperature to obtain a CuS-Ru-NO solution. Then, 400 μL of phase change material containing IR780 (1.5 mg / mL) is rapidly added to the CuS-Ru-NO mixed solution preheated to 50-55°C. After sonication for 10-15 min, the mixture is placed in an ice bath. Under rapid cooling conditions, the PCM solidifies instantaneously, 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, with each polycyclic ring linked to a hydrophilic sulfate group. Therefore, it is an amphiphilic tricarbon cyanine dye with relatively low toxicity to humans. Under near-infrared laser irradiation, ICG can be excited to generate ROS, achieving phototransformation through phototransformation (PDT). This invention can also use ICG as a photosensitizer. First, 10 mL of the above-mentioned HMCuS NPs and 100 mg of L-Arg were stirred overnight at room temperature to obtain a CuS-Arg solution. Then, 400 μL of phase change material containing ICG (1.5 mg / mL) was rapidly added to the CuS-Arg mixed solution preheated to 50-55°C. After sonication for 10-15 min, the mixture was placed in an ice bath. Under rapid cooling, the PCM solidified instantaneously, encapsulating the drug. Finally, the prepared CuS-Arg@PCM-ICG nanoparticles were dialyzed overnight to remove impurities.

[0054] Application Example 1

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

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

[0057] To investigate the effect of concentration on the photothermal effect of CAPI NPs, pure water was used as a control group. 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) was measured. One mL of each sample was irradiated with an 808 nm laser (1 W / cm²). 2 (10 min), record the temperature every 30 seconds and plot the temperature rise curve.

[0058] To investigate the photothermal stability of CAPI NPs, 1 mL of 0.2 mg / mL CAPI NPs solution was used, and the solution was heated to 808 nm at 1.0 W / cm². 2 Near-infrared laser irradiation was performed for 10 minutes each time, followed by 10 minutes of light avoidance, for a total of 4 cycles. The temperature was recorded every 30 seconds during the cycle to observe the temperature changes.

[0059] S2. Examination of photodynamic performance.

[0060] 1 Determination of continuous O2 release: A 5 mM stock solution of singlet oxygen green fluorescent probe (SOSG) was prepared using methanol solution, shaken well, and stored at -20°C protected from light. Before use, thaw at 4°C, and dilute an appropriate amount of the stock solution with deionized water to a 1 μM working solution. Mix 2 mL of SOSG with 2 mL of CAPI NPs and analyze using a near-infrared laser (808 nm, 1 W / cm²). 2 Irradiate for 10 min, then collect 200 μL of sample every 1 min into a 96-well plate, setting up three replicates. Use SOSG-containing pure water as a blank control. Measure the fluorescence intensity (λE) using a microplate reader. X 504nm,λE M (525nm).

[0061] 1 O2-responsive release determination: Prepare a 5 mM stock solution of SOSG with methanol, shake well, and store at -20°C protected from light. Before use, thaw at 4°C, and dilute an appropriate amount of the stock solution with deionized water to a 1 μM working solution. Mix 2 mL of SOSG with 2 mL of CAPI NPs and analyze using a near-infrared laser (808 nm, 1 W / cm²). 2 Irradiation was performed, with a switching cycle every 1 minute. At each time point, 200 μL of sample was collected into a 96-well plate, with three replicates. Pure water containing SOSG was used as a blank control. Fluorescence intensity (λE) was measured using a microplate reader. X 504nm,λE M (525nm).

[0062] Evaluation of S3 and NO release performance.

[0063] To construct the NO standard curve: Remove Griess Reagent I and II and allow them to return to room temperature. Dilute the standards with deionized water to concentrations of 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 20 μM, 40 μM, 60 μM, and 80 μM. Add 50 μL / well of the standard to each well of a 96-well plate. Then add 50 μL / well of room temperature Griess Reagent I and Griess Reagent II to each well. Set up three replicates. After incubating at 37°C for 10 min, measure the absorbance at 540 nm. Plot the standard curve based on the test results.

