Nano regulator as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, and discloses a nano regulator and a preparation method and application thereof.The preparation method of the nano regulator comprises the following steps that an organic ligand is added into a metal salt solution, a coordination reaction is conducted, a metal organic framework crystal is obtained, metal salt comprises copper acetylacetonate and zinc salt, and the metal organic framework crystal is obtained; or the metal salt comprises copper acetylacetonate and cobalt salt; freeze-drying the metal organic framework crystal to obtain nanoparticles; the preparation method comprises the following steps: dispersing nanoparticles in a solvent, then adding indocyanine green, mixing, and carrying out ultrafiltration on the obtained mixed solution to obtain the space-time activatable nano regulator. According to the invention, the light stability of indocyanine green and the enrichment capacity of the drug at a tumor can be effectively improved, copper ions in tumor cells can perform chemical kinetics through Fenton reaction to kill the tumor cells, copper overload in the cells is caused to induce copper death of the cells, and in cooperation with photothermal therapy guided by NIR two-region imaging, the drug has a good application prospect. The tumor treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a nanometer regulator, a preparation method and an application thereof. Background Art

[0002] Photothermal therapy (PTT) is an important noninvasive cancer treatment strategy that utilizes photoabsorbers to convert light energy into heat, raising the local temperature above 42°C and thereby killing cancer cells. Near-infrared (NIR) light has facilitated the development of PTT due to its low tissue adsorption, deep penetration, and low phototoxicity. Inorganic media that absorb near-infrared light, such as gold nanostructures, carbon nanotubes, and copper sulfide nanoparticles (NPs), have been widely used as PTT agents. However, these inorganic media are non-biodegradable and exhibit long-term toxicity, severely limiting their clinical application. While indocyanine green (ICG), the only NIR agent approved by the US FDA, offers higher photothermal conversion efficiency and improved biocompatibility, ICG is susceptible to photodegradation, has a rapid blood clearance rate (ultra-short half-life of 2-4 minutes), and exhibits a low tumor accumulation rate, making it ineffective for PTT.

[0003] In addition, the dense extracellular matrix, high interstitial fluid pressure, and inherent heterogeneity of tumor cells within the tumor microenvironment severely limit the diffusion and enrichment of photothermal agents such as ICG. These limitations often lead to the formation of therapeutic cold spots, providing opportunities for tumor stem cells to evade treatment, ultimately leading to tumor recurrence. In addition, the high temperature stimulation induced in the initial stage of photothermal therapy triggers the cell's self-protection mechanism. For example, the intracellular heat shock protein increases during heat stress, which in turn affects the therapeutic effect. As a commonly used treatment strategy, small molecule heat shock protein inhibitors often lead to unsatisfactory drug targeting and enrichment in tumor cells due to their poor water solubility, high cytotoxicity, and especially the asynchronous lag in therapeutic effect.

[0004] Therefore, how to improve the photostability and tumor accumulation of the photothermal agent ICG and enhance the targeting and enrichment effect of the drug in tumor cells is an urgent problem to be solved. Summary of the Invention

[0005] In view of this, the present invention provides a nano-modulator and its preparation method and application to solve the problems of poor photostability, low tumor accumulation rate, and poor targeting and enrichment effect of indocyanine green as a photothermal preparation in the existing tumor treatment process.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a method for preparing a nano-modifier, comprising the following steps:

[0008] (1) adding an organic ligand to a metal salt solution to carry out a coordination reaction to obtain a metal organic framework crystal, wherein the metal salt comprises copper acetylacetonate and a zinc salt, or the metal salt comprises copper acetylacetonate and a cobalt salt;

[0009] (2) freeze-drying the metal organic framework crystals to obtain nanoparticles;

[0010] (3) The nanoparticles are dispersed in a solvent, and then indocyanine green is added and mixed, and the resulting mixture is ultrafiltered to obtain a nano-regulator.

[0011] Preferably, the mass ratio of the organic ligand to the metal salt is 1:0.5-3.

[0012] Preferably, the mass ratio of the zinc salt or cobalt salt to the copper acetylacetonate is 1:0.5-2.

[0013] Preferably, the zinc salt includes one or more of zinc acetylacetonate, zinc nitrate, zinc sulfate and zinc perchlorate.

[0014] Preferably, the cobalt salt includes one or more of cobalt acetylacetonate, cobalt nitrate, cobalt sulfate and cobalt chloride.

[0015] Preferably, the organic ligand is imidazole or an imidazole derivative.

[0016] Preferably, the imidazole derivative includes one or more of 2-methylimidazole, 2-ethylimidazole and benzimidazole.

[0017] Preferably, the mass ratio of the metal organic framework crystal to indocyanine green is 1:8-10.

