Divalent inorganic metal ion / photosensitizer protein nanoparticle, and preparation therefor and use thereof

Abstract

Disclosed in the present invention are a divalent inorganic metal ion / photosensitizer protein nanoparticle, and a preparation therefor and the use thereof. In the present invention, a new technical train of thought is utilized to effectively solve the present defect whereby the immunotherapy effect can only be exerted when a delivered high-valence inorganic oxide is reduced to a divalent state in cells. The nanoparticle is successfully prepared at room temperature in a water solution with a certain pH, and the divalent inorganic metal ion and the photosensitizer entrapped in the nanoparticle form a complex. The nanoparticle has the advantages of stable metal-ion valence and small size, can penetrate deep into tumor tissue via a blood vessel wall, releases the divalent inorganic metal ion after being quickly dissociated in the subacid environment of a lysosome, and plays a phototherapy role. The nanoparticle can damage a lysosome membrane, and breaks through a lysosome barrier to directly transfer Mn2+ to cytoplasm, so that after illumination, the release of double-stranded DNA is induced, cGAMP is generated in-situ in the cytoplasm, a STING signal pathway is activated, the immune response rate is improved, and the treatment effect of the tumor immunotherapy is enhanced. Experiments verify that tumor growth can be completely suppressed in a mouse in-situ breast cancer model.

Description

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

[0001] The present invention belongs to biomedicine technology, and specifically relates to divalent inorganic metal ion / photosensitizer protein nanoparticles 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 advantages such as broad-spectrum anti-tumor activity, low toxic side effects, effective inhibition of tumor metastasis and recurrence, clearance of difficult-to-find micro-lesions, and significant extension of patients' median survival. It has made great progress in the clinical treatment of tumors (Immunity, 2020, 52(1): 17-35.). Currently, most tumor immunotherapies focus on enhancing T cell responses. For example, immune checkpoint inhibitors (ICIs) 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, T cells specifically expressing chimeric antigen receptors (CARs) modified by genetic engineering (Nature Reviews Clinical Oncology, 2020, 17(7): 418-434.) can directly target and attack tumor cells, achieving good clinical efficacy. However, due to the low response rate of single-agent ICIs 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 attracted 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 potent anti-tumor immune response. It has special advantages in treating various tumor models and improving 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.Synthetic small molecule STING agonists, such as acridones (dimethylpyrazol) and aminobenzimidazoles (diABZI), have many drawbacks, including poor solubility, off-target effects, and significant 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 delivery properties still significantly 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 a key component of current tumor treatment methods. Indocyanine green (ICG), a commonly used photosensitizer in tumor treatment, primarily acts photodynamically, with a photothermal effect. It can kill cancer cells by converting light energy into heat energy under an external light source (near-infrared light). The core of photodynamic therapy involves irradiating the tumor site with near-infrared laser light in the wavelength range of 650 to 1350 nm. After exposure to the near-infrared laser, the photosensitizer accumulated in the tumor absorbs photons and transitions from the ground state (S0) to the singlet excited state (S1) or to the triplet excited state via intersystem crossing. 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 effect, 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 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. However, due to Mn 2+It is difficult to break through the intracellular delivery barrier, and the low content of double-stranded DNA in tumors limits the application of this strategy. For example, the existing technology uses a 37°C incubation method to adsorb manganese ions with bovine serum albumin. However, after the nanoparticles are taken up into the cells, they are mainly distributed in the lysosomes and lack the cytoplasmic transport properties. 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, and 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 existing methods is usually oxidized to a high-valent form, such as manganese dioxide (positive tetravalent). MnO2 has a mainly photothermal effect and has strong reducing properties under acidic conditions. It can produce oxygen in the body, which helps to alleviate the hypoxic microenvironment of tumors. It can also be slowly converted into MnO2 with STING activation function in cells. 2+ However, the entrapment of nanoparticles makes it difficult to release from the carrier and transform smoothly, which limits their effectiveness. Therefore, it is urgent to develop a novel nanoparticle with cytoplasmic transport properties while maintaining Mn 2+ Valence delivery strategy. Technical issues

[0005] In order to solve the above problems, the purpose of the present invention is to provide a divalent inorganic metal ion / photosensitizer protein nanoparticles and their 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 irradiation, oxidative stress damages the lysosomal membrane, causing the drug in the lysosome to diffuse into the cytoplasm (this process is called light-induced drug cytoplasmic transport), and further diffuse to the cell nucleus to kill tumor cells, exerting the effect of photodynamic therapy. At the same time, Mn 2+Generate double-stranded DNA required for STING activation, efficiently activate the STING signaling pathway, improve the immune response rate, and play an immunotherapy role. 2+ Co-delivery of TNF-α and ICG can achieve efficient tumor treatment through multiple means. Technical Solutions

[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 aforementioned divalent inorganic metal ion / photosensitizer protein nanoparticles, comprising the following steps: adding a photosensitizer solution to a mixture of divalent inorganic metal ions and protein, stirring the mixture to react, and obtaining the divalent inorganic metal ion / photosensitizer protein nanoparticles; and further, stirring the mixture to react and purify the mixture to obtain the 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 the mixture of divalent inorganic metal ions and protein.