[0064] Evaluation of NO continuous release performance of CAPI NPs: Take 0.2 mg / mL CAPI NPs solution, and use 808 nm, 1 W / cm 2 Laser irradiation. At 0, 10, 15, 20, 25, and 30 minutes, 100 μL of sample was extracted and placed into 96-well plates, with three replicates for each sample. Then, 50 μL of Griess Reagent I and Griess Reagent II were added sequentially to each well. After incubation at 37°C for 10 minutes, the absorbance was measured at 540 nm. The NO release was calculated based on the NO standard curve. A CAPI NPs solution without NIR irradiation was used as a control group, which was identical to the control group except for the absence of NIR irradiation.

[0065] Evaluation of NO-responsive release performance of CAPI NPs: A 0.2 mg / mL CAPI NPs solution was used, and the solution was analyzed at 808 nm and 1.0 W / cm². 2 Laser irradiation was performed, with the laser switched on and off every 5 minutes in an "On, Off" cycle. At each time point, 100 μL of sample was extracted and placed into a 96-well plate, with each sample having three replicates. Then, 50 μL of Griess Reagent I and Griess Reagent II were added to each well sequentially. After incubation at 37°C for 10 minutes, the absorbance was measured at 540 nm, and the NO release was calculated based on the NO standard curve. A CAPI NPs solution without NIR laser irradiation was used as a control group, which was identical to the control group except for the absence of NIR laser irradiation.

[0066] Figure 4 (a) indicates 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 light-dark cycles, the heating and cooling curves are basically symmetrical, with no significant temperature change, indicating that CAPI NPs possess excellent photothermal stability and photothermal regeneration. Figure 4 (c) It can be seen that CAPI NPs were not generated in the group that was not exposed to near-infrared light. 1 O2. And at 808nm, 1W / cm 2 Under laser irradiation, CAPI NPs can be detected 1 The fluorescence of O2 indicates the formation of ROS. More importantly, CAPI NPs can be continuously generated with increasing irradiation time. 1 O2 indicates that CAPI NPs are suitable as photosensitizers for photodynamic therapy. Figure 4 (d) It can be seen that there is almost no ROS generation under the absence of near-infrared light irradiation; ROS is generated immediately after laser irradiation, and no ROS is generated after the laser is turned off. The above pattern is observed in several cycles. Therefore, CAPI NPs generate singlet oxygen with near-infrared light responsiveness. Figure 4 (e) shows that NO production is almost undetectable in CAPI NPs without near-infrared light irradiation. However, at 808 nm, 1 W / cm²... 2 Under laser irradiation, NO generation can be detected in CAPI NPs. More importantly, CAPI NPs can continuously generate NO with increasing irradiation time, which is consistent with the trend of ROS generation by CAPI NPs. Figure 4 As shown in (f), almost no NO is produced without near-infrared light irradiation; NO is produced immediately after laser irradiation is applied, and NO production stops after the laser is turned off. The above pattern is observed in several cycles. Therefore, CAPI NPs have near-infrared light responsiveness in NO production, which provides a basis for achieving precise control of NO dosage.

[0067] Application Example 2

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

[0069] This invention uses the mouse breast cancer 4T1 cell line as a model and utilizes the fluorescence properties of the IR780 to measure the cell uptake using flow cytometry.

[0070] First, cells in the logarithmic growth phase were collected, washed twice with PBS, digested with trypsin, and digested with complete culture medium to stop the digestion. After centrifugation and dispersion, the cells were seeded into 6-well plates and cultured in an incubator. After the cells had completely adhered to the plate, the old culture medium was discarded, and the cells were washed twice with PBS. 2 mL of CAPI NPs and IR780 (IR780 equivalent) solutions prepared with complete culture medium were added to each well, and the cells were co-incubated. After 4 hours, the culture medium in the wells was aspirated, and the cells were washed several times with PBS. After digestion, the cell suspension was collected by centrifugation and analyzed.

