Oxygen-carrying biomimetic mineralization nano-platform for remodeling tumor hypoxic microenvironment, preparation and application in tumor treatment

Abstract

The invention belongs to the technical field of medicines, and relates to an oxygen-carrying biomimetic mineralization nano platform for remodeling a tumor hypoxic microenvironment, preparation and application in tumor treatment. The nano platform is formed by packaging oxygen-enriched hemoglobin in a metal organic framework. The self-oxygen-carrying biomimetic mineralization nano-platform disclosed by the invention can effectively overcome immunosuppression in a tumor by remodeling a tumor hypoxic microenvironment, so that an autoimmune system is awakened to achieve an effect of directly treating the tumor. Furthermore, the platform can also achieve a synergistic treatment effect on any one or more of radiotherapy, chemotherapy, photodynamic therapy and immunotherapy of the tumor by increasing the oxygen content of the tumor part, and has important significance in inhibiting tumor growth and metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to a self-oxygen-carrying biomimetic mineralized nanoplatform for remodeling the tumor hypoxic microenvironment, its preparation, and its application in tumor treatment. Background Art

[0002] The hypoxic microenvironment is closely related to the occurrence, development, invasion, and metastasis of tumors. First, hypoxia has an obvious resistance to various oxygen-consuming treatment methods, such as radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy. Second, hypoxia can induce the up-regulation of hypoxia-inducible factor-1 (HIF-1) expression, thereby causing an increase in the expression of multiple downstream drug-resistant genes, resulting in a decrease in the sensitivity of tumor cells to therapeutic drugs. In addition, hypoxia also promotes the up-regulation of the expression of immunosuppressive factors such as programmed death ligand (PD-L1) by tumor cells and induces the formation of an immunosuppressive microenvironment, thereby enabling tumor cells to escape the attack of the immune system and promoting tumor growth and spread. Therefore, developing a targeted and efficient novel oxygen transport system for reversing the tumor hypoxic microenvironment is of great significance for improving the direct and synergistic treatment effects of hypoxic tumors, inhibiting tumor growth, and metastasis.

[0003] Currently, there are mainly two categories of oxygenation modes based on nanomaterials to relieve tumor hypoxia. The first category is endogenous oxygen production, that is, the enzyme-catalyzed oxygen production strategy, which in-situ regenerates oxygen through a catalytic reaction. Due to abnormal metabolism, the intracellular hydrogen peroxide level in tumor cells is higher than that in normal tissues. Therefore, catalase can use hydrogen peroxide as a substrate to catalyze oxygen production, but the amount of oxygen produced by this method is easily limited by the H 2 O 2 content in the tumor site and it is difficult to achieve continuous oxygen supply. The second category is exogenous oxygen supply, that is, the oxygen direct delivery strategy, which dissolves oxygen in a nanoscale oxygen carrier in an oxygen-rich environment. After reaching the lesion site, oxygen is released by simple diffusion in a hypoxic environment to achieve an oxygen-increasing effect.

[0004] Hemoglobin (Hb) is a functional protein in red blood cells that realizes the oxygen transport function with blood circulation in red blood cells and can reversibly bind to oxygen according to different oxygen partial pressures. However, free Hb has an extremely short circulation time in the blood and is easily recognized and rapidly cleared by the human endothelial reticuloendothelial system. Therefore, in order to increase the biological stability of Hb in the body and the precise targeting of oxygen release to tumors, it is urgent to develop and construct a functionalized nanoplatform with tumor microenvironment responsiveness to achieve efficient loading and delivery of oxygen-rich hemoglobin.

