Nanometer autophagy induction preparation, preparation method and application thereof, and pharmaceutical composition

Abstract

The invention relates to the technical field of nano materials, in particular to a nano autophagy induction preparation, a preparation method and application thereof and a pharmaceutical composition. The nano autophagy induction preparation comprises a composite material with a core-shell structure and a substance which is fixed on the composite material and can generate NO through photolysis, in the composite material, the core is caffeic acid modified up-conversion nanoparticles, and the shell is an MOFs material; the metal center of the MOFs material is copper ions, and organic ligands are selected from one or more of carboxylic acid organic ligands. The nano autophagy induction preparation has a light and valence state conversion function, generates UV under excitation of near-infrared light, reduces Cu < 2 + > into Cu < + >, can release carboxylic acid organic ligands and reduced Cu < + >, can generate NO through photolysis of substances capable of generating NO through photolysis, can be used for inducing excessive autophagy, and provides a powerful synergistic anti-tumor photoimmunotherapy effect for resisting tumor development.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and particularly relates to a nano-autophagy inducer, a preparation method and application thereof, and a pharmaceutical composition. Background Art

[0002] Cancer immunotherapy is one of the most promising strategies for treating tumors and has attracted extensive attention. Inducing tumor immunogenic cell death (ICD) is a widely used method by which tumor cells can release damage-associated molecular patterns (DAMPs) after encountering stimuli, thereby stimulating the infiltration of immune cells into the tumor to kill residual tumor cells. Currently, various ICD inducers, including metal-based anticancer drugs, photothermal molecules, photodynamic molecules, small molecules generating NO gas, etc., have been applied to ICD-based tumor immunotherapy. However, the weak stimulation of the above-mentioned ICD inducers on the immune response limits the current clinical efficiency of ICD inducers, which is attributed to the low immunogenicity of tumor cells and insufficient presentation of tumor endogenous antigens in dying tumor cells. Therefore, developing strategies to enhance ICD efficiency and improve tumor immunogenicity has become the main research direction for improving the overall efficacy of immunotherapy.

[0003] Autophagy plays a key role in promoting small molecule-induced ICD in tumor cells. During cell death, activated autophagy promotes the transport of tumor cytoplasmic components to lysosomes for hydrolysis, thereby enhancing the processing of endogenous antigens and their presentation on MHC-I molecules. In addition to antigen processing, autophagy also promotes the secretion of ATP by dying tumor cells, serving as a "find me" signal for dendritic cell (DC) precursors and stimulating the infiltration of cytotoxic T lymphocytes into tumors. Therefore, autophagy activation is crucial for the presentation of tumor antigens and the recruitment of immune cells, which are important aspects of an effective anti-tumor immune response. However, in the context of tumor treatment, autophagy is a double-edged sword. Mild autophagy can provide a protective effect against treatment, while excessive autophagy triggers cell death. Unfortunately, the use of autophagy inducers alone usually only triggers a mild and tumor-protective autophagy response, which is not sufficient to reach the excessive autophagy threshold required to induce cell death, thus limiting its effect on inducing tumor cell death. Therefore, developing new strategies to reliably induce excessive autophagy by combining different inducers is crucial for enhancing ICD and improving anti-tumor treatment.

[0004] To achieve excessive autophagy for therapeutic benefits, it is necessary to effectively co - deliver different autophagy inducers to the tumor site. Although nano - carriers utilizing the enhanced permeability and retention (EPR) effect and active targeting show potential in drug delivery, specific design considerations are still required to induce strong autophagy. To avoid toxicity, maximize the payload, and simplify the synthesis process, carrier - free nano - carriers constructed from autophagy - inducing materials such as metal - organic frameworks (MOFs) have emerged as an attractive alternative. The inherent complexity of the autophagy pathway and the diversity of MOF structures require a strategic approach in the selection of metal ions and ligand components. In terms of delivery performance, metal ions, ligand molecules, and the delivered cargo should be precisely transported into the cytoplasm, where these components can rapidly initiate autophagy and induce more substrates to guide stronger ICD. In this regard, the main strategies for prolonging the circulation time and enhancing cancer cell uptake have not been fully successful. Overcoming lysosomal sequestration to facilitate cytoplasmic entry is crucial for the induction of strong autophagy. For MOFs, the ultimate task is to consider the release of active components, especially the valence state of metal ions, as lower valence states often exhibit better autophagy - inducing efficiency. Additionally, achieving tumor - specific autophagy activation without damaging normal tissues remains a key challenge. Summary of the Invention

[0005] An object of the present invention is to provide a nano - autophagy - inducing preparation, which has the functions of light and valence state conversion, generates ultraviolet light (UV) under near - infrared light excitation, such that Cu 2+ is reduced to Cu + , can release carboxylic acid - based organic ligands and the reduced Cu + , and can photolyze a substance that can generate NO by photolysis to generate NO, which can be used to induce excessive autophagy and provide a strong synergistic anti - tumor photo - immunotherapy effect against tumor progression.

[0006] The present invention first constructs a light - responsive nano - autophagy - inducing preparation using two autophagy inducers (MOF materials and a substance that can generate NO by photolysis). The nano - autophagy - inducing preparation of the present invention can comprehensively activate autophagy, leading to enhanced ICD, and provide a strong synergistic anti - tumor photo - immunotherapy effect against tumor progression.

[0007] To achieve the above object, in a first aspect of the present invention, a nano - autophagy - inducing preparation is provided, which includes a composite material having a core - shell structure and a substance that can generate NO by photolysis fixed on the composite material;

[0008] In the composite material, the core is an up - conversion nanoparticle modified with caffeic acid, and the shell is a MOF material; the metal center of the MOF material is copper ion, and the organic ligand is selected from one or more of carboxylic acid - based organic ligands.

[0009] The second aspect of the present invention provides a method for preparing the nano-autophagy inducing preparation described in the present invention, and the method includes:

[0010] Method 1: Contact a mixed solution containing upconversion nanoparticles modified with caffeic acid, copper ions and a hydrophilic polymer with a solution containing an organic ligand and a substance capable of generating NO through photolysis, and separate to obtain a solid; or

[0011] Method 2: Contact a mixed solution containing upconversion nanoparticles modified with caffeic acid, copper ions and a hydrophilic polymer with a solution containing an organic ligand, and separate the solid; mix the solid with a solution containing a substance capable of generating NO through photolysis, and separate to obtain a solid.

[0012] The third aspect of the present invention provides an application of the nano-autophagy inducing preparation described in the present invention in the preparation of a drug for treating tumors.

[0013] The fourth aspect of the present invention provides a pharmaceutical composition, which contains the nano-autophagy inducing preparation described in the present invention and a pharmaceutically acceptable excipient.

[0014] Through the above technical solutions, the nano-autophagy inducing preparation described in the present invention has functions of light and valence state conversion, can be used to induce excessive autophagy, and the nano-autophagy inducing preparation has high stability.

[0015] For the nano-autophagy inducing preparation described in the present invention, once triggered by near-infrared light, the near-infrared to ultraviolet (UV) conversion characteristic of upconversion nanoparticles (UCNP) enables Cu 2+ to be reduced to Cu + , and it can release a carboxylic acid organic ligand (such as 1,3,5-benzenetricarboxylic acid) and the reduced Cu + as well as a substance capable of generating NO through photolysis (such as BNN6) to generate NO through photolysis; Cu + binds to ULK1 to initiate the autophagy flux, the carboxylic acid organic ligand (such as BTC) causes depletion of cytoplasmic acetyl-CoA, and NO damages mitochondria to further provide autophagy substrates. This comprehensive autophagy activation leads to enhanced ICD, providing a strong synergistic anti-tumor photoimmunotherapy effect against tumor progression. For the nano-autophagy inducing preparation described in the present invention, after the MOFs material decomposes, the subsequently delivered NO precursor molecule can further cascade and amplify the autophagy effect of tumor cells to enhance the immunogenic death effect of tumors, which is different from the prior art that is difficult to activate the autophagy pathway in all pathways.

[0016] The nano-autophagy inducing preparation described in the present invention exhibits near-infrared light responsiveness.

[0017] In the present invention, when the MOF material is Cu - BTC, the present invention for the first time demonstrates that molecules with the function of inducing autophagy in cells by both Cu and BTC are delivered to tumors for anti - tumor purposes. Description of the Drawings

[0018] Figure 1 It is the TEM image of the nano - autophagy - inducing preparation prepared in Example 2;

[0019] Figure 2 It is the elemental distribution map of the nano - autophagy - inducing preparation prepared in Example 2;

[0020] Figure 3 It is the Zeta potential and particle size characterization map of UCBH and UCB prepared in Example 1 and Example 2;

[0021] Figure 4 It is the Zeta potential and particle size characterization map of UCBH prepared in Example 2 placed in a culture medium solution containing 10% serum for 1 - 7 days;

[0022] Figure 5 It is the diagram of the change of UCBH prepared in Example 2 in response to near - infrared light;

[0023] Figure 6 It is the evaluation diagram of the effect of the nano - autophagy - inducing preparations UCBH and UCB on killing three kinds of tumor cells in vitro after light irradiation;

[0024] Figure 7 It is the evaluation result diagram of the in vitro uptake of the nano - autophagy - inducing preparations UCBH and UCB;

[0025] Figure 8 It is the evaluation result diagram of the lysosomal escape of the nano - autophagy - inducing preparation UCBH after light irradiation;

[0026] Figure 9 It is the evaluation result diagram of the NO production of the photo - responsive nano - autophagy - inducing preparations UCBH and UCB after light treatment;