[0071] The cytotoxicity of the formulation was determined using the MTT assay to evaluate the in vitro antitumor effect of CAPI NPs. Logarithmic growth phase cells were collected, washed twice with PBS, digested with trypsin, and digestion was terminated with complete culture medium. After centrifugation, the cells were diluted with complete culture medium to form a cell suspension. Cells were then cultured at a concentration of 1×10⁻⁶ cells / mL. 4 Cells were seeded at the specified density in 96-well plates and incubated in an incubator. After the cells had fully adhered, the original culture medium was discarded, and 100 μL of different concentrations 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 was added to each well. Incubation continued for 24 h, with four replicates per group. The control group consisted of cells treated with blank culture medium. The light-treated group was incubated with the formulations for 24 h, followed by laser irradiation for 5 min (808 nm, 1 W / cm²). 2 Then, continue co-culturing in an incubator for 4 hours. Remove residual culture medium and add 100 μL of complete culture medium containing 10% MTT (5 mg / mL) to each well. After incubation for 4 hours, discard the culture medium and add 100 μL of DMSO to each well. Shake for 10 minutes in the dark to ensure complete dissolution of Formazan. Then, measure the absorbance (OD) at 490 nm using a microplate reader and calculate cell viability using the following formula:

[0072]

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

[0074] Live and dead cell staining: In addition to detecting cell viability using MTT assay, this experiment further explored the synergistic effect of PDT / PTT / NO multifunctionality of CAPI NPs using Calcein AM-propidium iodide (PI) co-staining. The experimental procedure is as follows: Cells were stained with 5 × 10⁻⁶ cells per well in a 24-well plate. 44T1 cells in logarithmic growth phase were seeded at a density of [number] wells and cultured overnight until cell attachment. Then, 1 mL of HMCuS, CPI, and CAPI solution (CuS equivalent, 20 μg / mL) was added to each well. The blank control group was added fresh culture medium. Incubation was performed for 24 h. The experimental groups were given 1.0 W / cm² of [treatment / conditioning]. 2 Irradiate the cells with 808nm NIR for 5 min, culture for 4 h, wash the cells twice with PBS to ensure the removal of active esterases in the culture medium, then add sufficient working solution to ensure that the monolayer of cells is submerged, incubate in an incubator for 20 min, wash out the working solution and stop the incubation, wash twice with PBS, and then observe using an inverted fluorescence microscope.

[0075] Apoptosis: Apoptosis was detected using Annexin V-FITC / PI double staining. Cells in logarithmic growth phase were washed twice with PBS, digested with trypsin, and digested with complete culture medium to terminate the digestion. After centrifugation and dispersion, cells were seeded into 6-well plates and cultured in an incubator. When the cells reached 80% confluence, the old culture medium was discarded, and the cells were washed twice with PBS. 2 mL of CuS, CPI, and CAPI solutions (CuS equivalent) were added, respectively. The culture medium served as a blank control. Cells were incubated for 8 hours. The light group received 808 nm (1 W / cm²) light. 2 Irradiate with laser for 5 min, then continue culturing for 15 h. Digest with trypsin without EDTA, centrifuge after terminating digestion with culture medium, wash twice with pre-cooled PBS, then resuspend cells in 100 μL of 1× Binding buffer. Add 5 μL of each of the two staining solutions to each group and incubate in the dark for 15 min. Finally, add 400 μL of 1× Binding buffer and analyze within 1 h.

[0076] Three control groups were set up: (1) blank control: normal cells without dye and no treatment; (2) dead cells with only Annexin V-FITC dye; (3) dead cells with only PI dye.

