Self-carried oxygen biomimetic mineralization nano-platform remodeling tumor hypoxic microenvironment, preparation and application in tumor treatment

Abstract

The application belongs to the technical field of medicine, and relates to a self-oxygen-carrying biomimetic mineralization nano platform for remodeling a tumor hypoxic microenvironment, and preparation and application in tumor treatment. The nano platform is that oxygen-rich hemoglobin is packaged in a metal organic framework. The self-oxygen-carrying biomimetic mineralization nano platform can not only effectively overcome the immunosuppression in the tumor by remodeling the tumor hypoxic microenvironment, so as to awaken the immune system to directly treat the tumor. Further, the platform can also play a synergistic treatment role on any one or more of radiotherapy, chemotherapy, photodynamic therapy and immunotherapy of the tumor by increasing the oxygen content of the tumor site, and has important significance for inhibiting tumor growth and metastasis.

Description

Technical Field

[0001] This invention belongs to the field of medical technology and relates to a self-contained oxygen-inspired biomimetic mineralization nanoplatform for reshaping the hypoxic microenvironment of tumors, its preparation, and its application in tumor treatment. Background Technology

[0002] The hypoxic microenvironment is closely related to the occurrence, development, invasion, and metastasis of tumors. First, hypoxia significantly inhibits resistance to various oxygen-consuming treatments, such as radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy. Second, hypoxia can induce upregulation of hypoxia-inducible factor-1 (HIF-1) expression, leading to increased expression of various downstream drug resistance genes and reduced sensitivity of tumor cells to therapeutic drugs. Furthermore, hypoxia also promotes the upregulation of immunosuppressive factors such as programmed death-ligand 1 (PD-L1) in tumor cells and induces the formation of an immunosuppressive microenvironment, allowing tumor cells to evade the immune system and promoting tumor growth and spread. Therefore, developing novel, targeted, and highly efficient oxygen delivery systems to reverse the hypoxic tumor microenvironment is of great significance for improving the direct and synergistic therapeutic effects of hypoxic tumors and inhibiting tumor growth and metastasis.

[0003] Currently, there are two main categories of oxygenation mechanisms based on nanomaterials to alleviate tumor hypoxia. The first is endogenous oxygen production, i.e., an enzyme-catalyzed oxygen production strategy, which regenerates oxygen in situ through catalytic reactions. Due to abnormal metabolism, tumor cells have higher intracellular hydrogen peroxide levels than normal tissues. Therefore, catalase can use hydrogen peroxide as a substrate to catalyze oxygen production. However, the amount of oxygen produced in this way is easily limited by the H2O2 content at the tumor site, making it difficult to achieve a continuous oxygen supply. The second category is exogenous oxygen supply, i.e., a direct oxygen delivery strategy. This involves dissolving oxygen in an oxygen-rich environment using nano-oxygen carriers. After reaching the lesion site, oxygen is released through simple diffusion in the hypoxic environment, thus achieving an oxygenation effect.

[0004] Hemoglobin (Hb) is a functional protein in red blood cells that transports oxygen through blood circulation. It can reversibly bind to oxygen based on varying oxygen partial pressures. However, free Hb has an extremely short circulation time in the blood and is easily recognized and rapidly cleared by the reticuloendothelial system. Therefore, to increase the biostability of Hb in vivo and its precise targeting of tumor oxygen release, it is urgent to develop and construct a functionalized nanoplatform responsive to the tumor microenvironment to achieve efficient loading and delivery of oxygen-rich hemoglobin.

[0005] Metal-organic frameworks (MOFs) are composed of inorganic and organic components and can be used as delivery carriers. They have good thermal and chemical stability. Generally speaking, there are three preparation methods for loading biomolecules onto MOFs: (1) Coupling: Biomolecules are mainly coupled to the MOF surface through two strategies: one is that the biomolecules are covalently linked to the MOF surface; the other is that the biomolecules are adsorbed onto the MOF surface through hydrogen bonds, van der Waals forces or electrostatic interactions. This loading method often exposes part of the biomolecule structure directly. Therefore, the stability and activity are easily affected by external factors. In addition, the limited anchoring points on the MOF surface will also reduce the loading efficiency. (2) Permeation: MOFs have ultra-high specific surface area and functional pores, which makes MOFs potentially valuable in the fields of gas adsorption and drug delivery. However, biomolecules are often large in size. How to prepare macroporous MOFs and embed biomolecules into the pores is very challenging. In addition, macroporous MOFs will also increase the risk of premature release of carried gases or drugs before reaching the lesion. (3) Encapsulation: Encapsulating biomacromolecules within a hollow MOF structure requires the MOF to possess a microcapsule-like structural pattern, which often necessitates the use of hard or soft templates. However, the template-based preparation process is quite complex. Therefore, the primary objective is to obtain an assembly capable of efficiently loading and delivering oxygenated hemoglobin and achieving the desired tumor microenvironment effect. Summary of the Invention