[0027] Figure 10 It is the evaluation result diagram of the killing effect of the nano - autophagy - inducing preparation at the two - dimensional cell level;

[0028] Figure 11 It is the evaluation result diagram of the killing effect of the nano - autophagy - inducing preparation at the three - dimensional cell level;

[0029] Figure 12 It is the WB analysis diagram of the autophagy occurrence of tumor cells after different treatments;

[0030] Figure 13 It is the TEM image of the ultra - thin section of tumor cells in different treatment groups;

[0031] Figure 14 ULK1 staining diagrams in tumor cells of different treatment groups;

[0032] Figure 15 Flow cytometry diagrams of mitochondrial membrane potential in tumor cells after different treatments;

[0033] Figure 16 Statistical charts of intracellular acetyl-CoA content in tumor cells after different treatments;

[0034] Figure 17 Flow cytometry diagrams and statistical analysis diagrams of CRT expression levels in tumor cells after different treatments;

[0035] Figure 18 CLSM diagrams of intracellular HMGB1 expression in tumor cells after different treatments;

[0036] Figure 19 Statistical charts of secreted ATP content in tumor cells after different treatments;

[0037] Figure 20 Flow cytometry diagrams and statistical analysis diagrams of DCs maturation in tumor cells after different treatments;

[0038] Figure 21 In vivo distribution diagrams and quantitative statistics of nano-autophagy inducer UCB and UCBH in pancreatic cancer-bearing mice;

[0039] Figure 22 Tumor suppression curves, tumor weights, and survival diagrams of nano-autophagy inducer in pancreatic cancer-bearing mice. Detailed implementation manners

[0040] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0041] The first aspect of the present invention provides a nano-autophagy inducer, which includes a composite material with a core-shell structure and a substance that can generate NO through photolysis and is fixed on the composite material;

[0042] In the composite material, the core is a coffee acid-modified upconversion nanoparticle, and the shell is a MOFs material; the metal center of the MOFs material is a copper ion, and the organic ligand is selected from one or more of carboxylic acid-based organic ligands. This nano autophagy inducer has the functions of light and valence state conversion and can be used to induce excessive autophagy. The nano autophagy inducer described in the present invention can comprehensively activate autophagy to enhance ICD and provide a strong synergistic anti-tumor photoimmunotherapy effect against tumor progression.

[0043] In the present invention, the metal center and the organic ligand form a metal-organic framework (MOFs) material with a structure of A 2 (OOCR) 4 wherein, R represents the aromatic ring part of the carboxylic acid-based organic ligand, and A represents a divalent metal ion; taking Cu-BTC as an example, R represents the aromatic ring part of 1,3,5-benzenetricarboxylic acid (BTC), and A represents a divalent Cu ion. Each Cu ion coordinates with four oxygen atoms, two of which come from the same BTC organic ligand, and the other two come from two different BTC organic ligands respectively to form an octahedral geometric configuration of the Cu-BTC metal-organic framework material.

[0044] In the nano autophagy inducer of the present invention, the content range of each substance is relatively wide. According to a preferred embodiment of the present invention, the content of the coffee acid-modified upconversion nanoparticle is 15-22 wt% of the total mass of the nano autophagy inducer; the content of the MOFs material is 50-70 wt% of the total mass of the nano autophagy inducer; the content of the substance that can generate NO by photolysis is 8-15 wt% of the total mass of the nano autophagy inducer.

[0045] According to a preferred embodiment of the present invention, the nano autophagy inducer further contains a coating material that coats the composite material. The coating material is selected from one or more of hyaluronic acid, cell membrane, folic acid, and targeting peptides, preferably hyaluronic acid; the coating material can not only extend the circulation time of the nano autophagy inducer in the blood in vivo, but also promote tumor accumulation and cell uptake through CD44 binding; it is beneficial to improve the anti-tumor photoimmunotherapy effect.

[0046] According to a preferred embodiment of the present invention, the content of the coating material is 5-12 wt% of the total mass of the nano autophagy inducer.

[0047] According to a preferred embodiment of the present invention, the content of caffeic acid-modified upconversion nanoparticles is 15-22 wt% of the total mass of the nano-autophagy inducer preparation, the content of MOFs material is 50-70 wt% of the total mass of the nano-autophagy inducer preparation, the content of the substance capable of generating NO by photolysis is 8-15 wt% of the total mass of the nano-autophagy inducer preparation, and the content of the coating material is 5-12 wt% of the total mass of the nano-autophagy inducer preparation. Preferably, the content of caffeic acid-modified upconversion nanoparticles is 15-20 wt% of the total mass of the nano-autophagy inducer preparation, the content of MOFs material is 50-70 wt% of the total mass of the nano-autophagy inducer preparation, the content of the substance capable of generating NO by photolysis is 8-15 wt% of the total mass of the nano-autophagy inducer preparation, and the content of the coating material is 5-12 wt% of the total mass of the nano-autophagy inducer preparation.

[0048] The content of caffeic acid-modified upconversion nanoparticles is 15-20% of the total mass of the nano-autophagy inducer preparation, the content of MOFs material is 55-68% of the total mass of the nano-autophagy inducer preparation, the content of the substance capable of generating NO by photolysis is 8-12% of the total mass of the nano-autophagy inducer preparation, and the content of the coating material is 7-12% of the total mass of the nano-autophagy inducer preparation.

[0049] In the present invention, the range of the types of substances capable of generating NO by photolysis is relatively wide. According to a preferred embodiment of the present invention, the substance capable of generating NO by photolysis is selected from one or more of N,N'-di-sec-butyl-N,N'-dinitroso-1,4-benzenediamine (BNN6), nitrosothiol, and nitrobenzene.

[0050] In the present invention, in the caffeic acid-modified upconversion nanoparticles, the mass ratio of caffeic acid to upconversion nanoparticles has a relatively wide range of options. According to a preferred embodiment of the present invention, the mass ratio of caffeic acid to upconversion nanoparticles is 1:5-20.

[0051] In the present invention, the upconversion nanoparticles are used to generate ultraviolet light by near-infrared light excitation to decompose the MOFs material. There is no particular limitation on the type of upconversion nanoparticles, as long as they can generate ultraviolet light (such as ultraviolet light around 365 nm) by near-infrared light (such as 980 nm laser) excitation.

[0052] Upconversion nanoparticles generally refer to those containing luminescent centers and matrix materials. The matrix materials include fluorides, oxides, etc. Among them, the fluorides can be, for example, one or more of NaYF, NaGdF, and LiYF; the oxides can be, for example, YO and / or ZrO. The upconversion nanoparticles can also reduce quenching caused by surface defects by coating an inert shell (such as a NaYF shell).

[0053] In the present invention, the luminescence center is, for example, Yb 3 +, Nd 3 +, Er 3 +, Tm 3 +, Ho3+ or a combination of one or more thereof, as long as it can generate ultraviolet light (e.g., ultraviolet light around 365 nm) by excitation with near-infrared light (e.g., 980 nm laser). According to a preferred embodiment of the present invention, by mass content, the upconversion nanoparticles contain 20-45% Yb, 0.1%-2% Tm, and 53%-81% matrix material; preferably, the upconversion nanoparticles contain 25%-35% Yb, 0.3-0.8% Tm, and 64.2%-74.7% matrix material.

[0054] According to a preferred embodiment of the present invention, in the MOFs material, the organic ligand is selected from at least one of trimesic acid, terephthalic acid, phthalic acid, biphenyl-3,4,5-tricarboxylic acid, 1,4-naphthalenedicarboxylic acid, biphenyldicarboxylic acid, and 3,3,5,5-biphenyltetracarboxylic acid, and preferably is trimesic acid.

[0055] According to a preferred embodiment of the present invention, in the MOFs material, the mass ratio of copper ions to the organic ligand is 1:1-5, preferably 1:1-3.

[0056] In the present invention, the fixation of the substance capable of generating NO by photolysis on the composite material means that in environments such as water, small molecule alcohols, chloroform, tetrahydrofuran, dichloromethane, dimethyl sulfoxide, serum-containing solution, blood, PBS buffer solution, DMEM, 1640 medium, etc., the substance capable of generating NO by photolysis can stably exist on the composite material, for example, the substance capable of generating NO by photolysis does not decompose or transfer; according to a preferred embodiment of the present invention, the substance capable of generating NO by photolysis is encapsulated in the cavity formed by the core-shell structure and / or the pore structure of the MOFs material.

[0057] According to a preferred embodiment of the present invention, the nano autophagy inducer has a size between 150 nm and 300 nm.

[0058] The second aspect of the present invention provides a preparation method of the nano autophagy inducer described in the present invention, and the method includes:

[0059] Method 1: Contact a mixed solution containing coffee acid-modified upconversion nanoparticles, copper ions, and a hydrophilic polymer with a solution containing an organic ligand and a substance capable of generating NO by photolysis, and separate to obtain a solid; or

[0060] Method 2: Contact the mixture of the upconversion nanoparticles modified with caffeic acid, copper ions and the hydrophilic polymer with the solution containing the organic ligand, and separate the solid; Mix the solid with the solution containing the substance capable of generating NO through photolysis, and separate to obtain the solid.

[0061] In the present invention, when a substance capable of generating NO through photolysis is added during the preparation of the composite material with a core-shell structure or after the composite material with a core-shell structure is prepared, and the substance capable of generating NO through photolysis is contacted with the composite material, the substance capable of generating NO through photolysis can be stably fixed on the composite material.

