A divalent inorganic metal ion / photosensitizer protein nanoparticle and its preparation and application

Abstract

The present invention discloses a divalent inorganic metal ion / photosensitizer protein nanoparticle and its preparation and application. The present invention adopts a new technical idea to effectively solve the problem that the high-valent inorganic oxides usually delivered need to be reduced to a divalent state in cells to exert the immunotherapy effect. The nanoparticles are successfully prepared at room temperature in a certain pH aqueous solution. The divalent inorganic metal ions encapsulated therein form a complex with the photosensitizer, which has the advantages of stable valence and small size, can penetrate deep into the tumor tissue through the blood vessel wall, and release the divalent inorganic metal ions after rapid dissociation in the slightly acidic environment of the lysosome, and exert a phototherapy effect, which can destroy the lysosomal membrane, break through the lysosomal barrier and directly release Mn 2+ It is transported to the cytoplasm and induces the release of double-stranded DNA after illumination, producing cGAMP in situ in the cytoplasm, activating the STING signaling pathway, increasing the immune response rate, and enhancing the efficacy of tumor immunotherapy. Experimental verification has shown that it can completely inhibit tumor growth in a mouse in situ breast cancer model.

Description

Technical Field

[0001] The present invention belongs to biomedical technology, and specifically relates to a divalent inorganic metal ion / photosensitizer protein nanoparticle and a preparation method and application thereof. Background Art

[0002] In recent years, immunotherapy, as a new method for tumor treatment, has rapidly become a research hotspot due to its broad-spectrum anti-tumor activity, low toxic side effects, effective inhibition of tumor metastasis and recurrence, clearance of hard-to-find micro-lesions, and significant extension of the median survival of patients. It has made great progress in the clinical treatment of tumors (Immunity, 2020, 52(1): 17-35.). Currently, tumor immunotherapy is mostly focused on enhancing T cell responses. For example, immune checkpoint inhibitors (ICI) regulate the function of tumor-infiltrating lymphocytes by targeting the programmed death 1 / programmed death ligand 1 (PD-1 / PD-L1) pathway (Nature Reviews Clinical Oncology, 2017, 14(4): 203-220.); for example, genetically engineered T cells that specifically express chimeric antigen receptors (CARs) (Nature Reviews Clinical Oncology, 2020, 17(7): 418-434.) can directly target and attack tumor cells and have achieved good clinical efficacy. However, due to the low response rate of single-agent ICI and the large differences in tumor antigens, only a small number of cancer patients benefit from these therapies. In recent years, immunotherapy based on the Stimulator of interferon genes (STING) signaling pathway has received widespread attention. It recognizes intracellular DNA through cyclic GMP-AMP synthase (cGAS) to generate the second messenger cyclic GMP-AMP (cGAMP), which then activates the STING protein on the endoplasmic reticulum, promotes the secretion of type I interferon (IFN) and drives a strong anti-tumor immune response. It has special advantages for a variety of tumor models and improves the anti-tumor immune response rate (Nature Reviews Immunology, 2021, 21(9): 548-569.). However, the endogenous STING agonist cGAMP has poor drugability, such as small molecular weight, negative charge, and easy degradation, which makes it difficult to load conventional carriers, has low targeting efficiency, and has weak cellular uptake ability.Artificially synthesized small molecule STING agonists, such as acridones (dimethylpyrazol) and aminobenzimidazoles (diABZI), have many defects such as poor solubility, off-target effects, and severe toxic side effects (PLoS One, 2014, 9(6):e99988; Nature, 2018, 564(7736):439-443.). Although the targeting of STING agonists can be improved to a certain extent by constructing nanoformulations such as polymer vesicles and liposomes, the lack of cytoplasmic transport properties still greatly limits the activation efficiency of the STING signaling pathway. Therefore, how to achieve efficient activation of the STING signaling pathway and significantly improve the immune response rate is a key issue facing the field of tumor immunotherapy.

[0003] Phototherapy is an important part of current tumor treatment methods. Indocyanine green (ICG), a commonly used photosensitizer in tumor treatment, mainly acts as a photodynamic agent and also has a photothermal effect. It can convert light energy into heat energy under an external light source (near-infrared light) to kill cancer cells. The core of its photodynamic therapy is to use a near-infrared laser with a wavelength range of 650 to 1350 nm to irradiate the tumor site. After irradiation with the near-infrared laser, the photosensitizer accumulated in the tumor site absorbs photons and transitions from the ground state (S0) to the singlet excited state (S1) or through intersystem crossing to the triplet excited state. Molecules in the singlet excited state are unstable and return to the ground state through non-radiative transitions and release heat, inducing apoptosis of tumor cells and exerting a photothermal therapeutic effect; or return to the ground state through radiative transitions and emit fluorescence, which can be used for in vivo and in vitro tracing; photosensitizers in the triplet excited state undergo direct energy transfer with ground state oxygen to generate singlet oxygen with cell-killing effects, generating oxidative stress to destroy tumor cells and exerting a photodynamic therapeutic effect (Chemical Reviews, 2005, 105(6): 2647-2694.).

[0004] Studies have shown that various divalent inorganic metal ions such as Co 2+ 、Zn 2+ , Cu 2+ and Mn 2+ etc., play an important role in the immune regulation process. Compared with other metal ions, Mn 2+ It can directly activate cGAS protein, and can also enhance the production and cytoplasmic enrichment of endogenous STING agonist cGAMP by enhancing cGAS's recognition of double-stranded DNA (dsDNA), thereby enhancing the activation efficiency of the STING signaling pathway. 2+It is difficult to break through the intracellular delivery barrier, and the low content of double-stranded DNA in the tumor limits the application of this strategy. For example, the existing technology uses bovine serum albumin to adsorb manganese ions using a 37°C incubation method. However, after the nanoparticles are taken up into the cells, they are mainly distributed in the lysosomes and lack the cytoplasmic transport characteristics. 2+ It is difficult to reach the target of the STING signaling pathway—the cytoplasm, resulting in poor STING activation effect. Kong et al. 2+ , DOX and PO4 3− Dispersed in DMEM culture medium, by controlling the feed ratio and reaction time, based on Mn 2+ Biomineralization can self-assemble to form MnP-DOX nanoparticles (ACS Biomaterials Science & Engineering, 2022, 8(5), 1930-1941.); Jiang et al. prepared manganese nanogels using MnCl2, Na3PO4 and NaOH as raw materials. By controlling OH − / PO4 3− The ratio of MnO2 to STING is greater than 2:1, which can significantly activate the type I interferon response (Cellular & Molecular Immunology, 2021, 18, 1222-1234.). However, the manganese element in the nanoparticles prepared by the existing methods is usually oxidized to a high-valent form, such as manganese dioxide (tetravalent), and MnO2 has a photothermal effect. It has strong reducing properties under acidic conditions and can produce oxygen in the body, which helps to alleviate the hypoxic microenvironment of tumors. It can also be slowly converted into Mn with STING activation function in cells. 2+ However, the inclusion of nanoparticles makes it difficult to release from the carrier and transform smoothly, which limits its effect. Therefore, it is urgent to develop a nanoparticle that has the characteristics of cytoplasmic transport and can maintain Mn 2+ Valence delivery strategy. Summary of the invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide a divalent inorganic metal ion / photosensitizer protein nanoparticle and its preparation method and application. The present invention adopts a new technical idea and is a low-toxic and non-invasive treatment method. 2+ After the ICG nanoparticles reach the tumor site through passive targeting, they enter the tumor cells through the energy-dependent pathway mediated by clathrin and are distributed in the lysosomes. Under light, oxidative stress damages the lysosomal membrane, causing the drugs in the lysosomes to diffuse into the cytoplasm (a process called light-induced drug cytoplasmic transport), and further diffuse to the cell nucleus to kill tumor cells, exerting a photodynamic therapy effect. At the same time, Mn 2+The double-stranded DNA required for STING activation is generated, the STING signaling pathway is efficiently activated, the immune response rate is improved, and the immunotherapy effect is exerted. 2+ Co-delivery with ICG achieves efficient tumor treatment through multiple means.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] A divalent inorganic metal ion / photosensitizer protein nanoparticle, comprising a divalent inorganic metal ion, a photosensitizer and a protein; the photosensitizer is an organic photosensitizer; the protein is a carrier protein; further preferably, the divalent inorganic metal ion comprises Mn 2+ 、Zn 2+ 、Co 2+ , Cu 2+ The photosensitizer includes one or more of indocyanine green, dihydrochlorin e6, and hematoporphyrin; the protein includes one or more of human serum albumin, bovine serum albumin, transferrin, and ovalbumin. The present invention combines a photosensitizer (such as indocyanine green, ICG) with a divalent metal ion (such as Mn 2+ ) are encapsulated together in protein nanoparticles, producing unexpected technical effects and achieving efficient tumor treatment with triple effects.

