Preparation method and application of iron MOF nanoparticles

Abstract

The invention discloses an iron MOF (Metal Organic Framework) nanoparticle and a preparation method thereof. Specifically, the preparation method comprises the following steps: by taking iron MOF, GOX, egg yolk lecithin, cholesterol, distearoyl phosphatidyl ethanolamine (DSPE), Ce6 and HCPT as reaction raw materials, preparing the Ce6-coated HGMOF nano-particles through a thin lipid membrane hydration and sequential extrusion method. The iron MOF nanoparticles can relieve hypoxia and have photodynamic and ferroptosis treatment effects at the same time.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically, the present invention relates to an iron MOF nanoparticle that can alleviate tumor hypoxia and enhance the therapeutic effect of synergistic photodynamic / ferroptosis / starvation therapy, as well as a preparation method and application thereof. Background Art

[0002] As a malignant disease, tumors seriously endanger human health and shorten human life. At the same time, with the rapid proliferation of tumors and the abnormal and twisted formation of new blood vessels, hypoxia has become one of the most common and basic characteristics of tumors. Tumor hypoxia can lead to the passivation of photodynamic therapy (PDT). Photodynamic therapy is an emerging tumor treatment method during which photodynamic therapy can treat tumors. Photosensitizers delivered to the tumor site can be activated by near-infrared irradiation at 660nm to convert oxygen into cytotoxic singlet oxygen ( 1 O 2 ). Photodynamic-induced oxidative stress produces reactive oxygen species (ROS) that can directly lead to tumor cell apoptosis and necrosis; therefore, adequate oxygen is essential for the efficacy of photodynamic therapy. The hypoxic nature of the tumor microenvironment severely limits the therapeutic efficiency of photodynamic therapy. In addition, the hypoxia-inducible factor 1α (HIF-1α) pathway activates angiogenesis in the presence of hypoxia, followed by the production of vascular endothelial growth factor (VEGF) by tumor cells and stromal cells, and the activated endothelial cells lead to abnormal tumor vasculature, further aggravating hypoxia, thus forming a vicious cycle. In various types of tumors, hypoxia provides an anti-apoptotic advantage, inhibits drug penetration, and promotes cancer progression and drug resistance. This may be because oxygen-dependent chemotherapeutics are more active when oxygen is available, and hypoxia directly inhibits the anti-tumor function of oxygen-dependent chemotherapeutics. Therefore, reversing the tumor hypoxic microenvironment is a key way to improve tumor synergy.

[0003] In addition to hypoxia, tumor cells transport and metabolize nutrients much faster than normal cells due to their rapid metabolism and proliferation. Most tumor cells produce energy through glycolysis rather than through respiratory metabolism. The metabolic mode of glycolysis frees tumor cells from oxygen dependence and promotes tumor cell growth and proliferation in a hypoxic environment. This is true even under normal oxygen levels, making cancer cells sensitive to changes in intracellular glucose concentrations. Given the key role of glucose in providing energy for tumor growth and metabolism, researchers often starve cancer cells (starvation therapy) by allowing glucose oxidase (GOX) to deplete intracellular glucose. To alleviate hypoxia, researchers introduced catalase, which can react with hydrogen peroxide (H 2 O 2 ) (provided by GOX consumption of glucose, provided by tumor cells) and converts it into O 2 To promote reoxygenation, thereby enhancing O 2 Dependence on photodynamics.

[0004] However, the instability of catalase often limits its use. In addition, the antioxidant system marked by reduced glutathione (GSH) becomes another important reason for tumor survival in the face of the threat of photodynamic oxidative stress, and tumor cells usually increase their own GSH levels to achieve intracellular homeostasis. GSH depletion is closely related to ferroptosis.

[0005] In view of this, there is an urgent need in this field to develop an anticancer drug with better comprehensive effects in reversing hypoxia in the tumor microenvironment and enhancing the synergistic therapeutic effect of tumors. Summary of the invention

[0006] One object of the present invention is to provide a nano drug that can reverse the hypoxia of the tumor microenvironment and enhance the synergistic therapeutic effect of tumors.