[0062] When a substance capable of generating NO through photolysis is added during the preparation of the composite material with a core-shell structure, the substance capable of generating NO through photolysis is encapsulated and fixed in the cavity formed by the core-shell structure and / or the pore structure of the MOFs material. After the composite material with a core-shell structure is prepared, when the substance capable of generating NO through photolysis is contacted with the composite material, the substance capable of generating NO through photolysis is fixed in the pore structure of the MOFs material.

[0063] According to a preferred embodiment of the present invention, the weight-average molecular weight of the hydrophilic polymer is 10,000-50,000 g / mol.

[0064] In the present invention, the type of the hydrophilic polymer can be selected from a relatively wide range. According to a preferred embodiment of the present invention, the hydrophilic polymer is selected from one or more of polyvinylpyrrolidone, polyvinyl alcohol and polyethylene glycol.

[0065] In the present invention, the amount of the hydrophilic polymer can be selected from a relatively wide range. According to a preferred embodiment of the present invention, the mass ratio of copper element to the hydrophilic polymer is 1:0.1-1.

[0066] In the present invention, in the said mixture, the mass ratio of copper element to the upconversion nanoparticles modified with caffeic acid is 1:0.005-0.03.

[0067] In the present invention, the concentration of copper ions in the said mixture can be selected from a relatively wide range. According to a preferred embodiment of the present invention, the concentration of copper ions is 1-50 g / L.

[0068] According to a preferred embodiment of the present invention, in the solution containing the organic ligand and the substance capable of generating NO through photolysis, the concentration of the organic ligand is 0.2-30 mg / mL, and the concentration of the substance capable of generating NO through photolysis is 0.2-10 mg / mL.

[0069] There is no special requirement for the dosage ratio of the solution containing the organic ligand and the substance capable of generating NO through photolysis to the mixture. It is determined according to the MOFs material. The mass ratio of copper ions to the organic ligand is 1:1-5, preferably 1:1-3.

[0070] In the present invention, in Method 1, when forming a core-shell structured composite material by contacting and encapsulating a substance capable of generating NO through photolysis within the composite material, the range of optional contact conditions is relatively wide. According to a preferred embodiment of the present invention, the contact conditions include: a temperature of 10 - 40°C and a time of 0.5 - 12 h; preferably, the contact is carried out under dynamic conditions, more preferably under stirring conditions, and even more preferably at a stirring speed of 500 - 2000 rpm / min.

[0071] In the present invention, in Method 2, when the core-shell structured composite material is formed by contacting, the range of optional contact conditions is relatively wide. According to a preferred embodiment of the present invention, the contact conditions include: a temperature of 10 - 40°C and a time of 0.5 - 12 h; preferably, the contact is carried out under dynamic conditions, more preferably under stirring conditions, and even more preferably at a stirring speed of 500 - 2000 rpm / min.

[0072] In the present invention, for the mixed solution, the solution containing the organic ligand and the substance capable of generating NO through photolysis, the types of solvents have a relatively wide range of options. According to a preferred embodiment of the present invention, in the mixed solution containing copper ions and hydrophilic polymers, the solvents are each independently selected from one or more of small molecule alcohols, chloroform, tetrahydrofuran, dichloromethane, and dimethyl sulfoxide. Preferably, the small molecule alcohols include at least one of methanol, ethanol, propanol, butanol, and pentanol.

[0073] In the mixed solution of the present invention, the solvent is used to form a liquid-phase contact environment, and there is no particular limitation on the amount of the solvent.

[0074] In the present invention, in Method 2, when a solid is mixed with a solution containing a substance capable of generating NO through photolysis, as long as the substance capable of generating NO through photolysis can be fixed onto the composite material, there is no particular requirement for the concentration of the solution containing the substance capable of generating NO through photolysis. According to a preferred embodiment of the present invention, the concentration is 0.2 - 10 mg / mL.

[0075] According to a preferred embodiment of the present invention, the present invention provides a method for preparing a nano-autophagy inducing preparation, which method includes:

[0076] Contacting a mixed solution containing coffee acid-modified upconversion nanoparticles, copper ions, and a hydrophilic polymer with a solution containing an organic ligand and a substance capable of generating NO through photolysis, and separating to obtain a solid;

[0077] In the mixed solution, the mass ratio of copper element to the upconversion nanoparticles modified with caffeic acid is 1:0.005 - 0.03, the mass ratio of copper element to the hydrophilic polymer is 1:0.1 - 1, and the concentration of copper ions is 1 - 50 g / L; in the solution containing the organic ligand and the substance capable of generating NO by photolysis, the concentration of the organic ligand is 0.2 - 30 mg / mL, and the concentration of the substance capable of generating NO by photolysis is 0.2 - 10 mg / mL.

[0078] According to a preferred embodiment of the present invention, in the above method, in the mixed solution, the mass ratio of copper element to the upconversion nanoparticles modified with caffeic acid is 1:0.01 - 0.015, the mass ratio of copper element to the hydrophilic polymer is 1:0.5 - 0.8, and the concentration of copper ions is 1 - 15 g / L; in the solution containing the organic ligand and the substance capable of generating NO by photolysis, the concentration of the organic ligand is 3 - 12 mg / mL, and the concentration of the substance capable of generating NO by photolysis is 2 - 9 mg / mL.

[0079] In the present invention, there is no particular limitation on the preparation method of the upconversion nanoparticles modified with caffeic acid. According to a preferred embodiment of the present invention, the preparation method of the upconversion nanoparticles modified with caffeic acid includes: mixing the mixed solution containing oleic acid-stabilized upconversion nanoparticles with the solution containing caffeic acid, separating, and washing. Preferably, the mixing conditions include: the mixing temperature is 10 - 40 °C, and the mixing time is 0.5 - 24 h.

[0080] In the oleic acid-stabilized upconversion nanoparticles, oleic acid is used to stabilize the upconversion nanoparticles, and the content of oleic acid can be the conventional content in the art.

[0081] In the present invention, the method further includes mixing the separated solid with the solution containing the coating material and separating.

[0082] In the present invention, there is no particular limitation on the mixing conditions, as long as the coating material can coat the composite material. For example, after mixing the separated solid with the solution containing the coating material, it can be left standing for a period of time. In the examples of the present invention, it is exemplified that the mixing is carried out by leaving it standing at room temperature for 20 min to illustrate the advantages of the present invention.

[0083] In the present invention, in the solution containing the coating material, there is no particular limitation on the concentration of the coating material. According to a preferred embodiment of the present invention, in the solution containing the coating material, the concentration of the coating material is 0.1 - 20 g / L.

[0084] In the present invention, there is no particular limitation on the separation method, as long as solid-liquid separation can be carried out, such as centrifugation and filtration. In the examples of the present invention, centrifugation is exemplified to illustrate the advantages of the present invention.

[0085] The third aspect of the present invention provides an application of the nano-autophagy inducer preparation of the present invention in the preparation of a drug for treating tumors. The nano-autophagy inducer preparation of the present invention can comprehensively activate autophagy to enhance ICD, and provides a strong synergistic anti-tumor photoimmunotherapy effect against tumor progression.

[0086] According to a preferred embodiment of the present invention, the tumor is one or more of pancreatic cancer tumors, breast cancer tumors, and liver cancer tumors; the nano-autophagy inducer preparation of the present invention is particularly suitable for activating autophagy in the aforementioned tumors.

[0087] The fourth aspect of the present invention provides a pharmaceutical composition containing the nano-autophagy inducer preparation of the present invention and pharmaceutically acceptable excipients.

[0088] In the present invention, the pharmaceutically acceptable excipients are selected from one or more of fillers, disintegrants, lubricants, and solvents.

[0089] According to a preferred embodiment of the present invention, the filler is selected from one or more of starch, lactose, dextrin, and sucrose.

[0090] According to a preferred embodiment of the present invention, the disintegrant is selected from one or more of starch, microcrystalline cellulose, calcium carboxymethylcellulose, and croscarmellose sodium.

[0091] According to a preferred embodiment of the present invention, the lubricant is selected from one or more of magnesium stearate, talc, polyethylene glycol, and sodium lauryl sulfate.

[0092] In the present invention, the types of solvents in the excipients have a relatively wide range of options. By way of example, but not limiting the scope of the present invention thereby, according to a preferred embodiment of the present invention, the solvent is selected from one or more of PBS and physiological saline.

[0093] In the present invention, there is no particular limitation on the dosage form of the drug prepared from the nano-autophagy inducer preparation, and it can be prepared into a corresponding dosage form according to the types of excipients, such as one or more of tablets, pills, powders, injections, and solutions.

[0094] In the context of the present invention specification, including the following examples, the various characterizations are as follows:

[0095] Measurement of the particle size and Zeta potential of the nano-autophagy inducer preparation:

[0096] The Zeta potential of the nanoautophagy-inducing preparation was tested using DLS. The nanoautophagy-inducing preparation was dispersed in deionized water, and the Zeta potential of the nanoautophagy agent was characterized using a Malvern laser particle size analyzer NANO ZS from the UK. The Zeta potential of the nanoautophagy agent at 100 μg / mL in ultrapure water with pH = 7 was measured at room temperature, and the average value was calculated by repeating the measurement three times.

[0097] Morphology and structure characterization of the nanoautophagy-inducing preparation:

[0098] The morphology of the nanoautophagy-inducing preparation was observed using TEM (120 kv). After determining the uniformly dispersed nanoautophagy-inducing preparation, HAADF-STEM was used to observe the elemental distribution of the nanoautophagy-inducing preparation.

