Metal polyphenol nano-material with photothermal effect and tumor microenvironment immunoregulation function as well as preparation method and application of metal polyphenol nano-material

Abstract

The invention discloses a metal polyphenol nano-material with a photothermal effect and a tumor microenvironment immunoregulation function as well as a preparation method and application of the metal polyphenol nano-material. The metal polyphenol nano-material is prepared by utilizing a complexation reaction of phenolic hydroxyl groups and metal ions; and the dioscin with an immune regulation function is embedded into the nano-particles while the material is self-assembled. The preparation process is simple and easy to implement, low in energy consumption, environment-friendly and convenient for large-scale production. The metal polyphenol nano material not only has good biocompatibility, but also shows a relatively strong tumor killing effect and an anti-tumor immune activation function in both in-vitro and in-vivo experiments, and shows a relatively strong photo-thermal and immune combined treatment effect on tumors.

Description

Technical Field

[0001] The present invention relates to a multifunctional nanomaterial with photothermal and immune combined therapeutic efficacy, and in particular to a metal polyphenol nanomaterial with photothermal effect and tumor microenvironment immunoregulatory function, and a preparation method and application thereof, belonging to the technical field of nanomaterials. Background Art

[0002] Malignant tumors (cancer) are currently one of the most deadly diseases worldwide. According to the World Health Organization (WHO), cancer is one of the leading causes of death worldwide, especially in developing countries. The incidence and mortality of cancer are rising worldwide. At present, the traditional means of clinical treatment of cancer mainly include surgery, radiotherapy and chemotherapy. Surgical treatment can remove tumor tissue and limit its growth, but this therapy may not completely eliminate all cancer cells, and residual cancer cells may grow again in the future. Radiotherapy uses ionizing radiation to inhibit the proliferation of cancer cells, but this method may cause a variety of complications and side effects. Chemotherapy is a systemic treatment method. Whether it is administered orally, intravenously or intracavitary, chemotherapy drugs can reach most organs and tissues in the body through blood circulation. For tumors that are easy to spread and cancers that have already metastasized, chemotherapy can provide better therapeutic effects, but while chemotherapy drugs kill tumor cells through blood circulation, they also cause damage to normal tissues. It is precisely because of the strong infiltration and invasiveness of tumor cells that the above traditional therapies are difficult to completely eliminate cancer cells, so preventing tumor recurrence and metastasis remains challenging.

[0003] Immunotherapy is a treatment that uses the body's own immune system to identify, attack and destroy cancer cells. This treatment approach aims to enhance or reactivate the immune system's anti-cancer ability to control or eliminate tumors. Despite this, immunotherapy still faces some challenges, including weak immune responses and severe adverse reactions, which are attributed to the lack of immunogenicity. Photothermal therapy (PTT) is an advanced medical technology that uses the targeting properties of specific drugs to concentrate efficient photothermal conversion materials on the lesion area. By irradiating the treatment area with near-infrared lasers, these photothermal materials are able to absorb laser energy and quickly convert it into heat energy. This local heating process does not cause harm to surrounding healthy tissues. At a temperature of 40°C, tumor cell proteins will denature, and when the temperature exceeds 55°C, they will suffer irreversible damage, resulting in cell lysis and inactivation. Although photothermal therapy can stimulate anti-tumor immune responses, local PTT monotherapy alone is usually not enough to prevent tumor metastasis and recurrence, which are often the main causes of death in cancer patients. In addition, studies have shown that tumor edges outside the PTT treatment area may grow faster due to the effects of hyperthermia. To overcome these challenges, researchers are exploring a variety of strategies, such as improving the photothermal conversion efficiency of photothermal converters (PTAs), increasing the PTA enrichment in tumor tissues, and combining PTT with other treatments (such as chemotherapy, radiotherapy, gene therapy, immunotherapy, etc.) to enhance the therapeutic effect and reduce side effects.

[0004] At present, there are no research reports on the use of dioscin, PD-L1 antagonist peptide and EGCG to prepare metal polyphenol nanomaterials for combined photothermal and immunotherapy. Summary of the invention

[0005] Studies have found that the use of photothermal therapy in combination with immunotherapy can improve the therapeutic effect of cancer. The reason is that the combined use of photothermal therapy and immunotherapy can improve the tumor microenvironment and promote anti-tumor immune response, thereby overcoming the shortcomings of single photothermal therapy. The main strategies include reducing immunosuppressive factors in the tumor, such as PD-1 / PD-L1 and CTLA-4, and activating immune cells such as NK cells, macrophages, DCs and T cells. In particular, the activation of T cells, which requires antigen-specific signals of TCR-MHC and co-stimulatory signals of CD28-B7, has a significant killing effect on tumor cells.

[0006] The tumor microenvironment (TME) is a complex ecosystem composed of tumor cells and a variety of non-tumor cells and molecules, which plays a vital role in tumor progression, metastasis and therapeutic response. In the TME, tumor-associated macrophages (TAMs) are the main tumor-infiltrating immune cells, which can be divided into classically activated M1 type and alternatively activated M2 type according to their activation state. M1 type TAMs have pro-inflammatory and anti-tumor properties, can secrete pro-inflammatory cytokines such as IL-12, TNF-α, IFN-γ, and exert tumor suppression effects by highly expressing nitric oxide synthase (iNOS), promote Th1 type immune response, and inhibit tumor progression. In contrast, M2 type TAMs produce anti-inflammatory cytokines such as IL-13, IL-10 and IL-4, highly express arginase (Arg-1) and mannose receptors (such as MR, CD206), thereby promoting tumor growth and metastasis. In many tumors, TAMs usually show M2 type, which is associated with poor prognosis of patients. Dioscin is a natural steroidal saponin extracted from a variety of plants, especially in the Dioscoreaceae family. It has been shown to have anti-tumor, anti-thrombotic, anti-allergic, anti-viral and anti-shock effects. Studies have shown that dioscin can promote the transformation of M2 macrophages to M1 macrophages, thereby changing the immunosuppressive state of TME to an immune-activated state. This transformation stimulates the maturation of dendritic cells (DCs), increases the expression of co-stimulatory molecules CD80 and CD86 on the surface of DCs, and then activates T cells to exert anti-tumor effects. Therefore, dioscin and its effects on TAMs polarization provide the possibility of developing new therapeutic strategies.

