Anti-tumor nano drug delivery system containing β-ketoenamine and preparation method and application thereof

Abstract

The present invention belongs to the technical field of biomedical materials, and specifically relates to an anti-tumor nano drug delivery system containing β-ketoenamine and a preparation method and application thereof. The anti-tumor nano drug delivery system is based on COF TFP‑DABP As a carrier, after loading the drug, RVG-29 modified with double-terminal amino-PEG was grafted on the surface to obtain OT@COF-RVG. The anti-tumor nano drug delivery system of the present invention uses a type I photosensitive covalent organic framework material containing a β-ketoenamine configuration as a carrier platform, integrates TMZ and OSMI-4 into the COF cavity, and then modifies the surface with polyethylene glycol and rabies virus peptide RVG-29, which can effectively activate non-oxygen-consuming photodynamic therapy to promote cell death, inhibit O-GlcNAc to promote lysosomal degradation of PD-L1, and enhance the effectiveness of immune checkpoint blockade therapy, thereby significantly improving its anti-tumor effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to an anti-tumor nano drug delivery system containing beta-ketoenamine, and a preparation method and application thereof. Background Art

[0002] Glioblastoma (GBM) is a common primary malignant brain tumor in the adult population. Although the standard treatment options for GBM include maximal surgical resection, postoperative radiotherapy and adjuvant chemotherapy, GBM patients face a grim prognosis. Programmed death ligand 1 (PD-L1) and its corresponding receptor programmed cell death 1 (PD-1) play a crucial role in T cell-mediated anti-tumor immunity. Immune checkpoint blockade (ICB) therapy using anti-PD-1 / PD-L1 antibodies has recently been recognized as a successful treatment option for various tumors. Nevertheless, the response to ICB treatment is suboptimal due to its tumor microenvironment (TME) showing limited cytotoxic T cell infiltration.

[0003] Covalent organic frameworks (COFs) are a unique class of crystalline porous polymer materials characterized by covalent bonding of small molecules and remarkable optical properties. This material has attracted widespread attention due to its unique properties, such as large specific surface area, ordered pores, easily functionalized structure, extremely low density, and excellent thermal and chemical stability. Photodynamic therapy (PDT) is a photochemotherapeutic modality that requires the activation of intracellular photosensitizers by specific excitation light, resulting in the conversion of tumor molecular oxygen into reactive oxygen species (ROS), leading to cell death. However, the common oxygen-consuming type II photodynamic therapy further aggravates the hypoxic microenvironment of the tumor, which seriously hinders its clinical translation.

[0004] PD-L1 and its corresponding PD-1 play crucial roles in T cell-mediated antitumor immunity. The regulation of lysosomal degradation of PD-L1 is affected by O-linked N-acetylglucosamine (O-GlcNAc) modification of hepatocyte growth factor-regulated tyrosine kinase substrate (HGS).

[0005] In view of this, developing a photodynamic therapy system that does not consume oxygen and can inhibit O-GlcNAc and promote lysosomal degradation of PD-L1 can effectively solve the above problems. Summary of the invention

[0006] In view of the deficiencies in the prior art, the purpose of the present invention is to provide an anti-tumor nano-drug delivery system containing β-ketoenamine, and a preparation method and application thereof. The anti-tumor nano-drug delivery system is a type I photosensitive covalent organic framework material carrier platform containing a β-ketoenamine configuration, which promotes cell death by activating non-oxygen-consuming photodynamic therapy, and promotes lysosomal degradation of PD-L1 by inhibiting O-GlcNAc, thereby enhancing the effectiveness of immune checkpoint blockade therapy; the present invention can significantly improve its anti-tumor effect by combining the above two treatment methods.

[0007] The present invention provides an anti-tumor nano drug delivery system containing β-ketoenamine, wherein the anti-tumor nano drug delivery system is based on COF TFP-DABP OT@COF-RVG was obtained by using RVG-29 (rabies virus peptide) modified with double-terminal amino-PEG (polyethylene glycol) as a carrier and then loading the drug on the surface.

[0008] Furthermore, the COF TFP-DABP It is a type I photosensitive covalent organic framework material carrier of β-ketoenamine configuration, which has a structure shown in formula (I):

[0009] .

[0010] Further, the drug is TMZ (temozolomide) and OSMI-4 (O-GlcNAc transferase inhibitor). Specifically, the drug has the following structure:

[0011] .

[0012] The present invention also provides a method for preparing an anti-tumor nano drug delivery system containing β-ketoenamine, which specifically comprises the following steps:

[0013] (1) Preparation of COF TFP-DABP DABP (4,4'-diaminobiphenyl) and TFP (1,3,5-triformylphloroglucinol) were first dissolved in a mixed solution of mesitylene / 1,4-dioxane, and a catalyst was added to obtain a reaction solution. The reaction solution was then subjected to three freeze-pump-thaw cycles after ultrasonic treatment for 5 min to 15 min, and then subjected to microwave radiation heating for 20 min to 40 min. Finally, the product was centrifuged, washed, and vacuum dried to obtain COF. TFP-DABP ;

[0014] (2) Preparation of OT@COF: First, add the drug-containing solution to COF TFP-DABPThe mixture was added to a phosphate buffer solution (PBS) (pH=7.4) to obtain a mixed solution, which was then reacted on a magnetic stirrer at room temperature for 6h~12h. The product was collected by centrifugation and dialyzed in PBS using a 3500Da dialysis bag for 12h~24h. The dialyzate was replaced every 6h. The dialyzate was collected by centrifugation and freeze-dried to obtain the product OT@COF.

[0015] (3) Preparation of OT@COF-RVG: First, RVG-29 and double-terminal amino-PEG were dissolved in DMSO (dimethyl sulfoxide) to obtain -NH2-RVG-29. Then, -NH2-RVG-29 was slowly dripped into OT@COF and stirred on a magnetic stirrer for 6 h~12 h. The product was collected by centrifugation and dialyzed in PBS using a 3500Da dialysis bag for 12 h~24 h. The dialysate was replaced every 6 h. The dialyzed product was collected by centrifugation and freeze-dried to obtain the product OT@COF-RVG.