[0099] The contents of organic ligands and substances that can generate NO through photolysis in the nanoautophagy-inducing preparation were determined using a UV-spectrophotometer; the contents of copper ions and upconversion nanoparticle elements in the nanoautophagy-inducing preparation were determined using an inductively coupled plasma mass spectrometer ICP-MS; the mass of the coating material was obtained by subtracting the total mass of the composite material and the mass of the substances that can generate NO through photolysis from the total mass of the nanoautophagy-inducing preparation.

[0100] Determination of the content of substances that can generate NO through photolysis (such as BNN6): Five standard solutions with different concentrations (1000, 500, 200, 100, 50, 20, 10, 5, 1 μg / mL) were prepared, and their absorbances at 360 nm were measured respectively. The standard curve was obtained by calculation and normalization. UCBH with a known mass was treated with concentrated hydrochloric acid and then diluted with deionized water. The absorbance value at 360 nm was measured, and the content of substances that can generate NO through photolysis (such as BNN6) in UCBH was calculated by combining with the standard curve.

[0101] Determination of the content of organic ligands (such as BTC): Five standard solutions with different concentrations (1000, 500, 200, 100, 50, 20, 10, 5, 1 μg / mL) were prepared, and their absorbances at 260 nm were measured respectively. The standard curve was obtained by calculation and normalization. The nanoautophagy-inducing preparation with a known mass was treated with concentrated hydrochloric acid and then diluted with deionized water. The absorbance value at 260 nm was measured, and the content of organic ligands (such as BTC) in the nanoautophagy-inducing preparation was calculated by combining with the standard curve.

[0102] Stability characterization of the nanoautophagy-inducing preparation

[0103] The particle size and zeta potential of the nano-autophagy inducing preparation were tested using DLS. Zeta potential and particle size test conditions: The Zeta potential of the nano-autophagy agent was characterized using a Malvern laser particle size analyzer NANO ZS from the UK. The Zeta potential or particle size of the nano-autophagy inducing preparation at 100 μg / mL in ultrapure water at pH = 7 was measured at room temperature, and the average value was calculated by repeating the measurement three times.

[0104] Characterization of the photoresponse performance of the nano-autophagy inducing preparation

[0105] 1. Characterization of the particle size change of the nano-autophagy inducing preparation before and after responding to near-infrared light:

[0106] To verify the NIR photoresponse performance of the nano-formulation nano-autophagy inducing preparation, TEM and DLS were used to test the morphological and particle size changes of the nano-autophagy inducing preparation before and after responding to NIR. The aqueous solution of the nano-autophagy inducing preparation (0.1 mL, 1 mg / mL) was irradiated with near-infrared light (980 nm, 5 min, 0.6 W / cm 2 ), after the irradiation ended, a part of the solution was diluted with deionized water at a ratio of 1:100 of the original solution for TEM observation, and the reaction solution was diluted with deionized water at a ratio of 1:50 of the original solution for DLS to detect the particle size.

[0107] 2. Characterization of the release of copper ions, organic ligands (such as BTC) and nitric oxide (NO) by the nano-autophagy inducing preparation in response to near-infrared light

[0108] ICP-MS and ultraviolet-spectrophotometer were used to investigate the ability of the nano-autophagy inducing preparation to release copper ions and organic ligands in response to near-infrared light. After the light treatment, the supernatant was collected by centrifugation, and the contents of copper element and organic ligand in the supernatant were determined by the above ICP-MS and ultraviolet-spectrophotometry respectively.

[0109] The ultraviolet-spectrophotometer was used to investigate the ability of the nano-autophagy inducing preparation to release NO in response to near-infrared light. As described in the above experiment, the aqueous solution of the nano-autophagy inducing preparation (0.1 mL, 1 mg / mL) was irradiated with near-infrared light (980 nm, 5 min, 0.6 W / cm 2 ), after the light treatment, the supernatant was collected by centrifugation, and the concentration of NO in the solution was measured using a commercial NO detection probe (2,3-diaminonaphthalene, DAN).

[0110] Fluorescent labeling of the nano-autophagy inducing preparation

[0111] The fluorescence staining method involved in subsequent cell experiments and animal experiments is as follows: Dilute the nano autophagy inducer preparation with MES buffer solution (1 mg of nano autophagy inducer preparation is resuspended in 1 ml of MES solution), add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (mass ratio, nano autophagy inducer preparation: NHS: EDC = 1:1:2), react at 37 °C for 4 h to obtain UCBH with activated carboxyl groups, and then resuspend with NaHCO 3 / Na2CO 3 buffer solution, and then add Cy7-PEG 2000 -NH 2 (mass ratio, nano autophagy inducer preparation: Cy7-PEG 2000 -NH 2 = 1:0.1).

[0112] In the present invention, including the following examples, in each statistical analysis chart, P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).

[0113] In the following examples, oleic acid-stabilized upconversion nanoparticles (UCNP-OA) were purchased from Xi'an Haoran Biotechnology Co., Ltd., product number R-UY450; according to elemental analysis, the upconversion nanoparticles UCNP contained 30% Yb, 0.5% Tm, and the rest were matrix materials.

[0114] In the following examples, the weight-average molecular weight of polyvinylpyrrolidone was 24000 g / mol.

[0115] Example 1

[0116] (1) Blend 3 mL of chloroform with 1 mL of UCNP-OA (10 mg / mL), then add 10 mL of a solution of dihydrocaffeic acid (DHCA) dissolved in tetrahydrofuran (concentration 20 mg / mL), and mix well by ultrasound. Subsequently, transfer to an oil bath at 45 °C and react for 24 hours. After the reaction is completed, collect UCNP-DHCA by high-speed centrifugation (10000 rpm / min, 30 min), wash three times alternately with DMF and ethanol, and finally dissolve in 5 mL of methanol for standby, with a final concentration of 2 mg / mL.

[0117] (2) Weigh 47 mg of copper nitrate, 24 mg of polyvinylpyrrolidone, 20 mg of BNN6, and 25.8 mg of trimesic acid. Then, dissolve the above copper ions and PVP together in 3 mL of methanol. Subsequently, add 100 μL of 2 mg / mL UCNP-DHCA. Then, transfer the mixed solution to a water bath for ultrasonic treatment for 5 min. Then, add a mixed solution of BTC and BNN6 dissolved in 3 mL of methanol and stir rapidly at room temperature for 10 min (1000 rpm / min). Subsequently, reduce the rotation speed to 500 rpm and react for 4 hours. When the reaction is completed, the reaction system shows sky blue. Centrifuge at high speed (10000 g, 10 min) to remove the excess components and obtain a precipitate nano-preparation, denoted as UCB. Wash the precipitate UCB three times with anhydrous ethanol and deionized water respectively. Finally, resuspend UCB in deionized water (concentration: 10 mg / mL) for standby.

[0118] By quantitative analysis using ICP-MS and ultraviolet-spectrophotometry, by mass ratio, copper accounts for 26.05% in UCBH, BTC accounts for 42.23%, BNN6 accounts for 11.32%, and UCNP-DHCA accounts for 20.4%.

[0119] Example 2

[0120] (1) Blend 3 mL of chloroform with 1 mL of UCNP-OA (10 mg / mL). Subsequently, add 10 mL of a solution of caffeic acid (DHCA) dissolved in tetrahydrofuran (concentration: 20 mg / mL) and mix well by ultrasonic treatment. Then, transfer it to an oil bath at 45 °C and react for 24 hours. After the reaction is completed, centrifuge at high speed (10000 rpm, 30 min) to collect UCNP-DHCA, and wash it three times alternately with DMF and ethanol. Finally, dissolve it in 5 mL of methanol for standby, with a final concentration of 2 mg / mL.

[0121] (2) Weigh 47 mg of copper nitrate, 24 mg of polyvinylpyrrolidone, 20 mg of BNN6, and 25.8 mg of trimesic acid. Then, dissolve the above copper ions and PVP together in 3 mL of methanol. Subsequently, add 100 μL of 2 mg / mL UCNP-DHCA. Then, transfer the mixed solution to a water bath for ultrasonic treatment for 5 min. Then, add a mixed solution of BTC and BNN6 dissolved in 3 mL of methanol and stir rapidly at room temperature for 10 min (1000 rpm). Subsequently, reduce the rotation speed to 500 rpm and react for 4 hours. When the reaction is completed, the reaction system shows sky blue. Centrifuge at high speed (10000 g, 10 min) to remove the excess components and obtain a precipitate nano-preparation UCB. Wash the precipitate UCB three times with anhydrous ethanol and deionized water respectively. Finally, resuspend UCB in deionized water (concentration: 10 mg / mL) for standby.

[0122] (3) Prepare a 1 mg / mL hyaluronic acid (HA) solution using deionized water; then, blend HA and UCB (1 mg / mL) at a volume ratio of 4:1; after reacting at room temperature for 20 minutes, centrifuge at high speed to remove excess HA molecules; when the reaction is complete, a solid is obtained, denoted as UCBH, which is a nanoautophagy-inducing preparation, and it is resuspended with deionized water for standby (concentration: 1 mg / mL).

[0123] The TEM image of the nanoautophagy-inducing preparation (UCBH) is as Figure 1 shown, where UCB has a typical core-shell structure, and the shell layer is a typical octahedral structure; the outermost layer is HA coating the UCB.

[0124] The elemental distribution map of the nanoautophagy-inducing preparation is as Figure 2 shown. The nanoautophagy-inducing preparation contains basic components of synthetic raw materials such as copper (Cu), nitrogen (N), oxygen (O), yttrium (Y), and ytterbium (Yb). And through elemental analysis, it can be known that each element is distributed on the nanoautophagy-inducing preparation in a specific distribution pattern, showing a typical core-shell structure.