[0007] PD-L1 antagonist peptides are a class of key immune checkpoint regulators that enhance the ability of immune cells T cells in the body to recognize and kill tumor cells by blocking the interaction between PD-L1 and PD-1. In the tumor microenvironment, tumor cells and tumor-associated antigen-presenting cells (APCs) usually highly express PD-L1, while tumor-infiltrating lymphocytes that are stimulated by tumor antigens for a long time highly express PD-1. The binding of PD-L1 to PD-1 induces apoptosis, dysfunction, and exhaustion of T cells, inhibits the activation, proliferation, and anti-tumor function of tumor antigen-specific CD8+T cells, and leads to tumor immune escape. To prevent this process, PD-L1 antagonist peptides competitively bind to PD-L1, effectively blocking the interaction between PD-L1 and PD-1, thereby preventing T cell inhibition and apoptosis. In addition, the targeting of PD-L1 antagonist peptides enables them to specifically recognize and bind to PD-L1 on tumor cells.

[0008] Metal polyphenol networks (MPNs) are a new type of biomedical material, which are formed by the coordination interaction between phenolic hydroxyl groups and multivalent metal ions. This material shows great potential for application in the biomedical field because they provide a fast and simple method to construct a multifunctional nanoplatform. As carriers of nanoparticles, MPNs not only have good biocompatibility, but also can control the release of bioactive ingredients under specific conditions during blood circulation. In addition, MPNs can be combined with polyphenols modified with PD-L1 antagonist peptides and metal ions through self-assembly to form nanoparticles that can encapsulate and carry active substances. Due to the relatively weak coordination effect of MPNs, they can easily decompose in the acidic microenvironment unique to tumors after targeting tumor cells, thereby releasing the encapsulated substances. This property, combined with the targeting ability of PD-L1 antagonist peptides, makes MPNs a powerful platform for drug delivery, especially in the fields of immunotherapy and tumor treatment.

[0009] There is no report on the combined use of dioscin, PD-L1 antagonist polypeptide, and metal polyphenol nanomaterials. The present invention further studies on the above research and provides a method for preparing a metal polyphenol nanomaterial with photothermal effect and tumor microenvironment immunomodulatory function and the resulting product. The method prepares a nanomaterial with targeted delivery and photothermal conversion function by rapid self-assembly of a metal polyphenol network, and loads dioscin with immunomodulatory function in the nanomaterial. The metal polyphenol nanomaterial can specifically target PD-L1 highly expressed on tumor cells, which not only improves the enrichment efficiency of the nanomaterial in the tumor, but also blocks the PD-1 / PD-L1-mediated immune escape pathway. Under near-infrared laser irradiation, the nanomaterial heats up the tumor locally through photothermal conversion, which can not only directly kill tumor cells, but also induce immunogenic cell death (ICD) effect, causing tumor cells to release related antigens and immunoactive molecules, thereby activating the immune system. In addition, the metal polyphenol nanomaterial releases dioscin in the acidic tumor microenvironment, which changes the tumor's immunosuppressive environment by remodeling tumor-associated macrophages, effectively enhancing the anti-tumor cell immune response, and thus enhancing the immunotherapy effect. The metal polyphenol nanomaterial has multiple functions and exhibits strong anti-tumor efficacy through synergistic photothermal therapy and immunotherapy.

[0010] The specific technical solutions of the present invention are as follows:

[0011] A method for preparing a metal polyphenol nanomaterial having a photothermal effect and tumor microenvironment immunomodulatory function, comprising the following steps:

[0012] (1) reacting eight-arm polyethylene glycol active ester, N-(2-aminoethyl)maleimide hydrochloride, and triethylamine in an organic solvent, and adding dopamine hydrochloride and triethylamine to continue the reaction to obtain dopamine-terminated eight-arm polyethylene glycol (PEG polyphenol);

[0013] (2) reacting the dopamine-terminated eight-arm polyethylene glycol obtained in step (1), the PD-L1 antagonist polypeptide and tri(2-carboxyethyl)phosphine in a PBS buffer to obtain a dopamine-terminated eight-arm PEG polyphenol (PA-PEG polyphenol) carrying a PD-L1 antagonist polypeptide;

[0014] (3) reacting the dopamine-terminated eight-arm PEG polyphenol with PD-L1 antagonist peptide obtained in step (2), tannic acid, diosgenin, metal salt and Tris-HCl buffer in a mixed solvent of water and DMSO to obtain a metal polyphenol nanomaterial with photothermal effect and tumor microenvironment immunomodulatory function.

[0015] Furthermore, in step (1), the molar ratio of eight-arm polyethylene glycol active ester, N-(2-aminoethyl)maleimide hydrochloride, dopamine hydrochloride, and triethylamine is 1:6:2:9-12, for example, 1:6:2:9, 1:6:2:10, 1:6:2:11, 1:6:2:12. Triethylamine is added twice, and in the first step reaction, the molar ratio of triethylamine to N-(2-aminoethyl)maleimide hydrochloride is preferably 1.5:1, and in the second step reaction, the molar ratio of triethylamine to dopamine hydrochloride is preferably 1.5:1.

[0016] Furthermore, in step (1), the molecular weight of the eight-arm polyethylene glycol active ester is 15000-30000.

[0017] Furthermore, in step (1), the organic solvent can be selected from DMF, DMSO, etc. The organic solvent is used as a reaction medium to ensure that the reaction is completed in a homogeneous phase, and its amount can be adjusted as needed.

[0018] Furthermore, in step (1), the reaction is carried out under the protection of an inert gas, and the inert gas may be nitrogen, argon, or the like.