[0005] Metal-organic framework materials (MOFs) are composed of inorganic and organic components and can be used as delivery carriers. They have good thermal and chemical stability. Generally, there are three preparation methods for loading biomacromolecules onto MOFs: (1) Coupling: Biomacromolecules are mainly coupled to the surface of MOFs through two strategies: one is the covalent connection of biomacromolecules to the surface of MOFs; the other is the adsorption of biomacromolecules on the surface of MOFs through hydrogen bonds, van der Waals forces or electrostatic interactions. This loading method often exposes some structures of biomacromolecules directly, so the stability and activity are easily affected by external factors. In addition, the limited anchoring sites on the surface of MOFs will also reduce the loading efficiency. (2) Penetration: MOFs have an extremely high specific surface area and functional pores, making MOFs have potential application value in the fields of gas adsorption and drug delivery. However, biomacromolecules are often large in size. How to prepare macroporous MOFs and embed biomacromolecules into the pores is a great challenge. In addition, macroporous MOFs will also increase the risk of premature release of the carried gas or drug before reaching the lesion. (3) Encapsulation: Encapsulating biomacromolecules in a hollow MOF structure requires the MOF to have the structural mode of microcapsules, which often needs to be realized by using hard templates or soft templates. However, the preparation process by the template method is relatively complex. Therefore, the primary goal is to achieve efficient loading and delivery of oxyhemoglobin after assembly and reach the tumor microenvironment for realization. Summary of the Invention

[0006] The purpose of the present invention is to provide a self-oxygen-carrying biomimetic mineralized nanoplatform for remodeling the tumor hypoxic microenvironment, its preparation and application in tumor treatment.

[0007] To achieve the above object, the technical solution adopted by the present invention is

[0008] A self-oxygen-carrying biomimetic mineralized nanoplatform for remodeling the tumor hypoxic microenvironment, wherein the nanoplatform is oxyhemoglobin encapsulated inside a metal-organic framework.

[0009] Furthermore, a reducing agent is used to reduce the hemoglobin loaded in the metal-organic framework to deoxyhemoglobin, and then it is fully adsorbed with oxygen under an oxygen flow and converted into oxyhemoglobin to obtain the self-oxygen-carrying biomimetic mineralized nanoplatform.

[0010] The metal-organic framework loaded with hemoglobin (Hb) is synthesized by a one-pot method from hemoglobin, metal ions and organic ligands. Among them, a deprotonating agent needs to be added during the synthesis process to induce the rapid crystallization of the metal-organic framework, and a stabilizer is added to prevent the aggregation of crystal seeds.

[0011] The deprotonating agent is selected from triethylamine, trioctylamine or sodium formate; the stabilizer is selected from polyvinylpyrrolidone K25, polyvinylpyrrolidone K30 or polyvinylpyrrolidone K60.

[0012] Furthermore, the framework is ZIF90, the molar ratio of the deprotonating agent to imidazole-2-carboxaldehyde (ICA) is 1:50 to 1:10; the concentration range of the stabilizer in the imidazole-2-carboxaldehyde (ICA) solution is 2 mg / mL - 10 mg / mL.

[0013] The responsiveness of the nanoplatform utilizes the differences in the physiological environments between tumor tissues and normal tissues to achieve targeted delivery or localized release of the carried drugs to tumors. This responsiveness can be reflected in the pH responsiveness to weak acidity in the tumor microenvironment, or the ATP responsiveness to high concentrations in tumor cells, or the dual responsiveness to both pH and ATP.

[0014] The nanoplatform for encapsulating biomacromolecules with universality can effectively maintain the biological activities of biomacromolecules.

[0015] The biomimetic mineralized nano metal-organic framework encapsulating oxyhemoglobin as a nanoplatform can achieve targeted and controllable delivery of oxygen in the tumor region through the responsiveness of the framework in the tumor microenvironment, thereby effectively alleviating tumor hypoxia.

[0016] A preparation method of the self-oxygen-carrying biomimetic mineralized nanoplatform for remodeling the tumor hypoxic microenvironment is to encapsulate oxyhemoglobin in a framework with tumor microenvironment responsiveness, thus obtaining the self-oxygen-carrying biomimetic mineralized nanoplatform.

[0017] An application of the self-oxygen-carrying biomimetic mineralized nanoplatform for remodeling the tumor hypoxic microenvironment, which is an application in a carrier for achieving targeted, controllable delivery and oxygen supply in the hypoxic tumor region.

[0018] An application of the self-oxygen-carrying biomimetic mineralized nanoplatform for remodeling the tumor hypoxic microenvironment, which is an application in combination with one or more tumor treatment methods as a tumor treatment drug.

[0019] The tumor treatment methods are radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy.

[0020] A tumor treatment drug contains the said platform.