[0077] like Figure 5As shown in (a), after co-incubation with cells for 4 hours, both IR780 and CAPI NPs showed increased fluorescence intensity. However, compared to free IR780, the fluorescence intensity of the CAPI group was higher, indicating that cells have some uptake capacity for the free drug, but a stronger uptake capacity for CAPI NPs. The cellular uptake capacity of nanoparticles is a crucial factor determining their efficacy. Generally, free IR780 enters cells via diffusion. Due to molecular polarity, IR780 is blocked by the lipid bilayer, resulting in low cellular uptake capacity. However, after being loaded into nanoparticles by a carrier, they can be internalized into cells. Because the contact area between the nanoparticles and cells is increased, the uptake efficiency is higher. The cytotoxicity of the formulation was determined using the MTT assay to evaluate the in vitro antitumor effect of CAPI NPs. Figure 5 As shown in (b), without NIR irradiation, the antitumor activity of the HMCuS NPs group, the HMCuS@PCM-IR780 (CPI NPs) group, and the CAPI NPs group increased with increasing concentration, with the CAPI group showing better tumor inhibition. After NIR irradiation, the cell survival rate of the Control+NIR group remained almost unchanged, while the survival rates of the HMCuS+NIR group, the CPI+NIR group, and the CAPI+NIR group all decreased compared to the unirradiated state, with the CAPI+NIR group showing the lowest cell survival rate. These results indicate that NIR irradiation is an important factor in improving drug efficacy, and that the multiple combination of PTT / PDT / NO in CAPI NPs has better antitumor efficacy compared to single PTT and PTT / PDT synergy. In addition to detecting cell survival rate by MTT assay, this experiment further explored the multifunctional synergistic effect of PTT / PTT / NO in CAPI NPs by Calcein AM-propidium iodide (PI) co-staining. Figure 5 As shown in (c), at 808 nm, 1.0 W / cm 2 After 5 minutes of near-infrared light irradiation, the Control+NIR group showed almost no dead cells, while the HMCuS+NIR and CPI+NIR groups showed a small number of dead cells with most cells surviving. The CAPI+NIR group, however, showed that most 4T1 cells failed to survive, with almost no green live cells visible in the entire field of view. All results were consistent with cytotoxicity and apoptosis assays, indicating that the PTT / PDT / NO synergistic effect of the CAPI+NIR group induced maximal apoptosis and necrosis, achieving the best anti-tumor effect. The potential mechanisms of cell death were investigated using the Annexin V-FITC / PI apoptosis detection kit. Figure 5As shown in (d), the double-stained cell signals were measured by flow cytometry. The treated cells were divided into four quadrants: Q1, Q2, Q3, and Q4, corresponding to the mechanically damaged cell / fragmentation group, late apoptosis / necrosis group, early apoptosis group, and survival group, respectively. Under near-infrared irradiation alone, almost no apoptosis was observed, indicating that NIR was not a factor inducing apoptosis. In the drug-treated groups, apoptosis was exacerbated after NIR irradiation. Compared to PBS+NIR and CPI+NIR, the CAPI+NIR group induced more late apoptosis or necrosis, with a total induced apoptotic cell proportion of nearly 50%, significantly higher than the PBS+NIR and CPI+NIR groups, demonstrating a strong apoptosis-inducing ability. In summary, CAPI NPs can effectively kill tumor cells under near-infrared irradiation, achieving a good synergistic therapeutic effect. The experimental results are consistent with the cytotoxicity experiment results. Compared with single PTT and PTT / PDT synergy, the multiple synergistic effect of PTT / PDT / NO generated by the CAPI+NIR group induced the maximum degree of 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, as follows: Figure 6 , Figure 7 and Figure 8 As shown.

[0080] To investigate the synergistic antitumor effects of CAPI NPs on a 4T1 tumor-bearing mouse model using PTT / PDT / NO, this invention establishes a 4T1 breast cancer tumor-bearing mouse model. When the tumor volume of the tumor-bearing mice reaches ~120 mm... 3 Then, subsequent in vivo experiments will be conducted.