[0019] Furthermore, in step (1), the reaction is carried out at 20-35°C, preferably at room temperature. The reaction time of the eight-arm polyethylene glycol active ester, N-(2-aminoethyl) maleimide hydrochloride and triethylamine is 5 to 8 hours, and the reaction is continued for 4 to 6 hours after adding dopamine hydrochloride and triethylamine. After the reaction, the resulting reaction solution is dialyzed in water with a pH of 4-5 to remove unreacted small molecules, and then lyophilized after dialysis to obtain dopamine-terminated eight-arm polyethylene glycol (PEG polyphenol).

[0020] Furthermore, in step (2), the molar ratio of dopamine-terminated eight-arm polyethylene glycol (PEG polyphenol) to the PD-L1 antagonist polypeptide is 1-2:6, and the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to the PD-L1 antagonist polypeptide is 1.5-2:1.

[0021] Further, in step (2), PEG polyphenol, PD-L1 antagonist polypeptide and tri(2-carboxyethyl)phosphine are first dissolved in PBS buffer to form solutions, and then the solutions are mixed, the concentration of PEG polyphenol solution is 10 mg / mL to 15 mg / mL, the concentration of PD-L1 antagonist polypeptide solution is 20 to 30 mg / mL, and the concentration of tri(2-carboxyethyl)phosphine solution is 5 to 10 mg / mL. Preferably, the pH of PBS buffer is 7.2-7.6.

[0022] Furthermore, in step (2), tri(2-carboxyethyl)phosphine (TCEP) is preferably present in the form of its hydrochloride. TCEP hydrochloride is a water-soluble small molecule compound that is widely used due to its stability in aqueous solution and low odor. In addition, TCEP has extremely high selectivity for the reduction of disulfide bonds and hardly reacts with other amino acids. This selectivity makes it more stable in a complex biomolecular environment and reduces potential interference with other chemical bonds (such as amide bonds).

[0023] Furthermore, in step (2), the reaction is carried out under the protection of an inert gas, and the inert gas may be nitrogen, argon, etc.

[0024] Furthermore, in step (2), the reaction is carried out at 20-35°C, preferably at room temperature. The reaction time is 10-14 hours. After the reaction, the obtained reaction solution is dialyzed in water with a pH of 4-5 to remove unreacted small molecules, and then freeze-dried after dialysis to obtain PA-PEG polyphenol.

[0025] Furthermore, in step (3), the mass ratio of PA-PEG polyphenol, diosgenin, metal salt and tannic acid is 1-2: 0.2-0.5: 2-4: 0.8-1.6.

[0026] Furthermore, in step (3), the metal salt is at least one of an iron salt, a copper salt, a manganese salt, a calcium salt, a cobalt salt, and a zinc salt, and the metal salt may be a chloride or a nitrate of each metal, such as ferric chloride, ferric nitrate, cupric chloride, and the like.

[0027] Furthermore, in step (3), the pH of the Tris-HCl buffer is 8.0 to 8.5, and the concentration is 0.5 to 0.6 M.

[0028] Furthermore, in step (3), preferably, PA-PEG polyphenol and metal salt are added in the form of aqueous solution, and dioscin and tannic acid are first dissolved in DMSO and then added in the form of solution. There is no special requirement for the concentration of the PA-PEG polyphenol aqueous solution, the DMSO solution of tannic acid, the DMSO solution of dioscin, and the aqueous solution of metal salt, as long as the volume ratio of water in the reaction system to DMSO in the reaction system is 57-60:40-43. The water in the reaction system refers to the sum of the water in which the PA-PEG polyphenol is dispersed, the water in the metal salt aqueous solution, and the water in the Tris-HCl buffer.

[0029] Furthermore, in step (3), the concentration of the aqueous metal salt solution can be 1 to 10 mg / mL, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, and 10 mg / mL.

[0030] Further, in step (3), the reaction is carried out at 20-35°C, preferably at room temperature. There is no special requirement for the mixing order of the raw materials. They can be mixed together or partially mixed first. One raw material can be added at once or in batches or in several times. Preferably, after each solution is added, it is stirred and mixed for 0.5 to 5 minutes to mix it evenly with other solutions in the system before adding another one. After all the raw materials are added, stir and react for 0.3 to 1 hour. Preferably, in step (3), after the PA-PEG polyphenol aqueous solution, the DMSO solution of diosgenin, and the DMSO solution of tannic acid are mixed, part of the metal salt aqueous solution and all of the Tris-HCl buffer are added first, stirred and reacted for a period of time, and then the remaining metal salt aqueous solution is added and stirred and reacted for a period of time.

[0031] Furthermore, in step (3), after the reaction, centrifugation is performed to obtain a precipitate, and the precipitate is washed to remove impurities, thereby obtaining the final metal polyphenol nanomaterial having a photothermal effect and tumor microenvironment immunomodulatory function. The centrifugation speed and time are generally 18000-21000 rpm and 20-25 min. Washing is performed with water, and the number of washing times is generally 1-2 times.

[0032] The present invention has the following beneficial effects:

[0033] 1. The metal polyphenol nanomaterial prepared by the present invention has a photothermal effect and tumor microenvironment immunomodulatory function, and exhibits a strong photothermal and immunotherapy combined treatment effect on tumors. The present invention utilizes the complexation reaction of phenolic hydroxyl groups and metal ions to prepare metal polyphenol nanomaterials, and while the material is self-assembled, diosgenin is embedded in nanoparticles to finally prepare a multifunctional composite nanomaterial.

[0034] 2. The preparation process of the nanomaterial prepared by the present invention is simple to operate, consumes less energy, is environmentally friendly, and is convenient for large-scale production. In addition, the nanomaterial is composed of non-toxic components, so it has good biocompatibility and in vivo safety.

[0035] 3. The nanomaterials prepared by the present invention can effectively target tumor cells, not only improving the enrichment efficiency of the materials at the tumor site, but also blocking the PD-1 / PD-L1 immune escape pathway of tumor cells. Moreover, both in vitro and in vivo experiments have shown strong tumor killing effects and anti-tumor immune activation functions.