[0006] The purpose of this invention is to provide a self-contained oxygen-inspired biomimetic mineralization nanoplatform for reshaping the hypoxic microenvironment of tumors, its preparation, and its application in tumor treatment.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A self-contained oxygen-inspired biomimetic mineralization nanoplatform for reshaping the hypoxic microenvironment of tumors, wherein the nanoplatform consists of oxygen-rich hemoglobin encapsulated within a metal-organic framework.

[0009] Furthermore, by using a reducing agent to reduce hemoglobin loaded in a metal-organic framework to deoxyhemoglobin, and then fully adsorbing oxygen under an oxygen flow to convert it into oxyhemoglobin, a self-oxygen-carrying biomimetic mineralization nanoplatform is obtained.

[0010] The metal-organic framework loaded with hemoglobin (Hb) is synthesized in a one-pot process from hemoglobin, metal ions, and organic ligands. During the synthesis process, a deprotonating agent is added to induce rapid crystallization of the metal-organic framework, and a stabilizer is added to prevent the aggregation of seed crystals.

[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 skeleton is ZIF90, the molar ratio of the deprotonating agent to imidazole 2-formaldehyde (ICA) is 1:50 to 1:10, and the concentration range of the stabilizer in the imidazole 2-formaldehyde (ICA) solution is 2 mg / mL to 10 mg / mL.

[0013] The responsiveness of the nanoplatform is achieved by utilizing the difference in physiological environment between tumor tissue and normal tissue to realize the targeted delivery or localized release of the drug carried on the tumor. This responsiveness can be reflected in the weakly acidic pH response in the tumor microenvironment, or the high concentration of ATP response in tumor cells, or the dual response of pH and ATP at the same time.

[0014] The aforementioned versatile nanoplatform for encapsulating biomolecules can effectively maintain the bioactivity of biomolecules.

[0015] The biomimetic mineralized nanometal-organic framework encapsulating oxygen-rich hemoglobin serves as a nanoplatform, enabling targeted and controllable delivery of oxygen to the tumor region through the framework's responsiveness to the tumor microenvironment, thereby effectively alleviating tumor hypoxia.

[0016] A method for preparing a self-oxygen-carrying biomimetic mineralization nanoplatform for reshaping the hypoxic tumor microenvironment involves encapsulating oxyhemoglobin in a framework that is responsive to the tumor microenvironment, thereby obtaining the self-oxygen-carrying biomimetic mineralization nanoplatform.

[0017] An application of the aforementioned self-contained oxygen biomimetic mineralization nanoplatform for reshaping the hypoxic tumor microenvironment, wherein the platform is used as a carrier for targeted, controllable delivery to hypoxic tumor regions and for oxygen supply.

[0018] An application of the aforementioned self-contained oxygen biomimetic mineralization nanoplatform for reshaping the hypoxic tumor microenvironment, wherein the platform is used in synergy with one or more tumor treatment methods as a therapeutic tumor drug.

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

[0020] A tumor treatment drug comprising the aforementioned platform.

[0021] The tumors mentioned include squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, gastric cancer, lung cancer, pancreatic cancer, esophageal cancer, nasopharyngeal carcinoma, 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 reshaping the hypoxic microenvironment of tumors provided by this invention has high safety and significant therapeutic effect.

[0023] Compared with the prior art, the advantages of the present invention are as follows:

[0024] (1) Biomimetic mineralized nanometal-organic frameworks with tumor microenvironment responsiveness can use oxygen-rich hemoglobin to deliver oxygen directly to the tumor area in a precise and controllable manner, thereby significantly increasing the oxygen content of the tumor site, serving as a self-carrying oxygen nanoplatform.