[0081] When the tumor volume in tumor-bearing mice reaches ~120 mm 3 Six tumor-bearing mice were selected, with three mice in each group. 200 μL of IR780 and CAPI NPs (IR780 equivalent, dose 10 mg / kg) were injected via the tail vein, respectively. Mice were anesthetized at predetermined time points (3, 6, 12, and 24 hours after injection), and the fluorescence distribution of the formulations in vivo was observed using an in vivo imaging system.

[0082] Two groups of mice were injected intravenously with 200 μL of IR780 and CAPI NPs (IR780 equivalent, dose 10 mg / kg), respectively. Mice were anesthetized at 3, 6, 12, and 24 hours post-administration. Fluorescence distribution images were acquired using an optical small animal imaging system with IR780 as a fluorescent probe. To test the in vivo organ distribution of the drugs, mice were sacrificed after 24 hours, and major organs (heart, liver, spleen, lungs, kidneys, and tumors) were removed.

[0083] To investigate the PTT effect of the formulation in vivo, tumors with a volume of ~120 mm were tested. 3 Two mice were selected and assigned to either the PBS group or the CAPI NPs group. 200 μL of PBS and CAPI NPs solution (10 mg / kg) were injected intravenously into each group of mice via the tail vein. After 24 hours, the mice were anesthetized and treated with 808 nm, 1.0 W / cm² solution. 2 The tumor site was irradiated with laser for 5 minutes. During the laser irradiation, images were taken every minute using an infrared thermal imager to record the temperature of the tumor surface.

[0084] An optical small animal imaging system was used, employing an IR780 as a fluorescent probe to acquire images of the animal's fluorescence distribution. Figure 6 (a) It can be seen that IR780 diffuses slowly in vivo, distributed throughout the body except for the tumor site. The fluorescence intensity reaches its peak at the tumor site after 12 hours, but the tail still retains a large amount of fluorescence. The fluorescence gradually weakens after 24 hours. CAPI NPs diffuse even faster in mice. Due to the EPR effect, the tumor site maintains strong fluorescence after 6 hours, and the fluorescence is almost exclusively concentrated in the tumor area after 12 hours. The fluorescence signal reaches its strongest after 24 hours. To test the organ distribution of the drug, mice were sacrificed after 24 hours, and major organs (heart, liver, spleen, lungs, kidneys, and tumors) were removed. Figure 6 As shown in (b), the fluorescence intensity of free IR780 at the tumor site was weaker than that of the CAPI group. These results indicate that CAPI NPs effectively accumulate at the tumor site 24 hours after injection. To investigate the PTT effect of the formulation in vivo, the temperature changes at the tumor site were compared 24 hours after tail vein injection of PBS and CAPI NPs solution (dose 10 mg / kg) followed by 5 minutes of NIR irradiation. Figure 6 (c) shows that the temperature at the tumor site in the PBS group only increased from 34.9℃ to 37.2℃, an increase of 2.3℃. In contrast, the temperature at the tumor site in mice injected via the tail vein of CAPI NPs continued to rise within 5 minutes of NIR irradiation, from 34.7℃ to 45.9℃, a total increase of 11.2℃, demonstrating a good photothermal warming effect. Therefore, its PTT effect can be utilized in anti-tumor therapy.