[0036] 4. The nanomaterials prepared by the present invention exhibit good photothermal effect under near-infrared light (808nm), causing local thermal effect on tumors without damaging normal tissues, inducing thermal ablation of tumor cells, and releasing tumor-associated antigens. This local thermal effect not only directly kills tumor cells, causing them to release tumor-associated antigens and immunostimulatory factors, but also activates tumor-specific immune responses by inducing immunogenic cell death (ICD), further enhancing anti-tumor immune responses.

[0037] 5. The nanomaterials prepared by the present invention release dioscin in the weakly acidic tumor microenvironment, thereby reshaping M1 tumor-associated macrophages into pro-inflammatory M2 macrophages, effectively improving the tumor immune microenvironment, and synergistically enhancing the anti-tumor immune response and immunotherapy effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the one-step preparation of the metal polyphenol nanomaterial with photothermal effect and tumor microenvironment immunoregulatory function prepared in Example 1.

[0039] Figure 2 is a transmission electron microscope image of PA-MPNs@Di prepared in Example 1.

[0040] Figure 3 Dynamic laser scattering results of MPNs@Di, PA-MPNs, and PA-MPNs@Di prepared in Example 1.

[0041] Figure 4 PA-MPNs@Di of different concentrations (0-200 μg / mL) prepared in Example 2 at (808 nm, 1.0 W / cm 2 )Photothermal curve under laser irradiation.

[0042] Figure 5 PA-MPNs@Di of different concentrations (0-200 μg / mL) prepared in Example 2 at (808 nm, 1.0 W / cm 2 )Thermal imaging under laser irradiation.

[0043] Figure 6 PA-MPNs@Di of different concentrations (0-200 μg / mL) prepared in Example 2 at (808 nm, 2.0 W / cm 2 )Photothermal curve under laser irradiation.

[0044] Figure 7 PA-MPNs@Di of different concentrations (0-200 μg / mL) prepared in Example 2 at (808 nm, 2.0 W / cm 2 )Thermal imaging under laser irradiation.

[0045] Figure 8 PA-MPNs@Di (100 μg / mL) prepared in Example 2 was irradiated with 808 nm laser (1.0 W / cm 2 ) After 10 min, the photothermal curves under six cycles of heating and cooling were closed.

[0046] Fig. 9 The figure shows the results of flow cytometric analysis of 4T1 cell uptake subjected to different treatments in Example 3.

[0047] Fig.10 The 4T1 cells in Example 4 were incubated with different concentrations of PA-MPNs@Di and the cells were exposed to laser light (808 nm, 2 W / cm 2 ) in the presence of .

[0048] Fig.11 4T1 cells treated with different materials (100 μg / mL) in Example 4 were irradiated with laser (808 nm, 2 W / cm 2 , 8min) after incubation for 4h and directly incubated for 4h without irradiation. The living cells and dead cells were stained with calcein-AM and PI, respectively. The scale bar represents 100μm.

[0049] Fig.12 This is a graph showing the results of flow cytometry detection of CD206 expression in RAW264.7 cells after treatment with different materials in Example 5.

[0050] Fig.13 This is a graph showing the results of flow cytometry detection of CD86 expression in RAW264.7 cells after treatment with different materials in Example 5.

[0051] Fig.14 PBS, MPNs@Di, PA-MPNs, and PA-MPNs@Di (200 μg / mL) were injected into the orthotopic 4T1 tumor-bearing mice in Example 6 for 1 h, and then the mice were irradiated with 808 nm laser (1.5 W / cm 2 ,5min) process.

[0052] Fig.15 PBS, MPNs@Di, PA-MPNs, and PA-MPNs@Di (200 μg / mL) were injected into the orthotopic 4T1 tumor-bearing mice in Example 6 for 1 h, and then the mice were irradiated with 808 nm laser (1.5 W / cm 2 ,5min) process.

[0053] Fig.16 This is a graph showing the changes in tumor growth curves of each group of mice in Example 7 over time.

[0054] Fig.17 The M2 type TAMs (CD206 + ) in percentage by quantity, expressed as F4 / 80 + CD11b + Cells were gated.

[0055] Fig.18 The M1 type TAMs (CD86 + ) in percentage by quantity, expressed as F4 / 80 + CD11b + Cells were gated.

[0056] Fig.19 It is the quantitative percentage of CD3+CD4+T cells in the tumor site in Example 7.

[0057] Fig. 20 The tumor site CD3+CD8 in Example 7 + T cell quantification percentage. DETAILED DESCRIPTION

[0058] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0059] In the following examples, the eight-arm polyethylene glycol active ester used was purchased from Jiankai Technology Co., Ltd. (Beijing) and had a molecular weight of 20,000.

[0060] Example 1

[0061] The preparation process of metal polyphenol nanomaterials with photothermal effect and tumor microenvironment immunomodulatory function for delivering photosensitizers and tumor microenvironment immunomodulators is as follows Figure 1 As shown, the specific steps are as follows:

[0062] 1. Synthesis of PEG polyphenols: Eight-arm polyethylene glycol active ester (300 mg, molecular weight 20000, 0.015 mmol) was dissolved in 5 mL DMF, N-(2-aminoethyl) maleimide hydrochloride (15.894 mg, 0.09 mmol) was dissolved in 1 mL DMF, and then these two solutions and triethylamine (18.76 μL, 0.135 mmol) were added to an eggplant-shaped reaction bottle at the same time. The resulting solution was stirred for 5 h under nitrogen protection, and then dopamine hydrochloride (5.689 mg, 0.03 mmol) and 6.25 μL triethylamine dissolved in 1 mL DMF were added, and the reaction was continued for another 4 h under nitrogen protection. After the reaction, the mixture was dialyzed in water at a pH of 4-5 for 48 h to remove unreacted small molecules, and then lyophilized after dialysis to obtain PEG polyphenols.