[0009] In the present invention, the divalent inorganic metal ion / photosensitizer protein nanoparticles are composed of a divalent inorganic metal ion, a photosensitizer, and a protein. The protein encapsulates the divalent inorganic metal ion and the photosensitizer. The particle size is 5 to 30 nm, and the drug loading is 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 ion or photosensitizer) / (mass of divalent inorganic metal ion / photosensitizer protein nanoparticles) in the purified sample × 100%.

[0010] In the present invention, proteins participate 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 exert photodynamic therapy; 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 taken into cells, they are primarily distributed in lysosomes, lacking cytosolic transport properties. Unlike existing technologies that use manganese dioxide to provide oxygen to alleviate the hypoxic tumor microenvironment, this invention has a strong ability to activate the STING signaling pathway, greatly enhancing the effectiveness of immunotherapy for tumors. This is a special case of photodynamic therapy supplemented with photothermal therapy and highly effective immunotherapy, achieving highly effective tumor treatment through the simultaneous use of multiple approaches.

[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 the 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 to 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 light therapy 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. A photosensitizer is added to allow the two to continue to grow in the protein cavity, 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 transport, 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 light exposure, 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 6-7; the protein solution is a protein aqueous solution with a concentration of 2-20 mg mL -1 The divalent inorganic metal ion solution is an aqueous solution of divalent inorganic metal ions. 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 to 10:1; the concentration of the divalent inorganic metal ion aqueous solution is 20 to 100 mM; the molar ratio of the divalent inorganic metal ion to the photosensitizer is 4 to 16:1; and the molar ratio of the protein to the divalent inorganic metal ion is (0.5 to 5):1, preferably (1 to 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 utilizes 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. Beneficial effects

[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 through precipitation methods, and modifies the surface of bovine serum albumin (BSA)-coated MnO2 nanoparticles. The existing technology all 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 enhance the photodynamic therapy effect of the photosensitizer, but it does not solve the problem of its poor cytoplasmic transport properties, let alone maintaining Mn 2+ The delivery requirements of valence state do 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 is the first to use a biomineralization method based on a protein hydrophobic cavity as a template to successfully prepare divalent inorganic metal ions and photosensitizer protein nanoparticles. The divalent inorganic metal ions and photosensitizer are both 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 present invention encapsulates the divalent inorganic metal ions and photosensitizers in the protein cavity, thereby enhancing the stability of the divalent inorganic metal ions and photosensitizers 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 present invention is completely different from the existing manganese dioxide in providing oxygen.

[0021] Compared with other STING agonist-loaded nanoparticles, the bivalent inorganic metal and photosensitizer protein nanoparticles provided by the present invention have the following significant advantages: 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 (average particle size determined by TEM is 9.74 nm, hydrated particle size is 30.2 ± 9.9 nm; drug loading Mn) are formed by combining with ICG anions in the protein cavity. 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 is activated by H in the slightly acidic environment of the tumor. + Can replace Mn 2+ Competitive binding with ICG converts Mn-ICG precipitate into a more stable ICG precipitate to form free Mn 2+Therefore, Mn-ICG@HSA is taken up by tumor cells and enters the lysosome (pH 5.5). The ICG-responsive temperature rise under light can produce a phototherapy effect and destroy the lysosome, releasing free Mn. 2+ , realize Mn 2+ Lysosomal escape and cytoplasmic transport, directly exerting the effect of Mn 2+ Inducing the release of double-stranded DNA, enriching cGAMP in situ in the cytoplasm, exerting the effect 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] FIG1 is a transmission electron microscope image of the nanoparticles according to Example 1 of the present invention.

[0023] FIG2 is an X-ray photoelectron spectrum of the nanoparticles according to Example 1 of the present invention.

[0024] FIG3 is a UV-visible absorption spectrum of indocyanine green (ICG) in the nanoparticles of Example 1 of the present invention.

[0025] FIG4 is a statistical diagram of the photothermal temperature rise curve of the nanoparticles in Example 1 of the present invention.