[0016] Furthermore, in step (1) of the above technical solution, the molar ratio of DABP to TFP is 3:2-12:7; the concentration of DABP in the reaction solution is 6.5 mg / mL-13.1 mg / mL; the catalyst is 6M acetic acid; and the volume ratio of mesitylene / 1,4-dioxane / acetic acid is 10:10:1-20:15:3.

[0017] Furthermore, in step (1) of the above technical solution, the temperature of the microwave radiation heating treatment is 100°C~120°C, and the stirring reaction is carried out for 20min~40min; the method for washing the product is: first, the product is centrifuged and washed with tetrahydrofuran, and then the product is centrifuged and washed with acetone, and the process is repeated 4~6 times, each washing consuming 20mL~25mL of tetrahydrofuran or acetone, and then the product is washed with tetrahydrofuran by Soxhlet extraction, and the tetrahydrofuran consumed is 150mL~250mL.

[0018] Furthermore, in step (2) of the above technical solution, the drug-containing solution is a solution of TMZ and OSMI-4 dissolved in DMSO; the concentration of TMZ in the mixed solution is 0.7 mg / mL to 1.5 mg / mL, the concentration of OSMI-4 is 0.4 mg / mL to 0.8 mg / mL, and the COF TFP-DABP The concentration is 1.0mg / mL~2.0mg / mL.

[0019] Furthermore, in step (3) of the above technical solution, the molar ratio of the RVG-29 and the double-terminal amino-PEG is 1:1.2~1:1.5; the mass ratio of the -NH2-RVG-29 and OT@COF is 1:2.5~1:5.0.

[0020] Furthermore, in the above technical solution, in each step, the centrifugal speed is 5000rpm-6000rpm, and the time is 5min-10min.

[0021] The present invention also provides an application of the anti-tumor nano drug delivery system containing beta-ketoenamine in the preparation of tumor treatment drugs, chemotherapy agents or photodynamic immunotherapy agents.

[0022] Advantages compared to the prior art:

[0023] 1. The present invention first prepares a pH-adapted excited state intramolecular proton transfer isomerization based on β-ketoenamine covalent organic framework nanocarrier platform COF TFP-DABP TMZ and OSMI-4 were then integrated into the COF cavity and modified on the surface with polyethylene glycol and rabies virus peptide RVG-29. The resulting OT@COF-RVG showed the potential to sensitize TMZ chemotherapy and initiate photodynamic therapy (PDT); at the same time, by inhibiting O-GlcNAc and promoting lysosomal degradation of PD-L1, OT@COF-RVG enhanced the effectiveness of immune checkpoint blockade therapy.

[0024] 2. The OT@COF-RVG prepared by the present invention is treated with light, which can significantly increase the level of reactive oxygen, thereby re-establishing the immune stimulation state, inducing immunogenic cell death, and having a significant anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a preparation flow chart of OT@COF-RVG of the present invention;

[0026] Figure 2 The scanning electron microscope (scale bar: 1 μm) and transmission electron microscope (scale bar: 0.1 μm) photos of the self-assembly process of OT@COF-RVG in the embodiment of the present invention are shown;

[0027] Figure 3 The particle size distribution of OT@COF-RVG during the self-assembly process of the embodiment of the present invention;

[0028] Figure 4 This is a potential diagram during the self-assembly process of OT@COF-RVG in an embodiment of the present invention;

[0029] Figure 5 This is a powder X-ray diffraction pattern of OT@COF-RVG during the self-assembly process of an embodiment of the present invention;

[0030] Figure 6 This is a Fourier transform infrared spectrum of OT@COF-RVG self-assembly process in an embodiment of the present invention;

[0031] Figure 7 This is a solid-state NMR spectrum of OT@COF-RVG during the self-assembly process of the embodiment of the present invention;

[0032] Figure 8 This is a test diagram of the specific surface area during the self-assembly process of OT@COF-RVG in an embodiment of the present invention;

[0033] Fig. 9 Evaluation of the drug loading performance of OT@COF-RVG in the embodiment of the present invention;

[0034] Fig.10 COF according to the present invention TFP-DABP Evaluation of photodynamic level in different ROS probe systems;

[0035] Fig.11 COF according to the present invention TFP-DABP Evaluation of photodynamic levels in different ROS capture agents;

[0036] Fig.12 This is the evaluation of the drug release effect of OT@COF-RVG in the embodiment of the present invention at different pH and under illumination / no illumination;

[0037] Fig.13 COF according to the present invention TFP-DABP Schematic diagram of theoretical calculation research on photodynamic performance;

[0038] Fig.14 This is an immunofluorescence image of the embodiment of the present invention verifying in vitro that COF-RVG is enriched in GBM cells;

[0039] Fig.15 The immunofluorescence image of the in vivo experiment verifying that COF-RVG can better pass through the blood-brain barrier and target GBM tissue in the embodiment of the present invention;

[0040] Fig.16 The immunofluorescence image of the in vitro experiment verifying that COF-RVG can better pass through the blood-brain barrier and target GBM cells in the embodiment of the present invention;

[0041] Fig.17 This is a diagram of the photodynamic therapy killing effect of OT@COF-RVG evaluated in vitro by an embodiment of the present invention;

[0042] Fig.18 This is a diagram showing the expression of CRT on GBM cells by immunofluorescence after OT@COF-RVG treatment in an embodiment of the present invention;

[0043] Fig.19 This is a graph analyzing the changes in ATP levels released by GBM cells after OT@COF-RVG treatment according to an embodiment of the present invention;

[0044] Fig. 20 This is a protein band diagram of the changes in the expression levels of CRT and HMGB-1 in GBM cells detected by Western Blot after OT@COF-RVG treatment in the embodiment of the present invention;

[0045] Fig.21 This is a protein band diagram of changes in PD-L1 and HGS expression levels in GBM cells detected by Western Blot after OT@COF-RVG treatment in an embodiment of the present invention;

[0046] Fig. 22 This is a comparative diagram of the therapeutic effect of OT@COF-RVG on GBM verified by in vivo experiments in the embodiments of the present invention;

[0047] Fig.23 This is a curve diagram of the changes in body weight and survival period of tumor-bearing mice after OT@COF-RVG treatment and an immunofluorescence diagram of the detection of related proliferation and apoptosis indicators in mouse brain tissues, which is verified by the in vivo experiments of the embodiments of the present invention;

