A responsive nano probe for diagnosis and treatment of calcium overload and ferroptosis synergistic photodynamic and a preparation method thereof

Abstract

The specification provides a calcium overload and iron death synergistic photodynamic diagnosis and treatment of responsive nano probes and preparation method. The method is to encapsulate porous iron-based metal nanoparticles (PEG-MOF-Fe) by calcium peroxide (HA-CaO2) nanoparticles, and to load the chemotherapeutic drug doxorubicin (DOX) into the pores to form calcium overload and iron death synergistic photodynamic diagnosis and treatment of responsive nano probes MOF-Fe@DOX-CaO2. With the long circulating effect of PEG, the nano probe is efficiently enriched in the tumor, and the tumor location is accurately determined by fluorescence / magnetic resonance multi-modal imaging. After entering the tumor tissue, CaO 2 Nanoparticles will dissolve in the low pH environment of the tumor, and then realize the DOX and Ca 2+ Site-controlled release. Under the co-catalysis of Ca 2+ , TCPP and Fe 2+ , calcium overload and iron death synergistic photodynamic co-action, the effect of efficient diagnosis and treatment of tumor is achieved.

Description

Technical Field

[0001] This invention belongs to the fields of advanced functional materials and biomedicine, specifically relating to a responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis and its preparation method. Background Technology

[0002] Cancer is a major global public health problem, and its complex pathogenesis and challenging treatment have made it a hot research topic. While surgical resection, radiotherapy, and chemotherapy are potential treatment options for cancer, further improvements are needed to achieve better treatment outcomes, higher survival rates, fewer side effects, and better prognoses. As a novel strategy for cancer treatment, photodynamic therapy (PDT) has become a research hotspot and attracted widespread attention in recent years due to its advantages such as minimal invasiveness, ease of operation, flexible treatment planning, strong efficacy, and high patient acceptability. During treatment, PDT utilizes photosensitizers to react with oxygen (O2) in the tumor microenvironment (TME) under specific wavelength light irradiation to generate reactive oxygen species (ROS), thereby achieving tumor cell death. This approach offers advantages such as spatiotemporal control and minimal side effects. However, the efficient application of PDT still faces two key challenges: first, the therapeutic effect of PDT on tumors is highly dependent on the local O2 supply, while most solid tumors have a hypoxic microenvironment, which significantly reduces the ROS levels generated after photosensitizer stimulation, weakening the ability to kill tumor cells; second, the high concentration of antioxidants such as glutathione (GSH) in the tumor microenvironment can clear some ROS, further reducing treatment efficiency. Furthermore, the relatively weak tumor selectivity in PDT may cause serious damage to normal tissues and skin. Therefore, how to effectively reduce the content of hydrogen peroxide (H2O2) and glutathione (GSH) in TME, and make full use of the acidic environment of TME to achieve a more ideal tumor treatment effect, is an important issue that urgently needs to be addressed.

[0003] Recently, CaO2 has been used as an O2-generating material in tumor therapy due to its high biocompatibility and efficient O2 release capacity. In specific microenvironments, CaO2 reacts with water to generate calcium ions (Ca2+). 2+ ( ) and hydrogen peroxide (H2O2), H2O2 can spontaneously decompose into oxygen. Meanwhile, Ca 2+ It directly participates in the production and regulation of ROS by regulating the expression of ROS-generating enzymes, and high levels of intracellular ROS contribute to cytoplasmic calcium... 2+ The accumulation of Ca2+. As a key intracellular molecule, excess Ca2+... 2+ It can interfere with mitochondrial metabolism, downregulate mitochondrial membrane potential, and reduce the production of adenosine triphosphate (ATP), thereby inducing concentration-dependent apoptosis. 2+Intracellular calcium overload is caused by calcium influx through cell membrane calcium channels, specifically targeting the mitochondria of tumor cells. Simultaneously, mitochondrial dysfunction further exacerbates ROS sensitivity, thereby enhancing the efficacy of PDT and forming a synergistic network of "calcium overload - mitochondrial damage - multi-pathway death".