[0026] FIG5 is a statistical diagram of active oxygen generation (A) and quantum yield (B) of nanoparticles according to Example 1 of the present invention.

[0027] FIG6 shows the Mn content of nanoparticles in Example 1 of the present invention. 2+ Release curve diagram.

[0028] FIG7 is a diagram showing that nanoparticles in Example 1 of the present invention promote the generation of reactive oxygen species in 4T1 cells.

[0029] FIG8 is a statistical diagram of the cell viability of 4T1 cells treated with nanoparticles, manganese dichloride and a physical mixture in Example 1 of the present invention, showing dark toxicity (A) and phototoxicity (B), respectively.

[0030] FIG9 is a diagram showing the nanoparticles promoting the release of dsDNA in 4T1 cells according to Example 1 of the present invention.

[0031] FIG10 is a statistical diagram showing that the nanoparticles in Example 1 of the present invention promote the secretion of type I interferon by DC2.4 cells.

[0032] FIG11 is a near-infrared in vivo fluorescence imaging diagram (A) and a fluorescence statistical diagram (B) of the nanoparticles of Example 1 of the present invention in 4T1 mouse orthotopic breast cancer.

[0033] FIG12 is a tumor section image observed by laser confocal microscopy after the treatment of orthotopic breast cancer in 4T1 mice with nanoparticles in Example 1 of the present invention. The samples are free rhodamine B, Abraxane labeled with rhodamine B, and Mn-ICG@HSA.

[0034] Figure 13 shows the tumor inhibition curves of mice bearing 4T1 orthotopic breast cancer and distal tumors treated with the nanoparticles and physical mixture of Example 1 of the present invention, which are the tumor growth curves of the proximal tumor (A) and the distal tumor (B), as well as the survival curve of the tumor-bearing mice (C).

[0035] FIG14 is a statistical diagram of type I interferon secretion in the tumor tissue of a 4T1 in situ breast cancer model of the nanoparticles and the physical mixture of Example 1 of the present invention.

[0036] Figure 15 shows the expression of immune cells in 4T1 orthotopic breast cancer and distal models of nanoparticles and physical mixtures in Example 1 of the present invention, which 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] FIG16 is a comparison of different divalent inorganic metal ion solutions, protein-divalent inorganic metal ion solution, and divalent inorganic metal ion-photosensitizer ICG solution in Example 2 of the present invention.

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

[0039] FIG18 is a comparison of different photosensitizer solutions, divalent manganese-photosensitizer precipitation, and protein nanoparticle solutions prepared with divalent manganese ions and different photosensitizers in Example 3 of the present invention.

[0040] FIG19 is a diagram showing the hydrated particle size of protein nanoparticle solutions prepared from divalent manganese ions and different photosensitizers in Example 3 of the present invention.

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

[0042] FIG. 21 shows different proteins encapsulating Mn in Example 4 of the present invention. 2+ -HPP precipitation hydration particle size diagram. Modes for Carrying Out the Invention

[0043] The specific embodiments of the present invention are further described in detail below 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 invention. The raw materials and reagents used in the present invention are all commercially available products and meet the conventional requirements of drugs. 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 were evaluated using one-way analysis of variance with a two-sided Student's t-test or a Tukey post hoc 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 generate a divalent metal ion (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 the 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, traditional delivery strategies 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, which can increase the activity of immune cells and catalyze cGAS to efficiently recognize dsDNA, accelerate the cytoplasmic enrichment of cGAMP, and promote the activation of immune cells through metal ions (such as Mn 2+ ) directly catalyzes the dsDNA induced by phototherapy, producing the STING agonist cGAMP in situ in the cytoplasm, and Mn 2+Further enhancing the activation effect of cGAMP on STING protein can significantly improve the activation efficiency of the 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, first, under near-neutral conditions, divalent inorganic metal ions bind to amino acid residues in proteins to form nucleation centers, and then a photosensitizer is introduced to react with the divalent inorganic metal to cause precipitation, so that the two are precipitated together in the hydrophobic cavity of the protein, thereby preparing a divalent inorganic metal ion and photosensitizer protein nanoparticle; solving the problem of water-soluble Mn 2+ It is difficult to form precipitation and cannot be effectively encapsulated in protein carriers, and it also solves the problem that the existing technology is to combine 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 and the mixed solution 2 are mixed and stirred to obtain mixed solution 3, and then stirred for reaction, and then purified by ultrafiltration and water washing 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 for realizing the stirring reaction and the control of the stirring reaction are conventional technologies; the purification is ultrafiltration and washing with water until neutrality is achieved to remove free small molecules and provide a stable environment for the nanoparticles. During ultrafiltration, the centrifuge speed is 1500 to 3000 rpm.