[0125] By quantitative analysis using ICP-MS and ultraviolet-spectrophotometry, by mass ratio, copper accounts for 23.71% in UCBH, BTC accounts for 38.44%, BNN6 accounts for 10.30%, UCNP-DHCA accounts for 18.57%, and HA accounts for 8.98%.

[0126] Dynamic light scattering (DLS) was used to characterize the Zeta potential and particle size of the nanoautophagy-inducing preparation (UCBH) and the nanoautophagy-inducing preparation (UCB), as Figure 3 shown. The particle size of UCBH is 210 nm, and the Zeta potential is -12.5 mV. The particle size of UCB is 200 nm, and the Zeta potential is -7 mV.

[0127] The premise for the nanoautophagy-inducing preparation to be effective after intravenous injection is that it can remain stable. Dispersing the nanoautophagy-inducing preparation in a medium containing 10% serum and placing it in an environment at 37 °C can simulate its physiological environment in the body. Samples were collected at different time points (0, 1, 2, 3, 4, 5, 6, 7 days), and DLS was used to measure the changes in its particle size and Zeta potential to determine its stability within 7 days. As Figure 4 shown, the results show that the particle size and potential of the nanoautophagy-inducing preparation remain basically stable within 7 days without large fluctuations, further proving its good stability and suitability for intravenous injection.

[0128] Apply 0.6 W / cm 2The nanoautophagy-inducing preparation was irradiated with NIR light (980 nm) for 5 min, and then TEM was used to observe the morphological changes and corresponding particle size changes before and after light irradiation, and the released Cu, BTC, and NO contents were measured. After light irradiation, the change diagram of the nanoautophagy-inducing preparation UCBH in response to near-infrared light is as shown in Figure 5 shown, and the TEM image is as shown in Figure 5 (A). The nanoautophagy-inducing preparation disintegrated. The photosensitivity of the nanoautophagy-inducing preparation can also be obtained from the change in particle size. After light irradiation, the particle size of the nanoautophagy-inducing preparation increased from about 200 nm to about 2000 nm, as shown in Figure 5 (B). By measuring the Cu, BTC, and NO contents in the supernatant of the centrifuged sample, it was found that copper, BTC, and NO were released from the nanoautophagy-inducing preparation after light treatment, as shown in Figure 5 (C), Figure 5 (D). Approximately 40% of Cu was released from the nanoautophagy-inducing preparation, and approximately 66% of BTC was released from the nanoautophagy-inducing preparation. In the supernatant, the NO content was about 3.1 μM.

[0129] Therefore, the above results comprehensively show that in the nanoautophagy-inducing preparation, BNN6 can be stably encapsulated in the cavity formed by the core-shell structure and / or the pore structure of the MOF material. The excellent light response performance of the nanoautophagy-inducing preparation lays a foundation for subsequent safe and efficient killing of tumors.

[0130] Example 3

[0131] (1) 3 mL of chloroform was blended with 1 mL of UCNP-OA (10 mg / mL), and then 10 mL of a caffeic acid (DHCA) solution dissolved in tetrahydrofuran (concentration: 20 mg / mL) was added, and the mixture was ultrasonically mixed. Subsequently, it was transferred to an oil bath at 45 °C and reacted for 24 hours. After the reaction was completed, UCNP-DHCA was collected by high-speed centrifugation (10000 rpm, 30 min), washed three times alternately with DMF and ethanol, and finally dissolved in 5 mL of methanol for standby, with a final concentration of 2 mg / mL.

[0132] (2) Weigh 47 mg of copper nitrate, 24 mg of polyvinylpyrrolidone, and 25.8 mg of trimesic acid. Then, dissolve the above copper ions and PVP together in 3 mL of methanol. Subsequently, add 100 μL of 2 mg / mL UCNP-DHCA. Then, transfer the mixed solution to a water bath for ultrasonic treatment for 5 min. Then, add a BTC mixed solution dissolved in 3 mL of methanol and stir rapidly at room temperature for 10 min (1000 rpm). Subsequently, reduce the rotation speed to 500 rpm and react for 4 hours. When the reaction is completed, the reaction system shows sky blue. Centrifuge at high speed (10000 g, 10 min) to remove the excess components and obtain the precipitate nanopreparation UCB. Wash the precipitate UCB three times with absolute ethanol and deionized water respectively, and finally resuspend UCB in deionized water (concentration: 10 mg / mL) for standby.

[0133] (3) Dissolve 20 mg of BNN6 in methanol to obtain a solution. Then, blend BNN6 and UCB (1 mg / mL) at a volume ratio of 4:1. After reacting at room temperature for 20 minutes, centrifuge at high speed to separate the solid, and resuspend it with deionized water for standby (concentration: 1 mg / mL).

[0134] By quantitative analysis using ICP-MS and ultraviolet-spectrophotometry, by mass ratio, in the solid, copper accounts for 24.29%, BTC accounts for 39.38%, BNN6 accounts for 8.1%, UCNP-DHCA accounts for 19.03%, and HA accounts for 9.2%.

[0135] Characterize the Zeta potential and particle size of the solid by dynamic light scattering (DLS), and the test results are shown in Table 1.

[0136] According to the method of Example 2, conduct a stability test on the solid material, and the test results are shown in Table 1.

[0137] According to the method of Example 2, irradiate the nano-autophagy induction preparation with NIR light (980 nm) of 0.6 W / cm 2 for 5 min, and test the contents of released Cu, BTC, and NO. The test results are shown in Table 1.

[0138] Example 4

[0139] According to the method of Example 2, the difference is that in step (2), 25 mg of nitrosothiol is used to replace 20 mg of BNN6; in step (3), folic acid is used to replace HA, and the other conditions are the same as in Example 2.

[0140] By quantitative analysis using ICP-MS and UV-spectrophotometry, by mass ratio, copper accounts for 23.88%, BTC accounts for 38.71%, nitrosothiol accounts for 12.4%, UCNP-DHCA accounts for 18.7%, and folic acid accounts for 6.31%.

[0141] Dynamic light scattering (DLS) was used to characterize the Zeta potential and particle size of the nanoautophagy-inducing preparation (UCBH) and the nanoautophagy-inducing preparation (UCB). The test results are shown in Table 1.

[0142] According to the method of Example 2, the stability test of the solid material was carried out. The test results are shown in Table 1.

[0143] According to the method of Example 2, 0.6 W / cm 2 of NIR light (980 nm) was irradiated on the nanoautophagy-inducing preparation for 5 min, and the contents of released Cu, BTC, and NO were tested. The test results are shown in Table 1.

[0144] Comparative Example 1

[0145] (1) 3 mL of chloroform was blended with 1 mL of UCNP-OA (10 mg / mL), and then 10 mL of a solution of caffeic acid (DHCA) dissolved in tetrahydrofuran (concentration: 20 mg / mL) was added, and the mixture was sonicated and mixed evenly. Subsequently, it was transferred to an oil bath at 45 °C and reacted for 24 hours. After the reaction was completed, UCNP-DHCA was collected by high-speed centrifugation (10000 rpm / min, 30 min), and washed three times alternately with DMF and ethanol, and finally dissolved in 5 mL of methanol for standby, with a final concentration of 2 mg / mL.

[0146] (2) 47 mg of copper nitrate, 24 mg of polyvinylpyrrolidone, and 25.8 mg of trimesic acid were weighed. Then, the above copper ions and PVP were dissolved together in 3 mL of methanol, and then 100 μL of 2 mg / mL UCNP-DHCA was added; then, the mixed solution was transferred to a water bath for sonication for 5 min; then, a BTC mixed solution dissolved in 3 mL of methanol was added, and the mixture was rapidly stirred at room temperature for 10 min (1000 rpm); subsequently, the rotation speed was reduced to 500 rpm and the reaction was carried out for 4 hours. When the reaction was completed, the reaction system showed sky blue, and excess components were removed by high-speed centrifugation (10000 g, 10 min) to obtain the precipitated nano-preparation UCB. The precipitated UCB was washed three times with anhydrous ethanol and deionized water respectively, and finally UCB was resuspended in deionized water (concentration: 10 mg / mL) for standby.

[0147] (3) Prepare a 1 mg / mL hyaluronic acid (HA) solution using deionized water; then, blend HA and UCB (1 mg / mL) at a volume ratio of 4:1; after reacting at room temperature for 20 minutes, centrifuge at high speed to remove excess HA molecules; when the reaction is complete, obtain a solid and resuspend it with deionized water for standby (concentration: 1 mg / mL).

[0148] (4) Dissolve 20 mg of BNN6 using methanol to obtain a solution; then, blend BNN6 and the solid obtained in step (3) (1 mg / mL) at a volume ratio of 4:1; after reacting at room temperature for 20 minutes, centrifuge to separate and obtain a solid, and resuspend it with deionized water for standby (concentration: 1 mg / mL).

[0149] By quantitative analysis using ICP-MS and ultraviolet-spectrophotometry, by mass ratio, the proportion of copper in UCBH is 26.43%, BTC is 42.85%, UCNP-DHCA is 20.7%, and HA is 10.02%.

[0150] Table 1

[0151] Example 3 Example 4 Comparative Example 1 Zeta potential, mV -10.3 -8.7 -8 Particle size, nm 193 156 203 Zeta potential, mV after 7-day stability test -10 -8.7 -8 Particle size, nm after 7-day stability test 193 156 203 Copper release amount after light treatment 35.72％ 31.66％ 43.89％ BTC release amount after light treatment 57.28％ 52.48％ 70.38％ NO content in supernatant after light exposure, uM 2.2 3.5 0

[0152] The anti-tumor characterization of the nano-autophagy inducing preparations prepared in each example is as follows:

[0153] 1. Cell culture

[0154] Select Pan02-luc pancreatic cancer cells for the main experiment. Pan02-luc pancreatic cancer cells are cultured using DMEM medium supplemented with 5% fetal bovine serum, 100 U / mL penicillin, and streptomycin, in an incubator at 37 °C and 5% CO 2 and passaged once every other day at a ratio of 1:3.