[0008] The present invention discloses a method for preparing the above-mentioned divalent inorganic metal ion / photosensitizer protein nanoparticles, comprising the following steps: adding a photosensitizer solution to a mixed solution of divalent inorganic metal ions and proteins, stirring and reacting, and obtaining divalent inorganic metal ion / photosensitizer protein nanoparticles; further, stirring and reacting, and purifying to obtain divalent inorganic metal ion / photosensitizer protein nanoparticles. Preferably, the pH of the protein solution is first adjusted, and then the divalent inorganic metal ion solution is added to obtain a mixed solution of divalent inorganic metal ions and proteins.

[0009] In the present invention, the divalent inorganic metal ion / photosensitizer protein nanoparticles are composed of divalent inorganic metal ions, photosensitizers and proteins. The proteins encapsulate divalent inorganic metal ions and photosensitizers, and have a particle size of 5 to 30 nm and a drug loading of 1% to 20%. The particle size is the transmission electron microscopy particle size, and the drug loading refers to the mass of (divalent inorganic metal ions or photosensitizers) / (divalent inorganic metal ions / photosensitizer protein nanoparticles) in the purified sample × 100%.

[0010] In the present invention, the protein participates in the formation of divalent inorganic metal ions and photosensitizer nanoparticles; the nanoparticles can accumulate in tumor cell lysosomes, destroy the lysosomal membrane and stress damage the cell nucleus after light exposure, and play a photodynamic therapy role; in particular, Mn 2+It is rapidly transported from lysosomes to the cytoplasm and reaches the target of the STING signaling pathway, thus solving the problem of only encapsulating Mn 2+ BSA-Man@Mn 2+ After nanoparticles are taken up and enter cells, they are mainly distributed in lysosomes, lacking the problem of cytoplasmic transport characteristics. Unlike the prior art that uses manganese dioxide to provide oxygen to alleviate the hypoxic microenvironment of tumors, the present invention has a good STING signaling pathway activation ability, greatly improving the effect of immunotherapy for tumors. This is a special case of photodynamic therapy supplemented by photothermal therapy and efficient immunotherapy, and multiple means are used simultaneously to achieve efficient tumor treatment.

[0011] In addition, the nanoparticles of the present invention have different technical ideas compared with other manganese oxide protein nanoparticles prepared by biomineralization. For example, the natural melanin / MnOx nanohybrid nanoparticles prepared by Guo et al. have a size of 125-158 nm; and the MnO2-encapsulated dopamine / ICG / BSA nanoparticles prepared by Xu et al. have a size of about 150 nm. In particular, the existing technologies all require the encapsulated manganese oxide (MnO2 or MnOx, manganese element is in a positive tetravalent or higher valence state) to be reduced to Mn after entering the cell. 2+ The divalent inorganic metal ion / photosensitizer protein nanoparticles of the present invention, with their size advantage (5-30 nm), can diffuse through tumor blood vessels and further penetrate deep into the tumor and directly release Mn 2+ play a role, especially to achieve Mn 2+ It is rapidly transported from lysosomes to the cytoplasm, reaching the target of the STING signaling pathway, and combined with phototherapy to achieve complete ablation of in situ breast cancer in mice.

[0012] The present invention uses protein as a carrier. Specifically, a divalent inorganic metal ion solution is mixed with a protein solution to allow the ions to enter the internal cavity of the protein, and a photosensitizer is added to allow the two to continue to grow in the inner cavity of the protein, thereby achieving the encapsulation of divalent inorganic metal ions and photosensitizers in the protein and maintaining the stability of the divalent state, thereby preparing divalent inorganic metal ion and photosensitizer protein nanoparticles, which effectively solves the current problems that STING agonists are difficult to load, lack the characteristics of cytoplasmic transmission, and have low STING activation efficiency. The divalent inorganic metal ion and photosensitizer protein nanoparticles provided by the present invention have good tumor targeting and promote Mn 2+ The transmission and accumulation in tumor tissues, combined with photosensitizers, can efficiently activate the STING signaling pathway in tumor tissues after illumination, thereby achieving anti-tumor immune activation.

[0013] In the present invention, sodium hydroxide solution and hydrochloric acid solution are used to adjust the pH of the protein solution to a pH of 6 to 7; the protein solution is a protein aqueous solution with a concentration of 2 to 20 mg mL -1 The divalent inorganic metal ion solution is a divalent inorganic metal ion aqueous solution. As is common knowledge, the divalent inorganic metal ion solution is obtained by dissolving a metal compound.

[0014] In the present invention, the volume ratio of the protein aqueous solution to the divalent inorganic metal ion aqueous solution is 1-10:1; the concentration of the divalent inorganic metal ion aqueous solution is 20-100 mM; the molar ratio of the divalent inorganic metal ion to the photosensitizer is 4-16:1; the molar ratio of the protein to the divalent inorganic metal ion is (0.5-5):1, preferably (1-3):1.

[0015] The present invention discloses the use of the above-mentioned divalent inorganic metal ion / photosensitizer protein nanoparticles in the preparation of a drug for improving the transport of divalent inorganic metal ions to the cytoplasm, and utilizing light to make the photosensitizer produce a photodynamic effect and achieve Mn 2+ It is rapidly transported from lysosomes to the cytoplasm, exerting phototherapeutic and immunotherapeutic effects.

[0016] The present invention discloses the application of the inorganic metal ion / photosensitizer protein nanoparticles in tumor immunotherapy, specifically in the preparation of tumor immunotherapy drugs.

[0017] In the present invention, the dosage form of the tumor immunotherapy drug with the above-mentioned inorganic metal ion / photosensitizer protein nanoparticles as active ingredients is not limited, and can be prepared into various preparations, such as injections for intravenous injection, subcutaneous injection, intratumor injection, etc.

[0018] The existing technology uses biomimetic mineralization to grow manganese dioxide in situ into albumin, and the photosensitizer (such as indocyanine green, ICG) molecules are loaded onto the protein through hydrophobic interaction and electrostatic adsorption to obtain the final nanosystem. The existing technology also prepares photosensitizers such as (dihydrochlorin e6 (Ce6)) photodynamic therapy systems by precipitation method, and modifies the surface of bovine serum albumin (BSA)-coated MnO2 nanoparticles. The existing technology uses the characteristics of manganese dioxide that it has strong reducing properties under acidic conditions and can produce oxygen in the body, in order to improve the photodynamic therapy effect of the photosensitizer, but it does not solve the problem of its poor cytoplasmic transmission characteristics, let alone maintaining Mn 2+ The valency delivery requirement does not provide any technical inspiration for achieving efficient activation of the STING signaling pathway and significantly improving the immune response rate.

[0019] The present invention adopts the biomineralization method based on the hydrophobic cavity of protein as a template for the first time, and successfully prepares divalent inorganic metal ion and photosensitizer protein nanoparticles, wherein both the divalent inorganic metal ion and the photosensitizer are encapsulated in the protein; the prepared nanoparticles have regular morphology, good size uniformity, good dispersibility, and their release behavior is pH responsive. In a slightly acidic medium of pH 5.5 simulating lysosomes, H + With divalent metal ions (such as Mn 2+ ) competes with photosensitizers (such as ICG) to achieve Mn 2+ Rapid release. In addition, nanoparticles have good tumor targeting and can achieve Mn 2+ The invention can effectively activate the STING signaling pathway, improve the immune response rate, and enhance the efficacy of tumor immunotherapy. The present invention encapsulates the divalent inorganic metal ions and the photosensitizer in the protein cavity, thereby enhancing the stability of the divalent inorganic metal ions and the photosensitizer in the solution and solving the problem of Mn 2+ As a STING agonist, it addresses issues such as delivery, uptake, drug release and cytoplasmic transport, thereby exerting its effect efficiently.