[0053] In the present invention, the solvent in the protein solution, divalent inorganic metal ion solution, and photosensitizer solution 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 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] Example 1

[0058] The specific steps for preparing divalent manganese ion and photosensitizer protein nanoparticles are as follows:

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

[0060] The prepared Mn-ICG@HSA was photographed by transmission electron microscopy, and the results are shown in Figure 1. The average particle size of the nanoparticles is (9.74±0.90) nm, and the particle size is uniform and evenly dispersed. In addition, the Mn 2+ The drug loading capacity of α-Glutathione is 2.2%, and the drug loading capacity of ICG is 6.2%.

[0061] Control 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. The specific steps were as follows: (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 shown in Figure 2. Compared with the free drug MnCl2, Mn 2++ICG physical mixture and Mn-ICG@HSA XPS spectrum peak shape is consistent with MnCl2, which proves 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 doublet binding energy was 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+ .

[0064] (2) ICG spectral analysis: Prepare the solution at a concentration of 10.0 mg mL -1 The ICG aqueous solution and Mn-ICG@HSA nanoparticle solution were prepared, and the absorption spectra from 300 to 900 nm were measured by UV-visible spectrophotometer. The results are shown in 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 the aqueous solution, its maximum absorption wavelength is red-shifted from 780 nm to 800 nm, proving that ICG is successfully encapsulated into the Mn-ICG@HSA nanoparticles.

[0065] 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 the solution was placed in a 2 mL round-bottom EP tube (n = 3). A 785 nm near-infrared laser was used to irradiate the solution. A digital thermometer probe was inserted into the solution and the solution temperature was recorded every 30 s. The temperature at the non-illumination and 0.15 W cm -2 , 0.3 W cm -2 and 0.5 W cm -2 The temperature rise of the solution within 5 min under the light power is shown in Figure 4. -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. As the light power increased, the photothermal warming effect of the nanoparticle solution also increased synchronously, indicating that Mn-ICG@HSA has a good photothermal warming effect in vitro.

[0066] 3. The photodynamic 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 1 μg mL-1 2.97 mL of the nanoparticle solution was placed in a quartz cuvette, and 30 μL of a 3 mM singlet oxygen probe (1,3-diphenylbenzofuran, DPBF) was added. Under stirring, the solution was irradiated with a 785 nm near-infrared laser and the wavelengths of the samples were recorded under non-illumination and 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 absorbance value (A) of the solution at 420 nm changes within 10 min under the illumination power. -2 The singlet oxygen quantum yield of Mn-ICG@HSA under the illumination 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: Δ = (Φ ΔIC G · 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: the photobleaching rate of DPBF; I: the absorbance rate of the test solution and the reference solution. The results are shown in Figure 5. Under non-illumination conditions, there is 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 was 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 different light irradiation powers were 0.11 ± 0.01, 0.16 ± 0.002, and 0.25 ± 0.02, respectively, indicating that Mn-ICG@HSA has a good ROS production ability under light irradiation, which is beneficial for killing tumor cells.

[0067] 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 A free MnCl₂ aqueous solution of the same concentration was prepared as a control (n=3). 1 mL of each MnCl₂ and Mn-ICG@HSA nanoparticle solution was sealed in a dialysis bag (molecular weight cutoff: 3500 Da) and placed in 10 mL of phosphate or acetate buffer at pH 7.4, pH 6.5, or pH 5.0. The system was shaken at 37°C and 120 rpm on a thermostatic shaker. The entire receiving medium was collected at time points of 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h, and 10 mL of fresh buffer was added. After sampling at all time points, the samples were filtered through a 0.22 μm microporous membrane. The released manganese from the receiving medium was measured by inductively coupled plasma optical emission spectrometry, and the cumulative release percentage was calculated. The results are shown in 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 rapidly 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% at 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 converted into a more stable ICG precipitate, and Mn 2+ Therefore, after being taken up by tumor cells, Mn-ICG@HSA is stored in lysosomes (pH 5.5). Under light, the ICG temperature rises in response to damage the lysosomes and quickly releases free Mn. 2+ , realize Mn 2+ Lysosomal escape and cytosolic trafficking.

