Application of metal organic framework nanomaterial as autophagy inhibitor in tumor immunotherapy

Abstract

The application discloses application of a metal organic framework nanomaterial as an autophagy inhibitor in tumor immunotherapy. 2+ and Cu 2+ The metal organic framework formed by the coordination self-assembly of the two metal elements through a disulfide bond has intramolecular pores, and forms a nanometer organic framework (FCMP) with good biocompatibility; the metal organic framework formed by the two metal elements of iron and copper has an effect of inhibiting autophagy of tumor cells, and can be used for tumor immunotherapy. The application further loads clinical drugs chloroquine (CQ) and an ultrasonic contrast agent perfluorohexane (PFH) by using the mesoporous advantage of the FCMP, and the prepared FCMP@CQ / PFH autophagy nanoinhibitor can release the drugs in a tumor microenvironment, and can achieve the effect of enhancing anti-tumor immunotherapy by synergistically inhibiting autophagy of tumor cells and jointly remodeling an immunosuppressive microenvironment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of a metal-organic framework nanomaterial as an autophagy inhibitor in tumor immunotherapy. Background Technology

[0002] Autophagy is an evolutionarily conserved process stimulated by cellular starvation and stress, degrading damaged or unwanted organelles and proteins in a lysosome-dependent manner. In mammalian cells, there are three types of autophagy: macroautophagy, microautophagy, and molecular chaperone-mediated autophagy. Macroautophagy (hereinafter referred to as autophagy) is a dynamic process involving multiple steps, including initiation, elongation, fusion, maturation, and degradation, precisely guided by numerous autophagy-associated group (ATG) proteins and multiple signaling pathways. Increasing evidence suggests that autophagy plays a crucial role in various diseases, including cancer, neurodegenerative diseases, and metabolic disorders. The role of autophagy in cancer is quite complex, and an important understanding is that it is a double-edged sword. In cancer cells, autophagy has a dual function: on the one hand, it supports tumor progression by providing energy under metabolic stress; on the other hand, inhibition of autophagy can impair cancer cell survival and promote cell death. Therefore, inhibiting autophagy has been used as a potential therapeutic strategy in various tumor models.

[0003] While studying the autophagy process in tumor cells, many researchers have also combined the study of nanomaterials with tumor cell autophagy. Metal-organic frameworks (MOFs), as a novel and excellent drug carrier composed of inorganic metal subunits and organic ligands, combine the advantages of traditional organic and inorganic carriers while overcoming their respective disadvantages. Previous studies have reported that many inorganic nanoparticles (NPs), such as iron oxide nanoparticles and Fe@Au nanoparticles, can disrupt autophagy flux, thereby inhibiting tumor growth. Among the many MOF nanomaterials, MOFs composed of single metal elements have been extensively studied in various biological fields due to their better flexibility and responsiveness to the tumor microenvironment compared to inorganic nanomaterials. For example, iron-based MOFs or copper-based MOFs are used for chemodynamic therapy or anti-tumor therapy through ferroptosis. However, no studies have yet found that organic frameworks composed of two metal elements can be used for the regulation of tumor cell autophagy.

[0004] Numerous studies have investigated the regulation of autophagy and immune control. Some evidence suggests that tumor cells express tumor-associated antigens on their surface via major histocompatibility complex class I (MHC-I), which can be effectively recognized by cytotoxic T lymphocytes (CTLs), leading to tumor regression. However, tumor cells often exhibit insufficient MHC-I expression due to excessive autophagy. Autophagy, a pervasive physiological process, plays a protective role by clearing misfolded proteins and damaged organelles. Therefore, tumor cells utilize autophagy to protect themselves from metabolic stress-induced apoptosis and necrosis. Recent research indicates that tumor cells engulf MHC-I in autophagosomes and then transport it to lysosomes for biodegradation. Thus, tumor cell autophagy is a key mechanism leading to the suppression of T-cell immune responses and intrinsic immune escape. This makes autophagy a promising target for cancer therapy: inhibiting cancer cell autophagy, enhancing surface MHC-I expression, and improving tumor cell immunotherapy. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a metal-organic framework nanomaterial.

[0006] Another object of the present invention is to provide the application of the metal-organic framework nanomaterials in the preparation of autophagy inhibitors.

[0007] Another object of the present invention is to provide the application of the metal-organic framework nanomaterials as autophagy inhibitors in the preparation of products for tumor immunotherapy.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A metal-organic framework nanomaterial is at least one of FCMP (iron-copper-based metal-organic framework nanoparticles), FCMP@CQ (iron-copper-based metal-organic framework nanoparticles loaded with chloroquine), FCMP@CQ / PFH (iron-copper-based metal-organic framework nanoparticles loaded with chloroquine and perfluorohexane), and fluorescent dye-labeled FCMP@CQ / PFH (iron-copper-based metal-organic framework nanoparticles loaded with dye, chloroquine, and perfluorohexane).

[0010] The FCMP nanoparticles were prepared by the following method:

[0011] (a) Add dopamine hydrochloride to a buffer solution, then add mesoporous metal-organic framework (FCM) aqueous solution, disperse evenly by ultrasonication, and then stir the reaction. After the reaction is completed, collect the product, centrifuge and wash to obtain the intermediate product.

[0012] (b) The intermediate product was resuspended in water and then added dropwise to an aqueous solution of mPEG-NH2 to continue the reaction (to modify FCM and PEG). After the reaction was completed, the product was collected, centrifuged and washed to obtain FCMP nanoparticles.

[0013] The mesoporous metal-organic framework (FCM) described herein can be prepared by referring to the method in Chinese patent application (application number 202010632300.7, entitled: A mesoporous metal-organic framework and its preparation method and application); it is formed by non-covalent bonding of disulfide monomers and metal ions, wherein the metal ion is Fe. 2+ and Cu 2+ The mesoporous metal-organic framework has a mesoporous structure, a particle size of 60–100 nm, and a specific surface area of ​​70–80 m². 2 / g, wherein the pore size of the mesopore is 7-10 nm; preferably, it is prepared by the following method:

[0014] (1) N,N-dimethylformamide (DMF) and anhydrous ethanol were mixed evenly to obtain a composite solution;

[0015] (2) Prepare Fe-containing solutions using composite solutions. 2+ Solution containing Cu 2+ Solution and disulfide bond monomer solution;

[0016] (3) Containing Fe 2+ Solution and Cu 2+ After mixing the solutions, disulfide monomer solution, polyvinylpyrrolidone and triethanolamine are added. After sonication to dissolve, the mixture is reacted at 135-155°C. After the reaction is complete, the mixture is cooled to room temperature, centrifuged and washed, and the precipitate is collected to obtain the mesoporous metal-organic framework (FCM).

[0017] The volume ratio of N,N-dimethylformamide to anhydrous ethanol in step (1) is (4-6):(2-4); preferably 5:3.

[0018] The Fe-containing element mentioned in step (2) 2+ The preferred solution is a ferrous chloride solution prepared using ferrous chloride tetrahydrate as a raw material.

[0019] The Fe-containing element mentioned in step (2) 2+ The preferred concentration of the solution is 50 mg / mL.

[0020] The Cu-containing element mentioned in step (2) 2+ The preferred solution is a copper chloride solution prepared using copper chloride dihydrate as a raw material.

[0021] The Cu-containing element mentioned in step (2) 2+ The preferred concentration of the solution is 50 mg / mL.

[0022] The disulfide monomer mentioned in step (2) is at least one of dithioglycolic acid and selenocysteine; preferably dithioglycolic acid.

[0023] The concentration of the disulfide monomer mentioned in step (2) is preferably 100 mg / mL.

[0024] In step (3), Fe 2+ and Cu 2+ The molar ratio is 1:(0.25~4); preferably 11:4.

[0025] The mass ratio of the disulfide monomer and polyvinylpyrrolidone mentioned in step (3) is 5.205:300.

[0026] The amount of triethanolamine used in step (3) is calculated as 38-40 μL of triethanolamine per milligram of disulfide monomer.

[0027] The polyvinylpyrrolidone mentioned in step (3) is polyvinylpyrrolidone K40.

[0028] The reaction time in step (3) is 12 to 24 hours; preferably 12 hours.

[0029] The preferred conditions for centrifugal washing in step (3) are: centrifugation at 14000 rpm for 15 min.