[0020] The existing technology uses manganese dioxide to provide oxygen, which is mainly used to alleviate the hypoxic microenvironment of tumors; the present invention combines light to achieve Mn 2+ The invention can transport manganese dioxide into the cytoplasm and damage the cell nucleus at the same time, thereby producing double-stranded DNA required for STING activation, thereby achieving efficient activation of the STING signaling pathway. The role of the present invention in providing oxygen is completely different from that of the existing manganese dioxide.

[0021] Compared with other STING agonist-loaded nanoparticles, the bivalent inorganic metal and photosensitizer protein nanoparticles provided by the present invention have significant advantages in that the preparation method is simple and controllable, the reaction conditions are mild, the drug can be released in response to the slightly acidic environment of the lysosome, the biosafety is good, the tumor targeting is high, the efficacy is good, and the side effects are small. 2+ The nanoparticles Mn-ICG@HSA (the average particle size determined by TEM is 9.74 nm, the hydrated particle size is 30.2 ± 9.9 nm; the drug loading Mn 2+ 2.2%, ICG 6.2%), with stable transmission of Mn 2+ The characteristics and size advantages of ICG can penetrate deep into the tumor. 2+ The bound ICG, due to the H + Can replace Mn 2+ Competitive binding with ICG, converting Mn-ICG precipitate into a more stable ICG precipitate to form free Mn 2+Therefore, the Mn-ICG@HSA taken up by tumor cells and entering the lysosomes (pH 5.5) can produce phototherapy effects due to the ICG-responsive temperature rise under light, destroy the lysosomes, and release free Mn 2+ , realize Mn 2+ Lysosomal escape and cytoplasmic transport directly play the role of Mn 2+ Inducing the release of double-stranded DNA, enriching cGAMP in situ in the cytoplasm, exerting the role of STING protein, promoting the secretion of IFN-β, thereby improving the immune response rate and achieving complete ablation of in situ breast cancer in mice. In addition, since the present invention can only release Mn under light conditions, 2+ , activating the STING signaling pathway and improving the side effects of traditional STING agonists caused by off-target effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron microscope image of the nanoparticles of Example 1 of the present invention.

[0023] Figure 2 This is the X-ray photoelectron spectrum of the nanoparticles of Example 1 of the present invention.

[0024] Figure 3 This is the UV-visible absorption spectrum of indocyanine green (ICG) in the nanoparticles of Example 1 of the present invention.

[0025] Figure 4 This is a statistical diagram of the photothermal heating curve of the nanoparticles in Example 1 of the present invention.

[0026] Figure 5 The graph shows the generation of active oxygen (A) and the quantum yield statistics (B) of the nanoparticles in Example 1 of the present invention.

[0027] Figure 6 Mn in the nanoparticles of Example 1 of the present invention 2+ Release curve diagram.

[0028] Figure 7 This is a diagram showing that nanoparticles in Example 1 of the present invention promote the generation of reactive oxygen species in 4T1 cells.

[0029] Figure 8 The cell survival rate statistics of nanoparticles, manganese dichloride and physical mixture on 4T1 cells in Example 1 of the present invention are dark toxicity (A) and phototoxicity (B), respectively.

[0030] Fig. 9 This is a diagram of nanoparticles promoting the release of dsDNA in 4T1 cells in Example 1 of the present invention.

[0031] Fig.10 This is a statistical diagram of the nanoparticles in Example 1 of the present invention promoting the secretion of type I interferon by DC2.4 cells.

[0032] Fig.11 This is the near-infrared in vivo fluorescence imaging image (A) and fluorescence statistical graph (B) of the nanoparticles in Example 1 of the present invention in 4T1 mouse orthotopic breast cancer.

[0033] Fig.12 This is a tumor section image observed under a laser confocal microscope after the nanoparticles in Example 1 of the present invention were used to treat 4T1 mice with orthotopic breast cancer. The samples are free rhodamine B, Abraxane labeled with rhodamine B, and Mn-ICG@HSA.

[0034] Fig.13 The tumor inhibition curves of the nanoparticles and physical mixture in Example 1 of the present invention in treating mice bearing 4T1 orthotopic breast cancer and distal tumors are shown, which are the tumor growth curves of the proximal tumor (A) and the distal tumor (B), and the survival curve of the tumor-bearing mice (C).

[0035] Fig.14 This is a statistical diagram of type I interferon secretion by nanoparticles and physical mixtures in Example 1 of the present invention in the tumor tissue of the 4T1 in situ breast cancer model.

[0036] Fig.15 The expression of immune cells in the 4T1 orthotopic breast cancer and distal models of the nanoparticles and physical mixtures in Example 1 of the present invention are natural killer cells (A), CD8 + Statistical graphs of the numbers of cytotoxic T cells (B), M1 macrophages (C), M2 macrophages (D), and myeloid-derived suppressor cells (E) in proximal and distal tumors.

[0037] Fig.16 The present invention compares different divalent inorganic metal ion solutions, protein-divalent inorganic metal ion solutions and divalent inorganic metal ion-photosensitizer ICG solutions in Example 2.

[0038] Fig.17 This is a comparison of precipitation formed by different divalent inorganic metal ions and photosensitizer ICG in different molar ratios in Example 2 of the present invention.

[0039] Fig.18 The present invention compares different photosensitizer solutions, divalent manganese-photosensitizer precipitation, and protein nanoparticle solutions prepared with divalent manganese ions and different photosensitizers in Example 3.

[0040] Fig.19 This is a diagram of the hydrated particle size of the protein nanoparticle solution prepared by divalent manganese ions and different photosensitizers in Example 3 of the present invention.

[0041] Fig. 20 The different protein solutions and Mn in Example 4 of the present invention 2+-Comparison of HPP protein nanoparticle solutions.

[0042] Fig.21 Different proteins encapsulate Mn in Example 4 of the present invention 2+ -HPP precipitation hydration particle size diagram. DETAILED DESCRIPTION

[0043] The following is a further detailed description of the specific embodiments of the present invention in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The raw materials and reagents used in the present invention are all commercially available products, meet the conventional requirements of drugs, and the specific preparation operations and performance tests used are conventional techniques; the animal experiments involved meet the requirements of animal experiments at Soochow University. All data are expressed as mean ± standard deviation, and the differences between the groups are evaluated using a one-way analysis of variance with a bilateral Student's t-test or a Tukey post-test. Statistical differences are defined as *P<0.05, **P<0.01, and ***P<0.001.

[0044] As an example, the present invention uses albumin as a carrier and a biomineralization method based on a single molecule protein template to synthesize divalent metal ions (such as Mn 2+ ) interacts with the amino acid residues of albumin to form nucleation centers, and the introduction of photosensitizers (such as ICG) and Mn 2+ The complex precipitates through electrostatic interaction in the albumin cavity and further grows. In the acidic environment of tumor cell mitochondria, the Mn in the complex 2+ Can be H + The reaction equation is as follows:

[0045]

[0046] The pH value of the control system of the present invention is adjusted by adding an appropriate amount of Mn 2+ It is combined with ICG that can be ionized into anions and then precipitated in the cavity of the protein carrier to construct a divalent inorganic metal ion and photosensitizer protein nanoparticle targeted drug delivery system. After illumination, Mn 2+ The cytoplasmic transport of the protein effectively exerts its STING signaling pathway activation ability and anti-tumor immune effect.

[0047] The existing reports on the efficacy of nanomedicines for STING activation are low, and they are clinically administered by intratumoral injection. However, the traditional delivery strategy cannot achieve lysosomal escape and cytoplasmic delivery of STING agonists, resulting in low efficiency of STING target activation. In the present invention, divalent inorganic metal ions, such as Mn 2+It is an inorganic metal ion with immunomodulatory properties. It can increase the activity of immune cells and catalyze cGAS to efficiently recognize dsDNA, accelerate the cytoplasmic enrichment of cGAMP, and 2+ ) directly catalyzes the dsDNA induced by phototherapy to produce the STING agonist cGAMP in situ in the cytoplasm, and Mn 2+ Further enhancing the activation effect of cGAMP on STING protein can greatly improve the activation efficiency of STING signaling pathway; ICG is a photosensitizer with high safety, photothermal and photodynamic effects, which can induce cell damage and achieve Mn after near-infrared light irradiation. 2+ lysosomal escape; albumin, transferrin and other proteins are a type of carrier material with good biocompatibility, which can induce divalent inorganic metal ions to bind to the hydrophobic cavity inside them. In the present invention, firstly, under near-neutral conditions, divalent inorganic metal ions bind to amino acid residues in proteins as nucleation centers, and then photosensitizers are introduced to react with divalent inorganic metals to cause precipitation, so that both are precipitated together in the hydrophobic cavity of proteins, and a divalent inorganic metal ion and photosensitizer protein nanoparticle is prepared; the water-soluble Mn 2+ It is difficult to form precipitation and cannot be effectively encapsulated in a protein carrier, and also solves the problem that the existing technology is to compound organic compounds and photosensitizers inside the protein.