[0068] 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 seeded into confocal glass bottom dishes (1×10 5 1 mL of conventional culture medium was added to each well. After overnight culture, 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 (ICG: 5 μg mL -1After the four samples were added and incubated for 6 h, the culture medium was removed and the 4T1 cells were washed three times with PBS. Fresh culture medium was added and the cells were illuminated (785 nm, 0.5 W cm -2 , 3 min); the non-illuminated group continued incubation. Immediately after illumination, the non-illuminated and illuminated groups underwent the following staining: 1) Add 10 μM dihydroethidium (DHE), a reactive oxygen species (ROS) probe, to the glass-bottomed dish. Incubate in the dark for 10 min, remove the stain, and wash three times with PBS. 2) Stain the cell nuclei with Hoechst 33342 for 6 min, remove the dye, and wash three times with PBS. Cell red fluorescence intensity was immediately observed using CLSM. The results are shown in Figure 7. The non-illuminated group exhibited only weak red fluorescence, while the illuminated group produced intense red fluorescence coinciding with the cell nucleus, indicating that cellular uptake of the Mn-ICG@HSA nanoparticles and illumination immediately generated significant amounts of ROS within the cells.

[0069] 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 96-well cell culture plates (5×10 3 Cells were plated at 400 μL per well (100 μL of single-cell suspension diluted in culture medium was added to each well and cultured overnight). Six groups were set up: MnCl2 non-illuminated group, ICG illuminated group, Mn+ICG non-illuminated group, Mn+ICG illuminated group, Mn-ICG@HSA non-illuminated group, and Mn-ICG@HSA illuminated group. The ratio of manganese to ICG in the physical mixture was consistent with that of Mn-ICG@HSA nanoparticles (Mn:ICG = 4:1). For each group, the following concentration gradient was set based on the manganese concentration: 0, 5, 10, 20, 50, 100, and 200 μM, corresponding to an ICG concentration gradient of 0, 1.25, 2.5, 5, 12.5, 25, and 50 μM. Before drug administration, the old culture medium was removed and 100 μL of drug-containing culture medium of different concentrations was added. Four replicate wells were set for each concentration. 12 h after 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 diluted in culture medium -1 Methylthialazole Tetrazolium (MTT) was incubated in the dark for 4 h, and the upper layer of liquid was discarded. 100 μL of 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 -2After 3 min, the cells were cultured for 12 h. The MTT dosing protocol was followed, and the cell viability was determined using a microplate reader. The following formula was used to calculate cell viability: Cell viability (%) = experimental group A / average absorbance of negative control group A0 × 100% (the group with a concentration of 0 μM was used as the negative control). The results are shown in Figure 8. Compared with MnCl2 and the physical mixture (Mn+ICG), Mn-ICG@HSA showed reduced cytotoxicity under non-illumination conditions. The IC values ​​of the three were significantly higher than those of the experimental group (A0). 50 The Mn concentrations of Mn-ICG@HSA were 71.1 mM, 64.9 mM and 100.9 mM, respectively (in terms of Mn concentration, Figure 8A). Compared with the ICG illumination group and the physical mixture illumination group, the cytotoxicity of Mn-ICG@HSA was enhanced after illumination (Figure 8B). 50 17.4 mg mL -1 , 8.4 mg mL -1 and 6.5 mg mL -1 (Measured in terms of ICG concentration). This demonstrates 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 while reducing side effects caused by off-target effects.

[0070] 7. The intracellular dsDNA release experiment of Mn-ICG@HSA prepared in Example 1 was conducted. The specific steps were as follows: 4T1 cells were seeded into confocal glass bottom dishes (1×10 5 After the cells attached 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, washing, 0.5 W cm -2After illumination for 3 min (the non-illuminated group continued incubation), 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 in primary antibody diluent was added to each dish, incubated at 4°C for 12 h, washed three times with PBS, and then FITC-conjugated fluorescent secondary antibody (FITC-AffiniPure Goat Anti-Rabbit IgG (H+L)) diluted in PBS was added and stained at room temperature in the dark for 45 min. The staining solution was removed and washed with PBS. 500 μL of Hoechst 33342 staining solution was immediately added to the glass-bottomed dish, stained for 6 min at room temperature in the dark, and then the staining solution was washed away. The green fluorescence intensity in the cells was immediately observed by CLSM. The results are shown in Figure 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.