[0025] (2) The above-mentioned oxygen-carrying nanoplatform can effectively overcome the immunosuppression in the tumor by reshaping the hypoxic microenvironment of the tumor, thereby awakening the body's own immune system and playing a direct role in treating the tumor.

[0026] (3) The above-mentioned oxygen-carrying nanoplatform can effectively increase the oxygen content of the tumor site by reshaping the hypoxic microenvironment of the tumor, thereby playing a synergistic role in the treatment of tumor radiotherapy, chemotherapy, photodynamic therapy and immunotherapy.

[0027] For radiotherapy: Oxygen molecules play a crucial role in the killing of tumor cells by ionizing radiation, but the hypoxic microenvironment prevalent in solid tumors limits the clinical efficacy of radiotherapy. By using the self-contained oxygen nanoplatform designed in this invention to regulate the hypoxic state of tumors, combined with radiotherapy, the sensitivity of tumors to radiotherapy can be increased without causing additional adverse reactions, thereby improving the radiotherapy effect.

[0028] For chemotherapy: Hypoxia increases the resistance of tumor cells to chemotherapy, leading to multidrug resistance and ultimately chemotherapy failure. By using the self-contained oxygen nanoplatform designed in this invention to regulate tumor hypoxia, combined with chemotherapy, it helps increase the tumor's sensitivity to chemotherapy, thereby improving the efficacy of chemotherapy.

[0029] For photodynamic therapy: Hypoxia severely limits the production of reactive oxygen species during photodynamic therapy and is accompanied by a rapid consumption of oxygen. Increased intracellular hypoxia directly leads to the failure of photodynamic therapy. By using the self-contained oxygen nanoplatform designed in this invention to regulate the hypoxic state of tumors, combined with photodynamic therapy, it helps to increase the sensitivity of photosensitizers to excitation light, thereby improving the efficacy of photodynamic therapy.

[0030] Regarding immunotherapy: Hypoxia causes cancer cells to produce adenosine, a signal transduction molecule that can block immune responses and mediate tumor immune tolerance, thereby allowing them to evade attacks from T cells and natural killer cells and promoting tumor development. By using the nano-oxygen-carrying platform designed in this invention to regulate tumor hypoxia, combined with immunotherapy, it helps to reawaken the anti-tumor capabilities of immune cells, thereby enhancing the efficacy of immunotherapy. Attached Figure Description

[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 of Hb and Hb@ZIF90;

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

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

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

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

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

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

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

[0041] Figure 11 Tumor growth inhibition diagrams for each group;

[0042] Figure 12 Images of CD8+ T cells detected in tumor tissues of each group. Detailed Implementation

[0043] The present invention will be further described below with reference to the embodiments and accompanying drawings. The embodiments described below are merely examples to illustrate the present invention, but the present invention is not limited thereto.

[0044] This invention utilizes the self-assembly process of biomineralization in organisms, employing deprotonating agents to induce rapid crystallization of metal-organic frameworks (MOFs) and using stabilizers to prevent seed crystal aggregation. A one-step method directly encapsulates bioactive macromolecules within a protective MOF shell, essentially creating a "MOF armor" that effectively maintains the tertiary structure of proteins and other biomolecules, significantly increasing the thermal stability and organic solvent resistance of the bioactive molecules. Furthermore, the degradation of specific MOF frameworks exhibits tumor microenvironment responsiveness. Therefore, encapsulating oxygen-rich hemoglobin within a metal-organic framework material allows for the full protection of hemoglobin's oxygen-carrying and oxygen-releasing functions through biomimetic mineralization, thus enabling a self-contained oxygen delivery system for precise oxygen delivery to tumor sites.

[0045] Example 1: Construction of a self-contained oxygen-inspired biomimetic mineralization nanoplatform to reshape the hypoxic tumor microenvironment

[0046] ZIF90 is a type of Zn 2+ The MOF formed by the coordination of 2-imidazolium formaldehyde not only has good biocompatibility, but also has dual tumor microenvironment pH / ATP responsiveness. Its skeleton is stable under physiological conditions, but it can undergo specific skeleton collapse in weakly acidic tumor sites and in mitochondria with high ATP concentration, thereby targeting and releasing drugs.