[0018] Preferably, the solvent for dispersing the nanoparticles includes one or more of water, anhydrous ethanol, methanol, and isopropanol.

[0019] Preferably, the metal salt solution is obtained by dissolving a metal salt in a solvent, and the solvent includes one or more of methanol, ethanol, isopropanol and water.

[0020] On the other hand, the present invention also provides a nano-regulator prepared by any of the methods described above.

[0021] In another aspect, the present invention further provides a use of a nano-modulator prepared by any of the methods described above in the preparation of a drug for treating tumors.

[0022] The present invention provides a nano-regulator and its preparation method and application. Compared with the prior art, its beneficial effects are:

[0023] The present invention loads indocyanine green onto a metal-organic framework, which can effectively improve the photostability and tumor accumulation ability of indocyanine green, reduce the clearance rate of the drug in the body, and prolong the retention time of the drug in the body, thereby achieving efficient photothermal therapy effects; at the same time, the metal-organic framework enables the nano-modulator to have better biocompatibility and tumor targeting effect, realizes the tissue penetration of the nano-modulator, improves the timeliness and efficacy of anti-tumor, and is applied to the field of photothermal therapy of solid tumors.

[0024] In addition, the present invention uses copper acetylacetonate as a metal salt, allowing the metal-organic framework to be loaded with a large amount of copper ions. The copper ions induce copper overload in tumor cells, thereby inducing copper cell death. Specifically, copper ion overload causes the aggregation of dihydroacyl S-acetyltransferase and the loss of iron-sulfur cluster proteins in tumor cells, thereby causing proteotoxic stress and ultimately leading to cell death. Furthermore, copper ions bind to glutathione to produce oxygen free radicals, which kill tumor cells using chemical kinetics and downregulate the expression of heat shock proteins, thereby weakening the cells' thermal repair ability. By inhibiting heat shock proteins, the therapeutic effect of low-temperature photothermal therapy is enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0026] Figure 1 Performance characterization diagrams of ZIF8, Pre-SANM and SANM;

[0027] Figure 2 This is a characterization diagram of the photothermal performance of SANM;

[0028] Figure 3 This is a diagram showing the anti-tumor effect of SANM combined with photothermal and copper death at the cellular level;

[0029] Figure 4 This is a diagram showing the effect of SANM anti-tumor therapy in activating tumor immunity;

[0030] Figure 5 This is a diagram showing the anti-tumor effect of SANM in a breast cancer model;

[0031] Figure 6 Schematic diagram of the in vivo biocompatibility of SANM in a breast cancer model;

[0032] Figure 7 This is the NIR-I fluorescence imaging effect of SANM on tissues and organs in tumor-bearing mice. DETAILED DESCRIPTION

[0033] The present invention will be described below by specific embodiment, and it will be appreciated by those skilled in the art that the specific embodiment below is only for illustrative purposes, and does not limit the scope of the present invention in any way. In addition, in the following examples, unless otherwise stated, the reagent and equipment used are all commercially available. If in the following examples, concrete treatment conditions and treatment process are not clearly described, then conditions and methods well known in the art can be adopted to process.

[0034] In one aspect of the present invention, the present invention provides a method for preparing a nano-regulator, comprising the following steps:

[0035] (1) adding an organic ligand to a metal salt solution to perform a coordination reaction to obtain a metal organic framework crystal, wherein the metal salt comprises copper acetylacetonate and a zinc salt, or the metal salt comprises copper acetylacetonate and a cobalt salt;

[0036] (2) freeze-drying the metal organic framework crystals to obtain nanoparticles;

[0037] (3) The nanoparticles are dispersed in a solvent, and then indocyanine green is added and mixed, and the resulting mixture is ultrafiltered to obtain a nano-regulator.

[0038] The present invention uses metal salts including copper acetylacetonate and organic ligands as raw materials, and obtains metal-organic framework powder, i.e., nanoparticles, through coordination reaction and freeze-drying. The divalent copper ions in the copper acetylacetonate give the material the ability to induce cell copper death. At the same time, the loaded indocyanine green can achieve the NIR-I fluorescence imaging effect of the nano-modulator at the tumor site in vivo, giving the nano-modulator photothermal properties, improving the timeliness and efficacy of anti-tumor treatment, so that the prepared nano-modulator can be used in the field of photothermal synergistic copper death treatment of solid tumors.

[0039] In the present invention, an organic ligand is first added to a metal salt solution to carry out a coordination reaction to obtain a metal organic framework crystal.

[0040] In some embodiments of the present invention, the mass ratio of the organic ligand to the metal salt is 1:0.5-3, specifically 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, etc. The organic ligand is imidazole or an imidazole derivative, and the imidazole derivative includes one or more of 2-methylimidazole, 2-ethylimidazole, and benzimidazole.