[0007] In a first aspect of the present invention, an iron MOF nanoparticle is provided, comprising: iron MOF, glucose oxidase GOX, photosensitizer Ce6, hydroxycamptothecin HCPT and egg yolk lecithin;

[0008] Among them, GOX is loaded in the iron MOF, and the outer layer of the iron MOF is encapsulated by liposomes loaded with photosensitizer Ce6 and hydroxycamptothecin HCPT;

[0009] Among them, the average particle size of the iron MOF is 123-128nm, and the average Zeta potential is 5.84±0.2mV; the average Zeta potential of the iron MOF loaded with GOX is 9.45±0.2mV; the average particle size of the iron MOF nanoparticles is 148-150nm; the average Zeta potential is 2.59±0.2mV; the average specific surface area is 1.8562m 2 / g.

[0010] In another preferred embodiment, the iron MOF nanoparticles are Ce6@HGMOF nanoparticles.

[0011] In the second aspect of the present invention, a method for preparing the iron MOF nanoparticles according to the first aspect of the present invention is provided, comprising the following steps:

[0012] (1) Synthesis of iron MOF: FeCl was pumped into 3 6H 2 The O solution was slowly added to the dithiodiacetic acid solution and magnetically stirred for about 20-50 minutes; when the liquid turned into a cream-yellow color, it was centrifuged at a speed of 8000-10000 rpm for 10 minutes, and the supernatant was removed to obtain the iron MOF;

[0013] (2) Synthesis of GMOF: Glucose oxidase GOX was dissolved in water, mixed with the iron MOF obtained in step (1), and mixed by magnetic stirring for 24 hours; centrifuged at a speed of 9000-12000 rpm, and centrifuged three times to obtain GMOF;

[0014] (3) Preparation of Ce package 6 -HCPT liposomes: egg yolk lecithin, cholesterol, distearoylphosphatidylethanolamine DSPE, photosensitizer Ce 6 and hydroxycamptothecin HCPT in an organic solution, and remove the solvent by rotary evaporation at 35-50° C.; hydrate the membrane liposomes with deionized water, and completely disperse them by contact ultrasonic treatment for 20 minutes to obtain a solution containing liposomes encapsulating Ce6-HCPT;

[0015] (4) The solution containing liposomes encapsulating Ce6-HCPT obtained in step (3) is mixed with the GMOF obtained in step (2), and the mixture is pressed repeatedly to obtain Ce6@HGMOF nanoparticles.

[0016] In another preferred embodiment, the preparation method of the liposome encapsulating Ce6-HCPT comprises: thoroughly mixing 200 mg egg yolk lecithin, 50 mg cholesterol, 4 mg DSPE, 5 mg Ce6 and 5 mg HCPT (ethanol / dichloromethane, 1:1) in 20 mL of dichloromethane solution, and removing the solvent by rotary evaporation at 40°C to obtain.

[0017] In another preferred embodiment, the method further comprises: after step (4), 6 @HGMOF nanoparticles were centrifuged at 2000-4000 rpm for 10 minutes, the supernatant was removed, and the particles were washed three times with deionized water to obtain Ce 6 @HGMOF nanoparticles.

[0018] In another preferred embodiment, in step (3), the organic solvent is selected from the following group: C1-C6 alcohol solvents, C1-C6 hydrocarbon solvents, or a combination thereof.

[0019] In another preferred embodiment, in step (3), the organic solvent is an ethanol / dichloromethane (1:1 v / v) solution.

[0020] In another preferred embodiment, the FeCl 3 6H 2 The concentration of O solution is 20-40 mg / mL; preferably 25 mg / mL.

[0021] In another preferred embodiment, the FeCl 3 6H 2The concentration of the O solution is 4 times that of the dithiodiacetic acid solution.

[0022] In another preferred embodiment, the FeCl 3 6H 2 O solution is 100 mg FeCl 3 6H 2 O was dissolved in 4 mL of deionized water.

[0023] In another preferred embodiment, the concentration of the dithiodiacetic acid solution is 5-9 mg / mL; preferably 6.25 mg / mL.

[0024] In another preferred embodiment, the dithiodiacetic acid solution is 100 mg of dithiodiacetic acid dissolved in 16 mL of water.

[0025] In the third aspect of the present invention, there is provided a use of the iron MOF nanoparticles according to the first aspect of the present invention for preparing a drug or preparation for treating cancer.

[0026] In another preferred embodiment, the cancer is esophageal cancer.

[0027] In another preferred embodiment, the iron MOF nanoparticles are used in combination with near-infrared light to treat cancer.

[0028] In another preferred embodiment, the wavelength of the near-infrared light is 500-800nm; preferably 600-700nm; more preferably 650-670nm.