[0071] 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 as follows: using a mouse IFN-β enzyme-linked immunosorbent assay (ELISA) kit to detect the secretion of IFN-β in the supernatant of DC2.4 after the treatment of Mn-ICG@HSA nanoparticles. The experimental method is as follows. DC2.4 were seeded in a 24-well plate (2×10 4Cells were grouped, dosed, and illuminated according to the Western Blot dosing protocol described above. Supernatants were collected 12 hours after illumination and centrifuged at 2000 rpm for 15 minutes at 4°C to remove cells and cell debris. Standard and sample wells were set up. The test samples were first diluted 5-fold with diluent. A series of standard solutions and test sample solutions were added to the enzyme-labeled plate. The solutions were added to the bottom of the wells and gently shaken to mix. The plate was sealed with sealing film and incubated at 37°C for 30 minutes. After incubation, 200 μL of wash buffer was added to each well, and the wells were washed five times. The liquid was discarded and the wells were dried. 50 μL of enzyme-labeled reagent was added to each well, and the wells were incubated at 37°C for 30 minutes. After incubation, the wells were washed five times and the color was developed. Add 50 μL of colorimetric reagent solution A and solution B to each well, gently shake and mix, develop at 37°C in the dark for 15 minutes, add 50 μL of stop solution to each well to terminate the reaction, and finally measure the absorbance of each well at 450 nm using a microplate reader. The concentration of IFN-β in each sample is calculated using the standard curve. The results are shown in Figure 10. There is no significant change in the IFN-β secretion in the PBS light-exposed group, indicating that light stimulation alone cannot induce IFN-β secretion; 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-light-irradiated group, due to the 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 increased to 2.8 times compared with 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.

[0072] 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 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 through the tail vein (MnCl2 and ICG solution were injected into the veins of both sides, not the same side or mixed solution). The other group was the nanoparticle group, which was injected with Mn-ICG@HSA nanoparticle solution through the tail vein. The injection dose of both groups was 3.0 mg kg -1 , ICG 10 mg kg -1. The mice were scanned using a small animal fluorescence imaging system before administration (0 h) and 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. The results are shown in Figure 11. No obvious fluorescence was observed in the abdomen of mice in both groups except for the tumor site. Moreover, the fluorescence intensity of the Mn-ICG@HSA nanoparticle group injected with the tail vein was approximately 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.

[0073] 10. The tumor penetration of Mn-ICG@HSA prepared in Example 1 was investigated. The specific steps were as follows: clinical albumin-bound paclitaxel (Abraxane) and Mn-ICG@HSA were labeled with Rhodamine B: 3 mL of freshly prepared Mn-ICG@HSA was mixed with 30 μL of Rhodamine B (5 mg mL -1 The mixture was 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 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 rhodamine B. -1 The dose was injected into the tail vein of tumor-bearing mice. Mice were sacrificed 24 hours later and tumors were collected. After embedding in paraffin, sections were sliced, incubated with CD31 antibody (green) at 4°C for 4 hours, and nuclear staining was performed (Hoechst 33342, 1 μg mL -1 ) and fluorescence intensity of the sections was observed using a confocal microscope. The results are shown in Figure 12. Due to poor tumor targeting, no significant red fluorescence was observed in the free rhodamine B group. Due to its size (~130 nm, much larger than the 9.74 nm nanoparticles of the present invention), the Abraxane group accumulated primarily in tumor blood vessels, making it difficult to penetrate deep into the tumor. However, in the Mn-ICG@HSA group, extensive red fluorescence was observed in the tumor tissue. This indicates that after targeting the tumor, Mn-ICG@HSA, thanks to its smaller size, can penetrate deep into the tissue through tumor blood vessels, facilitating its therapeutic effect.

[0074] 11. The Mn-ICG@HSA prepared in Example 1 was used to investigate the tumor inhibition of 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 (5×10 5Cells were treated 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. The injection dose of Mn in each group was 3.0 mg kg -1 , ICG 9.6 mg kg -1 Twelve hours after 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 the mice were recorded every two days, and the tumor growth of the mice was recorded within 30 days (when the proximal tumor volume was greater than 1500 mm 3 Mice were sacrificed and the tumor inhibition rate was calculated (tumor inhibition rate = (1 - tumor volume of experimental group / tumor volume of control group) × 100%). The survival status of the mice was monitored for 60 days and a survival curve was drawn. The results are shown in Figure 13. The growth of in situ tumors has a similar growth trend to that of distal tumors. There is no significant difference in the growth rate of tumors in the PBS illumination group, free drug group and Mn-ICG@HSA non-illumination group compared with 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. The inhibition rates of distal tumors were slightly lower than those of in situ, which were 43.7% and 50.3%, respectively. This shows that untreated distal tumors also have a tumor growth inhibition effect when the body's immune system is 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 pre-injection of Vc consumes 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+ Cytoplasmic transport of manganese ions is key to efficient activation of the cGAS-STING signaling pathway. The present invention's tumor treatment mechanism primarily relies on the photodynamic effect generated by illumination to achieve cytoplasmic transport of divalent manganese ions, with photothermal effects as a supplement. This significantly enhances immunotherapy and unexpectedly achieves complete ablation of in situ tumors, unlike other studies that primarily utilize photothermal therapy.