[0030] The mass ratio of the mesoporous metal-organic framework (FCM) and dopamine hydrochloride described in step (a) to the mPEG-NH2 described in step (b) is 1:1:2.

[0031] The conditions for ultrasonic dispersion described in step (a) are: ultrasonic power 600W, ultrasonic time 5-10min.

[0032] The buffer solution mentioned in step (a) is a Tris buffer solution; preferably a Tris buffer solution with pH 8.5 (concentration 10 × 10⁻⁶). -3 (mol / L).

[0033] The stirring reaction time in step (a) is 12 to 24 hours; preferably 12 hours.

[0034] The reaction time described in step (b) is 1 to 3 hours; preferably 2 hours.

[0035] The centrifugal washing conditions described in steps (a) and (b) are: centrifugation at 10,000 to 14,000 rpm for 10 to 15 minutes; preferably: centrifugation at 14,000 rpm for 10 minutes.

[0036] The centrifugal washing process described in steps (a) and (b) is performed at least three times.

[0037] The FCMP@CQ nanoparticles were prepared by the following method:

[0038] (I) Mesoporous metal-organic frameworks (FCM) were dispersed in water, then chloroquine (CQ) was added, the product was collected after thorough stirring, and centrifuged and washed to obtain FCM@CQ nanoparticles;

[0039] (II) Add dopamine hydrochloride to the buffer solution, then add FCM@CQ nanoparticle aqueous solution dropwise, disperse evenly by ultrasonication, and then stir the reaction. After the reaction is completed, centrifuge and wash the product, then add water to resuspend it, and add it dropwise to mPEG-NH2 aqueous solution to continue the reaction (to modify FCM and PEG). After the reaction is completed, collect the product, centrifuge and wash it to obtain FCMP@CQ nanoparticles.

[0040] The preferred mass ratio of mesoporous metal-organic framework (FCM) and chloroquine (CQ) in step (I) is 5:1.

[0041] The stirring time in step (I) is 10 to 14 hours; preferably 12 hours (stirring overnight).

[0042] The conditions for centrifugal washing in step (I) are: centrifugation at 10,000 to 14,000 rpm for 10 minutes; preferably: centrifugation at 14,000 rpm for 10 minutes.

[0043] The mass ratio of FCM@CQ nanoparticles, dopamine hydrochloride and mPEG-NH2 in step (II) is 1:1:2.

[0044] The conditions for ultrasonic dispersion described in step (II) are: ultrasonic power 600W, ultrasonic time 5-10min.

[0045] The buffer solution mentioned in step (II) is a Tris buffer solution; preferably a Tris buffer solution with pH 8.5 (concentration 10 × 10⁻⁶). -3 (mol / L).

[0046] The stirring reaction time in step (II) is 12 to 24 hours; preferably 12 hours.

[0047] The reaction time after adding the mPEG-NH2 aqueous solution in step (II) is 1 to 3 hours; preferably 2 hours.

[0048] The conditions for centrifugal washing described in step (II) are: centrifugation at 10,000 to 14,000 rpm for 10 to 15 minutes; preferably: centrifugation at 14,000 rpm for 10 minutes.

[0049] The centrifugal washing process described in step (II) is performed three or more times.

[0050] The FCMP@CQ / PFH nanoparticles were prepared by the following method:

[0051] (i) Mesoporous metal-organic frameworks (FCM) were dispersed in water, then chloroquine (CQ) was added, the product was collected after thorough stirring, and centrifuged and washed to obtain FCM@CQ nanoparticles;

[0052] (ii) FCM@CQ nanoparticles were freeze-dried, then placed in a sealed container, and vacuumed (air was removed from the container). Perfluorohexane (PFH) was then injected to obtain a mixture of FCM@CQ and PFH. The mixture of FCM@CQ and PFH was ultrasonically treated under vacuum conditions in ice water, and then a buffer solution was injected for further ultrasonic treatment to obtain FCM@CQ / PFH nanoparticles.

[0053] (iii) Add dopamine hydrochloride to a buffer solution, then add FCM@CQ / PFH nanoparticle aqueous solution dropwise, disperse evenly by ultrasonication, and then stir the reaction. After the reaction is complete, centrifuge and wash the product, then add water to resuspend it, and add it dropwise to mPEG-NH2 aqueous solution to continue the reaction (to modify FCM and PEG). After the reaction is complete, collect the product, centrifuge and wash it to obtain FCMP@CQ / PFH nanoparticles.

[0054] The mass ratio of mesoporous metal-organic framework (FCM) and chloroquine (CQ) in step (i) is 5:1.

[0055] The stirring time in step (i) is 10 to 14 hours; preferably 12 hours (stirring overnight).

[0056] The conditions for centrifugal washing in step (i) are: centrifugation at 10,000 to 14,000 rpm for 10 min; preferably: centrifugation at 14,000 rpm for 10 min.

[0057] The amount of perfluorohexane (PFH) used in step (ii) is calculated as 10 μL of perfluorohexane per milligram of mesoporous metal-organic framework (FCM) in FCM@CQ nanoparticles.

[0058] The buffer solution described in step (ii) is preferably a PBS buffer solution.

[0059] The conditions for ultrasonic treatment in step (ii) are: ultrasonic power 600W, ultrasonic time 5-10min; preferably: ultrasonic power 600W, ultrasonic time 5min.

[0060] The duration of the continued ultrasound treatment described in step (ii) is 5 to 15 minutes.

[0061] The mass ratio of FCM@CQ / PFH nanoparticles, dopamine hydrochloride, and mPEG-NH2 in step (iii) is 1:1:2.

[0062] The conditions for ultrasonic dispersion described in step (iii) are: ultrasonic power 600W, ultrasonic time 5-10min.

[0063] The buffer solution mentioned in step (iii) is a Tris buffer solution; preferably a Tris buffer solution with pH 8.5 (concentration 10 × 10⁻⁶). -3 (mol / L).

[0064] The stirring reaction time in step (iii) is 12 to 24 hours; preferably 12 hours.

[0065] The reaction time after adding the mPEG-NH2 aqueous solution in step (iii) is 1 to 3 hours; preferably 2 hours.

[0066] The conditions for centrifugal washing described in step (iii) are: centrifugation at 10,000 to 14,000 rpm for 10 to 15 minutes; preferably: centrifugation at 14,000 rpm for 10 minutes.

[0067] The centrifugal washing process described in step (iii) is performed three or more times.

[0068] The fluorescent dye-labeled FCMP@CQ / PFH nanoparticles are nanoparticles encapsulating dye and FCMP@CQ / PFH; preferably Cy5.5-FCMP@CQ / PFH nanoparticles, which are prepared by the following method: dissolving FCMP@CQ / PFH nanoparticles and Sulfo-cy5.5-NHS in a buffer solution, stirring and mixing in the dark at room temperature, and then centrifuging and washing to obtain Cy5.5-FCMP@CQ / PFH nanoparticles.

[0069] The preferred mass ratio of the FCMP@CQ / PFH nanoparticles to Sulfo-cy5.5-NHS is 1:5.

[0070] The buffer solution is a PBS buffer solution; preferably a PBS buffer solution with pH 8.0.

[0071] Unless otherwise specified, the water mentioned above is deionized water or ultrapure water.

[0072] The application of the metal-organic framework nanomaterials in the preparation of autophagy inhibitors.

[0073] The autophagy inhibitors described herein are used to inhibit autophagy in tumor cells.

[0074] The application of the metal-organic framework nanomaterials as autophagy inhibitors in the preparation of products for tumor immunotherapy.

[0075] The tumors mentioned are malignant tumors, including colorectal cancer and breast cancer.

[0076] The present invention has the following advantages and effects compared with the prior art:

[0077] 1. This invention provides a novel autophagy inhibitor based on metal-organic framework nanomaterials, which incorporates Fe... 2+ and Cu 2+ A biocompatible metal-organic framework (FCMP) is formed through disulfide bond coordination self-assembly. This iron-based and copper-based bimetallic element-based metal-organic framework itself has the effect of inhibiting autophagy in tumor cells and can be applied to the inhibition of autophagy in tumor cells. It achieves anti-tumor therapy by inducing the initial accumulation of autophagosomes in tumor cells, inhibiting the degradation of downstream autophagy, and blocking autophagy flux. Furthermore, it remodels the immune microenvironment by enhancing surface MHC-I expression and polarizing M2 macrophages into M1 macrophages.