[0063] 2. Synthesis of PA-PEG polyphenol: The above 50 mg PEG polyphenol was dissolved in 5 mL PBS (0.01 M, pH = 7.4), added to the reaction bottle, and then 1 mL PBS (0.01 M, pH = 7.4) dissolved PD-L1 antagonist polypeptide (23.7 mg, relative molecular mass 1658.79) was added, and then 1 mL PBS (0.01 M, pH = 7.4) dissolved tris (2-carboxyethyl) phosphine hydrochloride (6.14 mg, relative molecular mass 286.65) was added. The resulting solution was stirred for 12 h under nitrogen protection. After the reaction, the mixture was dialyzed in water at pH 4 to 5 for 24 h to remove unreacted small molecules. After dialysis, it was freeze-dried to obtain PA-PEG polyphenol.

[0064] 3. Preparation of metal polyphenol nanomaterial PA-MPNs@Di for delivering photosensitizers and tumor microenvironment immunomodulators and having photothermal effect and tumor microenvironment immunomodulatory function: Add the above-mentioned PA-PEG polyphenol (20 μL, 50 mg / mL) aqueous solution into a 5 mL sample bottle, then add tannic acid DMSO solution (80 μL, 10 mg / mL), stir at room temperature for 2 min, add 300 μL DMSO and diosgenin DMSO solution (50 μL, 5 mg / mL) after stirring evenly, stir at room temperature for 30 s, then add FeCl3·6H2O aqueous solution (200 μL, 5 mg / mL), stir at room temperature for 5 min, add Tris-HCl solution (150 μL, 0.5 M, pH = 8.0) after mixing, stir the mixed solution at room temperature for 20 min, then add 200 μL FeCl3·6H2O solution (5 mg / mL), and stir the reaction with a magnetic stirrer for 20 min. After the reaction, centrifugation (20000r / min, 20min) was performed to obtain a precipitate, which was washed once with pure water to remove excess PA-PEG polyphenols, FeCl3, tannic acid and dioscin to obtain PA-MPNs@Di.

[0065] The nanomaterials were prepared in the same manner as above, except that the dioscin solution was not added, and the resulting nanomaterials were named PA-MPNs. Similarly, the nanomaterials were prepared in the same manner as above, except that step 2 was omitted, i.e., PA-PEG polyphenol was replaced with PEG polyphenol, and the resulting nanomaterials were named MPNs@Di.

[0066] Figure 2 This is a transmission electron microscope picture of PA-MPNs@Di. It can be seen from the picture that the particle size of the nanomaterial PA-MPNs@Di is about 150nm. Figure 3 The dynamic laser scattering results of MPNs@Di, PA-MPNs, and PA-MPNs@Di. It can be seen from the figure that PA-MPNs@Di has an average hydrodynamic particle size of 152.6 nm and a relatively narrow particle size distribution, which is consistent with the above-mentioned transmission electron microscopy results. This shows that our nanomaterials have a smaller particle size and can be taken up by cells.

[0067] Example 2

[0068] Study on the photothermal performance of PA-MPNs@Di in vitro

[0069] In order to study the in vitro photothermal performance of PA-MPNs@Di at different concentrations, the PA-MPNs@Di nanoparticles prepared in Example 1 were dispersed in deionized water and diluted to 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively, and added to a 48-well plate. The photothermal effect of the nanomaterial was measured using an infrared thermal imager. When the light source intensity was set to 1.0 W / cm 2 and 2W / cm 2 , the near-infrared light wavelength is 808nm, the irradiation distance is 5cm, the volume of the particle solution is 0.5mL, and the 808nm near-infrared light is irradiated for 10 minutes.

[0070] The results are shown in the figure. Figure 4 and Figure 5 0.5 mL of PA-MPNs@Di with different concentrations (0-200 μg / mL) was exposed to near-infrared light (with a light source intensity of 1.0 W / cm 2 , near-infrared light wavelength 808nm, irradiation distance 5cm) under irradiation, different concentrations quickly rise in temperature over time, and a 200μg / mL nanoparticle solution can rise from 27℃ to 52℃ within 10 minutes. Similarly, Figure 6 and Figure 7 0.5 mL of PA-MPNs@Di with different concentrations (0-200 μg / mL) was exposed to near-infrared light (with a light source intensity of 2.0 W / cm 2Under the irradiation of near-infrared light with a wavelength of 808 nm and an irradiation distance of 5 cm, different concentrations rapidly heated up with time, and a 100 μg / mL nanoparticle solution could be heated from 27°C (0 min, i.e., room temperature) to 60°C within 10 minutes. This indicates that the PA-MPNs@Di particles prepared in Example 1 have a good photothermal effect.

[0071] Figure 8 PA-MPNs@Di (100 μg / mL) prepared in Example 1 was irradiated with 808 nm laser (1.0 W / cm 2 , 10min) irradiation. It can be seen from the figure that repeated 808nm laser irradiation did not destroy the photothermal effect of PA-MPNs@Di.

[0072] Example 3

[0073] In vitro cellular uptake assessment

[0074] Preparation of fluorescein isothiocyanate (FITC)-loaded PA-MPNs@Di: The PA-PEG polyphenol (20 μL, 50 mg / mL) aqueous solution prepared in Example 1 above was added to a 5 mL sample bottle, and then a DMSO solution of tannic acid (80 μL, 10 mg / mL) was added, and stirred at room temperature for 2 min. After stirring evenly, 300 μL of DMSO and a DMSO solution of diosgenin (50 μL, 5 mg / mL) were added, and stirred at room temperature for 30 s. Then, an aqueous solution of FITC (100 μL, 1 mg / mL) was added, and stirred at room temperature for 30 s. Then, an aqueous solution of FeCl3·6H2O (200 μL, 5 mg / mL) was added, and stirred at room temperature for 5 min. After mixing evenly, a Tris-HCl solution (150 μL, 0.5 M, pH = 8.0) was added, and the mixed solution was stirred at room temperature for 20 min. Then, 200 μL of FeCl3·6H2O solution (5 mg / mL) was stirred with a magnetic stirrer for 20 min. After the reaction, the precipitate was obtained by centrifugation (20000 r / min, 20 min), and the precipitate was washed once with pure water to remove excess PA-PEG polyphenols, FeCl3, tannic acid and dioscin, and FITC-loaded PA-MPNs@Di were obtained.