[0041] In some embodiments of the present invention, the metal salts are copper acetylacetonate and zinc salts, and the mass ratio of the zinc salt to copper acetylacetonate is 1:0.5-2, specifically 1:0.5, 1:1, 1:1.5, and 1:2, etc., wherein the zinc salt comprises one or more of zinc acetylacetonate, zinc nitrate, zinc sulfate, and zinc perchlorate. When the metal salts are copper acetylacetonate and zinc salts, what is actually obtained is a metal-organic framework crystal loaded with divalent copper ions, for example, ZIF-7, ZIF-8, ZIF-95, etc. loaded with divalent copper ions.

[0042] In some embodiments of the present invention, the metal salt includes copper acetylacetonate and a cobalt salt, and the mass ratio of the cobalt salt to the copper acetylacetonate is 1:0.5-2, specifically 1:0.5, 1:1, 1:1.5, and 1:2, etc., wherein the cobalt salt includes one or more of cobalt acetylacetonate, cobalt nitrate, cobalt sulfate, and cobalt chloride. When the metal salt is copper acetylacetonate and a cobalt salt, what is actually obtained is a metal-organic framework crystal loaded with divalent copper ions.

[0043] In some embodiments of the present invention, the metal salt solution is obtained by dissolving a metal salt in a solvent, and the solvent includes one or more of methanol, ethanol, isopropanol, and water. Specifically, there is no particular limitation on the preparation method of the metal salt and it can be adjusted according to actual conditions. For example, a copper salt and a zinc salt or a cobalt salt can be mixed and dissolved in a solvent to obtain a metal salt solution, or a copper salt and a zinc salt or a cobalt salt can be dissolved in a solvent to obtain a copper salt solution and a zinc salt or a cobalt salt solution, respectively, and then the copper salt solution and the zinc salt or the cobalt salt solution are mixed to obtain the metal salt solution.

[0044] In some embodiments of the present invention, the organic ligand is added directly to the metal salt solution in its original state, or is diluted or dissolved in a solvent and then added to the metal salt solution. The solvent includes one or more of methanol, ethanol, isopropanol, and water. This is not particularly limited and can be adjusted according to actual conditions.

[0045] It should be noted that experimental studies have found that only when organic ligands are added to the metal salt solution can copper ions be effectively loaded inside the metal organic ligand, thereby achieving the loading of a large amount of copper ions on the metal organic framework. The copper ions induce copper overload in tumor cells, thereby inducing copper death in tumor cells. When the metal organic framework is obtained by other means, such as adding metal salts or metal salt solutions to the organic ligand solution, adding metal salt solutions to the organic ligand, or mixing the organic ligand and the metal salt and then adding a solvent for dissolution, the copper ion loading amount will be greatly reduced, and the copper ion loading amount on the metal organic framework will be greatly reduced, and the effect of copper overload in tumor cells cannot be achieved, thereby reducing the tumor cell mortality rate.

[0046] In addition, the present invention does not impose any special restrictions on the amount of solvent added, as long as it can ensure that the solids therein can be completely dissolved, and can be adjusted according to actual conditions; and the conditions for the coordination reaction are also not particularly limited, and conventional coordination reaction conditions can be used, such as stirring at 1200 rpm for 5 minutes at room temperature, which can be adjusted according to actual conditions.

[0047] In some embodiments of the present invention, after the coordination reaction is completed, the reaction solution is ultrafiltered to obtain the metal-organic framework crystals, wherein the ultrafiltration is performed using an ultrafiltration tube or ultrafiltration membrane with a pore size of 3 kD.

[0048] In the present invention, after the metal organic framework crystals are obtained, the metal organic framework crystals are freeze-dried to obtain nanoparticles.

[0049] The present invention utilizes freeze-drying to treat metal-organic framework crystals. This method removes moisture from the material through sublimation in a low-temperature vacuum environment, maximizing the preservation of the material's active ingredients, physical structure, and chemical properties while extending its shelf life. Conventional freeze-drying methods are sufficient and are not particularly limited.

[0050] In the present invention, after obtaining the nanoparticles, the nanoparticles are dispersed in a solvent, and then indocyanine green is added and mixed. The resulting mixed solution is ultrafiltered to obtain the nano-regulator.

[0051] In some embodiments of the present invention, the mass ratio of the metal organic framework crystal to indocyanine green is 1:8-10, specifically 1:8, 1:9, 1:10, etc.

[0052] In the present invention, the metal-organic framework modified with indocyanine green has adjustable physical and chemical properties, is easily enriched at the tumor site, has good blood clearance ability, and combines the flexibility of the metal-organic framework itself with the photothermal advantages of the optoelectronic material, so that the prepared nano-regulator can effectively convert near-infrared light into thermal energy for killing cancer cells.