[0078] 2. In order to achieve better tumor treatment efficiency, this invention utilizes the mesoporous advantages of FCMP to load the clinical drug chloroquine (CQ) and the ultrasound contrast agent perfluorohexane (PFH). The FCMP@CQ / PFH autophagy nano-inhibitor releases the drugs in the tumor microenvironment, thereby enhancing the anti-tumor immunotherapy effect by synergistically inhibiting tumor cell autophagy and jointly reshaping the immunosuppressive microenvironment. Attached Figure Description

[0079] Figure 1 Transmission electron microscopy (TEM) image of FCMP@CQ / PFH nanoparticles.

[0080] Figure 2 This is a particle size distribution diagram of FCMP@CQ / PFH nanoparticles.

[0081] Figure 3 Potential diagrams for FCM, FCMP, FCMP@CQ, and FCMP@CQ / PFH nanoparticles.

[0082] Figure 4 The particle size distribution of FCMP@CQ / PFH nanoparticles in deionized water, RPMI 1640 medium containing 10% FBS, and PBS buffer solution is shown.

[0083] Figure 5The UV-Vis absorption spectra of CQ and FCMP, and FCMP@CQ / PFH nanoparticles are shown.

[0084] Figure 6 This is a drug release diagram of FCMP@CQ / PFH nanoparticles under different conditions.

[0085] Figure 7 The fluorescence results show the intensity of autophagy induced by FCMP nanoparticles in HeLa cells.

[0086] Figure 8 The figure shows the results of Western blot analysis of the varying LC3 and P62 protein induction in MC38 cells by different concentrations of FCMP nanoparticles.

[0087] Figure 9 The figure shows the results of Western blot analysis of the LC3 and P62 protein induction in MC38 cells at different time points using FCMP nanoparticles.

[0088] Figure 10 The image shows the Western blot results of phosphorylation of MTOR protein induced by FCMP nanoparticles in MC38 and 4T1 cells; where A is the electrophoresis result and B is the statistical result of the quantified Western blot band signal intensity of each sample.

[0089] Figure 11 The image shows the Western blot results of phosphorylation of the MTOR phosphorylated base P70S6K protein induced by FCMP nanoparticles in MC38 and 4T1 cells; where A is the electrophoresis result and B is the statistical result of the quantified Western blot band signal intensity of each sample.

[0090] Figure 12 Figure A shows the Western blot results of whether FCMP nanoparticles induced complete autophagic flux in MC38 cells; Figure B shows the results of treatment with the upstream autophagy inhibitor Wollman penicillin; Figure B shows the results of treatment with the downstream autophagy inhibitor bavarimic acid A1.

[0091] Figure 13 The image shows the results of a Western blot analysis to verify whether autophagy was complete in HeLa cells at different concentrations of FCMP nanoparticles.

[0092] Figure 14 Immunofluorescence image to detect whether FCMP nanoparticles induce complete autophagy in HeLa cells.

[0093] Figure 15Western blot results for detecting LC3 and P62 proteins in MC38 cells using control (PBS), sonication alone (US), free chloroquine (CQ), FCMP, FCMP@CQ nanoparticles, and FCMP@CQ / PFH nanoparticles + US.

[0094] Figure 16 The results of cell viability assays after MC38 cells were treated with free chloroquine (Free CQ), FCMP, and FCMP@CQ nanoparticles.

[0095] Figure 17 The image shows the cell viability results of MC38 cells after treatment with control (PBS), free chloroquine (CQ), FCMP, FCMP@CQ nanoparticles, and FCMP@CQ / PFH nanoparticles + US.

[0096] Figure 18 Colony formation of MC38 and 4T1 cells after treatment with control (PBS), ultrasound alone (US), free chloroquine (CQ), FCMP, FCMP@CQ nanoparticles, and FCMP@CQ / PFH nanoparticles + US.

[0097] Figure 19 The graph shows the Annexin-V FITC / PI detection results after MC38 cells were treated with control (PBS), ultrasound alone (US), free chloroquine (CQ), FCMP, FCMP@CQ nanoparticles, and FCMP@CQ / PFH nanoparticles + US.

[0098] Figure 20 Flow cytometry and statistical graphs of MHC-I expression were obtained after FCMP treatment of MC38 tumor cells for 24 hours; where A is the flow cytometry expression graph and B is the statistical result of the flow cytometry.

[0099] Figure 21 Flow cytometry results of different treatment groups of mouse bone marrow-derived macrophages; where A is the flow cytometry expression plot; and B is the statistical results of the flow cytometry.

[0100] Figure 22 Biodistribution map of Cy5.5-FCMP@CQ / PFH in MC38 tumor-bearing mice.

[0101] Figure 23 The figures show the results of the therapeutic experiment of nanoparticles in MC38 tumor-bearing mice; where A is the curve of weight change of mice in each group during the treatment period; B is the curve of tumor volume change of mice in each group during the treatment period; C is the tumor image of each group after the mice were sacrificed; and D is the tumor mass of each group after the mice were sacrificed.

[0102] Figure 24The image shows the HE staining results of mouse sections from different groups of MC38 tumor-bearing mice.

[0103] Figure 25 Immunohistochemical images of LC3 and P62 in tumor sections from different groups of MC38 tumor-bearing mice.

[0104] Figure 26 The graph shows the flow cytometry results of the proportion of macrophages in each group of MC38 tumor-bearing mice; where A represents the proportion of M2 macrophages and B represents the proportion of M1 macrophages.

[0105] Figure 27 This is a flow cytometry graph showing the percentage of T cells in each group of MC38 tumor-bearing mice; where A represents CD8. + T cell percentage; B represents CD4. + T cell percentage.

[0106] Figure 28 This is a flow cytometry graph showing the percentage of regulatory T cells in each group of MC38 tumor-bearing mice. Detailed Implementation

[0107] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions. Unless otherwise specified, all raw materials and reagents used in the following embodiments are commercially available.

[0108] Example 1: Preparation of FCMP, FCMP@CQ, FCMP@CQ / PFH, and Cy5.5-FCMP@CQ / PFH nanoparticles

[0109] (1) Preparation of FCMP nanoparticles

[0110] ①Preparation of FCM nanoparticles

[0111] Synthesized using a hydrothermal method, a solution of N,N-dimethylformamide (DMF) and anhydrous ethanol at a volume ratio of 5:3 was first prepared. Certain amounts of ferrous chloride tetrahydrate, copper chloride dihydrate, and dithioglycolic acid powder were weighed and dissolved in a stock solution containing 50 mg / mL copper chloride, 50 mg / mL ferrous chloride, and 100 mg / mL dithioglycolic acid using the prepared DMF / DMF solution. The solution was then sonicated to ensure complete dissolution. Ferric and copper ions were added at a molar ratio of 11:4, i.e., 124 μL of the ferrous chloride tetrahydrate stock solution, 38 μL of the copper chloride dihydrate stock solution, and 52 μL of the dithioglycolic acid stock solution were added to 5... 300 mg of polyvinylpyrrolidone K40 and 200 μL of triethanolamine were added to a 0 mL centrifuge tube. Finally, the volume was adjusted to 13.5 mL with a solution of N,N-dimethylformamide (DMF) and anhydrous ethanol in a volume ratio of 5:3. After thorough sonication, the solution was added to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene. The reaction was carried out at 150 °C for 12 h to obtain the reactant. After cooling to room temperature, the product was washed with anhydrous ethanol by centrifugation (14000 rpm, 15 min). The precipitate was collected to obtain the FCM product without PEG modification.

[0112] ②Preparation of FCMP nanoparticles by PEG modification

[0113] To encapsulate the FCM product with a PDA (polydopamine) nanofilm, 5 mg of dopamine hydrochloride (CAS No.: 62-31-7, molecular weight 189.64) was first dissolved in 10 ml of pH 8.5 Tris buffer solution (10 × 10⁻⁶). -3 M), to obtain dopamine hydrochloride solution (concentration of 0.5 mg / mL) -1 Next, the product FCM solution (5 mg FCM product dissolved in 5 mL deionized water, concentration 1 mg / mL) was added dropwise to the dopamine hydrochloride solution, and the mixture was sonicated (600 W power, 5-10 min time) and stirred for 12 h. The product was then centrifuged and washed (to remove unreacted dopamine hydrochloride), and resuspended in deionized water (concentration 1 mg / mL). This solution was then added dropwise to the mPEG-NH2 solution (Carbon Water Technology Co., Ltd., product number: 80010102.MW=5000) (10 mg mPEG-NH2 dissolved in 10 mL deionized water, concentration 1 mg / mL). -1 In the reaction, FCM and PEG were modified for 2 hours. The product was collected and washed three times with ultrapure water at 14000 rpm for 10 min (to remove unreacted PEG) to obtain FCMP nanoparticles.