[0075] The nanomaterial was prepared in the same manner as above, except that the dioscin solution was not added, and the obtained nanomaterial was named FITC-loaded PA-MPNs. Similarly, the nanomaterial was prepared in the same manner as above, except that the PA-PEG polyphenol was replaced with the PEG polyphenol prepared in Example 1, and the obtained nanomaterial was named FITC-loaded MPNs@Di.

[0076] Take the 4T1 cells with good growth status, resuspend and count, and then use 1×10 5The cells were plated in a 12-well plate at a concentration of 100 μg / mL of FITC-loaded MPNs@Di (dispersed with 1640 basal medium) to the MPNs@Di group, and 100 μg / mL of FITC-loaded PA-MPNs (dispersed with 1640 basal medium) to the PA-MPNs group, and 100 μg / mL of FITC-loaded PA-MPNs@Di (dispersed with 1640 basal medium) to the PA-MPNs group. The cells were incubated for 2 h and 4 h, respectively, in a dark environment. Then, the culture medium in each well was aspirated, and the plates were washed once with PBS. After adding 200 μL of trypsin digestion solution for 3 minutes, 500 μL of 1640 full culture medium was added to terminate the process. The 12-well plate was blown with a pipette to remove the cells attached to the wall. After centrifugation at 1200 rpm for 5 minutes, the plates were resuspended with 500 μL of PBS for flow cytometric analysis.

[0077] Fig. 9 The in vitro uptake of different nanomaterials by 4T1 cells measured by flow cytometry is shown. The experiment was divided into the Control group, MPNs@Di group, PA-MPNs group and PA-MPNs@Di group, and the uptake of MPNs@Di, PA-MPNs and PA-MPNs@Di nanomaterials was evaluated at 2 hours and 4 hours, respectively. As can be seen from the figure, the uptake rate of the control group is very low, and the in vitro cell uptake rates of the MPNs@Di group, PA-MPNs group and PA-MPNs@Di group at 2h and 4h are significantly different from those of the Control group, and the in vitro cell uptake rate at 4h is higher than that at 2h.

[0078] Example 4

[0079] In vitro cytotoxicity and in vitro live-dead double staining evaluation of PA-MPNs@Di

[0080] First, 4T1 cells were incubated in a 96-well plate at a density of 7000 cells per well for 24 hours. Then, PA-MPNs@Di of Example 1 was dispersed in 1640 basal medium at certain concentrations (0, 25, 50, 100, 200, 400 μg / mL) and added to the 96-well plate. The illumination group was illuminated with near-infrared light (light source intensity of 2.0 W / cm 2The well plates with different concentrations of PA-MPNs@Di nanomaterials were illuminated by irradiation with near-infrared light (wavelength 808nm, irradiation distance 5cm), and each well was illuminated for 8min. The non-illuminated group was not illuminated. After treatment, the 96-well plates were placed in a cell culture incubator for 24 hours, and the cell viability was determined using a standard CCK-8 assay.

[0081] Fig.10 The results of the in vitro cytotoxicity evaluation of PA-MPNs@Di on 4T1 cells with or without laser irradiation were determined by CCK8. The results showed that at a concentration of 25-50 μg / mL, PA-MPNs@Di did not show obvious cytotoxicity to 4T1 cells when laser irradiated. However, at a concentration of 100-400 μg / mL, PA-MPNs@Di showed obvious cell mortality to 4T1 cells when laser irradiated, among which the cell mortality was most significant at 200 μg / mL and 400 μg / mL. The experimental results showed that the cell mortality rate increased significantly with the increase of PA-MPNs@Di concentration. This indicates that high concentrations of nanomaterials can more effectively absorb near-infrared light and convert it into heat energy, thereby enhancing the photothermal effect. In addition, PA-MPNs@Di has good safety for cells when there is no near-infrared light 808nm irradiation, proving that PA-MPNs@Di has good biocompatibility and biosafety.

[0082] 4T1 cells were cultured at a rate of 1×10 5 The density was inoculated in a laser confocal culture dish and cultured in a 37°C incubator for 24 hours. The MPNs@Di, PA-MPNs, and PA-MPNs@Di prepared in Example 1 were diluted to 100 μg / mL with 1640 basal culture medium and set aside. After the cells were observed to be completely attached to the wall under an inverted microscope, group experiments were carried out and the cells were divided into 8 groups: (1) Blank control group (PBS group): 1 mL PBS was added; (2) PBS+L group: 1 mL PBS was added and then treated with light; (3) MPNs@Di group: 1 mL of 100 μg / mL MPNs@Di solution was added; (4) PA-MPNs group: 1 mL of 100 μg / mL PA-MPNs solution was added; (5) PA-MPNs@Di group: 1 mL of 100 μg / mL PA-MPNs@Di solution was added; (6) MPNs@Di+L group: 1 mL of 100 μg / mL MPNs@Di solution was added and then treated with light; (7) PA-MPNs+L group: 1 mL of 100 μg / mL PA-MPNs solution was added and then treated with light; (8) PA-MPNs@Di+L group: 1 mL of 100 μg / mL PA-MPNs@Di solution was added and then treated with light. For the light-exposed groups, near-infrared light (light source intensity 2.0 W / cm 2, wavelength 808nm, irradiation distance 5 cm) for 8 minutes. Then each group of laser confocal culture dishes containing 4T1 cells were placed in a 37°C cell culture incubator and incubated for 4 hours. After the incubation, the culture medium containing the nanomaterials was discarded and the culture dishes were rinsed with PBS. 2mM Calcein-AM solution and 15μL PI solution were added for staining, and incubated at 37°C in the dark for 30 minutes. Fix with paraformaldehyde for 15 minutes, wash with PBS, and observe using a laser confocal microscope.