[0048] Fig.24 This is an in vivo experiment of the embodiment of the present invention to verify that OT@COF-RVG activates CD8+T cells in vivo and downregulates PD-L1 immunofluorescence image;

[0049] Fig.25 This is a flow cytometry graph of an in vitro experiment to verify that OT@COF-RVG activates CD8+T cells and downregulates PD-L1 cells in vivo. DETAILED DESCRIPTION

[0050] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0051] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0052] The carrier COF of the present invention TFP-DABP TFP and DABP were used as raw materials in a system of mesitylene / 1,4-dioxane / acetic acid by high-temperature microwave reaction; then the glioblastoma chemotherapy drugs TMZ (temozolomide) and OSMI-4 (O-GlcNAc transferase inhibitor) were integrated into the COF under stirring at room temperature. TFP-DABPThe surface of the nanostructured nanoparticles was modified with double-terminal amino-PEG and rabies virus peptide RVG-29 to obtain the anti-tumor nano-drug delivery system OT@COF-RVG containing β-ketoenamine. The OT@COF-RVG showed the potential to enhance TMZ chemotherapy and initiate photodynamic therapy. It can inhibit O-GlcNAc to promote the lysosomal degradation of PD-L1, enhance the effectiveness of immune checkpoint blockade (ICB) therapy, and has a significant anti-tumor effect. The preparation flow chart of OT@COF-RVG is shown in the figure. Figure 1 shown.

[0053] The present invention is further described in detail below in conjunction with specific embodiments:

[0054] Example 1: Preparation method of OT@COF-RVG

[0055] The preparation method of OT@COF-RVG comprises the following specific steps:

[0056] (1) COF was synthesized by preparing a solution of DABP (55 mg, 0.3 mM) and TFP (42 mg, 0.2 mM) in a mixture of mesitylene / 1,4-dioxane / 6 M acetic acid (8.4 mL, 10:10:1) at a molar ratio of 3:2 under microwave heating conditions. TFP-DABP ; After ultrasonic treatment for 15 minutes, the reaction solution was subjected to three freeze-pump-thaw cycles, the thick-walled pressure bottle was closed, and the product was stirred at 120°C for 20 minutes using a microwave synthesizer and heated by microwave radiation; the product was first centrifuged and washed with THF, and then with acetone, and the washing was repeated three times, the powder was collected, and then washed using THF as a solvent using a standard Soxhlet method to remove any impurities adsorbed in the porous structure; finally, after vacuum drying at 60°C for 24 hours, an orange-yellow powder of COF was obtained. TFP-DABP , with a yield of 87.3%.

[0057] (2) 1 mL of TMZ (3.8 mg, 20 mM) and OSMI-4 (2.1 mg, 3.44 mM) solutions prepared in DMSO were added to 3 mL of COF TFP-DABP (5 mg) phosphate buffer solution (pH = 7.4), and reacted on a magnetic stirrer at room temperature for 12 h; the product was collected by centrifugation (5000 rpm, 5 min), and dialyzed in PBS for 24 h using a 3500 Da dialysis bag, with the dialysate replaced every 6 h. The dialyzed product was collected by centrifugation and freeze-dried for 6 h to obtain the product OT@COF, which was an orange-yellow powder with a yield of 91.8%.

[0058] (3) RVG-29 and double-terminal amino-PEG (MW 1000) were dissolved in DMSO at a molar ratio of 1:1.2, then slowly added dropwise to 5 mg of OT@COF and stirred on a magnetic stirrer for 12 h. The product was collected by centrifugation (5000 rpm, 5 min) and dialyzed in PBS using a 3500Da dialysis bag for 24 h. The dialysate was replaced every 6 h. The dialyzed product was collected by centrifugation and freeze-dried to obtain OT@COF-RVG with a yield of 81.3%.

[0059] The OT@COF-RVG synthesized in the embodiment of the present invention and its process products were characterized:

[0060] PBS-dispersed OT@COF-RVG dispersion (0.1 mg / mL) was prepared and then dropped on a silicon wafer and a copper mesh to obtain scanning electron microscopy (SEM) and transmission electron microscopy (TEM) samples. The performance characterization results are shown in Figure 2~Figure 9 shown.

[0061] in Figure 2 The electron microscope photographs of OT@COF-RVG prepared in the embodiment of the present invention and its process products are shown in FIG. TFP-DABP The SEM images show a cubic morphology (e.g. Figure 2 As shown in a in FIG), it remains unchanged after the drug co-loading process in step (2) (as shown in FIG. Figure 2 As shown in b in FIG), after the surface modification in step (3), the surface of the nanoparticles becomes rougher and the boundaries become more blurred (as shown in FIG. Figure 2 As shown in (c), after drug loading and peptide modification, the nanoparticles became elliptical with blurred boundaries and increased size.

[0062] Figure 3 and Figure 4 The particle size distribution and potential of OT@COF-RVG prepared in the embodiment of the present invention and its process product are shown in Figures 2 and 3. The dynamic light scattering measurement shows that after step (2) and step (3), the hydrodynamic diameter of OT@COF-RVG increases significantly ( Figure 3 ); After RVG-29 modification, the zeta potential of OT@COF turned from positive to negative, confirming the success of peptide grafting ( Figure 4 ).

[0063] Figure 5 The figure is a powder X-ray diffraction (PXRD) pattern of OT@COF-RVG prepared in the embodiment of the present invention and its process product. Figure 5 The COF shows a distinct peak that is unique to the β-ketoamine-linked crystalline porous framework. TFP-DABPPeaks appeared at 3.35° and 5.82°, corresponding to the (100) and (110) planes, respectively. Drug loading and surface peptide targeting reduced the COF. TFP-DABP of crystallinity.

[0064] Figure 6 Fourier transform infrared spectra (FT-IR) of OT@COF-RVG prepared in the present invention and its process products. TFP-DABP The characteristic vibration bands of OT@COF and OT@COF-RVG were found, and the characteristic C=C and CN stretching vibration peaks appeared at 1569 and 1257~1257cm, respectively. −1 .

[0065] Figure 7 OT@COF-RVG prepared in the embodiment of the present invention and its process products 13 In the C solid-state nuclear magnetic resonance (NMR) spectrum, a characteristic exocyclic C=C signal can be observed in the region of 103ppm-106ppm. In addition, a characteristic C=O signal generated by the irreversible tautomerism from enol to keto was detected at 184ppm.