[0114] (2) Preparation of FCMP@CQ nanoparticles

[0115] ③ Preparation of FCM@CQ nanoparticles

[0116] The FCM product obtained in step (1)① was washed with anhydrous ethanol to remove excess unreacted reagents, and then dispersed in ddH2O for later use. To obtain FCM loaded with chloroquine (CQ), CQ (1 mg / mL, 1 mL) was added to 5 mL of FCM dispersion (1 mg / mL, dissolved in ddH2O), and the resulting solution was stirred overnight. The product was washed three times by centrifugation at 14000 rpm for 10 min with ddH2O to obtain FCM@CQ nanoparticles.

[0117] ④ Preparation of FCMP@CQ nanoparticles by PEG modification

[0118] Following the method in step (1) ② above, replace the product FCM with the FCM@CQ nanoparticles prepared in step (2) ③, and the remaining steps are the same to prepare FCMP@CQ nanoparticles.

[0119] (3) Preparation of FCMP@CQ / PFH nanoparticles

[0120] ⑤ Preparation of FCMP@CQ / PFH nanoparticles

[0121] The FCM@CQ solution obtained in step (2) (quantitatively 5 mg of FCM) was freeze-dried overnight. The solid mixture was then placed in a 25 mL three-necked flask with a rubber stopper. The air in the flask was evacuated using a vacuum pump, and then 50 μL of the ultrasound contrast agent perfluorohexane (PFH) was injected. The PFH and FCM@CQ mixture was sonicated in ice water under vacuum (600 W for 5 min), and then 5 mL of PBS buffer was injected. The mixture was then sonicated again in ice water (600 W for 10 min) to obtain the FCM@CQ / PFH solution.

[0122] ⑥ Preparation of FCMP@CQ / PFH nanoparticles by PEG modification

[0123] Following the method in step (1) ② above, replace the product FCM with the FCMP@CQ / PFH nanoparticles prepared in step (3) ⑤, and the remaining steps are the same to prepare FCMP@CQ / PFH nanoparticles.

[0124] (4) Preparation of Cy5.5-FCMP@CQ / PFH nanoparticles

[0125] Sulfo-cy5.5-NHS (purchased from Chongqing Xinweichuang Biotechnology) (10 mg) and FCMP@CQ / PFH nanoparticles (NPs) (2 mg) prepared in step (3) were dissolved in 2 ml of PBS buffer solution (pH 8.0). The solution was stirred overnight at room temperature and protected from light. Finally, excess Sulfo-cy5.5-NHS was removed from the solution by centrifugation and washing to obtain Cy5.5-FCMP@CQ / PFH nanoparticles, which were stored at 4 °C protected from light.

[0126] Example 2 Characterization of nanoparticles

[0127] I. Morphology Characterization of Nanoparticles

[0128] 10 μL of the FCMP@CQ / PFH nanoparticles (1.0 mg / mL) prepared in Example 1 was pipetted onto a 400-mesh copper grid and placed in a clean, dry place overnight to air dry. The morphology of the nanoparticles was then observed using a transmission electron microscope. The results are as follows: Figure 1 As shown, the FCMP@CQ / PFH nanoparticles all exhibit a complete ring-shaped flower morphology under electron microscopy, and their size is around 140 nm.

[0129] II. Determination of Nanoparticle Size and Surface Potential

[0130] (1) The FCMP@CQ / PFH nanoparticles prepared in Example 1 were dispersed in ultrapure water at a concentration of 1.0 mg / mL, and the particle size was then measured using a nanoparticle size analyzer (Malvern ZS90). The results are as follows: Figure 2 As shown, dynamic light scattering (DLS) results indicate that the particle size of FCMP@CQ / PFH nanoparticles is around 140 nm.

[0131] (2) The FCM, FCMP, FCMP@CQ, and FCMP@CQ / PFH nanoparticles prepared in Example 1 were dispersed in ultrapure water at a concentration of 1.0 mg / mL, and the Zeta potential of each nanoparticle was measured. The results are as follows: Figure 3 As shown, the test structure of the Zeta potential reveals that each nanoparticle is negatively charged.

[0132] III. Stability Characterization of Nanoparticles

[0133] The FCMP@CQ / PFH nanoparticles prepared in Example 1 were dispersed at a concentration of 1.0 mg / mL in RPMI 1640 medium containing 10% (v / v) fetal bovine serum (FBS), PBS buffer (pH 7.4, 0.01 M), and deionized water, respectively. Figure 4The particles were incubated in Distilled Water at 37°C. The particle size was then measured using a Malvern ZS90 nanoparticle size analyzer at 0, 1, 2, 3, 4, 5, 6, and 7 days. The results are as follows: Figure 4 As shown, the particle size of FCMP@CQ / PFH nanoparticles did not change significantly within 7 days, indicating that the nanoparticles have good particle stability in RPMI 1640 medium containing 10% FBS, PBS buffer solution and deionized water.

[0134] IV. Ultraviolet-Vis Spectroscopy Detection

[0135] Three samples—free chloroquine (CQ), FCMP prepared in Example 1, and FCMP@CQ / PFH nanoparticles—were placed in a 96-well plate, and their absorption characteristics were detected using a UV spectrophotometer. The results are as follows: Figure 5 As shown, CQ exhibits a distinct absorption peak near approximately 342 nm, indicating its strong ultraviolet absorption capability. Compared to CQ, the absorption curve of FCMP nanoparticles shifts towards lower absorption overall and lacks a distinct absorption peak, indicating weaker absorption in the ultraviolet and visible light ranges. Compared to FCMP nanoparticles, FCMP@CQ / PFH nanoparticles show a slightly higher absorption curve and a small shoulder peak near 342 nm (indicated by the arrow), indicating that CQ was successfully loaded into FCMP@CQ / PFH and retained some of its characteristic absorption.

[0136] V. Drug Release Test

[0137] The release of CQ from FCMP@CQ / PFH at different pH values ​​(6.5 and 7.4) and different GSH concentrations (0 and 10 mM) was detected using UV-Vis spectroscopy. 1 mL of FCMP@CQ / PFH (CQ concentration: 0.4 mg / mL) was placed in a dialysis bag (MWCO 8000) and sonicated (US) at a frequency of 1 MHz and an intensity of 2 W / cm². 2 The reaction was carried out for 60 seconds. Next, the dialysis bag was immersed in 15 mL of PBS solution with different pH values ​​and GSH concentrations. Then, the mixture was stirred at 80 rpm at 37°C. At regular intervals, the filtrate from each group was collected, and 100 μL of the culture medium was analyzed using a microplate reader at 342 nm, and the results were calculated using a standard curve. An equal volume of fresh release culture medium was added simultaneously. Each assay was repeated three times. The amount of drug CQ was calculated, and finally, the drug release curve was prepared as shown in the figure. Figure 6 The results showed that CQ release was higher at pH 6.5, with GSH (10 mM), and after ultrasonic treatment.

[0138] Example 3: Investigation of the autophagy mechanism by nanoparticles

[0139] I. The strength of autophagosomes induced by FCMP nanoparticles

[0140] HeLa-LC3 cells (HeLa cells purchased from ATCC, stably transfected with GFP-LC3 plasmid (purchased from Sangon Biotech (Shanghai) Co., Ltd.)) were seeded in 35 mm glass dishes and cultured overnight. Before adding samples, the cells were replaced with fresh 1640 medium, and FCMP nanoparticles (prepared in Example 1) at a final concentration of 25 μg / ml and free chloroquine (CQ) at a final concentration of 4 μg / ml were added as positive controls. The negative control (Control) consisted of an equal volume of PBS buffer. After 24 h of cell culture, the fluorescence of the GFP reporter gene was observed under a fluorescence microscope and photographed. The results are as follows: Figure 7 The results showed that FCMP nanoparticles induced stronger autophagy in HeLa-LC3 cells compared to the Control group.