[0029] In another preferred embodiment, the irradiation power of the near-infrared light is 100 mW·cm -2 The irradiation time is 5 minutes.

[0030] In the fourth aspect of the present invention, a method for treating esophageal cancer is provided, wherein a therapeutically effective amount of the iron MOF nanoparticles described in the first aspect of the present invention is administered to a subject in need thereof, and the affected area is irradiated with near-infrared light, thereby treating cancer.

[0031] In another preferred embodiment, the wavelength of the near-infrared light is 500-800nm; preferably 600-700nm; more preferably 650-670nm.

[0032] In another preferred embodiment, the irradiation power of the near-infrared light is 100 mW·cm -2 The irradiation time is 5 minutes.

[0033] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the synthesis of Ce6@HGMOF and reversal of the tumor microenvironment for synergistic photodynamic / ferroptosis / starvation therapy against tumors.

[0035] Figure 2 Characterization of Ce6@HGMOF. (A) SEM of MOF (scale bar: 200nm (left), scale bar: 1μm (center), scale bar: 2μm (right)). (B) TEM of MOF (scale bar: 200nm) and (C) Ce6@HGMOF (scale bar: 200nm (left), scale bar: 100nm (right)). (D) Elemental analysis of Ce6@HGMOF (scale bar: 200nm). (E) Hydrodynamic size of MOF and Ce6@HGMOF. (F) Zeta potential of MOF, Ce6, GMOF and Ce6@HGMOF. (G) FTIR spectra of HCPT, Ce6, GOX, MOF and Ce6@HGMOF.

[0036] Figure 3 Performance tests of MOF and Ce6@HGMOF (A) N2 adsorption-desorption isotherms of MOF and Ce6@HGMOF. (B) HCPT release curve. (C) Iron ion release curve. (D) GSH consumption with or without MOF (n=3). (E) UV-visible spectra changes of TMB solution and H2O2 catalyzed by MOF. (F) pH values ​​of different reaction systems in phosphate buffer. (G) pH changes caused by the reaction of GOX with different concentrations of glucose. (H) UV-vis spectra of TMB solution in the presence of Ce6@HGMOF. (I) Under 660nm laser (100mW / cm -2 The 1O2 production efficiency was determined by DPBF under various conditions before and after irradiation (100 ng / min, 20 min).

[0037] Figure 4 MB was used to detect hydroxyl radicals (1. MB group, 2. MB+H 2 O 2 group, 3.MB+glucose group, 4.MB+MOF group, 5.MB+H 2 O 2 +MOF group, 6.MB+glucose+MOF group).

[0038] Figure 5Verification of in vivo synergistic effect. (A) The expression of HIF-1a, VEGF, PgpGPX4, SLC7A11, PKM2 and HK2 in tumor mice was determined by Western blotting. (B) Results of Western blotting analysis and semi-quantitative Western blotting (n=3). (1. Control group, 2. MOF. 3. Ce6@MOF+laser group, 4. Ce6@GMOF+laser group, 5. Ce6@HGMOF+laser group) (calculated according to MOF, 4 mg / kg, calculated according to Ce6, 0.4 mg / kg), (660nm, 100mW / cm -2 )Significance is defined as ns. Not significant, *p<0.05, **p<0.01. ***p<0.001.

[0039] Figure 6 C11-BODIPY fluorescent probe was used to detect the level of LPO in tumor cells (1. control group, 2. MOF group, 3. Ce6 + laser group, 4. Ce6@MOF + laser group, 5. Ce6@GMOF + laser group, 6. Ce6@HGMOF + laser group) (scale bar: 100μm).

[0040] Figure 7 The in vitro antitumor effect of Ce6@HGMOF. (A) Cytotoxicity of MOF group, Ce6+laser group, Ce6@MOF+laser group, Ce6@GMOF+laser group, and Ce6@HGMOF+laser group on EC109 cells after 48 hours of culture (n=3). (B) Staining of live and dead cells after different treatments (1. control group, 2. MOF group, 3. Ce6+laser group, 4. Ce6@MOF+laser group, 5. Ce6@GMOF+laser group, 6. Ce6@HGMOF+laser group) (scale bar: 200μm). (C) Flow cytometry detection of cell apoptosis after different treatments (1. control group, 2. MOF group, 3. Ce6+laser group, 4. Ce6@MOF+laser group, 5. Ce6@GMOF+laser group, 6. Ce6@HGMOF+laser group). (D) Statistical graph of cell apoptosis. (calculated according to MOF, 50 μg / mL), (660 nm, 100 mW / cm2, 5 min). Significance is defined as ns (not significant), *P<0.05, **P<0.01, ***P<0.001.