[0053] In some embodiments of the present invention, the solvent for dispersing the nanoparticles includes one or more of water, anhydrous ethanol, methanol, and isopropanol; the ratio of the nanoparticles to the solvent can be, for example, 1 mg / L, which is not particularly limited; the mixing can be stirring mixing and / or ultrasonic mixing at room temperature, and the mixing time can be, for example, 20 hours, which is not particularly limited; the ultrafiltration is carried out using ultrafiltration materials, for example, a 3kd ultrafiltration membrane or ultrafiltration tube can be used for treatment.

[0054] In another aspect of the present invention, the present invention also provides a nano-regulator prepared by any of the methods described above.

[0055] In another aspect of the present invention, the present invention also provides an application of a nano-modulator prepared by any of the methods described above in the preparation of drugs for treating tumors. Specifically, under the guidance of near-infrared fluorescence imaging, the nano-modulator is used to prepare drugs for treating tumors with photothermal synergistic copper death.

[0056] The present invention assembles a metal-organic framework modified with divalent copper ions and indocyanine green, which can effectively improve the photostability of indocyanine green and the drug enrichment ability in tumors. Copper ions can kill tumor cells through the Fenton reaction in tumor cells through chemical kinetics, while causing copper overload in the cells to induce copper cell death, and synergistically cooperate with photothermal therapy guided by NIR zone II imaging to improve the therapeutic effect of tumors.

[0057] The present invention prepares a spatiotemporally activatable tumor nano-modulator with tumor microenvironment stimulation response ability and glutathione consumption ability. Specifically, spatiotemporal activation means that the nano-modulator can be degraded in the specific acidic microenvironment of the tumor through light irradiation; in addition, the present invention utilizes the coordination ability of divalent copper ions to give the nano-modulator copper death induction ability, so that it can be used for photothermal synergistic copper death treatment of solid tumors.

[0058] The technical solutions of the present invention will be described clearly and completely below with reference to specific embodiments. The embodiments of the present invention are only examples, and all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0059] Example 1

[0060] This embodiment provides a method for preparing a nano-regulator, and the specific steps are as follows:

[0061] (1) copper acetylacetonate and zinc nitrate in a mass ratio of 0.5:1 are mixed and dissolved in ethanol to obtain a metal salt solution; benzimidazole is dissolved in ethanol to obtain an organic ligand solution, wherein the mass ratio of the total mass of copper acetylacetonate and zinc nitrate to benzimidazole is 1.5:1;

[0062] The organic ligand solution was added to the metal salt solution, stirred at 1200 rpm for 5 minutes at room temperature, and filtered using a 3 kd ultrafiltration tube to obtain a metal organic framework crystal loaded with copper ions.

[0063] (2) The metal organic framework crystals are freeze-dried to obtain nanoparticles (hereinafter referred to as Pre-SANM).

[0064] (3) The nanoparticles were dissolved in anhydrous ethanol and indocyanine green (ICG) was added. The mass ratio of nanoparticles to indocyanine green was 1:8. The mixture was stirred for 20 h. The resulting mixture was treated with a 3 kd ultrafiltration membrane to obtain a nano-modifier (hereinafter referred to as SANM).

[0065] Comparative Example 1

[0066] This comparative example provides a method for preparing a metal organic framework, and the specific steps are as follows:

[0067] Zinc nitrate was dissolved in ethanol to obtain a metal salt solution; benzimidazole was dissolved in ethanol to obtain an organic ligand solution, wherein the mass ratio of zinc nitrate to benzimidazole was 1:1; the organic ligand solution was added to the metal salt solution, stirred at 1200 rpm for 5 minutes at room temperature, and filtered through a 3kd ultrafiltration tube to obtain metal-organic framework crystals; the metal-organic framework crystals were freeze-dried to obtain metal-organic framework powder (hereinafter referred to as ZIF8).

[0068] Comparative Example 2

[0069] This comparative example provides a method for preparing a nanomaterial, and the specific steps are as follows:

[0070] (1) Dissolve zinc nitrate in ethanol to obtain a metal salt solution; dissolve benzimidazole in ethanol to obtain an organic ligand solution, wherein the mass ratio of zinc nitrate to benzimidazole is 1:1; add the organic ligand solution to the metal salt solution, stir at 1200 rpm for 5 minutes at room temperature, and filter through a 3 kd ultrafiltration tube to obtain a metal organic framework crystal.

[0071] (2) Freeze-drying the metal organic framework crystals to obtain nanoparticles.

[0072] (3) The nanoparticles were dissolved in anhydrous ethanol and indocyanine green (ICG) was added. The mass ratio of nanoparticles to indocyanine green was 1:8. The mixture was stirred for 20 h. The resulting mixture was treated with a 3 kd ultrafiltration membrane to obtain the nanomaterial (hereinafter referred to as ZIF8-ICG).