[0141] II. Do FCMP nanoparticles inhibit tumor cell autophagy?

[0142] (1) MC38 cells (purchased from ATCC) were seeded in 24-well cell culture plates at a density of approximately 8–10 × 10⁶ cells / well. 4 / well, incubated overnight, ready for use. Before adding samples, the cells were replaced with fresh 1640 medium, and FCMP nanoparticles (prepared in Example 1) were added at concentrations of 6.25, 12.5, 25, and 50 μg / ml, respectively. The negative control was an equal volume of PBS buffer. After 24 hours of cell culture, Western blot analysis was performed. Results are as follows: Figure 8 As shown, FCMP nanoparticles induced a gradient increase in the concentration of LC3 and P62 proteins in MC38 cells, indicating that FCMP nanoparticles induced the accumulation of autophagosomes in MC38 cells, but blocked the downstream autophagy flux.

[0143] (2) MC38 cells (purchased from ATCC) were simultaneously seeded in 24-well cell culture plates at a density of approximately 8–10 × 10⁶ cells / well. 4 / well, incubated overnight, ready for use. Before adding samples, the cells were replaced with fresh 1640 medium and 25 μg / ml of FCMP nanoparticles (prepared in Example 1) were added. The negative control was an equal volume of PBS buffer. Cells were cultured at 0, 6, 12, and 24 h, and then Western blot was performed. Results are as follows. Figure 9 As shown, FCMP nanoparticles induced a time-gradient increase in LC3 and P62 proteins in MC38 cells, indicating that FCMP nanoparticles induced the accumulation of autophagosomes in MC38 cells, but blocked the downstream autophagy flux.

[0144] III. Effects of FCMP Nanoparticles on the mTOR Signaling Pathway

[0145] MC38 and 4T1 cells (purchased from ATCC) were seeded into 24-well cell culture plates at a density of approximately 8–10 × 10⁶ cells / well. 4 / well, incubated overnight, ready for use. Before adding samples, the cells were replaced with fresh 1640 medium. FCMP nanoparticles (prepared in Example 1) at a final concentration of 25 μg / ml were added to both cell types. An equal volume of PBS buffer was used as a blank control. Western blot analysis and statistical analysis were then performed. Three parallel experiments were conducted. Results are shown below. Figure 10 and Figure 11 As shown, compared with the control group, FCMP nanoparticles downregulated the phosphorylation level of MTOR and its substrate P70S6K in both MC38 and 4T1 cells.

[0146] IV. Testing whether FCMP nanoparticles induce complete autophagic flux in MC38 cells

[0147] MC38 cells were seeded in 24-well cell culture plates at a density of approximately 8–10 × 10⁶ cells / well. 4 / well, incubated overnight, ready for use. Before adding samples, the cells were replaced with fresh 1640 medium, and then the following solutions were added: 25 μg / ml FCMP nanoparticles (prepared in Example 1), 1 mM autophagy inhibitor Wort (Wormaginycin), 400 nM autophagy inhibitor Baf A1 (Bavaroxycin A1, purchased from Sigma), 25 μg / ml FCMP + 1 mM Wort, and 25 μg / ml FCMP + 400 nM Baf A1 (all final concentrations). The blank control was an equal volume of PBS buffer. Western blot analysis was then performed. Results are shown below. Figure 12 As shown, the upstream autophagy inhibitor Wort can inhibit the effect of FCMP nanoparticles on the accumulation of autophagosomes, and the downstream inhibitor Baf A1 can increase the accumulation of downstream P62 protein levels, indicating that FCMP nanoparticles block the autophagy flux of tumor cells.

[0148] V. Testing whether FCMP nanoparticles induce complete autophagic flux in HeLa cells

[0149] (1) HeLa-LC3 cells {HeLa cells were purchased from ATCC and stably transfected with GFP-LC3 plasmid (purchased from Sangon Biotech (Shanghai) Co., Ltd.)} were seeded in 24-well cell culture plates at a density of approximately 3–5 × 10⁶ cells / well. 4 / well, incubated overnight, ready for use. Before adding samples, the cells were replaced with fresh DMEM medium, and FCMP nanoparticles (prepared in Example 1) were added to final concentrations of 6.25, 12.5, 25, and 50 μg / ml, respectively. An equal volume of PBS buffer was used as a blank control. Western blot analysis was then performed. Results are shown below. Figure 13 As shown, no free GFP was detected with increasing concentration, indicating that FCMP nanoparticles blocked autophagic flux in HeLa cells.

[0150] (2) HeLa-LC3 cells {HeLa cells were purchased from ATCC and stably transfected with GFP-mRFP-LC3 plasmid (purchased from Sangon Biotech (Shanghai) Co., Ltd.)} were seeded in 24-well cell culture plates at a density of approximately 3–5 × 10⁶ cells / well. 4 / well, incubated overnight, ready for use. Before adding samples, the cells were replaced with fresh DMEM medium, and FCMP nanoparticles (prepared in Example 1) were added to a final concentration of 25 μg / ml. An equal volume of PBS buffer was used as a blank control, and CQ was used as a positive control. Immunofluorescence imaging was then performed. Results are as follows: Figure 14 As shown, in cells treated with FCMP nanoparticles, mRFP (red fluorescence) and GFP (green fluorescence) representing the LC3 protein exhibited significant co-localization. GFP fluorescence was significantly reduced due to its quenching under the acidic conditions of autolysosomes, while red fluorescence remained relatively stable. The FCMP nanoparticle treatment group did not weaken either red or green fluorescence, indicating that FCMP nanoparticles blocked autophagy flux in HeLa cells.

[0151] VI. Study on the autophagy flux of each group of nanoparticles in MC38 cells

[0152] MC38 cells (purchased from ATCC) were seeded in 24-well cell culture plates at a density of approximately 8–10 × 10⁶ cells / well. 4 / well, incubate overnight, ready for use. Before adding samples, replace the cells with fresh 1640 medium and add the following separately: sonication (US), chloroquine (CQ) at a final concentration of 4 μg / ml, FCMP nanoparticles at a final concentration of 25 μg / ml, FCMP@CQ nanoparticles (added at a final CQ concentration of 4 μg / ml), and FCMP@CQ / PFH nanoparticles (prepared in Example 1, added at a final CQ concentration of 4 μg / ml) + sonication (US), wherein the sonication frequency is 1 MHz and the intensity is 2 W / cm². 2 The incubation period was 60 seconds. The negative control consisted of an equal volume of PBS. Cells were cultured for 24 hours, followed by Western blot analysis. Results are as follows: Figure 15As shown, CQ and FCMP nanoparticles induced an increase in LC3 and P62 proteins in MC38 cells, indicating that FCMP nanoparticles induced the accumulation of autophagosomes in MC38 cells, but blocked the downstream autophagy flux. FCMP@CQ further increased LC3 and P62 proteins, indicating that they synergistically increased the blocking autophagy flux.

[0153] Example 4: Detection of the cytotoxic and immunotherapeutic effects of nanoparticles at the cellular level.

[0154] I. Cytotoxicity assay of nanoparticles in cells

[0155] MC38 cells were seeded in 96-well plates, approximately 8,000 cells per well. The cells adhered to the plates in a 37°C, 5% CO2 incubator until use. Before adding samples, the cells were replaced with fresh 1640 medium. The following solutions were then added: sonication (US) alone, chloroquine at a final concentration of 4 μg / ml, FCMP nanoparticles at a final concentration of 25 μg / ml, FCMP@CQ nanoparticles (added at a final CQ concentration of 4 μg / ml), and FCMP@CQ / PFH nanoparticles (prepared in Example 1, added at a final CQ concentration of 4 μg / ml) + sonication (US) for 24 hours. The sonication frequency was 1 MHz and the intensity was 2 W / cm². 2 The reaction time was 60 seconds. 10 μL of CCK-8 assay reagent was added to each of 96 wells and incubated at 37°C for 1–2 hours in the dark. A negative control was prepared by adding an equal volume of PBS. The absorbance was measured at 450 nm using a microplate reader, and the viability of the nanomaterials in MC38 cells was calculated using Graphpad Prism. Three parallel experiments were performed. Results are shown below. Figure 16 and Figure 17 As shown, free CQ exhibited negligible cytotoxicity, and FCMP showed mild cytotoxicity. However, FCMP@CQ induced significant tumor cell killing at a CQ concentration of 4 μg / mL, validating the therapeutic effect of combined treatment on MC38 tumor cells. Compared with the free CQ group and other nanoparticle groups, the FCMP@CQ / PFH+US group showed significantly reduced cell activity, and exhibited the strongest anti-proliferative effect under US treatment.