[0041] Figure 8Cellular uptake and tumor targeting of Ce6@HGMOF. (A) Time-dependent cellular uptake of Ce6@HGMOF in EC109 cells (calculated as MOF, 50μg / mL) (scale bar: 25μm). (B) Statistical graph of cell fluorescence (n=3). (C) In vivo, fluorescence images and distribution of each organ in mice bearing EC109 tumors injected intravenously with free Ce6 and Ce6@HGMOF at different time intervals. (D) Animal fluorescence statistics (n=3). (calculated as MOF, 50μg / mL), (660nm, 100mW / cm-2, 5 minutes). Significance is defined as ns, not significant, *P<0.05, **P<0.01, ***P<0.001.

[0042] Fig. 9 The in vitro antitumor effect of Ce6@HGMOF. (A) Cytotoxicity of MOF group, Ce6+laser group, Ce6@MOF+laser group, Ce6@GMOF+laser group, and Ce6@HGMOF+laser group on EC109 cells after 48 hours of incubation (n=3). (B) Staining of live and dead cells after different treatments (1. PBS group, 2. MOF group, 3. Ce6+laser group, 4. Ce6@MOF+laser group, 5. Ce6@GMOF+laser group, 6. Ce6@HGMOF+laser group) (scale bar: 200μm). (C) Cell apoptosis was detected by flow cytometry after different treatments (1. PBS group, 2. MOF group, 3. Ce6+laser group, 4. Ce6@MOF+laser group, 5. Ce6@GMOF+laser group, 6. Ce6@HGMOF+laser group). (D) Statistical graph of apoptosis. (calculated according to MOF, 50 μg / mL), (660 nm, 100 mW / cm -2 , 5 minutes). Significance is defined as ns, not significant, *P<0.05, **P<0.01, ***P<0.001.

[0043] Fig.10 The content of MDA in mouse tumor tissue (1. control group, 2. MOF, 3. Ce6+laser group, 4. Ce6@MOF+laser group, 5. Ce6@GMOF+laser group, 6. Ce6@HGMOF+laser group) (n=3). Significance is defined as ns, no significance, *P<0.05, **P<0.01, ***P<0.001.

[0044] Fig.11To verify the synergistic effect. (A) Western blot detection of HIF-1α, VEGF, Pgp, GPX4, SLC 7A 11, PKM 2 and HK 2 in tumor-bearing mice. (B) Western blot analysis and semi-quantitative results of Western blot (n=3). (1. Control group, 2. MOF group, 3. Ce6@MOF+laser group, 4. Ce6@GMOF+laser group, 5. Ce6@HGMOF+laser group) (calculated according to MOF, 4 mg / kg; calculated according to Ce6, 0.4 mg / kg), (660 nm, 100 mW / cm -2 , 5 minutes). Significance is defined as ns, not significant, *P<0.05, **P<0.01, ***P<0.001.

[0045] Fig.12 Biosafety evaluation of Ce6@HGMOF. (A)-(F) Changes of ALT, AST, ALB, CREA, UN and UA in mouse serum (n=3). (1. Control group, 2. MOF group, 3. Ce6+laser group, 4. Ce6@MOF+laser group, 5. Ce6@GMOF+laser group, 6. Ce6@HGMOF+laser group) (calculated according to MOF, 4 mg / kg; calculated according to Ce6, 0.4 mg / kg), (660 nm, 100 mW / cm -2 , 5 minutes). Significance is defined as ns, not significant, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0046] After extensive and in-depth research, the inventors have provided for the first time an iron MOF nanoparticle with GOX and peroxidase-like effects, which can overcome the tumor hypoxic microenvironment. Specifically, the present invention has developed a photosensitizer composed of dihydrochlorin e 6 (Ce 6 ), GOX, the chemotherapeutic agent 10-hydroxycamptothecin (HCPT) and an iron-based metal organic framework (MOF) composed of a novel nanoparticle Ce 6 @HGMOF, to achieve the effect of synergistic photodynamic / ferroptosis / starvation therapy for cancer treatment. Iron MOF has peroxidase-like activity, which is pH-dependent. Under certain conditions, the enzyme activity increases with the decrease of pH. In addition, the metal ions and disulfide bonds in the iron MOF can also deplete GSH and react with H in the Fenton reaction. 2 O 2The reaction produces hydroxyl radicals (·OH), which induce ferroptosis in tumor cells and enhance the therapeutic effect of photodynamic therapy. The above process is usually accompanied by the decomposition of nanocarriers, which promotes the complete release of the drug HCPT in the carrier. Ce6@HGMOF realizes synergistic photodynamic / ferroptosis / starvation therapy in vitro and in vivo, and these therapies can promote each other to exert significant tumor inhibition. Based on this, the inventors completed the present invention.