[0004] With the rapid development of nanozymes, iron-based nanoparticles have become ideal candidate materials for regulating the tumor microenvironment due to their excellent catalytic activity and biocompatibility. Studies have shown that ferroptosis is a regulated form of cell death driven by iron-dependent oxidative damage, distinct from apoptosis, necrosis, autophagy, and other cell death mechanisms. The main characteristic of ferroptosis is the accumulation of ROS and lipid peroxide (LPO) products at levels sufficient to induce cell death. Within the tumor microenvironment (TME), Fe... 2+ Can pass through the Fenton reaction (Fe 2+ + H2O2 → Fe 3+ + OH − +·OH) catalyzes the generation of hydroxyl radicals (·OH) from H2O2. As a reactive oxygen species, ·OH can oxidize polyunsaturated fatty acids (PUFAs) to LPO. Furthermore, glutathione (GSH) in TME can also oxidize Fe... 3+ Reduced to Fe 2+ This further promotes the continuous Fenton reaction cycle that generates ROS. GSH depletion downregulates the activity of glutathione peroxidase 4 (GPX 4), which further promotes ferroptosis by inhibiting the detoxification effect of LPO.

[0005] In recent years, nanoscale metal-organic frameworks (MOFs) have been widely explored as promising nanomedicine platforms due to their excellent biocompatibility, high loading capacity, and feasible surface modification. Among them, porphyrin-based MOFs combine the photosensitizing properties of porphyrin molecules with the porous framework advantages of MOFs. They can effectively avoid the self-quenching of porphyrin photosensitizers and can also load chemotherapeutic drugs, potentially enabling integrated diagnosis and treatment. Simultaneously, the porosity of porphyrin MOF materials facilitates the loading of singlet oxygen (… 1 The diffusion of H2O2 in the porphyrin-metal-organic framework (MOFs) enhances the photodynamic therapy (PDT) effect, thereby exhibiting superior efficacy. Furthermore, iron-based MOFs (Fe-MOFs) also demonstrate the ability to react with H2O2 in the tumor microenvironment (TME) to induce ferroptosis in tumor cells. Finally, surface modification and alteration of MOFs can improve the water solubility and biocompatibility of the porphyrin-metal-organic framework, thereby enhancing the efficacy of PDT in multiple ways and demonstrating superior photodynamic therapy. Summary of the Invention

[0006] The purpose of this invention is to provide a responsive nanoprobe for synergistic photodynamic diagnosis and treatment of calcium overload and ferroptosis, and its preparation method. The preparation method is stable, reliable, and reproducible, and has the advantages of abundant yield, safe operation, and low cost. The prepared MOF-Fe@DOX-CaO2 nanoprobe can not only enhance T1 magnetic resonance imaging, but also effectively overcome the problem of fluorescence quenching of porphyrin molecules, thus constructing a dual-modal probe with high magnetic resonance imaging efficiency and long fluorescence imaging time, so as to achieve early and accurate diagnosis of tumors.

[0007] To achieve the above objectives, the technical solution adopted by this invention is: a method for preparing a responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis, comprising the following steps:

[0008] (1) Preparation of NH2-PEG: At room temperature, polyethylene glycol was dissolved in deionized water; then, ammonia was slowly added dropwise to the polyethylene glycol solution and the solution was stirred at room temperature for 24 h; after the reaction was complete, the solution was dialyzed in deionized water for 1-3 days using a dialysis bag with a molecular weight of 1000, and then freeze-dried to obtain aminated polyethylene glycol, denoted as NH2-PEG.

[0009] (2) Synthesis of PEG-MOF-Fe nanoparticles: Meso-tetra(4-carboxyphenyl)porphyrin, ferric chloride hexahydrate, benzoic acid and NH2-PEG were dissolved in N,N-dimethylformamide in sequence, and the mixed solution was stirred in the dark at 70-150℃ for 5-10 h; after the reaction was complete, the mixed solution was cooled to room temperature and dialyzed in deionized water for 1-3 days using a dialysis bag with a molecular weight of 5000 to obtain water-soluble porous iron-based metal nanoparticles, denoted as PEG-MOF-Fe nanoparticles;

[0010] (3) Drug loading on PEG-MOF-Fe nanoparticles: Doxorubicin was dissolved in an aqueous solution of PEG-MOF-Fe nanoparticles, so that doxorubicin was adsorbed into the pores of PEG-MOF-Fe through π-π interaction, thus obtaining MOF-Fe@DOX nanoparticles loaded with doxorubicin;

[0011] (4) Encapsulation of drug-loaded PEG-MOF-Fe nanoparticles: The prepared MOF-Fe@DOX nanoparticles were dispersed in anhydrous methanol, and sodium hyaluronate aqueous solution and calcium chloride aqueous solution were added. Then, hydrogen peroxide was slowly added to the mixture. After stirring for 2 h, ammonia was added dropwise to the above mixture to activate the reaction until a suspended solid was formed. After the reaction was completed for 2 h, the product was collected by centrifugation and washed twice with methanol to obtain a responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis, denoted as MOF-Fe@DOX-CaO2.