[0047] 1 mL of a 19.2 mg / mL ICA aqueous solution (with a molar ratio of protonating agent TEA to ICA of 1:30 and a final concentration of stabilizer PVP-K30 of 5 mg / mL) was mixed with 2 mL of a 1 mg / mL Hb aqueous solution. 1 mL of a 14.9 mg / mL zinc nitrate tert-butanol solution was added dropwise under 37°C water bath. After incubation for 2 h, centrifugation at 10000 rpm for 10 min was performed. The precipitate was then redispersed in 2 mL of pH 7.4 PBS solution to obtain hemoglobin (Hb)-loaded ZIF90 (Hb@ZIF90). The Hb encapsulation efficiency was 98.30 ± 2.46%, the drug loading was 13.86 ± 0.60%, the particle size was 250.4 ± 7.86 nm, and the potential was +3.4 ± 0.25 mV. The morphology was observed by SEM. Figure 1 The rough surface is due to the fact that, in addition to being directly embedded inside the skeleton, some of the Hb is also adsorbed onto the surface of ZIF90.

[0048] Take appropriate amounts of dried and ground ZIF90 and the above-obtained Hb@ZIF90, respectively, and place them in a specific surface area and pore size tester for vacuum degassing. The results are shown in Table 1 and... Figure 2 The BET adsorption results show that after Hb is loaded onto ZIF90, the surface area, pore volume and pore size are significantly reduced, which verifies the effective encapsulation of Hb by ZIF90.

[0049] Due to the Fe in Hb raw materials 2+ It will auto-oxidize into high-valence iron (Fe). 3+ This causes it to lose its ability to combine with or release oxygen. Therefore, a reducing agent is needed for reduction treatment. 2 mg of sodium dithionite SD(m) was added to the above Hb@ZIF90. Hb :m SD After mixing the components at a ratio of 1:1, stir for 5 minutes at 37°C in the dark to obtain ZIF90 loaded with deoxyhemoglobin (deoxy-Hb@ZIF90). Then, introduce an oxygen flow at a pressure of 1 MPa and equilibrate for 15 minutes to obtain ZIF90 loaded with oxyhemoglobin (oxy-Hb@ZIF90).

[0050] Example 2: Stability assessment of a self-contained oxygen-inspired biomimetic mineralization nanoplatform for reshaping the hypoxic tumor microenvironment.

[0051] (1) Hb structural stability

[0052] Circular dichroism (CD) is a rapid, simple, and accurate method for studying the molecular conformation of proteins and enzymes in solutions with low concentrations. Using an argon lamp as the light source and PBS as the baseline, free Hb and Hb@ZIF90 obtained in the above examples were scanned in the range of 190–250 nm. The results are shown in [Figure number missing]. 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 exhibits these two negative absorption peaks, but due to the hydrogen bonding between ZIF90 and the amino acid residues on the Hb surface, these two negative absorption peaks are red-shifted to 213 nm and 225 nm, respectively, but the peak shape remains unchanged, indicating that the secondary structure of Hb loaded with ZIF90 is basically consistent with that of free Hb.

[0053] (2) Oxygen carrying stability of oxy-Hb@ZIF90

[0054] The freshly prepared oxy-Hb@ZIF90 (Hb concentration of 1 mg / mL) according to the above embodiment was stored at 4°C in the dark, and the oxygen content of oxy-Hb@ZIF90 was measured after 0 days and 30 days using a tissue oxygen partial pressure analyzer. Figure 4 This indicates that the oxygen partial pressure of oxy-Hb@ZIF90 can be maintained at the same level within 0 to 30 days, meaning that oxy-Hb@ZIF90 has good oxygen-carrying stability.

[0055] (3) Oxy-Hb@ZIF90 oxygen release stability

[0056] The freshly prepared oxy-Hb@ZIF90 (Hb concentration of 1 mg / mL) according to the above embodiments was stored at 4°C in the dark. The oxygen concentration in the solution was detected at 0, 5, 15 and 30 days using deoxygenated pH 7.4 PBS as the medium. Figure 5 The results show that the oxygen content and oxygen release rate in oxy-Hb@ZIF90 remained essentially unchanged within 30 days of storage at 4℃ in the dark, demonstrating that oxy-Hb@ZIF90 has good oxygen release stability.