[0085] Figure 7 (a) is a schematic diagram of the animal experimental protocol for near-infrared photoresponsive photothermal / photodynamic / nitric oxide therapy nanomaterials and other experimental groups. Specific protocol: When the tumor volume in tumor-bearing mice reaches ~120 mm²... 3Mice were randomly divided into four groups of five each: (1) PBS+NIR group; (2) HMCuS+NIR group; (3) CPI+NIR group; and (4) CAPI+NIR group. After weighing, each mouse was injected with 200 μL of the solution via the tail vein. PBS was injected intravenously as a control group. The remaining groups were administered the same dosage (10 mg / kg, CuS equivalent). 24 hours after injection, each mouse's tumor site was treated with 808 nm, 1.0 W / cm² injection. 2 Mice were irradiated with laser for 5 minutes, and drugs were administered on days 0, 3, 6, and 9, respectively. NIR irradiation was performed on days 1, 4, 7, and 10. Tumor volume and mouse weight were then measured. All mice were sacrificed after 22 days. 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 CAPI+NIR group had the smallest tumor volume, while the PBS+NIR group had the largest and fastest average tumor volume growth, approximately 8 times the original volume. Other groups showed some inhibitory effect, with the CAPI+NIR group having a tumor volume nearly 4.6 times smaller than the HMCuS+NIR group and 3.3 times smaller than the CPI+NIR group. This indicates that the CAPI+NIR group had the best inhibitory effect on tumor growth. Simultaneously, the tumors removed after treatment were observed (…). Figure 7 (c) Consistent with the above results, the CAPI+NIR group had the smallest tumor volume and the best treatment effect, followed by the CPI+NIR group and the HMCuS+NIR group, while the PBS+NIR group had the largest tumor volume. This further demonstrates the anti-tumor efficacy of CAPINPs in vivo.

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

[0087] The proliferation of tumor cells was examined using Ki67 immunohistochemical staining in frozen sections of tumor tissue. Figure 7 (d) It can be seen that the tumor cell proliferation level in mice treated with PBS+NIR was significantly higher than that in other groups, which explains the rapid growth of tumors in this group throughout the treatment cycle. The tumor cell proliferation rate was reduced in the HMCuS+NIR and CPI+NIR groups. The proportion of Ki67-positive tumors was significantly reduced in the CAPI+NIR group, indicating the weakest tumor proliferation ability and effective inhibition of tumor cell proliferation. Hematoxylin-eosin (H&E) staining can be used to observe tumor cell apoptosis. 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 in turn. The CAPI+NIR group has the most severe 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 overall weight showed an upward trend and remained at a stable level. These results confirm that the preparation has low toxicity and no obvious side effects on the growth of mice.

[0089] To investigate the biocompatibility of CAPI NPs, a hemolysis test was used as an indicator of their hemotoxicity. 1 mL of fresh mouse blood was collected in an anticoagulant tube, 3 mL of PBS was added, and the mixture was centrifuged at 3000 rpm for 15 min. The supernatant was discarded, and the centrifugation and washing were repeated until the supernatant was clear and transparent with no further blood color. Then, a 2% erythrocyte suspension was prepared with PBS for later use. For the negative control group, an equal volume of 2% erythrocyte suspension was mixed with PBS. For the positive control group, an equal volume of 2% erythrocyte suspension was mixed with deionized water. For the experimental groups, suspensions with concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL were prepared using sterile PBS, and an equal volume of 2% erythrocyte suspension was added. The prepared solutions were incubated at 37°C for 2 h, centrifuged at 10000 rpm for 10 min, and the supernatant was analyzed at 541 nm using a microplate reader. Each group was tested in triplicate. The absorbance of the positive control tube was considered as 100% hemolysis, and the hemolysis rate was calculated using the following formula:

[0090]