[0066] Figure 8 The specific surface area test of OT@COF-RVG prepared in the embodiment of the present invention and its process products. The nitrogen adsorption-desorption isotherm data in the figure show that COF TFP-DABP The specific surface areas of OT@COF and OT@COF-RVG are 445.20 m 2 / g, 220.12m 2 / g and 190.5m 2 / g.

[0067] Fig. 9 The content of TMZ and OSMI-4 in OT@COF-RVG prepared in the embodiment of the present invention is tested. The HPLC data in the figure show that the loading efficiency of TMZ and OSMI-4 in OT@COF-RVG is 62.1% and 76.9%, respectively.

[0068] Example 2: COF TFP-DABP Photodynamic performance evaluation

[0069] The COF prepared in Example 1 TFP-DABP Photodynamic performance evaluation was performed.

[0070] 1. Use 2',7'-dichlorofluorescein (DCFH) as an indicator to measure COF TFP-DABPThe total amount of reactive oxygen species (ROS) generated under light was analyzed, and DHR123, TMB and ABDA were used as ROS probes to detect superoxide anions (O2 .- ), hydroxyl ( . OH) and singlet oxygen ( 1 O2). Fig.10 It can be seen that within 90 seconds of light irradiation, the fluorescence intensity of DCFH increased by 32 times (e.g. Fig.10 The fluorescence intensity of DHR123 (as shown in a) Fig.10 b) and the UV absorption intensity of TMB (as shown in Fig.10 c) reached 12 and 93 times higher than before irradiation, while only ABDA showed no significant change (as shown in Fig.10 Therefore, it can be concluded that the COF prepared in Example 1 of the present invention TFP-DABP Produces O2 .- and . The efficiency of OH is greatly improved.

[0071] 2. Electron spin resonance (ESR) testing was performed in the organic phase using 2,2,6,6-tetramethylpiperidine (TEMP) as a spin trapping agent to confirm the COF TFP-DABP Produced 1 O2. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was tested as a spin trapping agent in both organic and aqueous phases to confirm the COF TFP-DABP O2 produced .- and . OH. From Fig.11 It can be seen that in COF TFP-DABP In the presence of TEMP, the ESR spectrum after 120s of light irradiation is 1 O2 did not show significant changes (e.g. Fig.11 b); in COF TFP-DABP In the presence of DMPO, after 120s of light irradiation, obvious O2 .- (like Fig.11 c in the figure) and . OH (eg Fig.11 Therefore, the COF of the embodiment of the present invention TFP-DABP It can be used as a type I photosensitizer to induce type I photodynamic therapy.

[0072] 3. COF TFP-DABP The samples were placed in different pH conditions and drug release tests were performed under different pH conditions with or without illumination.

[0073] First, prepare the COF TFP-DABP(1mg / mL) PBS solution and PBS buffer solutions with different pH values ​​(pH=5.5, 6.2, 7.2, 7.4) were used to simulate different physiological environments. A full-spectrum emitting LED lamp (7W) was fixed above the test liquid (10cm) to simulate the lighting environment, and the light was turned off for the non-lighting group. TFP-DABP Dissolve or disperse in buffer solutions of different pH values ​​at a predetermined concentration to ensure that the concentration of the sample is consistent under each pH condition. Take samples from each sample at predetermined time points (0, 2, 4, 6, 8, 12, 24, 48, 96h), determine the drug concentration in the sample using high performance liquid chromatography, etc., record the drug concentration at each time point, and calculate the drug release rate and cumulative release amount. Analyze the data using statistical methods to compare the drug release behavior under different pH and light conditions. Fig.12 It can be seen that under pH 5.5 conditions, COF TFP-DABP The drug release efficiency of COF is as high as 87%, which is further increased to 94% under light; under the pH 7.2-7.4 conditions of normal cells and tissues, the drug release efficiency does not exceed 21%. TFP-DABP Combining light with an acidic environment facilitates drug release.

[0074] 4. COF TFP-DABP The energy levels of the singlet and triplet states of the keto form and the alcohol form in acidic and neutral conditions were calculated. Fig.13 It can be seen that the COF prepared in the embodiment of the present invention TFP-DABP The alcohol configuration exhibits additional intersystem crossing (ISC) with an energy gap as low as 0.118 eV, and the calculated spin-orbit coupling (SOC) value between alcohol S1 and T5 is 0.108 cm -1 , which is less than the SOC value of the keto-ISC channel. Therefore, COF TFP-DABP The efficient ISC process of the alcohol configuration from S1 to T5 should be the reason why the alcohol structure has excellent ROS generation ability.

[0075] Example 3: COF-RVG Targeted Test

[0076] The efficacy of GBM drug treatment depends on the ability of the drug to cross the blood-brain barrier and accumulate at the tumor site. RVG-29 peptide exhibits specific affinity for nicotinic acetylcholine receptors (nAchR) present on the outer membrane of GBM cells and brain microvascular endothelial cells, promoting its uptake by GBM cells.

[0077] The preparation method of COF-RVG in this embodiment is:

[0078] (1) COF was synthesized by preparing a solution of DABP (55 mg, 0.3 mM) and TFP (42 mg, 0.2 mM) in a mixture of mesitylene / 1,4-dioxane / 6 M acetic acid (8.4 mL, 10:10:1) at a molar ratio of 3:2 under microwave heating conditions. TFP-DABP ; After ultrasonic treatment for 15 minutes, the reaction solution was subjected to three freeze-pump-thaw cycles, the thick-walled pressure bottle was closed, and the product was stirred at 120°C for 20 minutes using a microwave synthesizer and heated by microwave radiation; the product was first centrifuged and washed with THF, and then with acetone, and the washing was repeated three times, the powder was collected, and then washed using THF as a solvent using a standard Soxhlet method to remove any impurities adsorbed in the porous structure; finally, after vacuum drying at 60°C for 24 hours, an orange-yellow powder of COF was obtained. TFP-DABP .

[0079] (2) RVG-29 and double-terminal amino-PEG (MW 1000) were dissolved in DMSO at a molar ratio of 1:1.2 and then slowly added dropwise to 5 mg COF. TFP-DABP The product was collected by centrifugation (5000 rpm, 5 min) and dialyzed in PBS for 24 h using a 3500 Da dialysis bag. The dialysate was replaced every 6 h. The dialyzed product was collected and freeze-dried to obtain COF-RVG.