[0156] II. Plate Cloning Assay for Nanoparticles in Cells

[0157] MC38 cells in logarithmic growth phase and in good growth condition were selected and washed, digested, centrifuged, resuspended, and counted according to standard methods. An appropriate amount of single-cell suspension (approximately 1000 cells / well) was evenly seeded into 6-well plates containing 2 mL of complete culture medium, mixing constantly to ensure a similar cell count in each well. The 6-well plates were incubated overnight in a cell culture incubator. After cell attachment, PBS, sonication alone (US), chloroquine (CQ), FCMP nanoparticles, FCMP@CQ, and FCMP@CQ / PFH treatment under sonication were added to the wells according to predetermined groups (CQ final concentration 4 μg / ml; sonication frequency 1 MHz, intensity 2 W / cm²). 2 After incubation for 60 seconds and 12 hours, the wells were removed and replaced with fresh complete culture medium. The plates were then returned to the incubator (37℃, 5% CO2) for another 12 days, with the culture medium changed once during this period. After 12 days of culture, visible cell clones (≥50 cells / clone) appeared in the control group (PBS group). The cells were then removed from the incubator to terminate the culture. The culture medium was aspirated from the wells, and the cells were gently washed twice with 1× PBS. The cells were then fixed with paraformaldehyde (4%) at room temperature for 10–15 minutes to remove methanol, and then gently washed twice with 1× PBS. Subsequently, crystal violet (1%) was used for staining at room temperature for 20–30 minutes. The crystal violet stain was recovered, and excess crystal violet was gently removed with 1× PBS. The wells were allowed to air dry, and finally, the stained clones on the wells were photographed. The results are as follows: Figure 18 As shown, compared with other treatment methods, FCMP@CQ / PFH+US can eliminate almost all colonies at extremely low concentrations, demonstrating that CQ-loaded nanoparticles (NPs) have excellent long-term cytotoxicity against tumor cells.

[0158] III. Testing the Annexin V-PI double staining method to detect the apoptotic effect of nanoparticles on cells.

[0159] MC38 cells were placed in 12-well plates and cultured overnight. Before adding samples, the cells were replaced with fresh 1640 medium. PBS, sonication alone (US), chloroquine (CQ), FCMP nanoparticles, FCMP@CQ, and FCMP@CQ / PFH nanomaterials under sonication (added at a final CQ concentration of 4 μg / ml; sonication frequency 1 MHz, intensity 2 W / cm²) were used. 2 After treatment (60 s), cells were collected and washed twice with PBS, then resuspended in 195 μL of Annexin V-FITC binding buffer. After gentle mixing, 5 μL of Annexin V-FITC was added to the cells, followed by 10 μL of PI staining solution. The mixture was gently mixed and incubated at room temperature in the dark for 20 minutes. Flow cytometry was then performed immediately. Triple-replica experiments were conducted. Results are shown below. Figure 19 As shown, compared with the control group, the apoptosis rate of FCMP@CQ / PFH combined with US reached the highest, which was about 2.2 times that of the free CQ group and about 1.4 times that of the FCMP group.

[0160] IV. Testing the effect of FCMP nanoparticles on increasing MHC-I levels in cells

[0161] MC38 and 4T1 cells (purchased from ATCC) were placed in 24-well plates and cultured overnight. Before adding samples, the cells were replaced with fresh 1640 medium and treated with PBS and FCMP nanoparticles (25 μg / mL) for 24 h. After incubation, the cells from each well were digested and collected into 1.5 mL centrifuge tubes, and washed once with PBS. The flow cytometry blocking antibody CD16 / 32 working solution (diluted to 1% (v / v) concentration with PBS buffer containing 1% (v / v) fetal bovine serum (FBS)) was added to the cell pellet. The plates were incubated at 4°C in the dark for 15 minutes. The working solution for the MHC-I flow cytometry antibody was prepared: anti-MHC-I antibody (FITC conjugated) (purchased from BioLegend) was prepared with PBS buffer containing 1% (v / v) FBS. After cell blocking, remove the cells from the refrigerator, add external standard flow cytometry antibody working solution to each tube, scrape the cells from the bottom of the centrifuge tubes onto the surface of a 96-well PCR rack for resuspending, and then incubate at 4°C in the dark for 30 minutes. After antibody incubation, add 1 mL of PBS to each tube for resuspending, centrifuge at 3000×g for 2 minutes at 4°C in a pre-chilled centrifuge, wash once with working solution (PBS containing 1% (v / v) fetal bovine serum (FBS), resuspend in 300 μL of working solution (PBS containing 1% (v / v) fetal bovine serum (FBS), and transfer the cells through a 200-mesh screen to flow cytometry tubes. Analyze using a flow cytometer. Three parallel experiments were set up. Results are as follows: Figure 20 As shown.

[0162] V. Detection of the effect of nanoparticles on the polarization of mouse bone marrow-derived macrophages (BMDM)

[0163] First, 8- to 10-week-old C57BL / 6 mice (purchased from Hunan Silek) were euthanized by cervical dislocation. The tibia and femur of the two hind legs were then removed using surgical scissors, taking care to disassemble the leg bones at the joints and avoid cutting them in the middle to expose the medullary cavity. The attached tissue and hair were shaved off the bones, and they were gently wiped with 75% alcohol wipes. The leg bones were then immersed in 2mL centrifuge tubes containing sterile PBS.

[0164] Subsequently, the leg bone was removed using autoclaved forceps in a laminar flow hood. The joints at both ends of the leg bone were severed using autoclaved sterile surgical scissors to expose the medullary cavity. 15 mL of DMEM complete culture medium was drawn into a 20 mL syringe, and the syringe needle was replaced with a 1 mL syringe needle. The syringe needle was inserted into the medullary cavity at both ends of the leg bone, and the syringe was gently moved up and down while pushing the needle to thoroughly flush out the bone marrow cells. The cells were collected in a 15 mL centrifuge tube. The collected cell suspension was centrifuged at 450 × g for 5 minutes at 4°C in a pre-chilled centrifuge, the supernatant was discarded, and 1 mL of erythrocyte lysis buffer was added for resuspending. The erythrocytes were lysed at room temperature in the dark for 2 minutes, and then 2 mL of DMEM complete culture medium was added for neutralization. The suspension was then centrifuged again at 450 × g for 5 minutes at 4°C in a pre-chilled centrifuge, and the supernatant was discarded. Bone marrow cells were filtered through a sterile 200-mesh screen and transferred to new centrifuge tubes for cell counting. The culture density of BMDM was 1×10⁻⁶. 6 BMDM was obtained by culturing in large petri dishes, with each dish containing 10 mL of culture medium.

[0165] The bone marrow cells were then transferred to a 37°C incubator with 5% carbon dioxide for further culture. The bone marrow cells differentiated into BMDM on day four, at which point the BMDM cells were adherent. The optimal time to obtain BMDM cells is between day 4 and day 6. The BMDM cells cultured to day 4 were digested and centrifuged (450×g, 5 minutes), followed by [further processing at 4×10⁻⁶]. 5 Inoculate 1 mL of each sample into 12-well plates. After thorough mixing by cross-shaking on a work surface, incubate at 37°C with 5% CO2. The next day, discard the supernatant and replace with DMEM complete medium containing IL-4 (20 ng / mL), incubating for 24 hours at 37°C with 5% CO2. Subsequently, discard the supernatant, replace with fresh medium, and use PBS, sonication alone (US), chloroquine (CQ), FCMP nanoparticles, FCMP@CQ / PFH, and FCMP@CQ / PFH nanomaterials under sonication (added at a final CQ concentration of 4 μg / mL; sonication frequency 1 MHz, intensity 2 W / cm²). 2 After treatment (60s), the sample was incubated again in a 37°C incubator with 5% carbon dioxide for 24 hours.

[0166] After incubation, cells from each well were digested and collected into 1.5 mL centrifuge tubes, and washed once with PBS. The CD16 / 32 flow cytometry blocking antibody working solution (diluted to 1% (v / v) concentration with PBS buffer containing 1% (v / v) fetal bovine serum (FBS)) was added to the cell pellet. The mixture was then incubated at 4°C in the dark for 15 minutes.