[0048] The present invention provides a method for preparing the above-mentioned divalent inorganic metal ion / photosensitizer protein nanoparticles, which comprises the following steps:

[0049] (1) adjusting the pH of the protein aqueous solution to facilitate the adsorption of divalent inorganic metal ions and proteins to obtain a mixed solution 1;

[0050] (2) mixing the divalent inorganic metal ion aqueous solution with the mixed solution 1 and stirring to obtain a mixed solution 2;

[0051] (3) The photosensitizer aqueous solution is mixed with the mixed solution 2 to obtain a mixed solution 3, and then after stirring for reaction, the mixed solution 3 is purified by ultrafiltration and washing with water to obtain divalent inorganic metal ion / photosensitizer protein nanoparticles.

[0052] In the present invention, the stirring reaction temperature is room temperature, the time is 1 to 6 hours, and the specific method of realizing the stirring reaction and the control of the stirring reaction are conventional technologies; the purification is ultrafiltration and washing to neutrality to remove free small molecules and provide a stable environment for nanoparticles. During ultrafiltration, the centrifuge speed is 1500 to 3000 rpm.

[0053] In the present invention, in the protein solution, the divalent inorganic metal ion solution and the photosensitizer solution, the solvent is deionized water or various aqueous solutions, the divalent manganese ion is manganese dichloride or other divalent manganese salt solution, the photosensitizer is indocyanine green, and the protein is human serum albumin.

[0054] In the present invention, the stirring reaction is carried out using a magnetic stirrer or the like, which is specifically a conventional technique.

[0055] Preferably, the divalent inorganic metal ion solution and the protein solution are mixed for 3 to 5 minutes, and the stirring reaction time after adding the photosensitizer is 1 to 6 hours.

[0056] In the present invention, sodium hydroxide aqueous solution or hydrochloric acid solution is used to adjust the pH value of the protein solution. Preferably, the concentration of the NaOH aqueous solution or the HCl solution is 2 mol / L.

[0057] Embodiment 1

[0058] The preparation of divalent manganese ion and photosensitizer protein nanoparticles, the specific steps are as follows:

[0059] Prepare 10 mg mL -1 30 mL of 10 mg mL HSA solution, 40 mM MnCl2 4H2O solution, 2.5 mM ICG solution and 2.0 M NaOH solution. -1 The HSA aqueous solution was placed on a magnetic stirrer, and the system was adjusted to pH 6.5 with NaOH solution. Then, 5.0 mL of MnCl2 solution was added under stirring. After stirring for 3 minutes, 5 mL of ICG solution was added and stirred at room temperature (300 rpm) for 4 h. Then, the solution was centrifuged at 4000 rpm for 5 min, and the supernatant was ultrafiltered and washed to neutrality (2000 rpm, 10 min) using an ultrafiltration tube with a molecular weight cutoff of 100 kD to remove free small molecules and provide a stable environment for the nanoparticles, thereby obtaining divalent manganese / photosensitizer protein nanoparticles (Mn-ICG@HSA), which were stored at 4°C in the dark.

[0060] The prepared Mn-ICG@HSA was photographed by transmission electron microscopy, and the results are as follows: Figure 1 The average particle size of the nanoparticles was (9.74±0.90) nm, with uniform particle size and even dispersion. In addition, the Mn 2+ The drug loading capacity of was 2.2%, and that of ICG was 6.2%.

[0061] Comparison example

[0062] On the basis of Example 1, the MnCl2 4H2O aqueous solution was omitted to obtain ICG@HSA nanoparticles.

[0063] 1. The components of Mn-ICG@HSA prepared in Example 1 were analyzed, and the specific steps were as follows:

[0064] (1) Analysis of manganese element and valence state: The nanoparticle solution (Mn-ICG@HSA) was freeze-dried, and the physical mixture precipitate formed by mixing MnCl2 powder, MnCl2 solution and ICG solution was used as a control; it was fully ground into fine powder, and the valence state of manganese element on the sample surface was analyzed by X-ray photoelectron spectroscopy (XPS). The results are as follows: Figure 2 Compared with free drug MnCl2, Mn 2+ +ICG physical mixture and Mn-ICG@HSA XPS spectrum peak shape is consistent with MnCl2, proving that Mn 2+ After binding with ICG, the valence state did not change. By fitting the XPS spectrum of Mn-ICG@HSA nanoparticles, the Mn 2p orbital double peak binding energies were 641.7 eV (Mn 2p 3 / 2 ) and 653.5 eV (Mn 2p 1 / 2 ), and an obvious satellite peak appears at the binding energy of 647 eV, proving that the manganese element in Mn-ICG@HSA exists in the form of Mn 2+ .

[0065] (2) ICG spectral analysis: Prepare the solution at a concentration of 10.0 mg mL -1 The absorption spectra of ICG aqueous solution and Mn-ICG@HSA nanoparticle solution at 300-900 nm were measured by UV-visible spectrophotometer. Figure 3 , Mn-ICG@HSA has a peak shape similar to that of ICG near 785 nm, and has a shoulder peak consistent with that of ICG. Due to the J aggregation of ICG in the nanoparticles in aqueous solution, its maximum absorption wavelength red-shifts from 780 nm to 800 nm, proving that ICG is successfully encapsulated in the Mn-ICG@HSA nanoparticles.

[0066] 2. The photothermal effect of Mn-ICG@HSA prepared in Example 1 was investigated. The specific steps were as follows: take the nanoparticle solution and dilute it with water to a final ICG concentration of 10 g mL -1 500 L of solution was placed in a 2 mL round-bottom EP tube (n=3). The solution was irradiated with a 785 nm near-infrared laser and a digital temperature probe was inserted into the solution. The solution temperature was recorded every 30 s. The non-illuminated, 0.15 W cm -2 , 0.3 W cm -2 and 0.5 W cm -2 The temperature rise of the solution within 5 minutes under the light power is shown in the following figure. Figure 4, 0.15W cm -2 , 0.3 W cm -2 , 0.5 W cm -2 After 5 minutes of irradiation, the solution temperature increased by 5.8°C, 11.1°C and 19.2°C, respectively. With the increase of light power, the photothermal warming effect of the nanoparticle solution also increased synchronously, indicating that Mn-ICG@HSA has a good photothermal warming effect in vitro.

[0067] 3. The photodynamic effect of Mn-ICG@HSA prepared in Example 1 was investigated. The specific steps were as follows: take the nanoparticle solution, dilute it with water to a final ICG concentration of 1 ug / mL, take 2.97 mL of the nanoparticle solution in a quartz cuvette, add 30 μL of a 3 mM singlet oxygen probe (1,3-diphenylbenzofuran, DPBF). Under stirring, irradiate with a 785 nm near-infrared laser, and record the non-illumination, 0.15 W cm -2 , 0.3 W cm -2 and 0.5 W cm -2 The absorbance value (A) of the solution at 420 nm changes within 10 min under the illumination power. In addition, the ICG aqueous solution was used as a control and the 0.5 W cm -2 The singlet oxygen quantum yield of Mn-ICG@HSA under light power. The absorbance value at 420 nm at time 0 is recorded as A0, and the absorbance values ​​at different time points are recorded as A i , with ln (A0 / A i ) is plotted against time point t, and the slope of the regression equation represents the photobleaching rate. The singlet oxygen quantum yield is calculated using the following formula: Φ Δ = (Φ ΔICG· W sample· I ICG ) / (W ICG· I sample ), I = 1-10 A785 ; where Φ ΔICG : The standard singlet oxygen quantum yield of ICG aqueous solution is 0.14; W: DPBF photobleaching rate; I: the absorption rate of the test solution and the reference solution. The results are shown in Figure 5 , under non-light conditions, no 1 O2 is produced. As the laser power increases, the singlet oxygen production increases. The quantum yield of Mn-ICG@HSA at different powers is further calculated by the formula. The results show that: taking ICG aqueous solution as a reference (Φ DICG = 0.14 ± 0.03), 0.15 W cm -2 , 0.3 W cm -2 , 0.5 W cm-2 The quantum yields of Mn-ICG@HSA under illumination powers were 0.11 ± 0.01, 0.16 ± 0.002, and 0.25 ± 0.02, respectively, indicating that Mn-ICG@HSA had a good ROS production ability under illumination, which was beneficial for killing tumor cells.