[0083] Fig.11 The results show that 4T1 cells treated with different materials (100 μg / mL) were stimulated by near-infrared light (light source intensity 2.0 W / cm 2 Confocal images of the cells in the MPNs@Di+L group, PA-MPNs+L group, and PA-MPNs@Di+L group treated with light, compared with the MPNs@Di group, PA-MPNs group, and PA-MPNs@Di group not treated with light. The cells in the illuminated group showed obvious cell death, as shown by the destruction of cell membrane integrity and a significant increase in the number of PI-stained positive cells. No significant difference was observed in the cell death of the blank control group with PBS added after light treatment and no light treatment. These results show that MPNs@Di, PA-MPNs, and PA-MPNs@Di materials have good photothermal or phototoxic effects under light conditions, and their optical properties are an important factor leading to cytotoxicity.

[0084] Example 5

[0085] Evaluation of in vitro macrophage polarization effects

[0086] Acquisition of M2 macrophages: RAW 264.7 cells were cultured at 3 × 10 5The cells were seeded at a density of 100 μg / mL in a 12-well plate and incubated for 24 hours to adhere to the wall. Then, the cells were divided into 6 groups: (1) Control group: no treatment; (2) IL-4 group: the original culture medium was replaced with DMEM culture medium containing IL-4 (25 ng / mL); (3) LPS group: the original culture medium was replaced with DMEM culture medium containing IL-4 (25 ng / mL); (4) MPNs@Di group: the original culture medium was replaced with DMEM culture medium containing IL-4 (25 ng / mL); (5) PA-MPNs group: the original culture medium was replaced with DMEM culture medium containing IL-4 (25 ng / mL); (6) PA-MPNs@Di group: the original culture medium was replaced with DMEM culture medium containing IL-4 (25 ng / mL). After 24 hours, the cells were observed using an inverted microscope to confirm the formation of M2 macrophages. Subsequently, the cells were collected with a cell scraper and resuspended in sterile PBS containing Anti-CD206-PE antibody. The cells were incubated at 4°C for 30 minutes, and then the expression of CD206 was detected by flow cytometry.

[0087] Repolarization of M2 macrophages: The M2 macrophages obtained by the above method were repolarized as follows: (1) Control group: 1 mL of DMEM basal medium was added; (2) IL-4 group: no treatment was performed; (3) LPS group: 1 mL of LPS (1 μg / mL) was added; (4) MPNs@Di group: 1 mL of 100 μg / mL MPNs@Di solution (dispersed with DMEM medium) was added; (5) PA-MPNs group: 1 mL of 100 μg / mL PA-MPNs solution (dispersed with DMEM medium) was added; (6) PA-MPNs@Di group: 1 mL of 100 μg / mL PA-MPNs@Di solution (dispersed with DMEM medium) was added. After incubation for 24 hours, the cells were collected and incubated with sterile PBS containing Anti-CD86-APC antibody at 4°C for 30 minutes. Finally, the expression of CD86 on the cell surface was detected by flow cytometry.

[0088] Fig.12 and Fig.13The expression results of CD206 and CD86 were detected by flow cytometry after RAW264.7 cells were treated with different materials. It can be seen from the figure that after IL-4 stimulation, the CD206 level increased significantly, confirming that the M2 macrophages were successfully stimulated, and the CD206 molecule level in the MPNs@Di group and PA-MPNs@Di group was significantly reduced compared with the IL-4 group, indicating that dioscin can downregulate M2 macrophages. In addition, the expression level of CD86 molecules in the MPNs@Di group and PA-MPNs@Di group was also significantly higher than that in the IL-4 group, while the expression level of CD86 in the PA-MPNs group and the Control group without dioscin was lower. The above experimental results show that PA-MPNs@Di can effectively induce the reprogramming of M2 macrophages, thereby improving the immunosuppression of the tumor microenvironment, and providing the possibility of exerting anti-tumor immune response in vivo.

[0089] Example 6

[0090] In vivo photothermal effect studies

[0091] Balb / c mice (20-25 g) bearing 4T1 tumors were used for in vivo photothermal effect testing. 6 4T1 cells were inoculated subcutaneously on the right side of the abdomen of mice, and the tumor volume was measured as A×B 2 / 2, where A is the length of the tumor and B is the width of the tumor. When the tumor grows to about 100-150 mm 3 At the same time, the mice were divided into four groups: PBS group, MPNs@Di group, PA-MPNs group and PA-MPNs@Di group, with one mouse in each group. The mice in the MPNs@Di group, PA-MPNs group and PA-MPNs@Di group were injected intratumorally with 100μL MPNs@Di solution (200μg / mL, dispersed in sterile PBS), PA-MPNs solution (200μg / mL, dispersed in sterile PBS) and PA-MPNs@Di solution (200μg / mL, dispersed in sterile PBS), respectively. The mice in the PBS group were injected intratumorally with 100μL PBS. One hour after the injection, the tumor was exposed to near-infrared light (light source intensity of 1.5W / cm 2 The tumor was irradiated for 5 min with near-infrared light (wavelength 808 nm, irradiation distance 3 cm). The temperature changes of the tumor were recorded using an infrared imaging camera.

[0092] We used the Balb / c mouse orthotopic breast cancer model to evaluate the in vivo photothermal effect of nanomaterials. MPNs@Di, PA-MPNs, and PA-MPNs@Di were injected into tumor-bearing mice and irradiated with 808 nm laser for 5 min (1.5 W / cm 2 ). The temperature changes on the tumor surface were monitored by infrared thermal imaging. Fig.14 and Fig.15 As shown in the figure, the surface temperature of the tumor treated with MPNs@Di, PA-MPNs, and PA-MPNs@Di increased to 57.6°C, 61.6°C, and 66.9°C, respectively, at 5 minutes. In contrast, the temperature of the tumor surface treated with PBS only increased to 43.7°C at 5 minutes. In summary, PA-MPNs@Di has the potential to become a new PTT agent.