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

[0092] Images and hemolysis rates of CAPI NPs incubated with fresh blood at different concentrations and then centrifuged are shown below. Figure 8 As shown in (b), CAPINPs did not exhibit significant hemolysis within the concentration range of 12.5-200 μg / mL, with hemolysis rates in all groups below 4%, indicating good blood compatibility. This provides a safety guarantee for the intravenous administration of CAPI NPs and its further clinical applications. After treatment, the major organs (heart, liver, spleen, lung, and kidney) of mice in each group were removed and subjected to H&E staining, as shown... Figure 8As shown in (c), no significant damage was observed in the major organs of either group of mice. The experimental results demonstrate that CAPI NPs are 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 this invention are used for combined tumor treatment with near-infrared (NIR) responsive photothermal therapy (PTT), photodynamic therapy (PDT), and gas therapy (GT). Compared with traditional radiotherapy and chemotherapy, photothermal therapy has advantages such as high tumor ablation efficiency, minimal trauma, high specificity, and fewer side effects, and has broad application prospects in medicine and clinical practice. Hollow mesoporous copper sulfide nanoparticles (HMCuS NPs) have strong light absorption in the near-infrared band, making them an ideal medium for photothermal therapy. Utilizing the high light absorption of HMCuS NPs in the near-infrared band, the high temperature generated by the photothermal effect induced by HMCuS NPs melts the phase change material (PCM), thereby precisely releasing L-arginine (L-Arg) and IR780 iodide (IR780) at the tumor site. Simultaneously, near-infrared light irradiation can excite IR780 to generate a large amount of singlet oxygen (…). 1 O2 can not only exert photodynamic therapy to kill cancer cells, but also oxidize L-Arg to generate nitric oxide (NO), which can be used for gas therapy. On the other hand, NO can inhibit cellular autophagy protection, downregulate the expression of heat shock proteins, make cells more sensitive to temperature, increase the sensitivity of tumor cells to reactive oxygen species (ROS), and destroy the antioxidant defense of intracellular glutathione. Ultimately, it is hoped that this will achieve both mild photothermal therapy for tumors and highly efficient photodynamic antitumor therapy.

Claims

1. A nanomaterial for combined tumor therapy based on near-infrared light response, characterized in that: The material has a spherical structure, which uses hollow mesoporous copper sulfide nanoparticles with photothermal properties as a carrier, loads nitric oxide donors, and encapsulates phase change materials containing photosensitizers. The nitric oxide donor is L-arginine; The photosensitizer is IR780; The phase change material is prepared by ultrasonically mixing 1-hexadecyl alcohol and oleic acid in anhydrous ethanol at a mass ratio of 140-180:30-40.

2. The nanomaterial for combined tumor therapy 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 therapy based on near-infrared light response according to claim 1, characterized in that: Nitric oxide donors account for 18-24% of the material mass.

4. The nanomaterial for combined tumor therapy 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; the phase change material accounts for 30-40% of the mass of the material.

5. A method for preparing nanomaterials for combined tumor therapy based on near-infrared light response as described in claim 1, characterized in that: Hollow mesoporous copper sulfide nanoparticles with photothermal properties were used as carriers to load nitric oxide donors, and then the loaded materials were wax-sealed with a phase change material containing photosensitizers. The specific preparation method is as follows: 1) Preparation method of hollow mesoporous copper sulfide nanoparticles: 200-260 mg of polyvinylpyrrolidone was dispersed in 20-30 mL of deionized water, and 120-150 μL of 0.5 mol / L copper chloride solution was added and mixed. After mixing, the system was adjusted to alkalinity with sodium hydroxide solution. Then, 3.2-6.4 μL of hydrazine hydrate and 150-200 μL of sodium sulfide aqueous solution were added to the system. After mixing, the system was heated in a water bath at 75-80 °C and refluxed for 2-3 h. 2) Add nitric oxide donor to the above solution and stir at room temperature for 24-48 h to obtain solution 1; 3) Add the photosensitizer to the phase change material and mix it evenly by ultrasonication to obtain solution 2; 4) Quickly add solution 2 to solution 1, which has been preheated to 50-55 ℃, and sonicate for 10-15 min. After sonication, place it in an ice bath and dialyze the dispersion overnight to remove impurities, thus obtaining the material.

6. The method for preparing nanomaterials for combined tumor therapy based on near-infrared light response according to claim 5, characterized in that: In step 3), the concentration of 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.

7. The application of the near-infrared light-responsive nanomaterial for combined tumor therapy as described in claim 1 in the preparation of antitumor drugs, characterized in that: The material can be used for anti-tumor therapy through a combination of photothermal therapy, photodynamic therapy, and gas therapy in response to near-infrared light.

Images