[0057] Example 3: Investigation of the oxygen release specificity of a self-contained oxygen-inspired biomimetic mineralization nanoplatform that reshapes the hypoxic tumor microenvironment.

[0058] oxy-Hb@ZIF90 (Hb concentration of 1 mg / mL) according to the above-described embodiment was placed in undeoxygenated PBS at pH 7.4 to examine the oxygen release of the carrier in simulated normal tissue. Then, oxy-Hb@ZIF90 was placed in deoxygenated PBS at pH 5.5 with concentrations of 0.4 mM and 4 mM ATP to examine the oxygen release of the carrier inside and outside simulated tumor cells, thereby comparing the oxygen release specificity of the self-carrying oxygen nanoplatform. Figure 6 (A) It is evident that in undeoxygenated pH 7.4 PBS, the oxygen concentration in the solution is only approximately 2 mg / L, which should be due to oxygen leakage adsorbed by the ZIF90 framework itself; while... Figure 6 (B) As can be seen, when the simulated tumor extracellular environment is reached (pH 5.5 + 0.4 mM ATP + hypoxia), the pH / ATP dual response of ZIF90 causes partial collapse of the framework. Under hypoxic conditions, oxy-Hb encapsulated within the ZIF90 framework releases some oxygen, approximately 6 mg / L. When taken into the simulated tumor intracellular environment (pH 5.5 + 4 mM ATP + hypoxia), the collapse of the ZIF90 framework intensifies further with increasing ATP concentration, and the oxygen release can increase to 9 mg / L. Therefore, the oxygen release from oxy-Hb@ZIF90 at the tumor site is specific.

[0059] Example 4: Application of a self-contained oxygen-inspired biomimetic mineralization nanoplatform for reshaping the hypoxic tumor microenvironment in direct and synergistic tumor therapy.

[0060] To investigate the effects of the designed carrier on direct and synergistic tumor therapy, while preparing the oxygen-carrying unit oxy-Hb@ZIF90 (Hb concentration of 1 mg / mL, as described above) as described in Example 1, a therapeutic unit integrating photodynamic therapy and chemotherapy was also prepared, namely DOX / HMME@ZIF90 which encapsulates HMME and is 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: 1 mL of 19.2 mg / mL ICA aqueous solution (where the molar ratio of deprotonating reagent TEA to ICA is 1:30, and the concentration of stabilizer PVP-K30 is 5 mg / mL) was mixed with 2 mL of 1 mg / mL HMME methanol solution. Then, 1 mL of 14.9 mg / mL zinc nitrate tert-butanol solution was added dropwise under a 37℃ water bath. After incubation for 30 min, the mixture was centrifuged at 10000 rpm for 10 min to obtain HMME@ZIF90, with an encapsulation efficiency of 72.33±3.46% and a drug loading of 16.93±2.55%.

[0062] The HMME@ZIF90 precipitate was redispersed in 2 mL of a 1 mg / mL DOX methanol solution 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 pH 7.4 PBS solution to obtain DOX / HMME@ZIF90 with a particle size of 273.1 ± 7.75 nm and a potential of +2.55 ± 0.29 mV. The DOX encapsulation efficiency was 91.19 ± 6.85%, and the drug loading was 18.24 ± 3.17%. The morphology was observed by SEM. Figure 7 It has a regular dodecahedral structure.

[0063] Take an appropriate amount of dried and ground DOX / HMME@ZIF90, place it in a surface area and pore size analyzer, and degas it under vacuum. The BET adsorption results are shown in Table 1 and 2. Figure 8 After ZIF90 adsorbs HMME and modifies its surface with DOX, the surface area, pore volume and pore size are significantly reduced, which can also verify the successful loading of drugs.