[0155] 2. Investigation of the uptake of nano-autophagy inducing preparations by Pan02-luc cells

[0156] Use CLSM to investigate the uptake of nano-autophagy inducing preparations by Pan02-luc cells. Use flow cytometry to investigate the endocytosis kinetics of Pan02-luc cells taking up CuNM (0, 2, 4, 6, 8, 12, 24, 48 h).

[0157] 3. Investigation of the lysosomal escape of nano-autophagy inducing preparations after light irradiation

[0158] The lysosomal escape of Pan02-luc cells treated with the nanoautophagy-inducing agent was examined using CLSM. Specifically, the nanoautophagy-inducing agent labeled with Cy5 was first co-incubated with Pan02-luc, followed by light treatment. Lysosomes in the cells were stained with a lysosome probe, and then the co-localization rate of the particles and lysosomes was observed using CLSM.

[0159] 4. Investigation of the production of NO by the nanoautophagy-inducing agent in tumor cells

[0160] The following groups were set up according to the experimental requirements: PBS, UC, UC+L, UCB+L, and UCBH+L. The concentration of UC was 100 μg / mL, and after culturing for 24 hours, light treatment was carried out. Subsequently, the cells were stained with a NO detection kit, and then the production of NO was evaluated using CLSM. Among them, UC is a composite material formed by coating UCNP with a MOF material formed by Cu and BTC.

[0161] 5. Investigation of the toxicity of the nanoautophagy-inducing agent on different tumor cells

[0162] HepG2 human liver cancer tumor cells, 4T1 mouse breast cancer cells, and Pan02-luc pancreatic cancer cells in the logarithmic growth phase were seeded into 96-well plates (1.5×10 4 cells / well), incubated overnight until they adhered to the wall. The following groups were set up according to the experimental requirements: UC, UC+L, UCB+L, and UCBH+L. Different groups and different concentrations of drugs (using the concentration of UC as the standard, ranging from 0 to 200 μg / mL) were added and co-incubated for 24 hours, followed by light treatment (980 nm, 0.6 W / cm 2 , 5 min), and then cultured for another 12 hours. Then, the cell viability was detected according to the kit.

[0163] 6. Investigation of the killing effect of the nanoautophagy-inducing agent at the two-dimensional cell level using CLSM

[0164] The following groups were set up according to the experimental requirements: PBS, UC, UC+L, UCB+L, and UCBH+L. The concentration of UC was 100 μg / mL, cultured for 24 hours, followed by light treatment, and then cultured for another 12 hours. Subsequently, the cells were stained with a cell viability detection kit, and then the proportion of dead cells was examined and analyzed using CLSM.

[0165] 7. Culture of three-dimensional cell spheroids

[0166] Agarose was added to serum-free DMEM medium, boiled, and the medium mixed with agarose was added to 96-well plates. After cooling and solidifying, it was reserved. The Pan02-luc cell suspension was prepared at 1×10 4Add it to the 96-well plate coated with agarose at a concentration of / mL, and add 200 μL of medium to each well. Incubate at 37 °C to form cell spheres, and subsequent experiments can be carried out after 4-5 days.

[0167] 8. Investigation of the killing effect of nano-autophagy inducing agents on three-dimensional cell spheres by CLSM

[0168] Grouping was carried out according to experimental needs: PBS, UC, UC+L, UCB+L, and UCBH+L. The concentration of UC was 100 μg / mL. After culturing for 24 hours, light treatment was carried out, and then cultured for another 12 hours. After collecting the cell spheres, low-speed centrifugation was carried out to collect the three-dimensional cell spheres, and then washed with PBS to remove the excess drugs. Subsequently, cell viability staining was performed, and the proportion of dead cells in the three-dimensional cell spheres was investigated and analyzed using CLSM.

[0169] 9. Investigation of the mechanism of nano-autophagy inducing agents in killing tumor cells

[0170] 9.1. Analysis of the expression of key proteins in the autophagy pathway

[0171] Western-Blot was used to determine the expression of autophagy pathway-related proteins. Grouping was carried out according to experimental needs: PBS, UC, UC+L, UCB+L, and UCBH+L. Pan02-luc pancreatic cancer cells in the logarithmic growth phase were inoculated into 48-well plates (1*10 5 cells / well), incubated overnight until adherent. Equal amounts of drugs from different groups were added (the concentration of UC was uniformly 100 μg / mL). After culturing for 24 hours, light treatment was carried out, and then cultured for another 12 hours. Then, whole cell proteins were extracted, and subsequent Western-Blot experiments were carried out to determine the content change of autophagy-related protein (LC3B).

[0172] 9.2. Morphological analysis of autophagosomes

[0173] After pancreatic cancer cells were treated with different drugs, the morphology of autophagosomes in pancreatic cancer cells was observed using a biological transmission electron microscope.

[0174] 9.3. Analysis of the activation of autophagy upstream protein ULK1 by copper ions

[0175] After pancreatic cancer cells were treated with different drugs, ULK1 in tumor cells was stained, and the expression content of ULK1 was analyzed and statistically analyzed using CLSM.

[0176] 9.4. Flow analysis of NO damage to tumor cell mitochondria

[0177] After pancreatic cancer cells were treated with different drugs, the mitochondrial membrane potential of the mitochondria in the tumor cells was stained, and a flow cytometer was used to analyze and statistically analyze the changes in the mitochondrial membrane potential.

[0178] 9.5. Investigation of the effect of BTC on intracellular acetyl-CoA content

[0179] After pancreatic cancer cells were treated with different drugs, the cells were collected, and the cells in different groups were treated with RIPA lysate. Subsequently, an acetyl-CoA detection kit was used to detect the changes in the intracellular acetyl-CoA content of different groups.

[0180] 10. Investigation of the activation of ICD effect by nano-autophagy inducer

[0181] 10.1. Investigation of the nano-autophagy inducer inducing the expression of calreticulin in tumor cells

[0182] After pancreatic cancer cells were treated with different drugs, the CRT in the tumor cells was stained, and a flow cytometer was used to analyze and statistically analyze the positive rate of CRT.

[0183] 10.2. Investigation of the nano-autophagy inducer inducing the migration of HMGB1 in tumor cells

[0184] After pancreatic cancer cells were treated with different drugs, the HMGB1 and cell nuclei in the tumor cells were stained, and CLSM was used to analyze and statistically analyze the intracellular expression content of HMGB1.

[0185] 10.3. Investigation of the nano-autophagy inducer inducing the secretion of ATP by tumor cells

[0186] After pancreatic cancer cells were treated with different drugs, the cell supernatant was collected, and then an ATP detection kit was used to detect the content of secreted ATP in different groups.

[0187] 10.4. Investigation of the nano-autophagy inducer promoting the maturation of DCs

[0188] After pancreatic cancer cells were treated with different drugs, the cells were collected and co-incubated with the extracted primary DCs cells for 24 hours; subsequently, flow antibodies were used to stain the DCs cells (CD11c, CD80, and CD86), and finally, a flow cytometer was used to evaluate the maturation of DCs in different groups.

[0189] 11. Animal experiment investigation of nano-autophagy inducer

[0190] 11.1. Construction of a pancreatic cancer mouse model

[0191] Female C57 black mice aged 4 - 6 weeks were selected for the experiment of normal wild - type mice. First, the mice were anesthetized and the hair on the back of the left leg was removed. Under sterile conditions, 2×10 6 Pan02 cells were injected subcutaneously. Three weeks later, a right - hand caliper was used to measure and observe the growth of pancreatic cancer tumors in the mice.

[0192] 11.2 Investigation of the enrichment of nano - autophagy - inducing preparations in tumor tissues

[0193] The tumor - bearing mice were divided into two groups: the UCB group and the UCBH group. Subsequently, 100 μL of UCBH / Cy7 (1 mg / mL) or UCB / Cy7 (1 mg / mL) was respectively injected into the tail vein of the mice with constructed pancreatic cancer. Then, small - animal in - vivo imaging was performed at seven time points of 1, 2, 4, 8, 12, 24, and 48 h to observe the distribution of nano - autophagy - inducing preparations in the in - situ pancreatic cancer mice, and software was used to quantitatively analyze the fluorescence intensity in major organs and tumor tissues.

[0194] 11.3 Investigation of the efficacy of nano - autophagy - inducing preparations on the pancreatic cancer tumor - bearing mouse model

[0195] First, a subcutaneous - tumor pancreatic cancer mouse model was constructed. Pan02 - luc tumor cells (1×10 6 cells / mouse) were inoculated under the armpits of C57 mice. After about three weeks of growth, when the size of the tumor grew to 100 mm 3 , drugs were administered through the tail vein (PBS, UC, UC + L, UCB + L, and UCBH + L), once every other day, for a total of three times, 100 μL / mouse. In the groups that required light treatment, near - infrared light treatment (980 nm, 0.6 W / cm 2 , 5 min / mouse) was performed 12 hours after intravenous drug administration. The tumor volume of the mice was measured every other day until the tumor volume was greater than 1200 mm 3 .