[0068] 4. The drug release behavior of Mn-ICG@HSA prepared in Example 1 was investigated. The specific steps were as follows: taking the nanoparticle solution, diluting it with water to Mn 2+ Final concentration: 100 g mL -1 At the same time, free MnCl2 aqueous solutions of the same concentration were prepared as controls (n=3). 1 mL of MnCl2 and Mn-ICG@HSA nanoparticle solutions were sealed in dialysis bags (molecular weight cutoff: 3500 Da) and placed in 10 mL of phosphate or acetate buffer media at pH 7.4, pH 6.5, and pH 5.0. The system was placed in a constant temperature shaker at 37°C and 120 rpm. All receiving media were collected at time points of 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h, and 10 mL of new buffer media was added. After sampling at all time points was completed, the samples were filtered with a 0.22 m microporous membrane, and the release of manganese in the receiving medium was determined by inductively coupled plasma emission spectrometry, and the cumulative release percentage of manganese was calculated. The results are shown in Figure 2. Figure 6 , pH 7.4 phosphate buffer (Na2HPO3 / NaH2PO3) was used to simulate the body fluid environment, pH 6.5 phosphate buffer (Na2HPO3 / NaH2PO3) was used to simulate the tumor microenvironment, and pH 5.5 acetic acid-acetate buffer (CH3COOH / CH3COONa) was used to simulate the lysosomal acidic environment. 2+ It can be quickly released from the dialysis bag by free diffusion, and the nanoparticles show acid-responsive release behavior, with release amounts of 28.7 ± 1.2%, 51.6 ± 3.5% and 78.0 ± 0.7% under pH 7.4, pH 6.5 and pH 5.5, respectively. This indicates that its acid responsiveness comes from the protonation of ICG, that is, in a slightly acidic environment, H + Competitive binding of ICG (replacing Mn 2+ ), Mn-ICG precipitate is transformed into a more stable ICG precipitate, while Mn 2+ Therefore, after being taken up by tumor cells, Mn-ICG@HSA in lysosomes (pH 5.5) is heated up in response to light, which can destroy the lysosomes and quickly release free Mn. 2+ , realize Mn 2+ Lysosomal escape and cytosolic transport.

[0069] 5. Experimental investigation on the promotion of ROS in 4T1 cells by Mn-ICG@HSA prepared in Example 1. The specific steps are as follows: 4T1 cells were inoculated into a confocal glass bottom dish (1×10 5 1 mL of conventional culture medium was added to each well. After overnight culture, four groups (ICG: 5 ug / mL) were set up: PBS non-illumination group, PBS illumination group, Mn-ICG@HSA nanoparticle non-illumination group, and Mn-ICG@HSA nanoparticle illumination group. After adding the four samples and incubating for 6 h, the culture medium was removed and the 4T1 cells were washed three times with PBS. Fresh culture medium was added and illuminated (785 nm, 0.5 W cm -2 , 3 min); the non-illuminated group continued to incubate. Immediately after illumination, the non-illuminated group and the illuminated group were stained as follows: 1) Added a 10 um active oxygen probe dihydroethidium (DHE) stain solution to the glass bottom dish, incubated for 10 min in the dark, removed the stain solution and washed three times with PBS; 2) Stained the cell nucleus with Hoechst 33342 for 6 min, removed the dye and washed three times with PBS. Immediately used CLSM to observe the red fluorescence intensity in the cells. The results are shown in Figure 7 The non-illumination group of nanoparticles showed only weak red fluorescence, while the illumination group of nanoparticles produced strong red fluorescence that coincided with the cell nucleus, indicating that after the cells took up Mn-ICG@HSA nanoparticles and were illuminated, a large amount of ROS could be immediately produced in the cells.

[0070] 6. The survival rate of 4T1 cells was investigated by using Mn-ICG@HSA prepared in Example 1. The specific steps were as follows: 4T1 cells were inoculated into a 96-well cell culture plate (5×10 3 Cells / well), 100 μL of single cell suspension diluted with culture medium was added to each well and cultured overnight. The following six groups were set up: MnCl2 non-illumination group, ICG illumination group, Mn+ICG non-illumination group, Mn+ICG illumination group, Mn-ICG@HSA non-illumination group and Mn-ICG@HSA illumination group. Among them, the ratio of manganese and ICG in the physical mixture was consistent with that of Mn-ICG@HSA nanoparticles (Mn∶ICG = 4∶1). Each group was calculated based on the concentration of manganese, and the following concentration gradients were set: 0, 5, 10, 20, 50, 100, 200 μM, and the corresponding ICG concentration gradients were: 0, 1.25, 2.5, 5, 12.5, 25, 50 μM. Before drug administration, the old culture medium was removed and 100 μL of different concentrations of drug-containing culture medium was added. Four replicate wells were set for each concentration. After 12 h of drug administration, the culture medium was removed and the cells were washed three times. The cells were illuminated (785 nm, 0.5 W cm -2 , 3 min). Add 0.5 mg mL-1 Methylthialazole Tetrazolium (MTT) was incubated in the dark for 4 h, and then the upper layer of liquid was discarded. 100 μL DMSO was added to each well, and the cells were shaken on a shaker for 10 min. The absorbance at 490 nm was read using a microplate reader. The illumination group was replaced with fresh culture medium and illuminated with a 785 nm laser (0.5 W cm -2 , 3 min), and then continue to culture for 12 h. According to the above MTT dosing regimen, the cell viability was determined by an enzyme-labeled instrument and calculated according to the following formula: Cell viability (%) = experimental group A / average absorbance of negative control group `A0 × 100% (with the concentration of 0 μM as the negative control). The results are shown in Figure 8 Compared with MnCl2 and physical mixture (Mn+ICG), Mn-ICG@HSA showed lower cytotoxicity under non-illumination conditions. 50 71.1 mM, 64.9 mM and 100.9 mM respectively (in terms of Mn concentration, Figure 8 Compared with the ICG illumination group and the physical mixture illumination group, the cytotoxicity of Mn-ICG@HSA was enhanced after illumination ( Figure 8 Middle B), three IC 50 17.4 mg mL -1 , 8.4mg mL -1 and 6.5 mg mL -1 (Measured in terms of ICG concentration). Therefore, it is shown that Mn-ICG@HSA nanoparticles have relatively low dark toxicity and high phototoxicity, indicating that Mn-ICG@HSA nanoparticles can effectively kill tumor cells under light conditions and reduce side effects caused by off-target effects.

[0071] 7. The intracellular dsDNA release experiment of Mn-ICG@HSA prepared in Example 1 was carried out. The specific steps were as follows: 4T1 cells were inoculated into a confocal glass bottom dish (1×10 5 After the cells adhered to the wall, four groups were set up: PBS non-illumination group, PBS illumination group, Mn-ICG@HSA nanoparticle non-illumination group, and Mn-ICG@HSA nanoparticle illumination group. PBS and Mn-ICG@HSA nanoparticles (ICG: 5 g mL -1 ) After incubation for 12 h, the cells were washed and the concentration was 0.5 W cm -2After 3 min of illumination (the non-illuminated group continued to incubate), the samples in each group were stained in the following order: 1) fixed with 4% paraformaldehyde (PFA) for 15 min and washed, permeabilized with 0.3% TritonX-100 for 15 min and washed, and blocked with 1% bovine serum albumin (BSA) for 30 min and washed; 2) anti-double-stranded DNA antibody diluted with primary antibody diluent was added to each dish, incubated at 4°C for 12 h, washed three times with PBS, and then FITC-labeled fluorescent secondary antibody (FITC-AffiniPure Goat Anti-Rabbit IgG (H+L)) diluted with PBS was added and stained at room temperature in the dark for 45 min, the staining solution was removed and washed with PBS, and 500 μL Hoechst 33342 staining solution was immediately added to the glass-bottomed dish, stained at room temperature in the dark for 6 min, and then the staining solution was washed away, and the green fluorescence intensity in the cells was immediately observed using CLSM. The results are shown in Fig. 9 Due to the characteristics of tumor cell genome instability and frequent micronucleus formation, the cells in the PBS non-illumination group and the Mn-ICG@HSA non-illumination group showed a small amount of green fluorescence. The green fluorescence of the nanoparticle illumination group increased to 2.2 times compared with the PBS non-illumination group, indicating that Mn-ICG@HSA under illumination has a promoting effect on increasing the production of dsDNA in the cytoplasm.