[0167] Prepare the external standard flow cytometry antibody working solution for detecting macrophage polarization: APC / Cyanine7 anti-mouse CD45.2, PE anti-mouse CD11b, FITC anti-mouse F4 / 80, Brilliant Violet 421 TM Anti-mouse CD86 antibody (purchased from BioLegend) was prepared using PBS buffer containing 1% (v / v) FBS. After cell blocking, the cells were removed from the refrigerator, and external standard flow cytometry antibody working solution was added to each tube. The cells at the bottom of the centrifuge tubes were scraped off and resuspended on the surface of a 96-well PCR rack, and then incubated at 4°C in the dark for 30 minutes. After antibody incubation, 1 mL of PBS was added to each tube for resuspending, and the tubes were centrifuged at 3000×g for 2 minutes at 4°C in a pre-chilled centrifuge. The supernatant was discarded, and the tubes were placed on ice.

[0168] After being labeled with an external standard, the cells need to be fixed and permeabilized using a cell fixation and permeabilization kit (BD Cytofix / Cytoperm). TM Fixation / Permeabilization Kit (PSK) is used for fixation and permeabilization. First, prepare the fixation / permeabilization solution and washing buffer (1×BD Perm / Wash) according to the instructions. TM Then, 100 μL of Fixation / Permeabilization solution was added to each tube of cells and the cells were resuspended and incubated at 4°C in the dark for 20 minutes. Subsequently, 250 μL of 1×BD Perm / Wash solution was added. TM Wash twice with buffer at 500×g for 5 minutes. Prepare the flow cytometry internal standard antibody working solution: use 1×BD Perm / Wash TM Dilute APC anti-mouseCD206 antibody (purchased from BioLegend) with buffer. Add flow cytometry internal standard antibody working solution to the cells, scrape the bottom cells from the centrifuge tubes onto the surface of a 96-well PCR rack, resuspend, and incubate at 4°C in the dark for 30 minutes. Finally, use 1×BD Perm / Wash... TM Wash once with buffer, resuspend in 300 μL PBS, pass through a 200-mesh screen and transfer to flow cytometry tubes. Analyze using a flow cytometer. Perform three replicate experiments. Results are as follows: Figure 21 As shown.

[0169] Example 5: Therapeutic effects of FCMP@CQ / PFH nanomaterials in animal studies

[0170] I. Detection of in vivo metabolic distribution of FCMP@CQ / PFH nanomaterials

[0171] Culture and expand MC38 cells to a sufficient number, perform routine PBS washing, trypsin digestion, centrifugation, resuspending and counting, and then divide the cells into 5×10⁶ cells. 5 Cells per mL were injected subcutaneously at a rate of 100 μL per mouse into the right axilla of C57BL / 6 mice (purchased from Hunan Slack, 20g female mice, 6-8 weeks old). The injection was administered when the tumor volume reached approximately 100 mm². 3 In tumor-bearing mice, cy5.5-FCMP@CQ / PFH (100 μL) prepared in Example 1 (concentration calculated as CQ: 2.5 mg / kg) was injected via tail vein. In-Vivo Xtreme was used to image and observe the metabolic distribution of FCMP@CQ / PFH in vivo at different time points (0, 0.5, 2, 4, 8, 12, 24, 36, 48, 72 h). Separately, mice were euthanized by cervical dislocation 24 h after tail vein injection of cy5.5-FCMP@CQ / PFH (100 μL), and tumors, hearts, livers, spleens, lungs, and kidneys were collected for in vitro fluorescence imaging. Results are as follows: Figure 22 As shown, the fluorescence intensity of nanoparticles in mouse tumor sites initially increased and then decreased over time. Increasing numbers of nanoparticles (NPs) were observed in the tumor sites starting 8 hours post-injection and remained well-developed for up to 72 hours, demonstrating good tumor targeting. In vitro images showed that the fluorescence intensity in tumors harvested 24 hours post-injection was significantly higher than in other organs (heart, liver, spleen, lung, and kidney), indicating that FCMP@CQ / PFH has a longer circulation time and better tumor-specific accumulation.

[0172] II. In vivo anti-tumor therapy experiments and immunotherapy effects

[0173] To investigate the tumor-suppressive effect of nanoparticles in vivo, we subcutaneously constructed a murine MC38 colorectal model in C57BL / 6 nude mice (purchased from Hunan Slack, weighing 20g, 6-8 weeks old, female) (construction method as described in step one above). When the tumor volume reached approximately 100mm², we... 3 Mice were randomly divided into six groups of five each: Control group, US group, CQ (chloroquine) group, FCMP group, FCMP@CQ group, and FCMP@CQ / PFH+US group. The equivalent injection dose of CQ, FCMP, and FCMP@CQ / PFH was 2.5 mg / kg (calculated based on CQ). The ultrasound frequency was 1 MHz and the intensity was 2 W / cm². 2The drug was administered for 60 seconds (CQ dose equivalent to 2.5 mg / kg in each drug group), once every 3 days for a total of 4 administrations; the control group received an equal dose of PBS buffer. Tumor volume and body weight were monitored in the tumor-bearing mice during treatment. Mice were sacrificed at the end of treatment (day 21). Tumors were excised and histologically stained, including LC3, P62, and H&E, to examine the anticancer effect. The drug was administered every other day, and tumor volume and body weight were recorded. On day 13 of treatment, mice were sacrificed, tumor tissue was excised, and weighed. Figure 23 As shown in Figure A, no significant changes in mouse body weight were observed throughout the treatment process, demonstrating that the components in each experimental group did not cause significant systemic toxicity to the mice, and also reflecting the good biocompatibility of the nanoparticles; Figure 23 The tumor growth curves in mice (B group) showed that, compared with the PBS, US, and CQ groups, the FCMP group and the FCMP@CQ / PFH group effectively inhibited tumor growth, while the FCMP@CQ / PFH+US group further inhibited tumor volume growth. Furthermore, consistent with the tumor volume data, Figure 23 Tumor weight measurements in mice in group D showed that, compared with other treatment groups, the FCMP group and the FCMP@CQ / PFH combined treatment group effectively inhibited tumor growth, and FCMP@CQ / PFH+US further reduced tumor weight. Heart, liver, spleen, lung, kidney, and brain organs were harvested from sacrificed mice and sectioned for HE staining observation. Figure 24 As shown, FCMP@CQ / PFH+US does not have a significant effect on the internal organs of mice. Figure 25 The results showed that, consistent with those obtained in cell lines, compared with other groups, the LC3 and P62 proteins in mouse tumor tissues treated with FCMP@CQ / PFH+US were significantly enhanced, and the combined treatment on the surface had the best effect on inhibiting autophagy.

[0174] As described above, tumors were isolated from different groups of mice. Figure 23C). Next, we cut open the tumor with scissors to make the tumor fragments as small as possible. These fragments were digested with digestive fluid. The above liquid was transferred to another centrifuge tube and centrifuged at 400g for 5 minutes at 4°C. Afterwards, the liquid was subjected to gradient centrifugation using different concentrations of isotonic Percoll solution. The cell population in the middle of the liquid was aspirated using a Pasteur tube, and the cells were centrifuged again under the same conditions. After centrifugation, erythrocyte (RBC) lysate was added to lyse the RBCs, the remaining cells were cleaned, and stained with different immunofluorescent antibodies: CD45.2-APC / Cyanine7, CD11c-PE, CD11b-BV510, F4 / 80-FITC, CD86-BV421, CD206-PE, CD3-FITC, CD8a-BV785, CD4-BUV563, Foxp3-APC, and CD25-PerCP594 (purchased from BioLegend). F4 / 80+, CD86, and CD206 were used to represent M1 and M2 phenotype macrophages. Treg cells were represented by CD3, Foxp3, and CD25, and cells stained with CD3 and CD4 or CD8a antibodies were identified as CD4+ T cells or CD8+ T cells, respectively. Flow cytometry was used for analysis. Results are as follows: Figure 26 , Figure 27 and Figure 28 As shown.