[0021] The said tumors include squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, gastric cancer, lung cancer, pancreatic cancer, esophageal cancer, nasopharyngeal cancer, liver cancer, rectal cancer, colon cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, and kidney cancer.

[0022] The method for directly or synergistically treating tumors by remodeling the tumor hypoxic microenvironment provided by the present invention has high safety and remarkable therapeutic effects.

[0023] Compared with the prior art, the advantages of the present invention are reflected in:

[0024] (1) The biomimetic mineralized nano-metal-organic framework with tumor microenvironment responsiveness can utilize oxygen-rich hemoglobin to precisely and controllably deliver oxygen to the tumor region, thereby significantly increasing the oxygen content at the tumor site and serving as a self-oxygen-carrying nano-platform.

[0025] (2) The above self-oxygen-carrying nano-platform can effectively overcome immunosuppression within the tumor by remodeling the tumor hypoxic microenvironment, thereby awakening the body's immune system to directly treat the tumor.

[0026] (3) The above self-oxygen-carrying nano-platform can effectively increase the oxygen content at the tumor site by remodeling the tumor hypoxic microenvironment, thereby playing a synergistic therapeutic role in radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy for tumors.

[0027] For radiotherapy: Oxygen molecules play an important role in the process of ionizing radiation to kill tumor cells, and the widespread hypoxic microenvironment in solid tumors limits the clinical radiotherapy efficiency. After regulating the tumor hypoxic state with the self-oxygen-carrying nano-platform designed by the present invention and combining with radiotherapy, the sensitivity of the tumor to radiotherapy can be increased without causing additional adverse reactions, thereby enhancing the radiotherapy effect.

[0028] For chemotherapy: Hypoxia increases the resistance of tumor cells to chemotherapy, leading to multidrug resistance in tumors and further resulting in the failure of chemotherapy. After regulating the tumor hypoxic state with the self-oxygen-carrying nano-platform designed by the present invention and combining with chemotherapy, it helps to increase the sensitivity of the tumor to chemotherapy, thereby enhancing the chemotherapy effect.

[0029] For photodynamic therapy: Hypoxia severely limits the generation of reactive oxygen species in photodynamic therapy and is accompanied by a sharp consumption of oxygen, and the aggravation of intracellular hypoxia directly leads to the failure of photodynamic therapy. After regulating the tumor hypoxic state with the self-oxygen-carrying nano-platform designed by the present invention and combining with photodynamic therapy, it helps to increase the sensitivity of photosensitizers to excitation light, thereby enhancing the photodynamic therapy effect.

[0030] For immunotherapy: Hypoxia causes cancer cells to produce adenosine, which, as a signal transduction molecule, can block the immune response and mediate the occurrence of tumor immune tolerance, thereby escaping the attack of T cells and natural killer cells and promoting tumor development. After regulating the tumor hypoxic state with the nano-oxygen-carrying platform designed by the present invention and combining with immunotherapy, it helps to reawaken the anti-tumor ability of immune cells, thereby enhancing the immunotherapy effect. Description of the Drawings

[0031] Figure 1 Scanning electron microscope image of oxy-Hb@ZIF90;

[0032] Figure 2BET adsorption curves of ZIF90 and Hb@ZIF90;

[0033] Figure 3 Circular dichroism spectra of Hb and Hb@ZIF90;

[0034] Figure 4 Oxygen-carrying stability curve of oxy-Hb@ZIF90;

[0035] Figure 5 Oxygen-releasing stability curve of oxy-Hb@ZIF90;

[0036] Figure 6 Specific oxygen-releasing curve of oxy-Hb@ZIF90;

[0037] Figure 7 Scanning electron micrograph of DOX / HMME@ZIF90;

[0038] Figure 8 BET adsorption curves of ZIF90 and DOX / HMME@ZIF90;

[0039] Figure 9 Detection graphs of ROS in tumor tissues of each group;

[0040] Figure 10 In vivo distribution map of DOX / HMME@ZIF90&oxy-Hb@ZIF90;

[0041] Figure 11 Tumor growth inhibition graphs of each group;

[0042] Figure 12 Detection graphs of CD8+ T cells in tumor tissues of each group. Detailed implementation manners

[0043] The present invention will be further described below in conjunction with embodiments and the accompanying drawings. The following embodiments only illustrate the present invention by way of example, but the present invention is not limited thereto.