[0064] Table 1

[0065]

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

[0067] (1) In vivo ROS detection

[0068] Establish a mouse 4T1 tumor model when the tumor volume reaches 100-150 mm. 3 The mice were divided into three groups: (Ⅰ) Control group, (Ⅱ) DOX / HMME@ZIF90 group, and (Ⅲ) DOX / HMME@ZIF90 & oxy-Hb@ZIF90 co-delivery group. Mice were injected with 100 μL of HMME (equivalent to 5 mg / kg of mouse body weight) via the tail vein. Eight hours later, each group of mice was injected intraperitoneally with the DCFH-DA fluorescent probe at a dose of 0.3 mg / kg. After 0.5 hours, the probe was detected at a wavelength of 630 nm and an intensity of 300 mW / cm². 2 Under laser conditions, the collimator was placed 3 cm away from the mice, and irradiation was carried out for 15 minutes. The mice in each group were then dissected, tumor tissue was removed, frozen sections were prepared, and the fluorescence intensity (DCF:E) was observed under a fluorescence microscope. X :504,E m :529; DAPI:E X 350,E m:460). By Figure 9 As can be seen, compared with the Control group and the DOX / HMME@ZIF90 group, the fluorescence intensity of the co-delivery group was significantly enhanced, demonstrating a substantial increase in ROS production at the tumor site. The reason for this is that when the co-delivery group was administered via tail vein injection, it targeted the tumor site through the EPR effect. Under the dual response of pH / ATP, the ZIF90 backbone collapsed, resulting in targeted drug release. Simultaneously, under the hypoxic conditions of the tumor microenvironment, the oxygen-carrying unit oxy-Hb@ZIF90 released a large amount of oxygen, thus significantly increasing in vivo ROS production, which greatly enhances the efficacy of photodynamic therapy for tumors.

[0069] (2) In vivo distribution investigation

[0070] Tumor-bearing mice were subjected to a tail vein injection of 100 μL (DOX equivalent to 5 mg / kg of mouse body weight) of the prepared DOX / HMME@ZIF90 & oxy-Hb@ZIF90 co-delivery group. The distribution in vivo was observed using a small animal in vivo imaging system at 1, 2, 4, 8, 12, 24, and 48 hours. X 480nm, E m (580nm). Figure 10 It is evident that the co-delivery group accumulated in the tumor site of mice 1 hour after tail vein injection. As time progressed, the fluorescence signal intensity in the tumor tissue continued to increase, reaching its peak at 8–12 hours. This indicates that the co-delivery group DOX / HMME@ZIF90&oxy-Hb@ZIF90 can target tumors based on the dual response of pH / ATP. This is an important prerequisite for achieving precise oxygen release and efficient therapeutic effect of the carrier at the tumor site, while reducing adverse reactions to other organs.

[0071] (3) Evaluation of antitumor effects

[0072] Tumor-bearing mice were divided into four groups: (I) Control group, (II) DOX group, (III) DOX / HMME@ZIF90 group, (IV) oxy-Hb@ZIF90 group (obtained in Example 1), and (V) DOX / HMME@ZIF90 & oxy-Hb@ZIF90 group. On days 1, 3, and 5, each group of mice received a tail vein injection of 100 μL of each formulation (DOX equivalent to 5 mg / kg of mouse body weight). Except for the Control group, all other groups underwent PDT treatment (630 nm, 300 mW / cm²) 8 hours after administration. 2 (15 min). The long axis (L) and short axis (S) of the mouse tumor were measured every other day for 24 days, according to the formula: V = L × S 2 / 2 Calculate tumor volume and plot a volume-time curve with time on the x-axis and tumor volume on the y-axis. See Figure 11(A). After all treatments were completed on day 24, mice in each group were sacrificed, and tumor tissue was removed and weighed. See below. Figure 11 (B) The results showed that the Control group had no inhibitory effect on tumor growth, while the tumor inhibition order of the other 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. This is attributed to the fact that this carrier can target and controllably deliver oxygen to the tumor site, significantly improving the hypoxic state of the tumor microenvironment and regulating immunosuppression, thereby directly treating the tumor and possessing significant clinical application value. Simultaneously, we also found that the combined delivery group DOX / HMME@ZIF90 & oxy-Hb@ZIF90 could maximally inhibit tumor growth, showing improved tumor inhibition ability compared to the oxy-Hb@ZIF90 group alone. This indicates that reshaping the hypoxic tumor microenvironment using a biomimetic mineralization nanoplatform can have a good synergistic and promoting effect on chemotherapy / photodynamic therapy, thus achieving a more ideal therapeutic effect.