[0075] 12. The Mn-ICG@HSA prepared in Example 1 was used to investigate the secretion of IFN-β 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 used to establish an orthotopic breast cancer mouse model. The mice were divided into groups according to the above tumor inhibition experiment and given medication. The injection dose of Mn 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, the water was absorbed with filter paper, and the tumor tissue was weighed. The tissue was cut into pieces with a diameter of 1-2 mm, and 1 mL of PBS was added. The mixture was homogenized in an ice bath for 5 minutes, and then centrifuged at 4°C and 3000 rpm for 20 minutes to collect the supernatant. The concentration of IFN-β in each sample was detected according to the mouse IFN-β ELISA kit. The results are shown in Figure 14. Compared with PBS, the IFN-β concentration in the tumor tissues of each administration group was increased, indicating that Mn alone can effectively inhibit the growth of IFN-β. 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 light treatment, respectively, indicating that Mn-ICG@HSA light therapy can greatly increase the secretion of IFN-β by enhancing the activation efficiency of the cGAS-STING signaling pathway.

[0076] 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 used to establish an orthotopic breast cancer mouse model. The mice were divided into groups according to the above tumor inhibition experiment and given medication. The injection dose of Mn 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 normal saline, minced 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 -1The cells were digested in a cell culture incubator with deoxyribonucleotidase I medium for 1 h. After digestion, the cells were squeezed and ground on a 70 μm cell strainer using the rubber tip on the inner core of a 10 mL syringe. The strainer was rinsed with 2 mL of pre-cooled FPBS solution, and the cells were collected and combined. The tumor tissue was ground and dispersed into a single-cell suspension and centrifuged at 1500 rpm at 4°C for 5 min. The supernatant was removed, and 2 mL of red blood cell lysis buffer was added. The cells were lysed at 4°C for 10 min. The upper blood layer was removed to obtain the tumor cell pellet. To detect different immune cells in tumor tissues, the following antibodies were used to label them: M2 / M1 macrophages were labeled with CD45-APC / cy7, CD11c-FITC, CD206-PE, or CD86-APC; myeloid-derived suppressor cells (MDSCs) were labeled with CD45-APC / cy7, CD11c-APC, and Gr-1-PE; natural killer cells (NK cells) were labeled with CD45-APC / cy7, CD3-APC, and CD335-PE; and cytotoxic T lymphocytes (CTLs) were labeled 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. Tumor cells were resuspended in FPBS and divided equally into five aliquots. Each sample was added with 300 μL of staining solution (except for the blank tube), stained, fixed, resuspended, and filtered. Finally, the cells were transferred to flow cytometer tubes for analysis. The results are shown in Figure 15. 1) NK cells also increased in the distal tumor without illumination. After Mn-ICG@HSA synergistic illumination treatment, the NK cell counts 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 tissue, systemically activate the body's innate immunity, and further induce NK cell infiltration in distal tumor tissue, thereby inhibiting the growth of distal tumors while treating the in situ tumor. 2) After illumination, CD8 + The number of CTLs increased significantly compared with that before light irradiation, indicating that light-induced IFN-β secretion can stimulate the proliferation of T cells; CD8 + The CTL percentages 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. +The increase in CTLs may be due to activated T cells in the lymph nodes, which migrate to the tumor site through the blood circulation. This further demonstrates 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 downward trend after treatment, decreasing from 34.9 ± 3.9% and 35.2 ± 4.3% to 8.4 ± 0.7% and 11.5 ± 1.2%, respectively. This study showed that Mn-ICG@HSA can effectively reduce the proportion of immunosuppressive cells in tumor tissue, such as M2 macrophages and MDSCs, while inducing TAMs to differentiate into M1 macrophages, which inhibit tumor growth, thereby improving the immunosuppressive tumor microenvironment. This indicates that Mn-ICG@HSA can significantly enhance the immune capacity of mice bearing 4T1 orthotopic breast cancer, thus verifying the immunological effects of Mn-ICG@HSA-induced STING activation.

[0077] Example 2

[0078] Nanoparticles were prepared using various divalent inorganic metal ions and photosensitizer ICG. The specific steps were as follows: First, 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; an aqueous solution of indocyanine green (ICG) photosensitizer with a concentration of 1 mM.