[0044] The present invention utilizes the self-assembly process of biomineralization in organisms, adopts a deprotonating reagent to induce the rapid crystallization of MOF, and uses a stabilizer to prevent the aggregation of crystal seeds. By using a one-step method, bioactive macromolecules are directly encapsulated in the protective shell of MOF, just like wearing a layer of "MOF armor", which better maintains the tertiary structure of biological macromolecules such as proteins and increases the thermal stability and organic solvent tolerance of bioactive molecules to a greater extent. At the same time, the degradation of a specific MOF skeleton has tumor microenvironment responsiveness. Therefore, encapsulating oxygen-rich hemoglobin in a metal-organic framework material can fully protect the biological functions of hemoglobin for oxygen-carrying and oxygen-releasing through a biomimetic mineralization method, so as to achieve the purpose of accurately delivering oxygen to the tumor site as a self-oxygen-carrying system.

[0045] Example 1 Construction of a Self-Oxygen-Carrying Bionic Mineralized Nanoplateform for Remodeling Tumor Hypoxic Microenvironment

[0046] ZIF90 is a kind of MOF self-assembled by Zn 2+ and 2-imidazolecarboxaldehyde through coordination. It not only has good biocompatibility but also has dual pH / ATP responsiveness in the tumor microenvironment. Its framework is stable under physiological conditions, but can specifically collapse in weakly acidic tumor sites and mitochondria with high concentrations of ATP, thereby targeting drug release.

[0047] Mix 1 mL of ICA aqueous solution with a concentration of 19.2 mg / mL (the molar ratio of the protonating reagent TEA to ICA in the ICA aqueous solution is 1:30, and the final concentration of the stabilizer PVP-K30 is 5 mg / mL) with 2 mL of Hb aqueous solution with a concentration of 1 mg / mL. Dropwise add 1 mL of tert-butanol solution of zinc nitrate with a concentration of 14.9 mg / mL under a 37 °C water bath and incubate for 2 h. After centrifuging at 10000 rpm for 10 min, redisperse the precipitate in 2 mL of PBS solution with pH 7.4 to obtain ZIF90 loaded with hemoglobin Hb (Hb@ZIF90). The encapsulation efficiency of Hb is 98.30 ± 2.46%, the drug loading is 13.86 ± 0.60%, the particle size is 250.4 ± 7.86 nm, the potential is +3.4 ± 0.25 mV, and the morphology observed by SEM is shown in Figure 1 The surface is rough because in addition to being directly embedded inside the framework, a part of Hb will also adsorb on the surface of ZIF90.

[0048] Take appropriate amounts of dried and ground ZIF90 and the above-obtained Hb@ZIF90 respectively, place them in a specific surface and pore size tester for vacuum degassing. It can be seen from the BET adsorption results in Table 1 and Figure 2 that after ZIF90 loads Hb, the surface area, pore volume and pore diameter are all greatly reduced, verifying the effective encapsulation of Hb by ZIF90.

[0049] Since the Fe 2+ in the Hb raw material will auto-oxidize to high-valent iron Fe 3+ , thus losing the ability to bind or release oxygen. Therefore, it is necessary to carry out reduction treatment with a reducing agent. Add 2 mg of sodium dithionite SD (m Hb :m SD = 1:1) to the above Hb@ZIF90, stir for 5 min in the dark at 37 °C to obtain ZIF90 loaded with deoxyhemoglobin (deoxy-Hb@ZIF90), and introduce an oxygen stream with a pressure of 1 MPa and balance for 15 min to obtain ZIF90 loaded with oxyhemoglobin (oxy-Hb@ZIF90).