[0175] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A metal-organic framework nanomaterial, characterized in that: The metal-organic framework nanomaterial is at least one of FCMP, FCMP@CQ, FCMP@CQ / PFH and fluorescent dye-labeled FCMP@CQ / PFH nanoparticles; The FCMP nanoparticles were prepared by the following method: (a) Add dopamine hydrochloride to a buffer solution, then add mesoporous metal-organic framework aqueous solution dropwise, disperse evenly by ultrasonication, and then stir the reaction. After the reaction is completed, collect the product, centrifuge and wash to obtain the intermediate product. (b) The intermediate product was resuspended in water and then added dropwise to an aqueous solution of mPEG-NH2 to continue the reaction. After the reaction was completed, the product was collected, centrifuged and washed to obtain FCMP nanoparticles. The FCMP@CQ nanoparticles were prepared by the following method: (I) Mesoporous metal-organic frameworks were dispersed in water, then chloroquine was added, the product was collected after thorough stirring, and centrifuged and washed to obtain FCM@CQ nanoparticles; (II) Add dopamine hydrochloride to the buffer solution, then add FCM@CQ nanoparticle aqueous solution dropwise, disperse evenly by ultrasonication, and then stir the reaction. After the reaction is completed, centrifuge and wash the product, then resuspend it in water, and add it dropwise to mPEG-NH2 aqueous solution to continue the reaction. After the reaction is completed, collect the product, centrifuge and wash it to obtain FCMP@CQ nanoparticles. The FCMP@CQ / PFH nanoparticles were prepared by the following method: (i) Mesoporous metal-organic frameworks were dispersed in water, then chloroquine was added, the product was collected after thorough stirring, and centrifuged and washed to obtain FCM@CQ nanoparticles; (ii) FCM@CQ nanoparticles were freeze-dried, then placed in a sealed container, vacuumed, and then injected with perfluorohexane to obtain a mixture of FCM@CQ and PFH; the mixture of FCM@CQ and PFH was ultrasonically treated under vacuum conditions in ice water, and then injected with a buffer solution for further ultrasonic treatment to obtain FCM@CQ / PFH nanoparticles. (iii) Add dopamine hydrochloride to a buffer solution, then add FCM@CQ / PFH nanoparticle aqueous solution dropwise, disperse evenly by ultrasonication, and then stir the reaction. After the reaction is complete, centrifuge and wash the product, then resuspend it in water, and add it dropwise to mPEG-NH2 aqueous solution to continue the reaction. After the reaction is complete, collect the product, centrifuge and wash it to obtain FCMP@CQ / PFH nanoparticles. The fluorescent dye-labeled FCMP@CQ / PFH nanoparticles are nanoparticles encapsulating dye and FCMP@CQ / PFH.

2. The metal-organic framework nanomaterial according to claim 1, characterized in that: The fluorescent dye-labeled FCMP@CQ / PFH nanoparticles are Cy5.5-FCMP@CQ / PFH nanoparticles, which are prepared by the following method: FCMP@CQ / PFH nanoparticles and Sulfo-cy5.5-NHS are dissolved in a buffer solution, stirred and mixed at room temperature in the dark, and then centrifuged and washed to obtain Cy5.5-FCMP@CQ / PFH nanoparticles; The mass ratio of the FCMP@CQ / PFH nanoparticles to Sulfo-cy5.5-NHS is 1:5; The buffer solution is a PBS buffer solution.

3. The metal-organic framework nanomaterial according to claim 1, characterized in that: The mesoporous metal-organic framework is formed by non-covalent bonding of disulfide monomers and metal ions, wherein the metal ion is Fe. 2+ and Cu 2+ The mesoporous metal-organic framework has a mesoporous structure, a particle size of 60–100 nm, and a specific surface area of ​​70–80 m². 2 / g, wherein the pore size of the mesopore is 7-10 nm; it is prepared by the following method: (1) Mix N,N-dimethylformamide with anhydrous ethanol until homogeneous to obtain a composite solution; (2) Prepare Fe-containing solutions using composite solutions. 2+ Solution containing Cu 2+ Solution and disulfide bond monomer solution; (3) Containing Fe 2+ Solution and Cu 2+ After mixing the solutions, disulfide monomer solution, polyvinylpyrrolidone and triethanolamine are added. After sonication to dissolve, the mixture is reacted at 135-155°C. After the reaction is complete, the mixture is cooled to room temperature, centrifuged and washed, and the precipitate is collected to obtain the mesoporous metal-organic framework.

4. The metal-organic framework nanomaterial according to claim 3, characterized in that: The volume ratio of N,N-dimethylformamide to anhydrous ethanol in step (1) is 4-6:2-4; The disulfide monomer mentioned in step (2) is at least one of dithioglycolic acid and selenocysteine; In step (3), Fe 2+ and Cu 2+ The molar ratio is 1:0.25 to 4; The mass ratio of the disulfide monomer and polyvinylpyrrolidone mentioned in step (3) is 5.205:300; The polyvinylpyrrolidone mentioned in step (3) is polyvinylpyrrolidone K40; The amount of triethanolamine used in step (3) is calculated based on 38-40 μL of triethanolamine per milligram of disulfide monomer; The reaction time described in step (3) is 12 to 24 hours.

5. The metal-organic framework nanomaterial according to claim 1, characterized in that: The mass ratio of the mesoporous metal-organic framework, dopamine hydrochloride, and mPEG-NH2 in step (a) is 1:1:2; The mass ratio of the mesoporous metal-organic framework to chloroquine in steps (I) and (i) is 5:1; The mass ratio of FCM@CQ nanoparticles, dopamine hydrochloride, and mPEG-NH2 in step (II) is 1:1:2; The amount of perfluorohexane used in step (ii) is calculated as 10 μL of perfluorohexane per milligram of mesoporous metal-organic framework in FCM@CQ nanoparticles. The mass ratio of FCM@CQ / PFH nanoparticles, dopamine hydrochloride, and mPEG-NH2 in step (iii) is 1:1:

2.

6. The metal-organic framework nanomaterial according to claim 1, characterized in that: The conditions for ultrasonic dispersion described in step (a) are: ultrasonic power 600W, ultrasonic time 5-10min; The buffer solution mentioned in step (a) is a Tris buffer solution; The stirring reaction time described in step (a) is 12–24 h; The reaction time described in step (b) is 1 to 3 hours; The centrifugal washing conditions described in steps (a) and (b) are: centrifugation at 10,000–14,000 rpm for 10–15 min; The centrifugal washing process described in steps (a) and (b) is performed at least three times.

7. The metal-organic framework nanomaterial according to claim 1, characterized in that: The stirring time described in step (I) is 10–14 hours; The conditions for centrifugal washing described in step (I) are: centrifugation at 10000-14000 rpm for 10 min; The conditions for ultrasonic dispersion described in step (II) are: ultrasonic power 600W, ultrasonic time 5-10min; The buffer solution mentioned in step (II) is a Tris buffer solution; The stirring reaction time described in step (II) is 12–24 h; The reaction time following the addition of the mPEG-NH2 aqueous solution in step (II) is 1–3 h. The centrifugal washing conditions described in step (II) are: centrifugation at 10,000–14,000 rpm for 10–15 min; The centrifugal washing process described in step (II) is performed three or more times.

8. The metal-organic framework nanomaterial according to claim 1, characterized in that: The stirring time described in step (i) is 10–14 h; The conditions for centrifugal washing described in step (i) are: centrifugation at 10000-14000 rpm for 10 min; The buffer solution mentioned in step (ii) is a PBS buffer solution; The conditions for ultrasonic treatment described in step (ii) are: ultrasonic power 600W, ultrasonic time 5-10min; The duration of continued ultrasonic treatment as described in step (ii) is 5–15 min; The conditions for ultrasonic dispersion described in step (iii) are: ultrasonic power 600W, ultrasonic time 5-10min; The buffer solution mentioned in step (iii) is a Tris buffer solution; The stirring reaction time described in step (iii) is 12–24 h; The reaction time following the addition of the mPEG-NH2 aqueous solution in step (iii) is 1–3 h. The centrifugal washing conditions described in step (iii) are: centrifugation at 10,000–14,000 rpm for 10–15 min; The centrifugal washing process described in step (iii) is performed three or more times.

9. The use of the metal-organic framework nanomaterials according to any one of claims 1 to 8 in the preparation of autophagy inhibitors.

10. The use of the metal-organic framework nanomaterials according to any one of claims 1 to 8 as autophagy inhibitors in the preparation of products for tumor immunotherapy.

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