[0072] 8. Experimental investigation on the promotion of extracellular type I interferon (IFN-β) secretion by dendritic cells (DC2.4) by Mn-ICG@HSA prepared in Example 1, the specific steps are: using mouse IFN-β enzyme-linked immunosorbent assay (ELISA) kit to detect the secretion of IFN-β in DC2.4 supernatant after the action of Mn-ICG@HSA nanoparticles, the experimental method is as follows. DC2.4 was inoculated in a 24-well plate (2×10 4Cells / well), after the cells adhere to the wall, group, administer and illuminate according to the above Western Blot dosing scheme. Collect the supernatant 12 hours after illumination, centrifuge at 4℃, 2000 rpm for 15 min, and remove cells and cell debris. Set standard wells and sample wells, dilute the sample to be tested 5 times with diluent, add a series of standard solutions and sample solutions to the enzyme-labeled plate, add the solution to the bottom of the well, and shake gently to mix. After sealing the plate with a sealing film, incubate at 37℃ for 30 minutes. After the incubation is completed, add 200 μL of washing solution to each well, wash 5 times, discard the liquid, and spin dry. Add 50 μL of enzyme labeling reagent to each well, incubate at 37℃ for 30 minutes, wash 5 times after the incubation is completed, and develop color. Add 50 μL of colorimetric reagent A and B to each well, shake gently to mix, color at 37°C in the dark for 15 minutes, add 50 μL of stop solution to each well to terminate the reaction, and finally use an ELISA reader to measure the absorbance of each well at 450 nm, and calculate the IFN-β concentration of each sample using the standard curve. Fig.10 , the IFN-β secretion in the PBS light-exposed group did not change significantly, indicating that light stimulation alone could not induce the secretion of IFN-β; 50 mM Mn 2+ Single administration can stimulate 4T1 cells to increase the secretion of IFN-β. Compared with PBS, the IFN-β secretion is increased to 1.9 times. In the Mn-ICG@HSA non-illumination group, due to Mn 2+ The slow diffusion of IFN-β can also increase the secretion of IFN-β; after light irradiation (0.5 W / cm 2 5 min), the IFN-β secretion in DC cells increased by 2.8 times compared with that in the PBS group, indicating that Mn-ICG@HSA combined with light can activate the STING signaling pathway in DC cells and promote the secretion of IFN-β.

[0073] 9. The Mn-ICG@HSA prepared in Example 1 was subjected to near-infrared in vivo fluorescence imaging experiments in 4T1 mouse breast cancer. The specific steps were as follows: 6-8 week old BALB / c female mice were inoculated with 4T1 cells (1×10 6 cells / mouse), establish an orthotopic breast cancer mouse model, and wait until the tumor tissue grows to 200 mm 3 The mice were randomly divided into two groups, three in each group. The abdomen of the mice was depilated before administration. One group was the free drug group, which was injected with free drug MnCl2 and ICG solution by tail vein (MnCl2 and ICG solution were injected into the veins of both sides, not the same side or mixed solution), and the other group was the nanoparticle group, which was injected with Mn-ICG@HSA nanoparticle solution by tail vein. The injection dose of both groups was Mn 3.0 mg kg -1 ,ICG10 mg kg -1The mice were scanned using a small animal fluorescence imaging system before administration (0 h), 4, 8, 12, 24, and 48 h after administration. After scanning at all time points, the fluorescence intensity of the tumor site at different time points was counted. Fig.11 No obvious fluorescence was observed in the abdomen of mice in the two groups except for the tumor site. Moreover, the fluorescence intensity of the Mn-ICG@HSA nanoparticle group injected by tail vein was about twice that of the free group within 12 h to 48 h, indicating that Mn-ICG@HSA has good tumor targeting and prolongs the retention time of the drug in the tumor.

[0074] 10. The tumor penetration of Mn-ICG@HSA prepared in Example 1 was investigated. The specific steps were as follows: labeling clinical albumin-bound paclitaxel (Abraxane) and Mn-ICG@HSA with rhodamine B: 3 mL of freshly prepared Mn-ICG@HSA and 30 μL of rhodamine B (5 mg mL -1 , dissolved in DMSO) and stirred at room temperature in the dark for 2 h, free rhodamine B was removed by ultrafiltration (1500 rpm, 10 min), and the mixture was collected for later use. Free rhodamine B and Abraxane labeled with rhodamine B and Mn-ICG@HSA were mixed at a concentration of 1 mg kg -1 The dose was injected into the tail vein of tumor-bearing mice. The mice were killed 24 hours later and the tumors were collected. After being embedded in paraffin, the sections were sliced ​​and incubated with CD31 antibody (green) at 4°C for 4 hours, and the cell nuclei were stained (Hoechst33342, 1 μg mL -1 ), and the fluorescence intensity of the slices was observed using a confocal microscope. Fig.12 In the free rhodamine B group, due to its poor tumor targeting, no obvious red fluorescence was observed in the tumor tissue; in the Abraxane group, due to its size (~130nm, much larger than the 9.74 nm of the nanoparticles of the present invention), most of it accumulated in the tumor blood vessels and was difficult to penetrate deep into the tumor. In the Mn-ICG@HSA group, a large area of ​​red fluorescence was observed in the tumor tissue, indicating that after Mn-ICG@HSA is targeted to the tumor site, it can rely on its smaller size advantage to penetrate deep into the tissue through the tumor blood vessels, which is conducive to exerting therapeutic effects.

[0075] 11. The Mn-ICG@HSA prepared in Example 1 was used to perform tumor inhibition experiments on 4T1 orthotopic and distal breast cancer models. The specific steps were as follows: 6-8 week old BALB / c female mice were inoculated with 4T1 cells (1×10 6 cells / mouse), establish an orthotopic breast cancer mouse model, and wait until the tumor tissue grows to 70 mm 3 4T1 cells (5×105 Cells / cell), and treatment was carried out on the second day: the following 8 groups were set up: ①PBS; ②PBS+light; ③MnCl2+ICG; ④MnCl2+ICG+light; ⑤ICG@HSA nanoparticles+light; ⑥Mn-ICG@HSA; ⑦Mn-ICG@HSA+light; ⑧Mn-ICG@HSA+vitamin C (Vc)+light. Drugs were administered by tail vein injection, and the injection dose of each group was 3.0 mg kg -1 , ICG 9.6 mg kg -1 12 h after drug administration, the tumor site was illuminated with a 785 nm laser (0.5 W cm -2 The proximal and distal tumor volumes and body weights of mice were recorded every two days, and the tumor growth of mice was recorded within 30 days (when the proximal tumor volume was greater than 1500 mm 3 After that, the mice were killed and the tumor inhibition rate was calculated (tumor inhibition rate = (1-tumor volume of experimental group / tumor volume of control group) × 100%). At the same time, the survival status of the mice was monitored for 60 days and a survival curve was drawn. The results are shown in Fig.13 The growth of in situ tumors had a similar growth trend to that of distal tumors. There was no significant difference in the tumor growth rate between the PBS illumination group, free drug group and Mn-ICG@HSA non-illumination group and the PBS group. The tumor growth inhibition rates of the free drug illumination group and ICG@HSA illumination group were 53.6% and 63.4%, respectively, and the distal tumor inhibition rates were slightly lower than those of the in situ, which were 43.7% and 50.3%, respectively, indicating that the untreated distal tumors also had a tumor growth inhibition effect when the body's immune system was activated. The in situ tumors in the Mn-ICG@HSA illumination group were completely ablated, and about 71% of the mice completely resisted the growth of distal tumors, indicating that the efficient activation of the cGAS-STING signaling pathway directly determines the degree of activation of the body's immune system and is also the key to the therapeutic efficacy of distal tumors. When Vc was pre-injected to consume the ROS generated by light, the proportion of mice resisting the growth of distal tumors dropped to 43%, indicating that the Mn of Mn-ICG@HSA 2+ The cytoplasmic transport of manganese ions is the key to the efficient activation of the cGAS-STING signaling pathway. The mechanism of the present invention for treating tumors is mainly the photodynamic effect generated by light to achieve the cytoplasmic transport of divalent manganese ions, with the photothermal effect as an auxiliary effect, and greatly promotes immunotherapy, unexpectedly achieving complete ablation of in situ tumors, which is different from the work of others who mainly use photothermal effect therapy.