[0050] Example 2 Investigation of the Stability of a Self-Oxygen-Carrying Biomimetic Mineralized Nanoplateform for Remodeling Tumor Hypoxic Microenvironment

[0051] (1) Hb Structural Stability

[0052] Circular dichroism (CD) is a rapid, simple, and accurate method for studying the molecular conformation in protein and enzyme solutions with relatively low concentrations. Using an argon lamp as the light source and PBS as the baseline, free Hb and Hb@ZIF90 obtained from the above examples were scanned in the range of 190 - 250 nm. The results are shown in Figure 3 . In the spectrum of free Hb, two distinct negative absorption bands were observed at 209 nm and 221 nm. The presence of these bands is related to the π→π* and n→π* electronic transitions of the porphyrin ring near the heme site in the Hb molecule. Hb@ZIF90 also has these two negative absorption peaks. However, due to the hydrogen bond interaction between ZIF90 and the amino acid residues on the Hb surface, these two negative absorption peaks were red-shifted to 213 nm and 225 nm respectively, but the peak shapes remained unchanged, indicating that the secondary structure of Hb loaded with ZIF90 is basically the same as that of free Hb.

[0053] (2) Oxygen-Carrying Stability of oxy-Hb@ZIF90

[0054] The freshly prepared oxy-Hb@ZIF90 (Hb concentration: 1 mg / mL) according to the above examples was stored in the dark at 4°C. The oxygen content of oxy-Hb@ZIF90 was measured on the 0th day and the 30th day of storage using a tissue oxygen partial pressure analyzer. Figure 4 The results showed that the oxygen partial pressure of oxy-Hb@ZIF90 could be maintained at the same level within 0 - 30 days, indicating that oxy-Hb@ZIF90 has good oxygen-carrying stability.

[0055] (3) Oxygen-Release Stability of oxy-Hb@ZIF90

[0056] The freshly prepared oxy-Hb@ZIF90 (Hb concentration: 1 mg / mL) according to the above examples was stored in the dark at 4°C. The oxygen concentration in the solution was detected using deoxygenated pH 7.4 PBS as the medium on the 0th, 5th, 15th, and 30th days. Figure 5 The results showed that within 30 days of storage in the dark at 4°C, there were basically no differences in the oxygen content and oxygen-release rate of oxy-Hb@ZIF90, demonstrating that oxy-Hb@ZIF90 has good oxygen-release stability.

[0057] Example 3 Investigation of the Oxygen-Release Specificity of a Self-Oxygen-Carrying Biomimetic Mineralized Nanoplateform for Remodeling Tumor Hypoxic Microenvironment

[0058] Take the oxy-Hb@ZIF90 prepared according to the above example (Hb concentration is 1 mg / mL), place it in PBS at pH 7.4 without deoxygenation to investigate the oxygen release of the carrier in the simulated normal tissue. Then, place oxy-Hb@ZIF90 in deoxygenated PBS at pH 5.5 with 0.4 mM and 4 mM ATP respectively to investigate the oxygen release of the carrier inside and outside the simulated tumor cells, so as to compare the oxygen release specificity of the self-oxygen-carrying nanoplatform. From Figure 6 (A), it can be seen that in the non-deoxygenated PBS at pH 7.4, the oxygen concentration in the solution is only about 2 mg / L, which should be caused by the leakage of oxygen adsorbed by the ZIF90 framework itself; while from Figure 6 (B), it can be seen that when reaching the simulated extracellular environment of tumor cells (pH 5.5 + 0.4 mM ATP + hypoxia), the pH / ATP dual responsiveness of ZIF90 will cause partial collapse of the framework. In the hypoxic state, oxy-Hb encapsulated in the ZIF90 framework releases some oxygen, about 6 mg / L; when taken into the simulated intracellular environment of tumor cells (pH 5.5 + 4 mM ATP + hypoxia), with the increase of ATP concentration, the collapse degree of the ZIF90 framework is further aggravated, and the oxygen release amount can increase to 9 mg / L. Thus, it can be seen that the oxygen release of oxy-Hb@ZIF90 at the tumor site is specific.

[0059] Application of the self-oxygen-carrying biomineralized nanoplatform for remodeling the tumor hypoxic microenvironment in the direct and synergistic treatment of tumors

[0060] To investigate the role of the designed carrier in the direct and synergistic treatment of tumors, while preparing the self-oxygen-carrying unit oxy-Hb@ZIF90 (Hb concentration is 1 mg / mL, as described above according to Example 1), a treatment unit integrating photodynamic therapy and chemotherapy was also prepared, that is, DOX / HMME@ZIF90 encapsulating HMME and surface-modified with DOX, and a co-delivery system DOX / HMME@ZIF90&oxy-Hb@ZIF90 was formed.