[0073] Performance Testing

[0074] 1. Characterization of Nano-modulators

[0075] (1) General performance characterization

[0076] like Figure 1 (a) shows the XRD spectrum of Pre-SANM, which reflects the polycrystalline diffraction pattern data of Pre-SANM. The XRD spectrum of the product obtained in the present invention is consistent with the standard card of the organic metal framework ZIF8-Cu, indicating that the present invention has obtained a metal organic framework loaded with copper ions. Figure 1(b) and Figure 1 (c) shows the XPS spectrum of Pre-SANM and the high-resolution XPS cu2p spectrum of Pre-SANM, respectively. It can be seen from the figure that a metal organic framework loaded with copper ions is obtained. Figure 1 (d) shows the UV / visible absorption spectra of ICG, ZlF8, Pre-SANM and SANM. It can be seen from the figure that ICG and SANM have corresponding characteristic peaks at 780nm, indicating that SANM has successfully loaded ICG. Figure 1 (a)-(d) illustrate the successful synthesis of metal-organic framework-loaded nano-modulators of copper ions and indocyanine green.

[0077] like Figure 1 (e) is a TEM image of SANM, from which it can be seen that the particle size of the SANM obtained by the present invention is uniform. Figure 1 (f) shows the particle size analysis of ZIF8, Pre-SANM and SANM. It can be seen from the figure that the particle size distribution of SANM is uniform, indicating that the prepared nano-regulator has good particle uniformity and stable structure.

[0078] like Figure 1 (g) shows the Zeta potential diagram of ZIF8, Pre-SANM and SANM. It can be seen from the figure that ZIF8, Pre-SANM and SANM are all positively charged and can be engulfed by tumor cells and enter the interior of tumor cells, achieving better therapeutic effects.

[0079] 3,3',5,5'-tetramethylbenzidine (TMB) was used to detect whether the drug generates hydroxyl radicals: SANM at different concentrations (10, 20, 40, 60, 80, 100 μg / mL) was dispersed in PBS (pH 5.6), and then TMB and 1 mM hydrogen peroxide were added to the solution in sequence with a final concentration of 0.5 mM. The sample was incubated at 37°C for 30 minutes, and the absorbance was recorded at 650 nm using a UV-visible spectrometer. Figure 1 (h) shows the TMB consumption graph of different concentrations of SANM. It can be seen from the figure that SANM can generate hydroxyl radicals and has chemokinetic therapeutic ability.

[0080] 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was used to detect the consumption of glutathione (GSH): SANM at different concentrations (0, 10, 20, 40, 80, 100 μg / mL) was dispersed in PBS (pH 5.6), and then glutathione with a final concentration of 1 mM and DTNB with a final concentration of 10 μM were added to the solution in sequence, and incubated at 37°C for 20 minutes and 5 minutes, respectively, and the absorbance was measured at 415 nm using a UV-visible spectrometer. Figure 1 (i) shows the glutathione consumption diagram of SANM at different concentrations. It can be seen from the figure that SANM has the ability to consume glutathione. SANM can achieve photothermal / photodynamic combined therapy by consuming glutathione, thereby improving the tumor treatment effect.

[0081] (2) Characterization of photothermal performance

[0082] SANM was taken and dispersed with water to obtain SANM solutions with concentrations of 0, 25, 50, 100, 200, and 400 μg / mL, respectively. The photothermal effects of SANM at different concentrations and different powers were evaluated using an infrared camera.

[0083] Specifically, 808 nm laser (1.0 W / cm 2 ) irradiated SANM with different concentrations for 10 minutes, and captured thermal images every 15 seconds using an infrared camera. The photothermal stability of SANM was studied by switching the laser on and off. After irradiating with 808 nm laser for 5 minutes, the laser was turned off to cool the sample for 5 minutes. This was considered a cycle. The same operation was repeated for 5 cycles, the temperature change was detected, and the photothermal conversion rate was calculated. In addition, the laser was switched on and off with 0.5, 1.0, 1.5, and 2.0 W / cm 2 The SANM solution with a concentration of 100 μg / mL was irradiated with an 808 nm laser for 10 min, and a thermal image was captured every 15 s using an infrared camera.

[0084] like Figure 2 (a) shows the different concentrations of SANM under 808 nm laser irradiation (1.0 W / cm 2 ) temperature rise curve. It can be seen from the figure that when the SANM concentration is 400 μg / mL, the 808 nm laser (1.0 W / cm 2 ) After 5 minutes of irradiation, the temperature increased from 26.4°C to 58.6°C, with a temperature change of 32.2°C. Generally speaking, when the temperature reaches 43°C, it is enough to destroy the cell structure of the tumor, and at the same time cause the denaturation of proteins and enzymes, leading to cell death. Therefore, SANM can effectively induce cell death. It can be seen from the figure that the photothermal performance of SANM is concentration-dependent, and the photothermal performance can be adjusted by adjusting its concentration.