[0012] Preferably, the concentration of polyethylene glycol in step (1) is 0.2 to 1 g / mL; the polyethylene glycol is one of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000 and polyethylene glycol 5000.

[0013] Preferably, the mass ratio of polyethylene glycol to ammonia in step (1) is 1:1 to 1:2.

[0014] Preferably, in step (2), the concentration of meso-tetra(4-carboxyphenyl)porphyrin is 0.5–1.5 mg / mL; the mass ratio between meso-tetra(4-carboxyphenyl)porphyrin and ferric chloride hexahydrate is 1:1–1:6; the mass ratio between meso-tetra(4-carboxyphenyl)porphyrin and benzoic acid is 1:15–1:60; and the mass ratio between meso-tetra(4-carboxyphenyl)porphyrin and NH2-PEG is 1:10–1:60.

[0015] Preferably, the PEG-MOF-Fe prepared in step (2) has a particle size of 100~200 nm and a surface charge of -20~-40 mV.

[0016] Preferably, in step (3), the concentration of the aqueous solution of PEG-MOF-Fe nanoparticles is 10~30 mg / mL; the mass ratio of PEG-MOF-Fe nanoparticles to doxorubicin is 1:2~3:1.

[0017] Preferably, the PEG-MOF-Fe nanoparticles prepared in step (3) have a drug loading of 45-70% for doxorubicin and an encapsulation rate of 40-60% for doxorubicin.

[0018] Preferably, in step (4), the concentration of the MOF-Fe@DOX nanoparticle solution is 10~20 mg / mL; the mass ratio of MOF-Fe@DOX nanoparticles to sodium hyaluronate is 10:1~80:1; the mass ratio of MOF-Fe@DOX nanoparticles to calcium chloride is 1:100~1:150; the nanoprobe prepared in step (4) is based on PEG-MOF-Fe nanoparticles as a framework, loaded with doxorubicin inside, and connected to calcium chloride on the surface.

[0019] This invention also proposes a responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis, prepared by the above-described method.

[0020] Preferably, the nanoprobe has a particle size of 200~300 nm and a surface charge of -2~-20 mV.

[0021] This invention encapsulates CaO2 nanoparticles onto the surface of a porous iron-based porphyrin metal-organic framework nanomaterial (Fe-MOF) containing polyethylene glycol (PEG), and encapsulates the chemotherapy drug doxorubicin (DOX) within the pores of the PEG-MOF-Fe nanoparticles, forming a responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis, denoted as MOF-Fe@DOX-CaO2. Leveraging the long circulation of PEG in the bloodstream, the MOF-Fe@DOX-CaO2 nanoprobe efficiently accumulates at tumor sites, and its location is accurately determined using fluorescence / T1MRI multimodal imaging technology. Once inside the tumor tissue, the unique low-pH environment of tumor cells specifically dissolves CaO2NPs, enabling the release of DOX and CaO2. 2+ The site-controlled release of H2O2. H2O2 in racemic tetra(4-carboxyphenyl)porphyrin (TCPP) and Fe 2+ Under the catalytic action of [a substance], highly toxic singlet oxygen is produced. 1 O2) and hydroxyl radicals (·OH) achieve the combined effect of photodynamic therapy (PDT) and ferroptosis. Furthermore, Fe 3+ The Fenton reaction consumes glutathione (GSH) in the tumor microenvironment (TME), further enhancing ferroptosis in tumor cells. Consuming glutathione (GSH) in the TME induces ferroptosis in tumor cells.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The nanoprobe prepared by the present invention is synthesized in three steps. The preparation method is stable, reliable and reproducible. It has the advantages of simple steps, abundant yield, safe operation and low economic cost.

[0024] (2) The nanoprobes prepared by the present invention have good biocompatibility and biosafety, and have both high catalytic activity and modifiable biochemical activity. They effectively improve the poor water solubility of traditional porphyrin-based organometallic framework materials, and are more conducive to in vivo delivery and clinical translation.

[0025] (3) The nanoprobe prepared by the present invention can be used as an organic diagnostic agent to realize dual-modal diagnosis and treatment of T1 magnetic resonance imaging and near-infrared fluorescence imaging: on the one hand, it overcomes the potential damage to liver and kidney function caused by traditional gadolinium-based contrast agents, and on the other hand, it effectively solves the problem of easy quenching of fluorescence of porphyrin molecules in water, realizes efficient near-infrared fluorescence imaging, and helps the early and accurate diagnosis of breast cancer and subsequent treatment guidance.