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

[0074] To verify whether the autoimmune system was activated, CD8 assays were performed on tumor tissue. + T-cell fluorescence detection was used to compare the recruitment levels of immune cells in each group. Tumor-bearing mice were divided into four groups: (I) Control group, (II) oxy-Hb@ZIF90 group, (III) DOX / HMME@ZIF90 group, and (IV) DOX / HMME@ZIF90&oxy-Hb@ZIF90 group. Mice in each group received 100 μL of each formulation via tail vein injection on days 1, 3, and 5. Groups (III) and (IV) underwent laser irradiation (630 nm, 300 mW / cm²) 8 hours after administration. 2 Mice in each group were euthanized (15 min) and tumor tissue was extracted and soaked in 4% paraformaldehyde for fluorescence detection on day 6. Figure 12 As can be seen, the fluorescence in the Control group was the weakest, proving that there was almost no CD8 in the tumor. + T cell aggregation, and the oxygen-carrying unit oxy-Hb@ZIF90 can induce some positive CD8 immune cells simply by improving the tumor hypoxic microenvironment. + T-cell infiltration demonstrated a certain degree of immunosuppression relief, comparable to the green fluorescence intensity produced by the treatment unit DOX / HMME@ZIF90 after photodynamic therapy and chemotherapy. However, the co-delivery group DOX / HMME@ZIF90 & oxy-Hb@ZIF90 showed enhanced green fluorescence signal after light irradiation, indicating that CD8+... +Both T cell infiltration and recruitment increased, indicating that the use of a self-oxygen-carrying biomimetic mineralization nanoplatform to improve tumor hypoxia has a good synergistic effect on reshaping the tumor immune microenvironment.

[0075] In summary, this invention encapsulates oxygen-rich hemoglobin within a metal-organic framework to create a platform that protects the oxygen-carrying and oxygen-releasing functions of hemoglobin. Utilizing the framework's dual pH / ATP responsiveness, it precisely and controllably delivers oxygen-carrying material to hypoxic tumor regions, significantly increasing oxygen levels at the tumor site. This self-oxygen-carrying biomimetic mineralization nanoplatform not only effectively overcomes tumor immunosuppression by reshaping the hypoxic tumor microenvironment, thereby awakening the body's own immune system and directly treating the tumor, but also, by increasing oxygen levels at the tumor site, synergistically enhances the therapeutic effect of one or more of radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy, playing a significant role in inhibiting tumor growth and metastasis.

Claims

1. A self-contained oxygen-inspired biomimetic mineralization nanoplatform for reshaping the hypoxic tumor microenvironment, characterized in that: The nanoplatform encapsulates oxygenated hemoglobin within a metal-organic framework; A reducing agent is used to reduce hemoglobin loaded in a metal-organic framework to deoxyhemoglobin, and then oxygen is fully adsorbed under an oxygen flow to convert it into oxyhemoglobin, thus obtaining an oxygen-carrying biomimetic mineralization nanoplatform. The metal-organic framework is ZIF-90; The metal-organic framework loaded with hemoglobin is synthesized in a one-pot process from hemoglobin, metal ions and organic ligands. During the synthesis process, a deprotonating agent is added to induce rapid crystallization of the metal-organic framework, and a stabilizer is added to prevent the aggregation of seed crystals.

2. The self-contained oxygen biomimetic mineralization nanoplatform for reshaping the hypoxic tumor microenvironment according to claim 1, characterized in that: The deprotonating agent is selected from triethylamine, trioctylamine, or sodium formate; the stabilizer is selected from polyvinylpyrrolidone K25, polyvinylpyrrolidone K30, or polyvinylpyrrolidone K60.

3. A method for preparing the self-contained oxygen-inspired biomimetic mineralization nanoplatform for reshaping the hypoxic tumor microenvironment as described in claim 1, characterized in that: By encapsulating oxyhemoglobin in a scaffold that is responsive to the tumor microenvironment, an oxygen-carrying biomimetic mineralization nanoplatform is obtained.

4. The application of the self-contained oxygen-bearing biomimetic mineralization nanoplatform for reshaping the hypoxic tumor microenvironment as described in claim 1 in the preparation of anti-tumor drugs, characterized in that: The platform is used in conjunction with one or more cancer treatment methods.

5. The application according to claim 4, characterized in that: The tumor treatment methods mentioned are radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy.

6. A tumor treatment drug, characterized in that: The platform as described in claim 1.

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