[0085] like Figure 2 (b) shows the temperature rise curve of SANM (100 μg / mL) under 808 nm laser irradiation with different powers. It can be seen from the figure that when the irradiation power is 2.0 W / cm 2When irradiated with 808nm laser for 5 minutes, the temperature increased from 26.4℃ to 71.5℃, with a temperature change of 46.1℃, which is enough to destroy the cell structure of the tumor and cause the denaturation of proteins and enzymes, leading to cell death.

[0086] like Figure 2 (c) shows the SANM (100 μg / mL) under 808 nm laser (1.0 W / cm 2 ) after five photothermal cycles. It can be seen from the figure that after five photothermal cycles, the temperature can still reach above 50°C and basically remains unchanged, indicating that SANM has good photothermal stability. The superior photothermal performance makes SANM suitable for tumor photothermal therapy applications.

[0087] like Figure 2 (d) shows the photothermal conversion efficiency curve of SANM, which is based on Figure 2 (c) As can be seen from the figure, the photothermal conversion efficiency of SANM is 19%, that is, the SANM of the present invention can achieve a good tumor photothermal treatment effect when the photothermal conversion efficiency is 19%.

[0088] 2. Characterization of the Antitumor Properties of Nanomodulators

[0089] (1) CCK8 drug toxicity detection

[0090] Mouse breast cancer 4T1 cells and normal tissue L929 cells were divided into two groups, material group and photothermal group, and inoculated into 96-well culture plates. Then, they were co-incubated with different concentrations of SANM (0, 6.25, 12.5, 25, 50, 100, 200, 400 μg / mL) for 24 hours. The cells in the illumination group were irradiated with 808 nm laser for 10 minutes, and the cells in the material group were not treated with other methods. The cell viability was determined by CCK-8 method, and the relative survival rate of the cells was calculated. The calculation method was: the OD value of the suspension at 450 nm was measured using a microplate reader (BioTek Synergy H1). Cell survival rate (%) = (N0-N t ) / N0×100%, where the OD value of the blank group is recorded as N0, and the OD value of the experimental group is recorded as N t .

[0091] like Figure 3 (a) shows the concentration-dependent cytotoxicity of 4T1 cells to SANM (the left side is the material group, and the right side is the photothermal group). It can be seen from the figure that SANM under light has a better therapeutic effect at the cellular level through chemical / photothermal treatment and copper death induction.

[0092] like Figure 3(b) shows the cytotoxicity of L929 cells in dependence on SANM concentration (the left side is the material group, and the right side is the photothermal group). It can be seen from the figure that the toxicity of SANM to normal cells is within a controllable range. Specifically, the concentration of SANM can be adjusted according to actual conditions to control the toxicity of SANM to normal cells.

[0093] (2) Detection of SANM drug uptake by cells

[0094] 4T1 cells were seeded in 6-well plates and incubated with 100 μg / mL fluorescein isothiocyanate (FITC)-modified SANM materials for 0 h, 2 h, 4 h, and 8 h, respectively. The 4T1 cell nuclei were stained with DAPI, and the cellular uptake of the materials was observed under confocal microscopy and flow cytometry.

[0095] like Figure 3 (c) shows the confocal laser scanning microscopy (CLSM) images of 4T1 cells at the time points (2h, 4h, and 6h) of FITC-SANM uptake (scale bar: 30μm); DAPI staining of 4T1 cell nuclei appears blue, and green cells are 4T1 cells after uptake of SANM drugs, which can intuitively show the cell uptake of drugs as the incubation time increases. Figure 3 (d) shows the proportion of 4T1 cells that took up SANM at the same time point detected by flow cytometry, indicating that SANM drugs have good targeting and enrichment performance on 4T1 cells.

[0096] (4) Live / dead tumor staining

[0097] 4T1 cells were seeded in 6-well plates and divided into four different treatment groups: phosphate buffer saline group (PBS group), Pre-SANM group, ZIF8-ICG+NIR group and SANM+NIR group, and cell apoptosis staining and flow cytometry were performed.

[0098] like Figure 3 (e) shows the flow cytometry apoptosis detection diagram after different groups of drug treatment at the cellular level, indicating that SANM photothermal therapy combined with copper death induction can significantly inhibit and eliminate the activity of tumor cells.