[0080] After the cell slides were placed in a six-well plate, 300,000 U251 and U87MG cells were added to each well and 30 μg / mL COF TFP-DABP The samples were co-incubated with COF-RVG for 4 h, fixed with 4% paraformaldehyde for 30 min, rinsed with PBS three times, and then permeabilized with 0.3% Triton X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 30 min. After rinsing with PBS, the nuclei were stained with Dapi (4',6-diamidino-2-phenylindole), and the slides were sealed and observed under a confocal microscope ( Fig.14 ). The results showed that GBM cell internalization was significantly enhanced, especially when RVG-29 peptide was added. To further prove the above conclusion, an orthotopic transplant tumor model was constructed in mice. After treatment with nanomaterials, immunofluorescence analysis of tissue sections in the core area of ​​the mouse tumor also supported this observation, revealing a significant increase in red fluorescence in the core area of ​​the mouse tumor treated with COF-RVG in the embodiment of the present invention ( Fig.15 ).

[0081] Laser scanning confocal microscopy (CLSM) was used to further analyze the cellular uptake ability of the embodiments of the present invention. TFP-DABPIn comparison, the internalization of COF-RVG was significantly enhanced, which may be due to the increased affinity between COF-RVG and nAChR receptors on GBM cells. In addition, the fluorescence intensity in U87MG and U251 cells showed significant dependence on time and concentration. The successful internalization of OT@COF-RVG by GBM cells is crucial for its anticancer efficacy.

[0082] The in vitro blood-brain barrier model in the embodiment of the present invention was developed using the Transwell (permeable cell culture chamber) system, in which one chamber was lined with a monolayer of brain microvascular endothelial cells (hCMEC / D3 or b.End3) and a lower chamber containing GBM cells (U87MG or GL261). After the nanomaterials were added to the upper chamber and incubated for 24 hours, the GBM cells were fixed and permeabilized to prepare slices, and the fluorescence signals of the GL261 or U87MG cells in the lower chamber were observed using CLSM. TFP-DABP In comparison, COF-RVG loaded with RVG-29 peptide showed stronger fluorescence intensity ( Fig.16 ).

[0083] The above experimental results show that RVG peptide enhances COF TFP-DABP The ability to cross the blood-brain barrier makes it more targeted.

[0084] Example 4: In vitro evaluation of the photodynamic level of OT@COF-RVG

[0085] To evaluate the ability of OT@COF-RVG to generate ROS in cells, fluorescent probes of DCFH-DA (fluorescent probe of reactive oxygen species ROS) and DHE (fluorescent probe of superoxide anion) were used to study the process of ROS generation in GBM cells.

[0086] The preparation method of COF-RVG is as follows: Example 3, the preparation method of OT@COF-RVG is as follows: Example 1, and the preparation method of T@COF-RVG is as follows:

[0087] (1) COF was synthesized by preparing a solution of DABP (55 mg, 0.3 mM) and TFP (42 mg, 0.2 mM) in a mixture of mesitylene / 1,4-dioxane / 6 M acetic acid (8.4 mL, 10:10:1) at a molar ratio of 3:2 under microwave heating conditions. TFP-DABP; After ultrasonic treatment for 15 minutes, the reaction solution was subjected to three freeze-pump-thaw cycles, the thick-walled pressure bottle was closed, and the product was stirred at 120°C for 20 minutes using a microwave synthesizer and heated by microwave radiation; the product was first centrifuged and washed with THF, and then with acetone, and the washing was repeated three times, the powder was collected, and then washed using THF as a solvent using a standard Soxhlet method to remove any impurities adsorbed in the porous structure; finally, after vacuum drying at 60°C for 24 hours, an orange-yellow powder of COF was obtained. TFP-DABP .

[0088] (2) Add 1 mL of TMZ (3.8 mg, 20 mM) solution prepared from DMSO to 3 mL of COF TFP-DABP (5 mg) phosphate buffer solution (pH = 7.4), and reacted on a magnetic stirrer at room temperature for 12 h; the product was collected by centrifugation (5000 rpm, 5 min), and dialyzed in PBS for 24 h using a 3500 Da dialysis bag, with the dialysate replaced every 6 h, and the dialyzed product was collected by centrifugation and freeze-dried for 6 h to obtain the product T@COF.

[0089] (3) RVG-29 and double-terminal amino-PEG (MW 1000) were dissolved in DMSO at a molar ratio of 1:1.2, then slowly added dropwise to 5 mg of T@COF and stirred on a magnetic stirrer for 12 h. The product was collected by centrifugation (5000 rpm, 5 min) and dialyzed in PBS using a 3500 Da dialysis bag for 24 h. The dialysate was replaced every 6 h. The dialyzed product was collected and freeze-dried to obtain T@COF-RVG.

[0090] The fluorescent probes of DCFH-DA and DHE were used to study the process of ROS production in GBM cells. First, U87MG and U251 cells in good condition were prepared. After trypsin digestion and centrifugation, they were spread into a 6-well plate with a 12-well cell slide placed in advance. The cells were shaken by the "cross method" to make the cells evenly distributed on the slide. When the cell density grew to about 50%, the experiment could be started. 50μg / mL of COF-RVG, T@COF-RVG, and OT@COF-RVG were added to the corresponding wells containing the slides, incubated for 6 hours, and washed with PBS three times, each time for 5 minutes, to remove the materials that did not enter the cells. 10μm / mL of DCFH-DA probe was added to each well, incubated at 37℃ for 30 minutes in the dark, and washed with PBS three times after incubation, each time for 5 minutes, to remove the dye that did not enter the cells. The illumination group was illuminated with 7W white light for 10 minutes, and the non-illumination group was incubated in the dark for 10 minutes. After the illumination, the culture medium was removed and the cells were washed with PBS three times for 5 minutes each time. After washing, 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30 minutes. After fixation, the cells were washed with PBS three times for 5 minutes each time. 50 μL of fluorescent antifade agent was dropped on the slide, and the cell slide was placed with the side containing the cells facing down. The cover was slowly closed to avoid bubbles. The excess fluorescent antifade agent around was absorbed by absorbent paper, and the slide was sealed with nail polish. After the nail polish was dried, the slide was prepared. The production of ROS in the cells was observed under a laser confocal microscope. 488 nm excitation light was used, and 529 nm emission light was collected to observe the green fluorescence in the cells.