[0076] 12. The Mn-ICG@HSA prepared in Example 1 was used to investigate the IFN-β secretion in the tumor tissue of mice bearing 4T1 orthotopic breast cancer. The specific steps were as follows: 6-8 week old BALB / c female mice were taken to establish an orthotopic breast cancer mouse model, and the mice were given medication according to the above tumor inhibition experiment. The injection dose of each group was 3.0 mg kg -1 , ICG 9.9 mg kg -1 On the third day after the last administration, the mice were killed and the tumor tissues were removed. 5 mL of 0.9% saline was added to wash the blood in the tissues, and the water was absorbed with filter paper. After weighing the tumor tissue, the tissue was cut into pieces with a diameter of 1-2 mm, 1 mL of PBS was added, and the mixture was homogenized in an ice bath for 5 min, followed by centrifugation at 4°C and 3000 rpm for 20 min, and the supernatant was collected. The concentration of IFN-β in each sample was detected according to the mouse IFN-β ELISA kit detection method. The results are shown in Fig.14 Compared with PBS, the IFN-β concentration in tumor tissues of each treatment group was increased, indicating that Mn 2+ Stimulation or inducing tumor tissue damage through light can cause the secretion of IFN-β; the free drug group and Mn-ICG@HSA group increased to 1.7 times and 2.7 times before illumination, respectively, indicating that Mn-ICG@HSA light therapy can greatly increase the secretion of IFN-β by improving the activation efficiency of the cGAS-STING signaling pathway.

[0077] 13. The Mn-ICG@HSA prepared in Example 1 was used to investigate the immune capacity of tumor tissue cells in mice bearing 4T1 orthotopic breast cancer. The specific steps were as follows: 6-8 week old BALB / c female mice were taken to establish an orthotopic breast cancer mouse model, and the mice were given medication according to the above tumor inhibition experiment. The injection dose of each group was 3.0 mg kg -1 , ICG 9.6 mg kg -1 On the third day after the last administration, the mice were killed and the tumor tissues were removed. The tumors were washed with saline, cut into 1-2 mm small tumor pieces and placed in a 12-well plate. 2 mL of 1 mg mL -1 Collagenase II and 0.1 mg mL -1Deoxyribonucleotidase I culture medium was used for digestion in a cell culture incubator for 1 h. After digestion, the rubber tip on the top of the inner core of a 10 mL syringe was used to squeeze and grind on a 70 um cell filter. The filter was rinsed with 2 mL of pre-cooled FPBS solution, and the tumor tissue was collected and combined. The tumor tissue was ground and dispersed into a single cell suspension, and centrifuged at 4°C and 1500 rpm for 5 min. The supernatant was removed, and 2 mL of red blood cell lysis buffer was added. The red blood cell was lysed at 4°C for 10 min, and the upper blood water was removed to obtain the tumor cell pellet. In order to detect different immune cells in tumor tissues, the following antibodies were used to mark them: M2 / M1 macrophages were marked with CD45-APC / cy7, CD11c-FITC, CD206-PE or CD86-APC; myeloid-derived suppressor cells (MDSCs) were marked with CD45-APC / cy7, CD11c-APC, and Gr-1-PE; natural killer cells (NK cells) were marked with CD45-APC / cy7, CD3-APC, and CD335-PE; cytotoxic T lymphocytes (CTLs) were marked with CD45-APC / cy7, CD3-APC, and CD8-PE. In addition, blank tubes for tumor cells and positive single-stained tubes for each antibody were prepared separately. The tumor cells were resuspended in FPBS and divided into 5 parts. 300L of staining solution was added to each sample (except the blank tube), and then stained, fixed, resuspended, filtered, and finally transferred to the flow cytometry loading tube for measurement. Fig.15 1) The NK cells in the distal tumors that were not irradiated also increased accordingly. After Mn-ICG@HSA irradiation synergistic treatment, the NK cells in the proximal and distal tumor tissues increased from 2.1 ± 0.2% and 2.1 ± 0.1% to 3.6 ± 0.2% and 3.1 ± 0.2%, respectively. This indicates that Mn-ICG@HSA can increase the proportion of NK cells in tumor tissues, systematically activate the body's innate immunity, and further induce the infiltration of NK cells in distal tumor tissues, thereby inhibiting the growth of distal tumors while treating in situ tumors; 2) CD8 + The number of CTLs increased significantly compared with that before light exposure, indicating that light-induced IFN-β secretion can stimulate the proliferation of T cells; CD8 + CTL increased from 2.4 ± 0.3% and 2.0 ± 0.3% to 17.9 ± 1.1% and 13.5 ± 1.4%, respectively, with an increase of 6.4 times and 5.8 times, respectively. +The increase in CTL may be due to the activated T cells in the lymph nodes, which migrate to the tumor site through the blood circulation, further indicating that Mn-ICG@HSA phototherapy can increase the number of tumor-infiltrating T lymphocytes and activate adaptive immunity to mediate a powerful tumor cell killing effect; ) After Mn-ICG@HSA phototherapy, the proportion of M1 macrophages in the tumor tissues on both sides increased significantly, by 5.5 times and 4.1 times, respectively; the proportion of M2 macrophages decreased significantly, by 76.6% and 63.1%, respectively; 4) Myeloid-derived suppressor cells MDSCs also showed a significant decrease trend after treatment, from 34.9 ± 3.9% and 35.2 ± 4.3% to 8.4 ± 0.7% and 11.5 ± 1.2%, respectively. This indicates that Mn-ICG@HSA can effectively reduce the proportion of immunosuppressive cells such as M2 macrophages and MDSCs in tumor tissues, and induce TAMs to differentiate into M1 macrophages that inhibit tumor growth, thereby improving the immunosuppressive tumor microenvironment. It can be seen that Mn-ICG@HSA can significantly improve the immune capacity of mice bearing 4T1 orthotopic breast cancer, thus verifying the immunological effect of Mn-ICG@HSA activating STING.

[0078] Embodiment 2

[0079] Nanoparticles are prepared using various divalent inorganic metal ions and photosensitizer ICG, and the specific steps are as follows:

[0080] First, prepare the concentration of 10 mg mL -1 Human serum albumin (HSA) aqueous solution: 100 mM Mn 2+ 、Zn 2+ , Cu 2+ 、Co 2+ Aqueous solution of divalent inorganic metal ions; Aqueous solution of indocyanine green (ICG) photosensitizer with a concentration of 1 mM.

[0081] Equal volumes of HSA solution and metal ion solution, and metal ion solution and ICG solution were mixed respectively, and centrifuged at 5000 rpm for 5 min to observe: 1) whether each divalent inorganic metal ion produces precipitation in water, HSA solution, and ICG solution, so as to examine whether various metal ions cause protein denaturation and whether they can produce precipitation with ICG through electrostatic interaction. The results are shown in Figure 2. Fig.16 Before adding albumin solution, Mn 2+ 、Zn 2+ It is a colorless, clear and transparent solution. 2+ and Co 2+ The blue and pink clear solutions were added in equal volumes of 10 mg mL -1After 50 mM metal ions were mixed with HSA solution, the solution remained clear and transparent, indicating that the metal ion solution at this concentration would not denature the protein. In addition, when the molar ratio of metal ions to photosensitizer solution was 50:1, obvious precipitation was observed, indicating that Mn 2+ 、Zn 2+ , Cu 2+ and Co 2+ It can form a precipitate with the photosensitizer solution through electrostatic interaction.

[0082] The above metal ion solution and photosensitizer ICG solution were further mixed at different molar ratios (1:1~16:1) to investigate the molar ratios of different metal ions and photosensitizer ICG to form precipitation. Fig.17 When the molar ratio of metal ions to ICG is increased to 4:1, Mn 2+ and Co 2+ It began to form a precipitate with ICG. When the molar ratio was further increased to 8:1, Zn 2+ and Cu 2+ It also began to form a precipitate with ICG, indicating that different divalent metal ions can form a precipitate with the photosensitizer ICG.