[0026] (4) The nanoprobes prepared in this invention can achieve a synergistic anti-tumor effect of calcium overload, ferroptosis and photodynamic therapy: CaO2 in the system can release Ca in the tumor microenvironment. 2+It induces calcium overload in tumor cells; MOF-Fe can catalyze the generation of hydroxyl radicals from H2O2 through the Fenton reaction, inducing ferroptosis in tumor cells; at the same time, porphyrin molecules can mediate the photodynamic effect to generate reactive oxygen species, achieving multi-mechanism synergistic killing of tumor cells and significantly improving the therapeutic effect. Attached Figure Description

[0027] Figure 1 This is a transmission electron microscope image of PEG-MOF-Fe nanoparticles in an embodiment of the present invention;

[0028] Figure 2 This is a DLS comparison chart of MOF, PEG-MOF, and PEG-MOF-Fe in an embodiment of the present invention;

[0029] Figure 3 This is a Zeta potential diagram of MOF, PEG-MOF, and PEG-MOF-Fe in an embodiment of the present invention;

[0030] Figure 4 The UV-Vis absorption spectra of TCPP, MOF, PEG-MOF, and PEG-MOF-Fe in the embodiments of the present invention are shown below.

[0031] Figure 5 This is a transmission electron microscope image of MOF-Fe@DOX-CaO2 nanoparticles in an embodiment of the present invention;

[0032] Figure 6 This is a DLS comparison chart of HA-CaO2, MOF-Fe@DOX, and MOF-Fe@DOX-CaO2 in the embodiments of the present invention;

[0033] Figure 7 This is a Zeta potential diagram of HA-CaO2, MOF, PEG-MOF, PEG-MOF-Fe, MOF-Fe@DOX, and MOF-Fe@DOX-CaO2 in the embodiments of the present invention;

[0034] Figure 8 The UV-Vis absorption spectra of HA-CaO2, MOF-Fe@DOX, and MOF-Fe@DOX-CaO2 in the embodiments of the present invention are shown below.

[0035] Figure 9 Extracellular singlet oxygen generation efficiency of MOF-Fe@DOX-CaO2 nanoparticles prepared in this embodiment of the invention: (A) Production efficiency of ABDA solution co-incubated with MOF-Fe@DOX-CaO2 at different times. 1 O2 performance comparison; (B) TCPP, PEG-MOF-Fe, MOF-Fe@DOX-CaO2 generation 1Performance comparison of O2; (C) Production of ABDA solution co-incubated with MOF-Fe@DOX-CaO2 under different treatment conditions 1 O2 performance comparison;

[0036] Figure 10 Extracellular hydroxyl radical generation efficiency of MOF-Fe@DOX-CaO2 nanoparticles prepared in the embodiments of the present invention: (A) UV-Vis absorption spectra of MB after blending with MOF-Fe@DOX-CaO2 under different conditions; (B) UV-Vis absorption spectra of MB after blending with different materials (TCPP, PEG-MOF-Fe, MOF-Fe@DOX-CaO2); (C) Comparison of the •OH generation performance of TCPP, PEG-MOF-Fe, and MOF-Fe@DOX-CaO2; (D) In ​​near-infrared laser (660nm 1W / cm) 2 UV-Vis absorption spectra of MB blended with MOF-Fe@DOX-CaO2 and H2O2 at different times under irradiation;

[0037] Figure 11 Extracellular glutathione consumption efficiency of MOF-Fe@DOX-CaO2 nanoparticles prepared in the embodiments of the present invention: (A) UV-Vis absorption spectra of DTNB at 485 nm after co-incubation with GSH by different nanoprobes (TCPP, PEG-MOF-Fe and MOF-Fe@DOX-CaO2); (B) Change of UV-Vis absorption spectrum of DTNB at 485 nm over time after co-culturing with GSH by MOF-Fe@DOX-CaO2 (18 mg / mL);

[0038] Figure 12 This is an extracellular fluorescence imaging image of the MOF-Fe@DOX-CaO2 nanoparticles prepared in the embodiments of the present invention;

[0039] Figure 13 The following are schematic diagrams of T1 magnetic resonance imaging of MOF-Fe@DOX-CaO2 nanoparticles prepared in the embodiments of the present invention: (A) T1 magnetic resonance imaging relaxation rate curve; (B) T1 magnetic resonance imaging signal diagram.

[0040] Figure 14 The cumulative release curves of iron ions from MOF-Fe@DOX-CaO2 nanoparticles prepared in the embodiments of the present invention under different pH conditions (5.5, 6.5, 7.4);

[0041] Figure 15 The oxygen release curves of MOF-Fe@DOX-CaO2 nanoparticles prepared in the embodiments of the present invention under different H2O2 concentrations are shown.