[0091] Secondly, prepare U87MG and U251 cells in good condition, digest with trypsin and centrifuge them, then spread them into a 6-well plate with a 12-well cell slide in advance. Shake the cells by the "cross method" to evenly distribute the cells on the slide. When the cell density grows to about 50%, the experiment can be started. 50μg / mL of COF-RVG, T@COF-RVG, and OT@COF-RVG were added to the corresponding wells containing the slides, incubated for 6 hours, and washed with PBS three times for 5 minutes each time to remove materials that did not enter the cells. 20μm / mL of DHE probe was added to each well, incubated at 37℃ for 30 minutes in the dark, and washed with PBS three times for 5 minutes each time after incubation to remove the dye that did not enter the cells. The illumination group was illuminated with 7W white light for 10 minutes, and the non-illumination group was incubated in the dark for 10 minutes. After the illumination, the culture medium was removed and the cells were washed with PBS three times for 5 minutes each time. After washing, 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30 minutes. After fixation, the cells were washed with PBS three times for 5 minutes each time. 50 μL of fluorescent antifade agent was dropped on the slide, and the cell slide was placed with the side containing the cells facing down. The cover was slowly closed to avoid bubbles. The excess fluorescent antifade agent around was absorbed by absorbent paper, and the slide was sealed with nail polish. After the nail polish was dried, the slide was prepared. The production of superoxide anion ROS in the cells was observed under a laser confocal microscope. 532 nm excitation light was used, and 590 nm to 610 nm emission light was collected to observe the red fluorescence in the cells.

[0092] The results are as follows Fig.17 As shown, exposure to light significantly increased the increase in the fluorescence levels of DCFH-DA and DHE, indicating that the OT@COF-RVG prepared in the embodiment of the present invention exhibited a significant cytotoxic effect on GBM cells.

[0093] Example 5: OT@COF-RVG in vitro experiment

[0094] 1. In vitro experiments verify that OT@COF-RVG promotes ICD

[0095] In order to evaluate the efficacy of OT@COF-RVG prepared in Example 1 in inducing ICD in vitro and the release of DAMP molecules, COF-RVG and T@COF-RVG prepared in Examples 3 and 4 were compared.

[0096] After laying cell slides in a six-well plate, 300,000 U251 and U87MG cells were added to each well and incubated with 10 μg / mL COF-RVG, T@COF-RVG, and OT@COF-RVG nanomaterials, respectively. The cells were fixed with 4% paraformaldehyde for 30 min, rinsed with PBS three times, and then permeabilized with 0.3% Triton X-100. After rinsing with PBS, the cells were blocked with 5% goat serum for 30 min. CRT antibody was added at a concentration of 1:50 and incubated overnight. The fluorescent secondary antibody was added on the next day and incubated for 30 min before preparation. The cells were observed by CLSM ( Fig.18 ), it was found that the OT@COF-RVG prepared in the embodiment of the present invention caused a significant increase in the exposure of CRT to tumor cells compared with the control group.

[0097] GBM cells were co-incubated with 10 μg / mL COF-RVG, T@COF-RVG, and OT@COF-RVG nanomaterials, and the ATP secretion level in the cell supernatant was detected using an ATP kit ( Fig.19 ), it was found that the ATP content produced by OT@COF-RVG prepared in the embodiment of the present invention was also higher.

[0098] In addition, GBM cells were co-incubated with 10 μg / mL COF-RVG, T@COF-RVG, and OT@COF-RVG nanomaterials, and then radioimmunoprecipitation lysis buffer mixed with 1% phenylmethylsulfonyl fluoride was added to the culture dish. The lysate was collected and the cells were broken using an ultrasonic disruptor. The lysate was centrifuged at 14,000 rpm in a refrigerated centrifuge for 15 minutes to obtain protein. The cell protein was quantified by the BCA method, and 5 times the sodium dodecyl sulfate protein loading buffer was added and boiled to denature the protein. An equal amount of protein was loaded, and the protein was fixed on a PVDF (polyvinylidene fluoride) membrane by electrophoresis and transfer. The antigen epitope was blocked in skim milk, and the corresponding primary antibody dilution was used for antibody incubation. After rinsing, the corresponding secondary antibody was used for incubation and then developed in ECL luminescent solution. The expression of internal reference and target protein was detected respectively. Compared with the other groups ( Fig. 20 ), it was found that the OT@COF-RVG and T@COF-RVG groups in the embodiments of the present invention showed a significant decrease in HMGB-1 protein expression, while CRT protein expression showed an opposite trend. The results show that OT@COF-RVG can effectively trigger immunogenic cell death in GBM cells.

[0099] 2. In vitro experiments verified that OT@COF-RVG downregulated PD-L1

[0100] O-GlcNAc modification of HGS inhibits the interaction between PD-L1 and the endosomal sorting machinery, resulting in the obstruction of PD-L1 degradation from early endosomes to lysosomes, which ultimately leads to the upregulation of PD-L1 cell membrane protein and the inhibition of T cell anti-tumor function.

[0101] GBM cells were incubated with 10 μg / mL COF-RVG, T@COF-RVG, OT@COF-RVG nanomaterials and 10 μg / mL OT@COF-RVG plus 20 μM / mL chloroquine (a lysosomal degradation pathway inhibitor), and then RIPA (radioimmunoprecipitation lysis buffer) solution mixed with 1% PMSF (phenylmethylsulfonyl fluoride) was added to the culture dish. The lysate was collected and the cells were broken using an ultrasonic disruptor. The lysate was centrifuged at 14,000 rpm in a refrigerated centrifuge for 15 min to obtain protein. The cell protein was quantified by the BCA method, and 5 times the sodium dodecyl sulfate protein loading buffer was added and boiled to denature the protein. Take an equal amount of protein for loading, fix the protein on the PVDF membrane by electrophoresis and transfer operation; block the antigen epitope in skim milk, incubate the antibody with the corresponding primary antibody diluent, rinse and incubate with the corresponding secondary antibody, then develop in ECL luminescent solution, and detect the expression of internal reference and target protein. It is found that OT@COF-RVG in the embodiment of the present invention can effectively inhibit the expression of PD-L1 by inhibiting the endosomal recycling of PD-L1. At the same time, Western blotting was used to evaluate the expression of HGS in GBM cells co-incubated with 10μg / mL COF-RVG, T@COF-RVG, and OT@COF-RVG, respectively ( Fig.21 ), indicating that reducing O-GlcNAc levels can enhance HGS expression. These findings indicate that OT@COF-RVG in the present invention effectively inhibits PD-L1 expression by disrupting the endosomal sorting pathway of PD-L1.