[0083] Embodiment 3

[0084] Divalent inorganic metal ions (Mn 2+ ) is combined with various photosensitizers to prepare nanoparticles. The specific steps are as follows:

[0085] Prepare the solution at a concentration of 10 mg mL -1 HSA aqueous solution, concentration 100 mM Mn 2+ The aqueous solution of divalent metal ions and the aqueous solution of ICG, chlorin e6 (Ce6) and hematoporphyrin (HPP) photosensitizer with a concentration of 1 mM were mixed with equal volumes of divalent metal ions and photosensitizers such as ICG, Ce6 and HPP aqueous solution to investigate the effects of various photosensitizers in water, divalent inorganic metal ions (Mn 2+ Take 3mL of 10 mg mL -1 HSA solution, adjust the solution pH to 6.5, add 1 mL of 16 mM MnCl2 solution, stir and react for 3 min, then add 1 mL of 1 mM ICG, Ce6 and HPP solution, stir and react for 2 h, observe the precipitation of the solution after centrifugation, and take the supernatant for determination. The hydrated particle size of the nanoparticles was determined using a Malvern particle size potentiometer. The results are shown in Fig.18 , when Mn 2+After mixing with ICG, Ce6, and HPP at a mass ratio of 16 / 1, a clear precipitate was observed after centrifugation. When human serum albumin was used to encapsulate the precipitate, the solution was clear and transparent, and no clear precipitate was observed after centrifugation, indicating that protein nanoparticles were formed. The particle size of the nanoparticles was further measured, and the results were as follows: Fig.19 The hydrated particle sizes of Mn-ICG@HSA, Mn-Ce6@HSA and Mn-HPP@HSA are 23.8 ± 8.1 nm, 27.3 ± 8.7 nm and 30.2 ± 9.9 nm, respectively.

[0086] Embodiment 4

[0087] Preparation of divalent inorganic metal ion Mn using different carrier proteins 2+ and photosensitizer hematoporphyrin (HPP) protein nanoparticles, the specific steps are as follows:

[0088] Prepare the solution at a concentration of 10 mg mL -1 HSA, bovine serum albumin (BSA), transferrin (TRF), ovalbumin (OVA) and 100 mM Mn 2+ Aqueous solution and 1mM HPP aqueous solution. Investigate whether the four proteins produce precipitation in water and the four protein nanoparticle solutions to verify the formation and encapsulation effect of nanoparticles. Mn / HPP precipitation was used as a model drug, and four proteins were used to encapsulate the precipitation. First, 3 mL of different protein aqueous solutions (HSA, BSA, TRF, OVA) were added to a 10 mL centrifuge tube, the pH of the solution was adjusted to 6.5, and 0.5 mL of Mn was added. 2+ The solution was stirred for 3 minutes, 0.5 mL of HPP solution was added, and the solution was stirred for 2 h at room temperature. The precipitation of the solution was observed after centrifugation, and the supernatant was measured. The hydrated particle size was measured using a Malvern particle size potentiometer. The results are shown in Fig. 20 When HSA, BSA, TRF, and OVA were used to encapsulate Mn / HPP, the nanoparticle solution was clear and no obvious precipitation was observed after centrifugation, indicating that protein nanoparticles were formed. The particle size of the protein nanoparticles was further measured. The results are as follows Fig.21 The hydrated particle sizes of Mn-HPP@BSA, Mn-HPP@TRF, and Mn-HPP@OVA are 29.4 ± 9.3 nm, 94.4 ± 22.7 nm, and 85.6 ± 25.9 nm, respectively. Among them, the hydrated particle size of Mn-HPP@BSA (29.4 ± 9.3 nm) is very similar to that of Mn-HPP@HSA (30.2 ± 9.9 nm).

[0089] The present invention adopts a new technical idea to effectively solve the problem that the high-valent inorganic oxides usually need to be reduced to a divalent state in cells to work. By precisely controlling the reaction pH in an aqueous solution, a protein nanoparticle encapsulating a divalent inorganic metal ion and an organic photosensitizer is successfully prepared at room temperature. The preparation conditions are mild, the method is simple, and the particle size is uniform and controllable. The obtained nanoparticles with good tumor targeting have the characteristics of stably maintaining divalent inorganic metal ions and size advantages, and can penetrate deep into tumor tissues through the blood vessel wall. In the slightly acidic environment of the lysosome (pH 5.5), the divalent inorganic metal ions / organic photosensitizers in the nanoparticles quickly dissociate and release divalent inorganic metal ions (such as Mn 2+ ), and exert phototherapy effect under light, and destroy lysosomal membrane, break through lysosomal barrier and directly transfer Mn 2+ It is transported to the cytoplasm, and then induces the release of double-stranded DNA after light exposure, produces cyclic guanosine-adenosine dinucleotide (cGAMP) in situ in the cytoplasm, activates the interferon gene stimulator (STING) signaling pathway, improves the immune response rate, enhances the efficacy of tumor immunotherapy, and can completely inhibit tumor growth in a mouse orthotopic breast cancer model; importantly, it solves the problem of water-soluble Mn 2+ The problem of difficulty in forming precipitation and inability to be effectively encapsulated in protein carriers also solves the problem that the prior art all uses organic compounds and photosensitizers to complex inside proteins. In particular, the present invention solves the problem that the existing nanomedicine treatment for STING activation is inefficient and is clinically administered by intratumoral injection.

Claims

1. A divalent inorganic metal ion / photosensitizer protein nanoparticle, characterized in that: The invention comprises a divalent inorganic metal ion, a photosensitizer and a protein, wherein the protein encapsulates the divalent inorganic metal ion and the photosensitizer; the preparation method of the divalent inorganic metal ion / photosensitizer protein nanoparticles comprises the following steps: adding a photosensitizer solution into a mixed solution of the divalent inorganic metal ion and the protein, stirring the mixture for reaction, and obtaining the divalent inorganic metal ion / photosensitizer protein nanoparticles; the divalent inorganic metal ion is Mn 2+ .

2. The divalent inorganic metal ion / photosensitizer protein nanoparticles according to claim 1, characterized in that: The photosensitizer is an organic photosensitizer; the protein is a carrier protein; the particle size of the nanoparticles is 5 to 30 nm, and the drug loading is 1% to 20%.

3. The divalent inorganic metal ion / photosensitizer protein nanoparticles according to claim 2, characterized in that: The photosensitizer includes one or more of indocyanine green, dihydrochlorin e6, and hematoporphyrin; the protein includes one or more of human serum albumin, bovine serum albumin, transferrin, and ovalbumin.

4. The method for preparing the divalent inorganic metal ion / photosensitizer protein nanoparticles according to claim 1, characterized in that: The following steps are involved: The photosensitizer solution is added into the mixed solution of the divalent inorganic metal ion and the protein, and stirred for reaction to obtain the divalent inorganic metal ion / photosensitizer protein nanoparticles.

5. The method for preparing divalent inorganic metal ion / photosensitizer protein nanoparticles according to claim 4, characterized in that: The pH value of the protein solution is first adjusted, and then a divalent inorganic metal ion solution is added to obtain a mixed solution of divalent inorganic metal ions and proteins; after stirring and reacting, the mixture is purified to obtain divalent inorganic metal ion / photosensitizer protein nanoparticles.

6. The method for preparing divalent inorganic metal ion / photosensitizer protein nanoparticles according to claim 5, characterized in that: The pH value of the protein solution is adjusted to 6-7; the stirring reaction temperature is room temperature and the time is 1-6 hours.

7. The method for preparing divalent inorganic metal ion and photosensitizer protein nanoparticles according to claim 5, characterized in that: The concentration of protein solution is 2-20 mg mL -1 ; The concentration of the divalent inorganic metal ion solution is 20-100 mM.

8. The method for preparing divalent inorganic metal ion and photosensitizer protein nanoparticles according to claim 5, characterized in that: The volume ratio of the protein solution to the divalent inorganic metal ion solution is 1-10:1; the molar ratio of the divalent inorganic metal ion to the photosensitizer is 4-16:

1.

9. Use of the divalent inorganic metal ion / photosensitizer protein nanoparticles according to claim 1 in the preparation of anti-tumor drugs.

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