[0042] Figure 16 Evaluation of the fluorescence imaging level of MOF-Fe@DOX-CaO2 nanoparticles prepared in the embodiments of the present invention at the cellular level: (A) Fluorescence imaging of MOF-Fe@DOX-CaO2 nanoparticles of different concentrations in 4T1 cells; (B) Qualitative analysis of fluorescence imaging of MOF-Fe@DOX-CaO2 nanoparticles of different concentrations in 4T1 cells.

[0043] Figure 17 The MOF-Fe@DOX-CaO2 nanoparticles prepared in this embodiment of the invention were used to study the ROS generation capacity of MOF-Fe@DOX-CaO2 aqueous solutions of different concentrations under light / no light conditions by reactive oxygen species fluorescent probe (DCFH-DA) staining.

[0044] Figure 18 The expression of lipid peroxides (LPO) in the MOF-Fe@DOX-CaO2 nanoparticles prepared in this embodiment of the invention at the cellular level: (A) CLSM observation of cellular LPO using the BODIPY-C11 probe; (B) Semi-quantitative statistical results of BODIPY fluorescence intensity. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0046] All raw materials and reagents in the embodiments of this application were purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0047] Example 1

[0048] A method for preparing a responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis is as follows:

[0049] (1) Preparation of NH2-PEG: At room temperature, 5g of polyethylene glycol 2000 (Mw=2000) was dissolved in 10mL of deionized water. After complete dissolution, ammonia was slowly added dropwise to the solution. The reaction solution was stirred at room temperature for 24h. After the reaction was complete, the solution was dialyzed in deionized water for 3 days using a dialysis bag with a molecular weight of 1000 and then freeze-dried to obtain aminated polyethylene glycol (NH2-PEG) particles.

[0050] (2) Synthesis of PEG-MOF-Fe nanoparticles: 0.026 g of racemic tetra(4-carboxyphenyl)porphyrin (TCPP), 0.061 g of ferric chloride (III) hexahydrate (FeCl3·6H2O), 0.703 g of benzoic acid and 0.650 g of amino-modified polyethylene glycol (NH2-PEG) were dissolved in 25 mL of N,N-dimethylformamide (DMF) to obtain a mixed solution. The mixed solution was heated to 95 °C and stirred at 500 rpm in the dark for 7 h. After the reaction was complete, the mixed solution was cooled to room temperature and dialyzed in deionized water for 3 days using a dialysis bag with a molecular weight of 5000 to obtain PEG-MOF-Fe nanoparticles.

[0051] (3) Drug loading on PEG-MOF-Fe nanoparticles: 20 mg of doxorubicin was dissolved in 2 mL of PEG-MOF-Fe nanoparticle aqueous solution with a concentration of 20 mg / mL, so that doxorubicin was adsorbed into the pores of PEG-MOF-Fe nanoparticles through π-π interaction, and DOX-loaded PEG-MOF-Fe (MOF-Fe@DOX) nanoparticles were obtained.

[0052] (4) Encapsulation of drug-loaded PEG-MOF-Fe nanoparticles: 0.6 mg of prepared MOF-Fe@DOX nanoparticles were completely dispersed in 0.3 mL of anhydrous methanol. Under vigorous stirring, 0.02 mL of sodium hyaluronate aqueous solution (0.5 mg / mL) and 1 mL of calcium chloride aqueous solution (0.625 mol / L) were added to the above solution. Under continuous stirring, 2.4 mL of 30% hydrogen peroxide was slowly added to the solution. After stirring for 2 h, 4 mL of ammonia water was added to the above mixed solution to activate the reaction until a suspended solid was formed. After stirring for 2 h, the product was collected by centrifugation and washed twice with methanol to obtain a responsive nanoprobe (MOF-Fe@DOX-CaO2) for synergistic photodynamic therapy of calcium overload and ferroptosis.

[0053] As shown in the figure, the successfully prepared PEG-MOF-Fe nanoparticles are spindle-shaped and nearly spherical with a particle size of approximately 200 nm, exhibiting relatively uniform size and good dispersibility. Figure 1 ), hydrated particle size approximately 200 nm ( Figure 2 And the surface carries a negative charge. Figure 3 PEG-MOF-Fe nanoparticles exhibit a characteristic ultraviolet absorption peak around 425 nm. Figure 4 ).