[0196] Experimental results

[0197] 1. Investigation of the universality of the anti - tumor effect of nano - autophagy - inducing preparations UCBH and UCB in vitro

[0198] After successfully preparing the nanoformulation CuNM, its killing effect was further evaluated at the cellular level. First, the killing effect of UCBH on three different cancer cell lines (HepG2 human liver cancer tumor cells, 4T1 mouse breast cancer cells, and Pan02-luc pancreatic cancer cells) was analyzed, and CCK-8 cytotoxicity analysis was performed. By co-incubating different concentrations of UC, UC+L, UCB+L, or UCBH+L with different types of tumor cells and adding CCK-8 reagent, and detecting the absorbance value at a wavelength of 450 nm, the cell survival rate at the corresponding concentration could be obtained. The results of the evaluation of the in vitro killing effect of the three tumor cells after light irradiation are as follows Figure 6 shown. In all cancer cell lines, the survival rate of tumor cells after treatment with UC basically remained above 80%. When light irradiation was added, UC could kill about 30-40% of the tumor cells. With the loading of BNN6, the tumor cell killing rate of the UCB+L group could reach about 40-50%. Notably, when the nanoformulation was modified with HA, the killing effect on tumor cells was increased to about 80%, demonstrating that the photo-responsive nanoformulation constructed in the present invention can effectively kill tumor cells under light irradiation conditions.

[0199] 2. Analysis of the endocytosis of the nano autophagy inducer in the pancreatic cancer cell line Pan02-luc

[0200] Based on the pancreatic cancer cell line, the specific mechanism of the strong killing effect of the nano autophagy inducer was further investigated. First, after co-incubating the nano autophagy inducers UCBH and UCB labeled with Cy5 with Pan02-luc cells respectively, Figure 7 is the result diagram of the in vitro uptake evaluation of the photo-responsive nano autophagy inducer. Figure 7 (A) CLSM images of UCB and UCBH after being taken up by tumor cells; Figure 7 (B) is the statistical analysis diagram of the endocytosis kinetics of UCB and UCBH being taken up by tumor cells. It can be found by CLSM that UCBH can be taken up by Pan02-luc cells in large amounts compared with UCB, and the analysis results also confirm that the endocytosis of UCBH by pancreatic cancer cells is time-dependent and reaches a basic saturation state at 24 h.

[0201] 3. Investigation of the lysosomal escape of the nano autophagy inducer after light irradiation

[0202] After successfully demonstrating that the nanoformulation can be effectively taken up by tumor cells, the next step is to investigate the situation where the nanoformulation breaks through the lysosomal restriction and enters the cytoplasm. First, after co-incubating the nanoformulation UCBH labeled with Cy5 with Pan02-luc cells, light treatment was performed, and then a lysosomal dye was used to stain the cell lysosomes, as Figure 8As shown, CLSM can reveal that the nanoautophagy-inducing agent UCBH can effectively break through the lysosomal restriction after light irradiation, with the co-localization rate decreasing by 28% from 78%, demonstrating that light can effectively promote the lysosomal escape of the nanoagent UCBH.

[0203] 4. Investigation of the production of NO by the nanoautophagy-inducing agent on tumor cells

[0204] After successfully demonstrating that UCBH can effectively induce breakthrough of lysosomal restriction under light irradiation (L), the next step is to verify the production of NO by the nanoautophagy-inducing agent under light conditions. After DAF-FM DA staining, CLSM was used to evaluate the intracellular NO production. The results are as Figure 9 shown. Only the UCBH and UCB treatment groups could detect NO signals, while no obvious NO signals were detected in the treatment groups without loading the BNN6 molecule (PBS, UC, UC+L). It is worth noting that due to the modification of the HA molecule on UCBH, it promoted the uptake of a large amount of UCBH by tumor cells, thereby achieving the strongest NO signal.

[0205] 5. Investigation of the killing effect of the nanoautophagy-inducing agent at the two-dimensional cell level

[0206] To more intuitively observe the cell survival situation, a kit was used to stain the living and dead cells. The evaluation results of the killing effect are as Figure 10 shown. The cell death situation in each group was consistent with the results shown by CCK8. The number of live cells was ranked in the following order: PBS > UC > UC+L > UCB+L > UCBH+L. The above results demonstrate that UCB, which is highly taken up by tumor cells, can effectively kill tumor cells under light conditions, while UCBH can effectively kill more tumor cells under light conditions.

[0207] 6. Investigation of the killing effect of the nanoautophagy-inducing agent on three-dimensional cell spheroids

[0208] Although the nanoagent has been proven to have the ability to potently kill tumor cells at the two-dimensional cell level, the two-dimensional cell culture mode is flat and stretched, and there is generally no overlap or coverage between tumor cells. Therefore, the investigation of the killing effect on three-dimensional cell spheroids can be more accurately used to evaluate the efficacy of the nanoagent. In the 3D multicellular tumor spheroid (MCTS) model, because it is closer to the actual in vivo tumor growth situation, this model was selected as a suitable in vitro model to study the in vitro efficacy of the nanoagent CuNMCN. The evaluation results of the killing effect are as Figure 11As shown, the killing effects of different drugs on 3D cell spheroids vary. In the cell spheroids treated with nano-autophagy inducer UCB and UCBH under light (L), only a small number of green live cells were observed, which proves that UCB and UCBH also have the ability to potently kill tumor cells at the three-dimensional cell level, laying a foundation for subsequent animal experiments.

[0209] 7. Investigation of the mechanism of tumor cell killing by nano-autophagy inducer

[0210] 7.1 Analysis of the expression of key proteins in the autophagy pathway

[0211] The key autophagy protein LC3B in tumor cells of different treatment groups was detected using western-blot (WB) technology. The detection results are as Figure 12 shown. With the successive involvement of single variable conditions, including light (L), BNN6 molecule loading (UCB + L), and HA molecule modification (UCBH + L), the expression level of LC3B showed an increasing trend successively, proving that the nano-autophagy inducer can significantly induce excessive autophagy in tumor cells.

[0212] 7.2 Morphological analysis of autophagosomes

[0213] To further observe the autophagy induction in tumor cells by the nano-autophagy inducer after light irradiation, ultra-thin section analysis of tumor cells in different treatment groups was carried out next. The TEM images are as Figure 13 shown. A small number of autophagosomes were present in the tumor cells of the PBS group and the UC group, and the intracellular mitochondrial structure was intact; with the addition of light (L), the number of autophagosomes in the UC + L group gradually increased and the mitochondrial structure was deformed; further, when the BNN6 molecule was added (UCB + L), the number of autophagosomes further increased and the content in the autophagosomes also increased significantly; finally, when the HA molecule was successfully modified onto the preparation, the number of autophagosomes in the (UCBH + L) treatment group reached the maximum and no obvious mitochondrial structure was observed. The above results together prove that the nano-autophagy inducer can effectively induce excessive autophagy under light, providing a basis for subsequent activation of anti-tumor immune responses.

[0214] 7.3 Analysis of the activation of the autophagy upstream protein ULK1 by copper ions

[0215] After preliminarily proving that the nano-autophagy inducer can effectively induce autophagy after light treatment, the next step is to specifically analyze the effect of the key components in the nano-autophagy inducer on autophagy. First, we investigated the effect of copper ions in the nano-autophagy inducer on ULK1, the key enzyme for autophagy activation. Immunofluorescence staining was used to stain ULK1 in tumor cells of different treatment groups, asFigure 14 As shown, after light irradiation (UC+L), the intracellular expression level of ULK1 was slightly enhanced. With the addition of BNN6 molecules (UCB+L) and HA molecules (UCBH+L), the intracellular ULK1 expression level in tumor cells gradually increased. The above results prove that the photoinduced reduction of divalent copper ions to monovalent copper ions is very important for the rapid and effective activation of autophagy.

[0216] 7.4 Flow cytometry analysis of NO damaging tumor cell mitochondria

[0217] Secondly, we further investigated the effect of NO produced by the nano-autophagy inducer after light irradiation on intracellular mitochondria. A mitochondrial membrane potential detection kit and flow cytometry were used to investigate the changes in mitochondrial membrane potential of tumor cells after different treatments. As Figure 15 shown, with the intervention of light irradiation, BNN6 molecules (UCB+L), and HA molecules (UCBH+L), the mitochondrial membrane potential was significantly affected, promoting its transformation from the aggregated form to the monomer form, proving that NO produced by the nano-autophagy inducer can significantly damage the mitochondrial structure of tumor cells and provide sufficient substrates for autophagy.

[0218] 7.5 Investigation of the effect of BTC on intracellular acetyl-CoA content

[0219] Finally, we also investigated the effect of BTC molecules released by the nano-autophagy inducer after light irradiation on cell autophagy. As a substrate of citric acid, BTC molecules will affect the content of intracellular acetyl-CoA, and thus provide enzyme and substrate guarantees for the further enhancement of autophagy. Therefore, we used an ELISA detection kit to evaluate the intracellular acetyl-CoA content. As Figure 16 shown, with the intervention of light irradiation, BNN6 molecules (UCB+L), and HA molecules (UCBH+L), the content of acetyl-CoA was significantly affected, proving that BTC molecules released by the nano-autophagy inducer can significantly damage tumor cell mitochondria and change cell metabolism to significantly enhance autophagy in tumor cells.

[0220] 10 Investigation of the ICD effect activated by the nano-autophagy inducer

[0221] 10.1 Investigation of the nano-autophagy inducer inducing tumor cells to express calreticulin

[0222] Given the important role of autophagy in tumor immunogenic death and successfully demonstrating that the nano-autophagy inducer can significantly induce excessive autophagy in cells at the in vitro cell level, the next step is to investigate the situation of UCBH activating the ICD effect in tumor cells. Flow cytometry was used to investigate the CRT expression level in tumor cells of different treatment groups. As Figure 17As shown, with the intervention of light, BNN6 molecule (UCB+L), and HA molecule (UCBH+L), the expression level of CRT was significantly affected, demonstrating that excessive autophagy significantly induced ICD in tumor cells.