[0099] 4T1 cells were treated with PBS, Pre-SANM, ZIF8-ICG+NIR, and SANM+NIR, respectively, to obtain cell suspensions, which were then stained using a live / dead staining kit. 1 mL of the cell suspension was stained with 1.5 μL of calcein AM and 1.5 μL of propidium iodide (PI) in the dark for 15 min, then washed twice with phosphate buffer, placed on a glass slide, and fluorescence images were captured using a positioning fluorescence microscope.

[0100] like Figure 3(f) shows a live / dead staining diagram of cells. It can be seen from the figure that the killing ability of tumor cells in the PBS group, Pre-SANM group, ZIF8-ICG+NIR group and SANM+NIR group increases in sequence. It can be seen that compared with the PBS group, the SANM+NIR group has the best killing ability for tumor cells, indicating that SANM photothermal therapy combined with copper death induction can significantly inhibit and eliminate the activity of tumor cells.

[0101] After breast cancer model mice were established, 200ul of different drugs were injected intravenously (PBS group, 100μg / mL Pre-SANM group, 100μg / mL ZIF8-ICG+NIR group, 100μg / mL SANM+NIR group). After 28 days, the spleen and lymph nodes were collected for flow cytometry detection of the proportion of infiltrating lymphocytes. At the same time, immunofluorescence staining was used to explore the number of infiltrating lymphocytes in the tumor site (CD4, CD8) and the immunogenic death of tumor cells after treatment (CRT, HMGB1).

[0102] like Figure 4 (ad) are schematic diagrams of the pathological analysis of tumor-infiltrating immune cells in breast cancer model mice. As can be seen from the figure, compared with the control group, the number of infiltrating lymphocytes in the tumor of the SANM group increased, and the tumor suffered immunogenic death after synergistic treatment, indicating that the synergistic treatment effect can activate the immunity in the tumor microenvironment to a certain extent, and SANM can effectively reduce the size of the tumor.

[0103] 3. In vivo research on nano-modulator photothermal therapy

[0104] The experiment used ICR mice (male, 16-20g) to establish a mouse breast cancer model, also known as a tumor-bearing mouse model. The mice were divided into four groups: a blank group, a Pre-SANM group, a ZIF8-ICG+NIR group, and a SANM+NIR group, with five mice in each group.

[0105] (1) Tumor size and volume detection: After the tumor-bearing mouse model was established, 200 μl of different drugs were injected intravenously (PBS group, 100 μg / mL Pre-SANM group, 100 μg / mL ZIF8-ICG+NIR group, 100 μg / mL SANM+NIR group). The tumor volume was measured and analyzed on days 0, 3, 6, 9, 12, 15, 18, 20, 22, 24, 26, and 28, and the tumor weight of the mice was measured after the treatment.

[0106] like Figure 5(a)-(c) show the tumor size change and tumor weight of breast cancer model mice after treatment with PBS, ZIF8-ICG+NIR group, Pre-SANM and SANM+NIR group, respectively. It can be seen from the figure that compared with the control group, the tumor volume and weight were significantly reduced after SANM+NIR treatment, indicating that SANM can effectively inhibit the development of solid tumors, promote the immunogenic death of tumor cells, and activate the immune response of the tumor microenvironment.

[0107] (2) Changes in mouse body weight: After the tumor-bearing mouse model was established, it was treated with different treatment groups (PBS group, 100 μg / mL Pre-SANM group, 100 μg / mL ZIF8-ICG+NIR group, and 100 μg / mL SANM+NIR group). The weight of the mice was measured and analyzed on days 0, 3, 6, 9, 12, 15, 18, 20, 22, 24, 26, and 28.

[0108] like Figure 5 (d) shows the weight changes of breast cancer model mice after treatment with PBS, ZIF8-ICG, Pre-SANM and SANM, respectively. It can be seen from the figure that after treatment with SANM+NIR, the weight of the mice does not decrease, but gradually increases, indicating that SANM has low toxicity and side effects and good biocompatibility.

[0109] (3) Histological analysis: After the tumor-bearing mouse model was established, it was treated with different treatment groups (PBS group, 100 μg / mL Pre-SANM group, 100 μg / mL ZIF8-ICG+NIR group, and 100 μg / mL SANM+NIR group). Then, the tumor tissues and organs of different groups were collected and fixed with 4% paraformaldehyde solution. Histological evaluation was performed by H&E and TUNEL staining, and the proportion of lymphocyte infiltration in the tumor was measured by immunofluorescence. Histological images were taken using a localized fluorescence microscope.

[0110] like Figure 5 (e) shows the HE and Tunel pathological analysis images of the heart, liver, spleen, lung, kidney, and tumor of breast cancer model mice after treatment with PBS, ZIF8-ICG+NIR, Pre-SANM, and SANM+NIR, respectively. It can be seen from the figure that there is no obvious abnormality in the organ morphology and cell structure of mice in different treatment groups. The tissue structure of the heart, liver, spleen, lung, and kidney of mice in each treatment group is normal, and no obvious inflammatory reaction occurs, indicating that the drug has good biocompatibility and no obvious toxicity. It can be seen from the HE and Tunel pathological analysis images that SANM has a good therapeutic effect on tumors in vivo under NIR light.