[0054] After the PEG-MOF-Fe nanoparticles were prepared, CaO2 nanoparticles were encapsulated onto the surface of PEG-MOF-Fe, and the chemotherapy drug DOX for treating tumors was encapsulated into the MOF pores to form a responsive nanoprobe for photodynamic therapy synergistic between calcium overload and ferroptosis, denoted as MOF-Fe@DOX-CaO2.

[0055] Test results show that the prepared MOF-Fe@DOX-CaO2 particles are spherical with a diameter of approximately 50 nm and have relatively uniform size. Figure 5 The hydrated particle size of the MOF-Fe@DOX-CaO2 nanoprobe is approximately 800 nm. Figure 6 ), surface carries negative charge ( Figure 7 Furthermore, MOF-Fe@DOX-CaO2 nanoparticles exhibit a characteristic ultraviolet absorption peak around 230 nm. Figure 8 ).

[0056] like Figure 9 As shown, in H2O2 and near-infrared light (660 nm, 1 W / cm²), 2 Under the combined effect of MOF-Fe@DOX-CaO2, 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) blended with MOF-Fe@DOX-CaO2 showed the fastest rate of decrease in UV absorbance at 380 nm and 400 nm, indicating that its... 1 O2 generation capacity is higher than TCPP and PEG-MOF-Fe. (1W / cm²) in the near-infrared region at 660 nm. 2 Under irradiation, the absorption peak at 650 nm of MOF-Fe@DOX-CaO2 aqueous solution (pH=5.5) with added H2O2 continuously decreased over time. Figure 9 (C) indicates that MOF-Fe@DOX-CaO2 can effectively generate 1 O2. Next, the effect of illumination time on the production of MOF-Fe@DOX-CaO2 was investigated. 1 The impact of O2 efficiency. (By...) Figure 9 (A) indicates that in H2O2 and near-infrared light (660 nm, 1 W / cm²), 2 In the presence of MOF-Fe@DOX-CaO2, the UV absorption of ABDA co-incubated with MOF-Fe@DOX-CaO2 decreased significantly with time, indicating that MOF-Fe@DOX-CaO2... 1 O2 yield increases with prolonged exposure time.

[0057] In addition, this invention uses methylene blue (MB) to verify the ability of the MOF-Fe@DOX-CaO2 nanoprobe to generate ·OH. Under different test conditions, the combination of H2O2 and GSH caused the absorption peak of MB in the MOF-Fe@DOX-CaO2 solution to decrease the fastest at 650 nm. Figure 10 (A) Comparing the MB absorption spectra of different nanomaterials, the MB absorption peak of the MOF-Fe@DOX-CaO2 group showed a more significant attenuation. Figure 10 (B) indicates that MOF-Fe@DOX-CaO2 can more efficiently trigger the generation of •OH and degrade MB. Notably, near-infrared light (660 nm, 1 W / cm²) shows… 2 Irradiation further accelerated the decline in MB absorption, possibly due to enhanced photo-induced peroxidase activity. When exposed to near-infrared light in the presence of both H₂O₂ and GSH, the absorbance of MB mixed with MOF-Fe@DOX-CaO₂ decreased significantly over time. Figure 10 (D) indicates that the generation of ·OH by MOF-Fe@DOX-CaO2 is time-dependent. After determining the optimal conditions and duration for ·OH generation by MOF-Fe@DOX-CaO2, we compared the ·OH generation capabilities of different nanoprobes. Figure 10 As shown in (C), the UV absorbance of MB co-incubated with MOF-Fe@DOX-CaO2 decreased the fastest at 662 nm, indicating that MOF-Fe@DOX-CaO2 has a higher ·OH production capacity than TCPP or PEG-MOF-Fe.

[0058] Furthermore, this invention uses 5,5α-dithiodithio-2-nitrobenzoic acid (DTNB), a glutathione indicator, as a marker to verify the GSH consumption efficiency of MOF-Fe@DOX-CaO2. Figure 11 As shown in (A), when MOF-Fe@DOX-CaO2 nanoparticles were incubated with GSH, the absorbance of DTNB decreased faster than that of TCPP and PEG-MOF-Fe alone, indicating a higher glutathione consumption rate. Furthermore, with increasing co-incubation time, the UV absorbance of GSH co-incubated with MOF-Fe@DOX-CaO2 gradually decreased, indicating a time-dependent consumption of GSH. Figure 11 (B)). These results indicate that MOF-Fe@DOX-CaO2 can not only generate a large amount of ROS under near-infrared light irradiation, but also through Fe... 2+ The Fenton reaction consumes GSH and induces ferroptosis in tumor cells.