[0223] 10.2 Investigation of the induction of HMGB1 migration in tumor cells by nano-autophagy-inducing agents

[0224] Secondly, we further investigated the effect of excessive autophagy generated after the nano-autophagy-inducing agent was irradiated with light on HMGB1 migration. Immunochemical staining technique and CLSM were used to investigate the distribution of HMGB1 in tumor cells after different treatments. As Figure 18 shown, with the intervention of light, BNN6 molecule (UCB+L), and HA molecule (UCBH+L), the expression location of HMGB1 was significantly affected, that is, it gradually transferred from the nucleus to the cytoplasm, demonstrating that excessive autophagy induced by UCBH+L significantly promoted ICD in tumor cells.

[0225] 10.3 Investigation of the induction of ATP secretion in tumor cells by nano-autophagy-inducing agents

[0226] Finally, we also investigated the effect of excessive autophagy generated after the nano-autophagy-inducing agent was irradiated with light on ATP secretion. Therefore, an ELISA detection kit was used to evaluate the content of secreted ATP. As Figure 19 shown, with the intervention of light, BNN6 molecule (UCB+L), and HA molecule (UCBH+L), the content of ATP was significantly affected, demonstrating that excessive autophagy induced after the nano-autophagy-inducing agent was irradiated with light significantly promoted ICD in tumor cells.

[0227] 10.4 Investigation of the promotion of DCs maturation by nano-autophagy-inducing agents

[0228] After successfully demonstrating that the nano-autophagy-inducing agent can enhance ICD by inducing excessive autophagy, the next step was to evaluate the effect of tumor cells in different treatment groups on activating the immune system, that is, promoting the maturation and activation of DCs. First, primary bone marrow DCs cells were extracted, and then the differently treated tumor cells were co-incubated with DCs, and flow cytometry was used to investigate the maturation of DCs. As Figure 20 shown, with the intervention of light (UC+L), BNN6 molecule (UCB+L), and HA molecule ((UCBH+L)), the ability of tumor cells to promote the maturation of DCs cells was gradually enhanced, reaching up to 51.52% at most, demonstrating that UCBH-induced excessive autophagy can effectively promote the maturation of DCs.

[0229] 11 Results of the investigation of the animal experiment effect of the nano-autophagy-inducing agent

[0230] 11.1 Investigation of the enrichment of nano-autophagy inducing agents in tumor tissues

[0231] After preliminarily demonstrating that nano-autophagy inducing agents can effectively induce excessive autophagy in vitro and then cause a strong ICD effect, we further investigated the in vivo distribution of nano-autophagy inducing agents. As Figure 21 shown, compared with UCB without modified HA molecules, UCBH can effectively enrich in the tumor part after being modified with HA molecules with targeting functions. And after circulating in vivo for 12 hours, the enrichment amount of nano-autophagy inducing agents in the tumor site reaches the maximum. With the extension of time, the fluorescence intensity of nano-autophagy inducing agents gradually weakens.

[0232] 11.2 Pharmacodynamic investigation of nano-autophagy inducing agents on a pancreatic cancer-bearing mouse model

[0233] After preliminarily demonstrating that nano-autophagy inducing agents can effectively enrich in the tumor site and inspired by the excellent anti-tumor effect in vitro, we applied it to a pancreatic cancer-bearing mouse model to explore the effect of its light-controlled immunotherapy. As Figure 22 shown, with the intervention of light (UC+L), BNN6 molecules (UCB+L), and HA molecules ((UCBH+L)), the tumor inhibition efficiency gradually increases, and it can ensure that 80% of the mice survive within 60 days, proving that UCBH can effectively kill tumors by inducing excessive autophagy under light.

[0234] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A nano autophagy inducing preparation, characterized in that: The nano autophagy inducing preparation comprises a composite material with a core-shell structure and a substance fixed on the composite material capable of generating NO by photolysis; In the composite material, the core is an upconversion nanoparticle modified with caffeic acid, and the shell is an MOFs material; the metal center of the MOFs material is a copper ion, and the organic ligand is selected from one or more carboxylic acid organic ligands.

2. The nano autophagy inducing preparation according to claim 1, wherein The content of the upconversion nanoparticles modified with caffeic acid is 15-22wt% of the total mass of the nano-autophagy inducing preparation, preferably 15-20wt%; The content of MOFs material is 50-70wt% of the total mass of the nano-autophagy inducing preparation, preferably 55-68wt%; The content of the substance capable of generating NO by photolysis is 8-15wt% of the total mass of the nano-autophagy inducing preparation, preferably 8-12wt%; Preferably, the nano autophagy inducing preparation further comprises a coating material for coating the composite material, and the coating material is selected from one or more of hyaluronic acid, cell membrane, folic acid and targeting peptide, preferably hyaluronic acid; More preferably, the content of the coating material is 5-12 wt % of the total mass of the nano autophagy inducing preparation.

3. The nano autophagy inducing preparation according to claim 1 or 2, wherein: The substance capable of generating NO by photolysis is selected from one or more of N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine, nitrosothiol and nitrobenzene; and / or In the caffeic acid-modified upconversion nanoparticles, the mass ratio of caffeic acid to upconversion nanoparticles is 1:5-20; and / or The upconversion nanoparticles are capable of generating ultraviolet light by near-infrared light excitation. Preferably, the upconversion nanoparticles contain 20-45% Yb, 0.1%-2% Tm, and 53%-81% matrix material by mass content; more preferably, the upconversion nanoparticles contain 25%-35% Yb, 0.3-0.8% Tm and 64.2%-74.7% matrix material; and / or The organic ligand is at least one selected from trimesic acid, terephthalic acid, phthalic acid, biphenyl-3,4,5-tricarboxylic acid, 1,4-naphthalene dicarboxylic acid, biphenyl dicarboxylic acid and 3,3,5,5-biphenyl tetracarboxylic acid, preferably trimesic acid; and / or In MOFs materials, the mass ratio of copper ions to organic ligands is 1:1-5.

4. The nano autophagy inducing preparation according to claim 1 or 2, wherein: The substance capable of generating NO by photolysis is encapsulated in the cavity formed by the core-shell structure and / or the pore structure of the MOFs material; and / or The nano autophagy inducing agent has a size between 150nm-300nm.

5. The method for preparing the nano autophagy inducing preparation according to any one of claims 1 to 4, characterized in that: The method includes: Method 1: a mixed solution containing upconversion nanoparticles modified with caffeic acid, copper ions and a hydrophilic polymer is contacted with a solution containing an organic ligand and a substance capable of generating NO by photolysis, and a solid is obtained by separation; or Method 2: contacting a mixture of upconversion nanoparticles modified with caffeic acid, copper ions and a hydrophilic polymer with a solution containing an organic ligand to separate the solid; mixing the solid with a solution containing a substance that can generate NO by photolysis to separate the solid.

6. The preparation method according to claim 5, wherein: The weight average molecular weight of the hydrophilic polymer is 10000-50000 g / mol; and / or The hydrophilic polymer is selected from one or more of polyvinyl pyrrolidone, polyvinyl alcohol and polyethylene glycol; and / or In method 1 and method 2, the contacting independently comprises: a temperature of 10-40° C. and a time of 0.5-12 h; preferably, the contacting is carried out under dynamic conditions, more preferably, the contacting is carried out under stirring conditions, and more preferably, the stirring speed is 500-2000 rpm / min; and / or In the mixed solution, the solution containing the organic ligand and the substance capable of generating NO by photolysis, the solvents are independently selected from one or more of small molecule alcohols, chloroform, tetrahydrofuran, dichloromethane and dimethyl sulfoxide. Preferably, the small molecule alcohol includes at least one of methanol, ethanol, propanol, butanol and pentanol.

7. The preparation method according to claim 5 or 6, wherein: In method 1 and method 2, in the mixed solution, the mass ratio of copper element to the hydrophilic polymer is 1:0.1-1; The mass ratio of copper element to caffeic acid modified upconversion nanoparticles is 1:0.005-0.03; the concentration of copper ions is 1-50g / L; and / or In method 1, in the solution containing the organic ligand and the substance capable of generating NO by photolysis, the concentration of the organic ligand is 0.2-30 mg / mL, and the concentration of the substance capable of generating NO by photolysis is 0.2-10 mg / mL; and / or In method 2, the concentration of the organic ligand in the solution containing the organic ligand is 0.2-30 mg / mL; In the solution containing the substance capable of generating NO by photolysis, the concentration of the substance capable of generating NO by photolysis is 0.2-10 mg / mL.

8. The preparation method according to claim 5 or 6, wherein: The method for preparing the caffeic acid modified upconversion nanoparticles comprises: mixing a mixed solution of upconversion nanoparticles stabilized by oleic acid with a solution containing caffeic acid, separating, and washing. Preferably, the mixing conditions include: a mixing temperature of 10-40° C. and a mixing time of 0.5-24 h; and / or The method further comprises mixing the separated solid with a solution containing the coating material, and separating the solid; preferably, in the solution containing the coating material, the concentration of the coating material is 0.1-20 g / L.

9. Use of the nano-autophagy inducing preparation according to any one of claims 1 to 4 in the preparation of a drug for treating a tumor, preferably, the tumor is one or more of a pancreatic cancer tumor, a breast cancer tumor and a liver cancer tumor.

10. A pharmaceutical composition, characterized in that Contains the nano autophagy inducing preparation according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

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