[0102] Example 6: OT@COF-RVG in vivo experiment

[0103] 1. In vivo experiments verify the therapeutic effect of OT@COF-RVG on GBM

[0104] To further explore the effects of different nanomaterial groups (COF-RVG, T@COF-RVG, OT@COF-RVG) on GBM progression, a mouse model was constructed by injecting luciferase-labeled GL261 cells into the cerebroventricular cavity of C57BL / 6 mice. Bioluminescence imaging was performed on the sixth day after implantation and then repeated every 5 days. Seven days after tumor implantation, 1 mg / kg of each nanomaterial was injected into the tail vein of each experimental group once a day for 3 days. Fig. 22As shown. Tumor in vivo imaging showed that the use of OT@COF-RVG in the present invention resulted in a significant reduction in tumor volume compared to the rest of the groups. This finding suggests that treatment with OT@COF-RVG in the present invention effectively prevented the progression of GBM.

[0105] In addition, the Ki-67 and cleaved caspase 3 levels in each experimental group were detected by tissue immunofluorescence for immunofluorescence evaluation of tumor proliferation and apoptosis rate. Fig.23 The results in a showed that Ki-67 positivity was significantly decreased and Cleaved caspase 3 positivity was significantly increased in the OT@COF-RVG group. Then, similar results were achieved by terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling (TUNEL) assay, as shown in Fig.23 As shown in Figure c, the OT@COF-RVG group showed the highest positive rate, indicating that OSMI-4 can enhance the apoptotic effect of TMZ on GBM cells in vivo. Starting from day 10 after tumor implantation, the body weight of mice was monitored every 3 days. Fig.23 As can be seen in b, the minimal fluctuation in the body weight of mice in the OT@COF-RVG group was in contrast to the significantly faster weight loss observed in the PBS group. Ultimately, the survival rate of mice in the OT@COF-RVG group exceeded that of the other groups (e.g. Fig.23 These findings indicate that OT@COF-RVG of the present invention can enhance the efficacy of TMZ in treating GBM by targeting O-GlcNAc in GBM cells.

[0106] 2. OT@COF-RVG activates CD8+T cells and downregulates PD-L1 in vivo

[0107] After the nude mice with transplanted tumors were treated with each nanomaterial, brain tissue was taken to make frozen sections. After the sections were taken out of the freezing environment, they were placed in a humidified box and rewarmed at room temperature for 3min~5min; 4% paraformaldehyde fully covered the cells, fixed at room temperature for 15min, and washed the cells 3 times with PBS, each time for 5min; wiped off the residual moisture on the sections, and drew a closed circle around the tissue with a tissue pen; 0.1~0.2% Triton X-100 covered the tissue, incubated at room temperature for 20min, and washed 3 times with PBS, each time for 5min; 3%~5% BSA (bovine serum albumin) completely covered the sample and incubated at room temperature for 30min~60min; the blocking solution diluted the primary antibody, mixed it and covered the sample, incubated at 4℃ overnight, rewarmed at room temperature for 20min the next day, and washed 3 times with PBS, each time for 5min; the secondary antibody was diluted, incubated at room temperature in the dark for 1h, and washed 3 times with PBS, each time for 5min; the residual liquid on the sections was dried with absorbent paper, and Dapi / antiquenching agent two-in-one sealing solution was added on the sections, and coverslips were covered to avoid bubbles. Confocal microscopy revealed a significant upregulation of CD8 expression in GBM tumor regions after treatment with OT@COF-RVG and T@COF-RVG (e.g. Fig.24 The results of in vivo experiments showed that OSMI-4 effectively reduced the expression of endogenous PD-L1, thereby enhancing ICB treatment. Immunofluorescence analysis of mouse tissue sections showed that PD-L1 expression in the GBM tumor region was significantly reduced after treatment with OT@COF-RVG orthotopically transplanted tumor mice (as shown in Figure 2A). Fig.24 (b) in Figure 1). Mature dendritic cells (mDC) are a significant indicator of enhancing T cell-mediated anti-tumor immunity by effectively presenting tumor antigens. The present invention used flow cytometry to examine the expression of mDC biomarker CD86 in tumors treated with various nanoparticles, and found that the number of CD86-positive mDC cells in the OT@COF-RVG treatment group increased significantly.

[0108] CD8 and CD4 T lymphocyte detection: First, after anesthetizing and killing the mice, the spleen was removed from the left upper abdomen of the mice, placed in a 5mL centrifuge tube containing 2mL PBS, and placed on ice for later use. Wash the spleen, put it in a clean cell culture dish, add 3mL PBS and grind the spleen. After grinding the spleen into a homogenate, collect the cell suspension and remove the connective tissue fragments through a 70μm cell sieve, and collect the filtrate. Centrifuge the filtrate at 450g for 10min, discard the supernatant, add 3mL red blood cell lysis buffer, and lyse for 10min. If the lysis is not sufficient, it can be lysed multiple times. Add an equal amount of PBS to stop the lysis, centrifuge at 450g, 4℃, 5min, and discard the supernatant. Resuspend the cell pellet with 100μL PBS, add CD3, CD4, and CD8 flow cytometry antibodies, incubate at room temperature for 1h, centrifuge at 450g for 5min after staining, add 1mL PBS to wash once, centrifuge at 450g for 5min again, discard the supernatant, and obtain 500μL PBS resuspended cell fluid. The cell suspension was added to the flow tube, and the number of CD8 and CD4 positive T lymphocytes was observed. Flow cytometric analysis showed that compared with other treatment groups, the number of CD8 positive T lymphocytes in the tumor area of ​​the orthotopically transplanted tumor mice treated with OT@COF-RVG and T@COF-RVG in the embodiment of the present invention was significantly increased (e.g. Fig.25 (as shown in a in the figure).