[0059] To verify the in vitro magnetic resonance and fluorescence imaging capabilities of MOF-Fe@DOX-CaO2, this invention prepared a series of MOF-Fe@DOX-CaO2 solutions with varying concentration gradients and performed magnetic resonance and fluorescence imaging on them. Figure 12 It is known that the fluorescence signal intensity of the MOF-Fe@DOX-CaO2 nanoprobe gradually increases with increasing Fe concentration. For example... Figure 13 (A) It is known that the T1 WI signal of an aqueous solution of MOF-Fe@DOX-CaO2 nanoparticles increases with increasing Fe concentration, indicating that MOF-Fe@DOX-CaO2 has significant T1 imaging capability. Calculations show that the T1 relaxation rate of MOF-Fe@DOX-CaO2 nanoparticles is 5.151 mM. -1 s -1 ( Figure 13 (B) The relaxation rate is slightly higher than that of clinically used Gd-type contrast agents. These experimental results demonstrate that the MOF-Fe@DOX-CaO2 nanoprobe has excellent magnetic resonance and fluorescence imaging capabilities.

[0060] like Figure 14 As shown, the iron ion release rate continuously increased over time, exhibiting a clear pH dependence: the stronger the acidity, the faster the iron ion release rate and the higher the total release amount. In the weakly acidic tumor microenvironment at pH 5.5, the iron ion release rate was approximately 45% after 10 h; approximately 37% at pH 6.5; and approximately 35% under physiological pH 7.4 conditions. This result indicates that the MOF-Fe@DOX-CaO2 nanoprobe has a significant response to acidic environments and can release iron ions more efficiently under weakly acidic conditions, demonstrating good pH responsiveness.

[0061] At the same time, such as Figure 15 As shown, the oxygen production performance of MOF-Fe@DOX-CaO2 exhibits significant H2O2 concentration- and time-dependent characteristics. Under H2O2-free conditions, the oxygen production remains essentially constant at the baseline level. In contrast, with the addition of H2O2 (10, 20, and 40 mM), the oxygen concentration continuously increases over time, and the higher the H2O2 concentration, the faster the oxygen production rate and the higher the final oxygen concentration. These results indicate that MOF-Fe@DOX-CaO2 can catalyze oxygen production in response to H2O2, demonstrating significant potential in alleviating tumor hypoxia.

[0062] To verify the intracellular fluorescence imaging capability of MOF-Fe@DOX-CaO2, this invention prepared MOF-Fe@DOX-CaO2 solutions with different concentration gradients and performed intracellular fluorescence imaging on them. Figure 16As shown, the fluorescence signal intensity of MOF-Fe@DOX-CaO2 in 4T1 breast cancer cells increases with increasing TCPP concentration. These experimental results demonstrate that the MOF-Fe@DOX-CaO2 nanoprobe still exhibits excellent fluorescence imaging capabilities at the cellular level.

[0063] In vitro test results show that the water-soluble MOF-Fe@DOX-CaO2 nanoprobe can generate ROS under near-infrared light irradiation, thereby modulating the tumor microenvironment. Therefore, this invention uses a reactive oxygen species fluorescent probe to assess the ROS generation level of this probe at the cellular level, and the results are as follows: Figure 17 As shown in the figure, compared with the unilluminated group, 4T1 cells under near-infrared light irradiation showed obvious green fluorescence, and the fluorescence intensity increased with the increase of MOF-Fe@DOX-CaO2 concentration. This indicates that 4T1 cells co-incubated with MOF-Fe@DOX-CaO2 can only produce ROS under near-infrared light irradiation, and the concentration of ROS produced increases with the increase of MOF-Fe@DOX-CaO2 concentration.

[0064] A key characteristic of ferroptosis is the accumulation of iron-dependent lipid peroxides (LPO). Therefore, this invention uses the lipid peroxidation sensor BODIPY to assess changes in intracellular LPO. CLSM images show ( Figure 18 (A)), CLSM corresponds to 4T1 cells Figure 18 In (A), the green fluorescence intensity, indicating LPO, increases dose-dependently with increasing MOF-Fe@DOX-CaO2 concentration. Meanwhile, semi-quantitative statistical results on BODIPY fluorescence intensity ( Figure 18 (B) Further evidence shows that MOF-Fe@DOX-CaO2 treatment can significantly increase the level of intracellular lipid peroxidation, and this effect is enhanced with increasing drug concentration, indicating that the nanoplatform can effectively induce ferroptosis in tumor cells.