[0061] The preparation method of DOX / HMME@ZIF90 is as follows: Mix 1 mL of ICA aqueous solution with a concentration of 19.2 mg / mL (where the molar ratio of the deprotonating reagent TEA to ICA is 1:30 and the concentration of the stabilizer PVP-K30 is 5 mg / mL) with 2 mL of HMME methanol solution with a concentration of 1 mg / mL. Dropwise add 1 mL of tert-butanol solution of zinc nitrate with a concentration of 14.9 mg / mL under a 37 °C water bath. After incubation for 30 min, centrifuge at 10000 rpm for 10 min to obtain HMME@ZIF90, where the encapsulation efficiency of HMME is 72.33 ± 3.46% and the drug loading is 16.93 ± 2.55%.

[0062] The HMME@ZIF90 precipitate was redispersed in 2 mL of a methanol solution of DOX with a concentration of 1 mg / mL and incubated at room temperature for 24 h. After centrifugation at 10,000 rpm for 10 min, the precipitate was redispersed in 2 mL of PBS solution with pH 7.4 to obtain DOX / HMME@ZIF90, with a particle size of 273.1 ± 7.75 nm and a zeta potential of +2.55 ± 0.29 mV. The encapsulation efficiency of DOX was 91.19 ± 6.85%, and the drug loading was 18.24 ± 3.17%. The SEM observation of the morphology is shown in Figure 7 , which is a regular dodecahedron structure.

[0063] An appropriate amount of dried and ground DOX / HMME@ZIF90 was placed in a specific surface area and pore size analyzer for vacuum degassing. The BET adsorption results are shown in Table 1 and Figure 8 . After ZIF90 adsorbed HMME and was surface-modified with DOX, the surface area, pore volume, and pore diameter all decreased significantly, which can also verify the successful loading of the drug.

[0064] Table 1

[0065]

[0066] The above-prepared DOX / HMME@ZIF90 and oxy-Hb@ZIF90 were mixed evenly at a volume ratio of 1:1 to obtain a co-delivery nanosystem. The particle size of DOX / HMME@ZIF90&oxy-Hb@ZIF90 was 263.2 ± 9.84 nm, and the zeta potential was +6.66 ± 0.36 mV.

[0067] (1) In vivo ROS detection

[0068] A 4T1 tumor model in mice was established. When the tumor volume reached 100 - 150 mm 3 , the mice were grouped. The groups were set as follows: (Ⅰ) Control group, (Ⅱ) DOX / HMME@ZIF90 group, and (Ⅲ) DOX / HMME@ZIF90&oxy-Hb@ZIF90 co-delivery group. 100 μL (HMME equivalent to 5 mg / kg of mouse body weight) was injected into the tail vein of the mice. After 8 h, the mice in each group were intraperitoneally injected with DCFH-DA fluorescent probe at a dose of 0.3 mg / kg. After 0.5 h, under the laser conditions of a wavelength of 630 nm and an intensity of 300 mW / cm 2 , with the collimator 3 cm away from the mice, irradiated for 15 min. Then, the mice in each group were dissected, the tumor tissues were taken out, frozen sections were made, and the fluorescence intensity was observed under a fluorescence microscope (DCF: E X :504, E m :529; DAPI: E X :350, E m: 460). From Figure 9 It can be seen that compared with the Control group and the DOX / HMME@ZIF90 group, the fluorescence intensity of the co-delivery group was significantly enhanced, proving that the ROS production in the tumor site increased substantially. The reason for the analysis is that when the co-delivery group was administered by tail vein injection, it targeted the tumor site relying on the EPR effect. Under the dual response of pH / ATP, the ZIF90 skeleton collapsed and the drug was released in a targeted manner. At the same time, under the condition of hypoxia in the tumor microenvironment, the self-oxygen-carrying unit oxy-Hb@ZIF90 released a large amount of oxygen. Therefore, the ROS production in vivo was significantly increased, which greatly improved the effect of tumor photodynamic therapy.