[0109] Activation of DC cells in mouse lymph nodes: First, after anesthetizing and killing mice, mesenteric lymph nodes were removed from the mouse mesentery, placed in a 5mL centrifuge tube containing 2mL PBS, and placed on ice for later use. Wash the lymph nodes, place them in a clean cell culture dish, add 3mL PBS and grind the lymph nodes. After grinding the lymph nodes into a homogenate, collect the cell suspension and remove connective tissue fragments through a 70μm cell sieve, and collect the filtrate. Centrifuge at 450g for 5min, remove the supernatant, resuspend the cell pellet with 100μL PBS, add CD11c, CD80, and CD86 flow cytometry antibodies, incubate at room temperature for 1h, centrifuge at 450g for 5min after staining, add 1mL PBS to wash once, centrifuge at 450g again for 5min, discard the supernatant, and resuspend the cells with 500μL PBS. Add the cell suspension to the flow tube, and observe the number of CD86 and CD80 positive T lymphocytes on the machine. Flow cytometry analysis showed that compared with the other treatment groups, the expression of CD86 in the tumor area of ​​the orthotopically transplanted tumor mice treated with OT@COF-RVG and T@COF-RVG in the present invention was significantly increased (e.g. Fig.25 (as shown in b in the figure).

[0110] The above experimental results show that OT@COF-RVG of the present invention has the ability to induce a strong immune response and inhibit the expression of PD-L1.

[0111] In summary, the anti-tumor nano-drug delivery system containing β-ketoenamine prepared by the present invention is based on a type I photosensitive covalent organic framework material carrier platform containing β-ketoenamine configuration. By integrating TMZ and OSMI-4 into the COF cavity and then modifying the surface with polyethylene glycol and rabies virus peptide RVG-29, it can effectively activate non-oxygen-consuming photodynamic therapy to promote cell death, inhibit O-GlcNAc to promote lysosomal degradation of PD-L1, and enhance the effectiveness of immune checkpoint blockade therapy; by combining the above two treatment methods, its anti-tumor effect can be significantly improved.

[0112] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-tumor nano drug delivery system containing β-ketoenamine, characterized in that: The anti-tumor nano drug delivery system is based on COF TFP-DABP As a carrier, after loading the drug, RVG-29 modified with double-terminal amino-PEG was grafted on the surface to obtain OT@COF-RVG; The COF TFP-DABP It is a type I photosensitive covalent organic framework material carrier of β-ketoenamine configuration, which has a structure shown in formula (I): 。 2. The anti-tumor nano drug delivery system containing β-ketoenamine according to claim 1, characterized in that: The drugs are TMZ and OSMI-4.

3. The method for preparing an anti-tumor nano drug delivery system containing β-ketoenamine according to any one of claims 1 to 2, characterized in that: The specific steps include: (1) Preparation of COF TFP-DABP DABP and TFP were first dissolved in a mixed solution of mesitylene / 1,4-dioxane, and a catalyst was added to obtain a reaction solution. The reaction solution was then subjected to three freeze-pump-thaw cycles after ultrasonic treatment for 5 min to 15 min, and then subjected to microwave radiation heating for 20 min to 40 min. Finally, the product was centrifuged, washed, and vacuum dried to obtain COF. TFP-DABP ; (2) Preparation of OT@COF: First, add the drug-containing solution to COF TFP-DABP phosphate buffer solution to obtain a mixed solution, which was then reacted on a magnetic stirrer at room temperature for 6 h to 12 h, the product was collected by centrifugation, and dialyzed in PBS for 12 h to 24 h using a 3500 Da dialysis bag, with the dialysate replaced every 6 h, the dialyzed product was collected by centrifugation, and freeze-dried to obtain the product OT@COF; (3) Preparation of OT@COF-RVG: First, RVG-29 and double-terminal amino-PEG were dissolved in DMSO to obtain -NH2-RVG-29. Then, -NH2-RVG-29 was slowly dripped into OT@COF and stirred on a magnetic stirrer for 6 h~12 h. The product was collected by centrifugation and dialyzed in PBS using a 3500Da dialysis bag for 12 h~24 h. The dialysate was replaced every 6 h. The dialyzed product was collected by centrifugation and freeze-dried to obtain the product OT@COF-RVG.

4. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of DABP to TFP is 3:2-12:7; the concentration of DABP in the reaction solution is 6.5 mg / mL-13.1 mg / mL; the catalyst is 6M acetic acid; and the volume ratio of mesitylene / 1,4-dioxane / acetic acid is 10:1:1-20:15:

3.

5. The preparation method according to claim 3, characterized in that: In step (1), the temperature of the microwave radiation heating treatment is 100°C to 120°C, and the stirring reaction is carried out for 20min to 40min. The method for washing the product is: first, centrifugally washing the product with tetrahydrofuran, and then centrifugally washing the product with acetone, repeating 4 to 6 times, each washing consuming 20mL to 25mL of tetrahydrofuran or acetone, and then washing the product with tetrahydrofuran by Soxhlet extraction, consuming 150mL to 250mL of tetrahydrofuran.

6. The preparation method according to claim 3, characterized in that: In step (2), the drug-containing solution is a solution of TMZ and OSMI-4 dissolved in DMSO; the concentration of TMZ in the mixed solution is 0.7 mg / mL to 1.5 mg / mL, the concentration of OSMI-4 is 0.4 mg / mL to 0.8 mg / mL, and the COF TFP-DABP The concentration is 1.0mg / mL~2.0mg / mL.

7. The preparation method according to claim 3, characterized in that: In step (3), the molar ratio of the RVG-29 to the double-terminal amino-PEG is 1:1.2-1:1.5; the mass ratio of the -NH2-RVG-29 to OT@COF is 1:2.5-1:5.

0.

8. The preparation method according to claim 3, characterized in that: In each step, the centrifugal speed is 5000 rpm~6000 rpm, and the time is 5 min~10 min.

9. Use of the anti-tumor nano drug delivery system containing β-ketoenamine as claimed in any one of claims 1 to 2 or the anti-tumor nano drug delivery system containing β-ketoenamine prepared by the preparation method as claimed in any one of claims 3 to 8 in the preparation of tumor treatment drugs, chemotherapy agents or photodynamic immunotherapy agents.

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