[0111] 4. Biocompatibility testing of nano-modulators.

[0112] (1) Hemolytic test: Mouse blood was centrifuged (1500 rpm) for 10 minutes to obtain red blood cells. After washing twice with PBS, the red blood cells were suspended in 5% (v / v) PBS to obtain a PBS suspension; SANM was dispersed in 1 mL PBS (pH 7.4) to obtain a SANM dispersion; SANM dispersion (500 μL) and PBC suspension (500 μL) were added to a 2 mL test tube. The positive control was polyethylene glycol octylphenyl ether (Triton X-100, 0.1%), and the negative control was PBS. After incubation at 37°C for 2 hours, the solution was centrifuged at 1500 rpm for 10 minutes, and then each solution (100 μL) was transferred to a 96-well culture plate, and the absorbance of the solution was recorded at a wavelength of 540 nm using an enzyme reader. Hemolysis rate (%) = (A s -A p ) / (A t -A p )×100%, where A s is the absorbance value of SANM dispersion, A t is the absorbance value of Triton, A p is the absorbance value of PBS.

[0113] like Figure 6 (a) and Figure 6 (b) is a schematic diagram of the hemolysis rate of SANM at different concentrations. It can be seen from the figure that the hemolysis rate of SANM at a concentration of 400 μg / mL is less than 5%, which is within the allowable range of the ISO 10993-4 standard. SANM has no obvious toxic effects on the mouse blood system, indicating the safety and availability of SANM for intravenous injection.

[0114] 5. Detection of NIR-I Fluorescence Imaging Performance of Nano-modulators

[0115] The experiment used ICR mice (male, 16-20g) to establish a mouse breast cancer model. The mice were divided into two groups: an ICG group and a SANM group, with 5 mice in each group. After the tumors reached a certain volume, 100uL of 1mg / mL ICG and 1mg / mL SANM were injected into the tail vein of the mice, respectively. Drug metabolism in the mice was monitored in real time using near-infrared imaging.

[0116] like Figure 7 (a) and Figure 7 (b) shows the NIR-I fluorescence imaging effect of SANM on tissues and organs in tumor-bearing mice. It can be seen from the figure that SANM is enriched in the tumor site 8 hours after injection of SANM drug, indicating that SANM can accurately target solid tumor sites.

[0117] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a nano-regulator, characterized in that: The following steps are involved: (1) adding an organic ligand to a metal salt solution to carry out a coordination reaction to obtain a metal organic framework crystal, wherein the metal salt comprises copper acetylacetonate and a zinc salt, or the metal salt comprises copper acetylacetonate and a cobalt salt; (2) freeze-drying the metal organic framework crystals to obtain nanoparticles; (3) The nanoparticles are dispersed in a solvent, and then indocyanine green is added and mixed, and the resulting mixture is ultrafiltered to obtain a nano-regulator.

2. The method for preparing the nano-regulator according to claim 1, wherein The mass ratio of the organic ligand to the metal salt is 1:0.5-3; The mass ratio of the zinc salt or cobalt salt to the copper acetylacetonate is 1:0.5-2.

3. The method for preparing the nano-regulator according to claim 1, wherein The zinc salt includes one or more of zinc acetylacetonate, zinc nitrate, zinc sulfate and zinc perchlorate.

4. The method for preparing the nano-regulator according to claim 1, wherein The cobalt salt includes one or more of cobalt acetylacetonate, cobalt nitrate, cobalt sulfate and cobalt chloride.

5. The method for preparing the nano-regulator according to claim 1, wherein The organic ligand is imidazole or an imidazole derivative; The imidazole derivatives include one or more of 2-methylimidazole, 2-ethylimidazole and benzimidazole.

6. The method for preparing the nano-regulator according to claim 1, wherein The mass ratio of the metal organic framework crystal to indocyanine green is 1:8-10.

7. The method for preparing the nano-regulator according to claim 1, wherein: The solvent for dispersing the nanoparticles includes one or more of water, anhydrous ethanol, methanol, and isopropanol.

8. The method for preparing the nano-conditioning agent according to any one of claims 1 to 7, characterized in that: The metal salt solution is obtained by dissolving a metal salt in a solvent, and the solvent includes one or more of methanol, ethanol, isopropanol and water.

9. A nano-regulator prepared by the method according to any one of claims 1 to 8.

10. Use of a nano-modulator prepared by the method according to any one of claims 1 to 8 in preparing a drug for treating tumors.

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