[0065] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. A method for preparing a responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis, characterized in that, Includes the following steps: (1) Preparation of NH2-PEG: At room temperature, polyethylene glycol was dissolved in deionized water; then, ammonia was slowly added dropwise to the polyethylene glycol solution, and the mixture was stirred at room temperature for 24 h; after the reaction was complete, the mixture was dialyzed in deionized water for 1-3 days using a dialysis bag with a molecular weight of 1000, and then freeze-dried to obtain aminated polyethylene glycol, denoted as NH2-PEG; (2) Synthesis of PEG-MOF-Fe nanoparticles: Meso-tetra(4-carboxyphenyl)porphyrin, ferric chloride hexahydrate, benzoic acid and NH2-PEG were dissolved in N,N-dimethylformamide in sequence, and the mixed solution was stirred in the dark at 70-150℃ for 5-10 h; after the reaction was complete, the mixed solution was cooled to room temperature and dialyzed in deionized water for 1-3 days using a dialysis bag with a molecular weight of 5000 to obtain water-soluble porous iron-based metal nanoparticles, denoted as PEG-MOF-Fe nanoparticles; (3) Drug loading on PEG-MOF-Fe nanoparticles: Doxorubicin was dissolved in an aqueous solution of PEG-MOF-Fe nanoparticles, so that doxorubicin was adsorbed into the pores of PEG-MOF-Fe through π-π interaction, thus obtaining MOF-Fe@DOX nanoparticles loaded with doxorubicin; (4) Encapsulation of drug-loaded PEG-MOF-Fe nanoparticles: The prepared MOF-Fe@DOX nanoparticles were dispersed in anhydrous methanol, and sodium hyaluronate aqueous solution and calcium chloride aqueous solution were added. Then, hydrogen peroxide was slowly added to the mixture. After stirring for 2 h, ammonia was added dropwise to the above mixture to activate the reaction until a suspended solid was formed. After the reaction was completed for 2 h, the product was collected by centrifugation and washed twice with methanol to obtain a responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis, denoted as MOF-Fe@DOX-CaO2.

2. The method for preparing the responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis according to claim 1, characterized in that, In step (1), the concentration of polyethylene glycol is 0.2 to 1 g / mL; the polyethylene glycol is one of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000 and polyethylene glycol 5000.

3. The method for preparing the responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis according to claim 1, characterized in that, In step (1), the mass ratio of polyethylene glycol to ammonia is 1:1 to 1:

2.

4. The method for preparing the responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis according to claim 1, characterized in that, In step (2), the concentration of meso-tetra(4-carboxyphenyl)porphyrin is 0.5–1.5 mg / mL; the mass ratio between meso-tetra(4-carboxyphenyl)porphyrin and ferric chloride hexahydrate is 1:1–1:6; the mass ratio between meso-tetra(4-carboxyphenyl)porphyrin and benzoic acid is 1:15–1:60; and the mass ratio between meso-tetra(4-carboxyphenyl)porphyrin and NH2-PEG is 1:10–1:

60.

5. The method for preparing the responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis according to claim 1, characterized in that, The PEG-MOF-Fe nanoparticles prepared in step (2) have a particle size of 100~200 nm and a surface charge of -20~-40 mV.

6. The method for preparing the responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis according to claim 1, characterized in that, In step (3), the concentration of the aqueous solution of PEG-MOF-Fe nanoparticles is 10~30 mg / mL; the mass ratio of PEG-MOF-Fe nanoparticles to doxorubicin is 1:2~3:

1.

7. The method for preparing the responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis according to claim 1, characterized in that, The PEG-MOF-Fe nanoparticles prepared in step (3) have a drug loading capacity of 45-70% for doxorubicin and an encapsulation efficiency of 40-60% for doxorubicin.

8. The method for preparing the responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis according to claim 1, characterized in that, In step (4), the concentration of the MOF-Fe@DOX nanoparticle solution is 10~20 mg / mL; the mass ratio of MOF-Fe@DOX nanoparticles to sodium hyaluronate is 10:1~80:1; the mass ratio of MOF-Fe@DOX nanoparticles to calcium chloride is 1:100~1:150; the nanoprobe prepared in step (4) is based on PEG-MOF-Fe nanoparticles as a framework, loaded with doxorubicin inside, and connected to calcium chloride on the surface.

9. A responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis, prepared by the preparation method of any one of claims 1-8.

10. The responsive nanoprobe for synergistic photodynamic therapy of calcium overload and ferroptosis according to claim 9, characterized in that, The nanoprobe has a particle size of 200~300 nm and a surface charge of -2~-20 mV.

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