[0069] (2) In vivo distribution investigation

[0070] Take tumor-bearing mice, and inject 100 μL of the above-prepared DOX / HMME@ZIF90&oxy-Hb@ZIF90 co-delivery group by tail vein (DOX is equivalent to 5 mg / kg of the mouse body weight). Observe the in vivo distribution at 1, 2, 4, 8, 12, 24, and 48 h using a small animal in vivo imager (E X : 480 nm, E m : 580 nm). From Figure 10 It can be seen that the co-delivery group accumulated in the tumor site of mice 1 h after tail vein injection. As time extended, the fluorescence signal intensity in the tumor tissue continued to increase and reached the strongest at 8-12 h, indicating that the co-delivery group DOX / HMME@ZIF90&oxy-Hb@ZIF90 can target tumors relying on the dual responsiveness of pH / ATP. This is an important prerequisite for realizing precise oxygen release of the carrier at the tumor site, efficiently exerting the therapeutic effect, and reducing the adverse reactions to other organs.

[0071] (3) Evaluation of anti-tumor effect

[0072] Take tumor-bearing mice and set up: (Ⅰ) Control group, (Ⅱ) DOX group, (Ⅲ) DOX / HMME@ZIF90 group, (Ⅳ) oxy-Hb@ZIF90 (obtained in Example 1) group, and (Ⅴ) DOX / HMME@ZIF90&oxy-Hb@ZIF90 group. Inject 100 μL of each preparation into each group of mice by tail vein on the 1st, 3rd, and 5th days (DOX is equivalent to 5 mg / kg of the mouse body weight). Except for the Control group, PDT treatment (630 nm, 300 mW / cm 2 , 15 min) was carried out 8 h after administration in the remaining groups. Measure the long diameter (L) and short diameter (S) of the mice tumors every other day for 24 days. Calculate the tumor volume according to the formula: V = L×S 2 / 2, and draw a volume-time curve with time as the abscissa and tumor volume as the ordinate. See Figure 11(A). After the treatment was completely finished on the 24th day, the mice in each group were sacrificed, and the tumor tissues were taken out for weighing, as shown in Figure 11 (B). As can be seen from the results, the Control group had no inhibitory effect on tumor growth. The tumor inhibition order of the remaining groups was: DOX / HMME@ZIF90&oxy-Hb@ZIF90 group > oxy-Hb@ZIF90 group > DOX / HMME@ZIF90 group > DOX group. Among them, the oxy-Hb@ZIF90 group showed a strong inhibitory effect on tumor growth. The reason was analyzed that this vector could target and controllably deliver oxygen to the tumor site, significantly improve the hypoxic state of the tumor microenvironment, regulate the immunosuppression of the microenvironment, and thus play a direct role in treating tumors, with great clinical application value. At the same time, we also found that the combined delivery group DOX / HMME@ZIF90&oxy-Hb@ZIF90 could inhibit tumor growth to the greatest extent. Compared with the simple oxy-Hb@ZIF90 group, the tumor inhibitory ability was improved, indicating that after remodeling the tumor hypoxic microenvironment using the biomimetic mineralization nanoplatform, it could play a good synergistic and promoting role in the chemotherapy / photodynamic therapy of tumors, thus obtaining a more ideal therapeutic effect.

[0073] (4) Research on anti-tumor immune mechanism

[0074] To verify whether the body's immune system was activated, CD8 + T cell fluorescence detection was performed on the tumor tissues to compare the recruitment degree of immune cells in each group. The tumor-bearing mice were divided into 4 groups, namely (Ⅰ) Control group, (Ⅱ) oxy-Hb@ZIF90 group, (Ⅲ) DOX / HMME@ZIF90 group, and (Ⅳ) DOX / HMME@ZIF90&oxy-Hb@ZIF90 group. Each group of mice was intravenously injected with 100 μL of each preparation through the tail vein on the 1st, 3rd, and 5th days. Groups (Ⅲ) and (Ⅳ) needed to be irradiated with laser (630 nm, 300 mW / cm 2 ², 15 min) 8 h after drug administration, and the mice in each group were dissected on the 6th day, the tumor tissues were taken out, soaked in 4% paraformaldehyde, and fluorescence detection was performed. As Figure 12 can be seen, the fluorescence of the Control group was the weakest, proving that there was almost no aggregation of CD8 + T cells in the tumor. The oxygen-carrying unit oxy-Hb@ZIF90 alone could induce the infiltration of a part of positive immune cells CD8 + T cells by simply improving the tumor hypoxic microenvironment, proving that it had a certain effect of relieving immunosuppression, and its degree was equivalent to the green fluorescence intensity generated by the treatment unit DOX / HMME@ZIF90 after photodynamic and chemotherapy; after the co-delivery group DOX / HMME@ZIF90&oxy-Hb@ZIF90 was irradiated with light, the green fluorescence signal was enhanced, indicating that CD8 +Both the degree of T cell infiltration and recruitment increased. It can be seen that after using the self-oxygen-carrying biomimetic mineralized nanoplatform to improve the tumor hypoxic state, it has a good synergistic effect on remodeling the tumor immune microenvironment.