[0093] Example 7

[0094] In vivo therapeutic studies

[0095] In order to verify whether PA-MPNs@Di inhibits the growth of mouse tumors under light conditions and establish a tumor model, we selected 6-8 week-old Balb / c mice with a body weight difference of less than 1g between individuals. After the mice were stable in the animal room for 1 week, 1×10 6 4T1 cells were added, and the day was determined as day 0. The tumor growth of the mice was observed and the tumor volume of the mice grew to 100 mm. 3 ,The mice were randomly divided into 8 groups: PBS group, PBS+Laser group, MPNs@Di group, MPNs@Di+Laser group, PA-MPNs group, PA-MPNs+Laser group, PA-MPNs@Di group, and PA-MPNs@Di+Laser group, with 5 mice in each group. On the 7th day, MPNs@Di, PA-MPNs, and PA-MPNs@Di nanomaterials were dispersed in PBS (pH7.4, 0.01M), and 100μL of different materials (3mg / mL) were injected into the tail vein. After 12h, 808nm infrared light was irradiated according to the groups, with a power of 1.5W / cm 2 The injection and irradiation were repeated on the 10th day, and the whole experimental period was 23 days. Starting from the 0th day, the tumor volume of mice was weighed every two days, and the average value of each group was taken.

[0096] from Fig.16 It can be seen that the PA-MPNs@Di+L group has the most significant inhibitory effect on mouse tumors. The reason for this phenomenon is that the PA-MPNs@Di nanomaterials enhance the immune response of photothermal therapy (PTT) under 808nm infrared light irradiation, and the PD-L1 antagonist peptide loaded on it plays a targeting role. Fig.17 The M2 TAMs (F4 / 80 + CD11b + As a gate control, CD206 + The data of proportion in gate control, Fig.18 M1-type TAMs (F4 / 80 +CD11b + As a gate, CD86 + The experimental results were consistent with expectations. Dioscin can promote the polarization of TAMs, and light exposure may accelerate the cleavage of nanomaterials, resulting in a significant increase in the M1 / M2 ratio of the PA-MPNs@Di+L group and the MPNs@Di+L group. In contrast, the non-light-exposed group and the PA-MPNs+L group had a weaker effect on the repolarization of TAMs in tumor tissues due to the lack of dioscin.

[0097] To further verify the improving effect of dioscin on the tumor immunosuppressive environment, we dissected the tumor tissues of mice in different treatment groups and analyzed the infiltrating T cells therein. Fig.19 and Fig. 20 The results showed that the tumor-infiltrating T lymphocytes (CD3 + CD4 + and CD3 + CD8 + ) were significantly higher than those in other groups. In particular, the number of cytotoxic T lymphocytes (CD3 + CD8 + ) in tumor tissues had an average ratio of 34.66%, which was significantly higher than that of other groups. In summary, the photothermal therapy combined with immunotherapy strategy adopted by the nanomaterials in the PA-MPNs@Di+L group showed great potential in cancer treatment models.

Claims

1. A method for preparing a metal polyphenol nanomaterial having photothermal effect and tumor microenvironment immunomodulatory function, characterized in that The following steps are involved: (1) reacting an eight-arm polyethylene glycol active ester, N-(2-aminoethyl)maleimide hydrochloride, and triethylamine in an organic solvent, and adding dopamine hydrochloride and triethylamine to continue the reaction to obtain dopamine-terminated eight-arm polyethylene glycol; (2) reacting the dopamine-terminated eight-arm polyethylene glycol obtained in step (1), the PD-L1 antagonist polypeptide and tri(2-carboxyethyl)phosphine in a PBS buffer to obtain a dopamine-terminated eight-arm PEG polyphenol with a PD-L1 antagonist polypeptide; (3) reacting the dopamine-terminated eight-arm PEG polyphenol tannic acid with PD-L1 antagonist peptide obtained in step (2), diosgenin, metal salt and Tris-HCl buffer in a mixed solvent of water and DMSO to obtain a metal polyphenol nanomaterial with photothermal effect and tumor microenvironment immunomodulatory function.

2. The preparation method according to claim 1, characterized in that: In step (1), the molar ratio of eight-arm polyethylene glycol active ester, N-(2-aminoethyl)maleimide hydrochloride, dopamine hydrochloride and triethylamine is 1:6:2:9-12.

3. The preparation method according to claim 1, characterized in that: In step (2), the molar ratio of dopamine-terminated eight-arm polyethylene glycol to the PD-L1 antagonist polypeptide is 1-2:6, and the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to the PD-L1 antagonist polypeptide is 1.5-2:

1.

4. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of dopamine-terminated eight-arm PEG polyphenol with PD-L1 antagonist peptide, diosgenin, metal salt and tannic acid is 1-2: 0.2-0.5: 2-4: 0.8-1.

6.

5. The preparation method according to claim 1 or 4, characterized in that: In step (3), the metal salt is at least one of an iron salt, a copper salt, a manganese salt, a calcium salt, a cobalt salt, and a zinc salt, and the metal salt is a chloride or a nitrate of each metal.

6. The preparation method according to claim 1 or 4, characterized in that: In step (3), the pH of the Tris-HCl buffer is 8.0 to 8.5, and the concentration is 0.5 to 0.6 M.

7. The preparation method according to claim 1 or 4, characterized in that: In step (3), the volume ratio of water in the reaction system to DMSO in the reaction system is 57-60:40-43.

8. The preparation method according to claim 1, characterized in that: The method comprises at least one of the following conditions: Condition 1, in step (1), the reaction is carried out under the protection of an inert gas, the reaction time of the eight-arm polyethylene glycol active ester, N-(2-aminoethyl)maleimide hydrochloride and triethylamine is 5 to 8 hours, and the reaction is continued for 4 to 6 hours after adding dopamine hydrochloride and triethylamine; Condition 2: In step (2), the reaction is carried out under the protection of an inert gas, and the reaction time is 10 to 14 hours; Condition 3: In step (3), each solution is stirred for 0.5 to 5 minutes before adding another solution. After all solutions are added, the reaction is stirred for 0.3 to 1 hour.

9. The metal polyphenol nanomaterial having photothermal effect and tumor microenvironment immunoregulatory function obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the metal polyphenol nanomaterial with photothermal effect and tumor microenvironment immunomodulatory function as claimed in claim 9 in the preparation of tumor drugs or cancer drugs.

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