[0079] Equal volumes of HSA solution and metal ion solution, and metal ion solution and ICG solution were mixed, centrifuged at 5000 rpm for 5 min, and the following observations were made: 1) whether each divalent inorganic metal ion produced precipitation in water, HSA solution, and ICG solution, thereby investigating whether various metal ions cause protein denaturation and whether they can produce precipitation with ICG through electrostatic interaction. The results are shown in Figure 16. Before adding albumin solution, Mn 2+ 、Zn 2+ It is a colorless, clear and transparent solution. 2+ and Co 2+ They were blue and pink clear transparent solutions, and 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 through electrostatic interaction with the photosensitizer solution.

[0080] 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 precipitates. 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.

[0081] Example 3

[0082] Divalent inorganic metal ions (Mn 2+ ) were combined with various photosensitizers to prepare nanoparticles. The specific steps were as follows: the concentration of 10 mg mL -1 HSA aqueous solution with a concentration of 100 mM Mn 2+ Aqueous solutions of divalent metal ions and 1 mM aqueous solutions of ICG, chlorin e6 (Ce6) and hematoporphyrin (HPP) photosensitizers were prepared. Equal volumes of divalent metal ions and photosensitizers such as ICG, Ce6 and HPP were mixed to investigate the effects of various photosensitizers in water, divalent inorganic metal ions (Mn 2+ Take 3 mL 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 measurement. The hydrated particle size of the nanoparticles was measured using a Malvern particle size potentiometer. The results are shown in Figure 18. When Mn 2+After mixing with ICG, Ce6, and HPP at a 16:1 ratio, a clear precipitate was observed after centrifugation. When this precipitate was encapsulated with human serum albumin, the solution became clear and transparent, and no significant precipitation was observed after centrifugation, indicating the formation of protein nanoparticles. Further measurement of the nanoparticle size reveals the following results, as shown in Figure 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.

[0083] Example 4

[0084] Preparation of divalent inorganic metal ions Mn using different carrier proteins 2+ and photosensitizer hematoporphyrin (HPP) protein nanoparticles, the specific steps are as follows:

[0085] Prepare the mixture to a concentration of 10 mg mL -1 HSA, bovine serum albumin (BSA), transferrin (TRF), ovalbumin (OVA) four kinds of protein aqueous solution, and the concentration of 100mM Mn 2+ Aqueous solution and 1mM HPP aqueous solution. Investigate whether the four proteins produce precipitation in water and the four protein nanoparticle solutions, so as to verify the formation and encapsulation effect of nanoparticles. Mn / HPP precipitation was used as a model drug, and the 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, and 0.5 mL of HPP solution was added. The reaction was stirred at room temperature for 2 hours. The solution was centrifuged to observe the precipitation, and the supernatant was collected for analysis. The hydrated particle size was determined using a Malvern particle size analyzer. The results are shown in Figure 20. When HSA, BSA, TRF, and OVA were used to encapsulate Mn / HPP, the nanoparticle solutions were clear, and no obvious precipitation was observed after centrifugation, indicating the formation of protein nanoparticles. The particle size of these protein nanoparticles was further measured, as shown in Figure 21. The hydrated particle sizes of Mn-HPP@BSA, Mn-HPP@TRF, and Mn-HPP@OVA were 29.4 ± 9.3 nm, 94.4 ± 22.7 nm, and 85.6 ± 25.9 nm, respectively. The hydrated particle size of Mn-HPP@BSA (29.4 ± 9.3 nm) was very similar to that of Mn-HPP@HSA (30.2 ± 9.9 nm).

[0086] The present invention adopts a new technical idea to effectively solve the problem that the current delivery of high-valent inorganic oxides requires reduction to a divalent state in cells to work. By precisely controlling the reaction pH in an aqueous solution, a protein nanoparticle encapsulating divalent inorganic metal ions and an organic photosensitizer was 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 have good tumor targeting properties and size advantages. They can penetrate deep into tumor tissue 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 play a phototherapy role under light, and destroy the lysosomal membrane, break through the 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 existing technology uses organic compounds and photosensitizers to combine inside proteins. In particular, the present invention solves the problem that the existing nanomedicines used for STING activation have low therapeutic efficiency and are clinically administered by intratumoral injection.

Claims

1. A divalent inorganic metal ion / photosensitizer protein nanoparticle, characterized in that: Including divalent inorganic metal ions, photosensitizers and proteins.

2. The divalent inorganic metal ion / photosensitizer protein nanoparticles according to claim 1, characterized in that: The protein encapsulates divalent inorganic metal ions and photosensitizers; 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 divalent inorganic metal ions include 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.

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 a drug for improving the transport of divalent inorganic metal ions to the cytoplasm.

10. 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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