[0075] In summary, the platform obtained by encapsulating oxyhemoglobin inside the metal-organic framework in the present invention protects the oxygen-carrying and oxygen-releasing functions of hemoglobin. By using the pH / ATP dual responsiveness of the framework, oxygen is precisely and controllably delivered to the hypoxic tumor area, thus significantly increasing the oxygen content in the tumor site. The self-oxygen-carrying biomimetic mineralized nanoplatform of the present invention can not only effectively overcome the immunosuppression in tumors by remodeling the tumor hypoxic microenvironment, thereby awakening the body's own immune system to directly treat tumors. Further, this platform can also play a synergistic therapeutic role in any one or more of radiotherapy, chemotherapy, photodynamic therapy and immunotherapy of tumors by increasing the oxygen content in the tumor site, which is of great significance for inhibiting tumor growth and metastasis.

Claims

1. A self-oxygen-carrying biomimetic mineralized nano-platform for reshaping the tumor hypoxic microenvironment, characterized by: The nanoplatform is oxygen-rich hemoglobin encapsulated inside a metal-organic framework.

2. The self-oxygen-carrying biomimetic mineralized nano-platform for reshaping the tumor hypoxic microenvironment according to claim 1, characterized in that: The hemoglobin loaded in the metal organic framework is reduced to deoxyhemoglobin by using a reducing agent, and then fully adsorbs oxygen under an oxygen flow and is converted into oxygenated hemoglobin to obtain an oxygen-carrying bionic mineralized nano-platform.

3. The self-oxygen-carrying biomimetic mineralized nano-platform for reshaping the tumor hypoxic microenvironment according to claim 1, characterized in that: The metal organic framework loaded with hemoglobin (Hb) is synthesized from hemoglobin, metal ions and organic ligands through a one-pot method, wherein a deprotonating agent needs to be added during the synthesis process to induce rapid crystallization of the metal organic framework, and a stabilizer is added to prevent aggregation of crystal seeds.

4. The self-oxygen-carrying biomimetic mineralized nano-platform for reshaping the tumor hypoxic microenvironment according to claim 3, characterized in that: The deprotonating agent is selected from triethylamine, trioctylamine or sodium formate; the stabilizer is selected from polyvinyl pyrrolidone K25, polyvinyl pyrrolidone K30 or polyvinyl pyrrolidone K60.

5. A method for preparing the self-oxygen-carrying biomimetic mineralized nano-platform for reshaping the tumor hypoxic microenvironment according to claim 1, characterized in that: Oxygenated hemoglobin is encapsulated in a skeleton responsive to the tumor microenvironment, resulting in an oxygen-carrying biomimetic mineralized nanoplatform.

6. An application of the self-oxygen-carrying biomimetic mineralized nano-platform for reshaping the tumor hypoxic microenvironment according to claim 1, characterized in that: The platform is used in achieving targeted, controllable delivery and oxygen supply to hypoxic tumor areas.

7. An application of the self-oxygen-carrying biomimetic mineralized nano-platform for reshaping the tumor hypoxic microenvironment according to claim 1, characterized in that: The platform is used in conjunction with one or more tumor treatment methods as a drug for treating tumors.

8. The use of the self-oxygen-carrying biomimetic mineralized nano-platform for reshaping the tumor hypoxic microenvironment according to claim 7, characterized in that: The tumor treatment methods include radiotherapy, chemotherapy, photodynamic therapy and immunotherapy.

9. A tumor treatment drug, characterized in that: A platform comprising the method of claim 1.

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