[0047] the term

[0048] PDT Photodynamic Therapy

[0049] MOF Metal Organic Framework

[0050] GSH Glutathione

[0051] O 2 oxygen

[0052] H 2 O 2 Hydrogen Peroxide

[0053] ROS Reactive oxygen species

[0054] 1O 2 Singlet oxygen

[0055] GOX Glucose oxidase

[0056] OH Hydroxyl radical

[0057] HCPT Hydroxycamptothecin

[0058] Ce6 Chlorin e6

[0059] DPBF 1,3-Diphenylisobenzofuran

[0060] TMB Tetramethylbenzidine

[0061] MB Methylene Blue

[0062] LPO Lipid peroxidation

[0063] MDA Malondialdehyde

[0064] SEM Scanning Electron Microscope

[0065] TEM Transmission electron microscopy

[0066] BET surface area

[0067] FTIR Fourier transform infrared spectroscopy

[0068] HIF-1α Hypoxia-mediated factor-1

[0069] VEGF Vascular endothelial growth factor

[0070] Pgp P-glycoprotein

[0071] PKM2 M2-type pyruvate kinase

[0072] HK2 Hexokinase

[0073] GPX4 glutathione peroxidase 4

[0074] SLC7A11 solute carrier family 7 member 11

[0075] Tunel terminal uridine nucleotide terminal labeling

[0076] CD31 Platelet endothelial cell adhesion molecule-1

[0077] Iron MOF nanoparticles

[0078] Hypoxia is of great significance in tumor treatment, and it is considered to be an important cause of tumor proliferation, angiogenesis and drug resistance. With the deepening of research, hypoxia and tumor treatment have received more and more attention, so reversing the tumor hypoxic microenvironment is crucial to improving the effect of tumor treatment. Compared with the prior art, in the present invention, the applicant has designed a multifunctional drug delivery system based on self-oxygen supply, which integrates photodynamics, ferroptosis and "starvation therapy" to achieve synergistic treatment of tumors.

[0079] Specifically, the present invention provides an iron MOF nanoparticle that alleviates tumor hypoxia while enhancing the therapeutic effect of synergistic photodynamic / ferroptosis / starvation therapy.

[0080] The iron MOF nanoparticles of the present invention are composed of iron MOF, glucose oxidase GOX, and photosensitizer Ce. 6 , hydroxycamptothecin HCPT and egg yolk lecithin, iron MOF is mixed with GOX to obtain GMOF loaded with GOX; Ce6 and HCPT are wrapped in egg yolk lecithin, and then mixed with GMOF to obtain a GMOF with lecithin on the outside and GOX and Ce on the inside. 6 Iron MOF nanoparticles with HCPT.

[0081] Among them, the average particle size of the iron MOF is 123-128nm, and the average Zeta potential is 5.84±0.2mV; the average Zeta potential of the iron MOF loaded with GOX is 9.45±0.2mV; the average particle size of the iron MOF nanoparticles is 148-150nm; and the average Zeta potential is 2.59±0.2mV.

[0082] In the present application, Ce6 and Ce6 have the same meaning, both representing the photosensitizer dihydrochlorin.

[0083] use

[0084] The iron MOF nanoparticles provided by the present invention can alleviate tumor hypoxia and simultaneously enhance the therapeutic effect of synergistic photodynamic / ferroptosis / starvation therapy.

[0085] Specifically, the nanoparticles of the present invention firstly reduce the pH in the tumor through "starvation therapy", COX consumes glucose in the tumor to produce H 2 O 2 Secondly, the acidic microenvironment can further enhance the peroxidase activity of the nanocarriers, which is related to the H produced in the tumor. 2 O 2 React, generate oxygen and enhance photodynamics. The change of hypoxic environment can slow down the growth of tumor blood vessels to a certain extent and improve the drug-resistant microenvironment. Finally, this nanoparticle can also deplete GSH and induce ferroptosis in tumor cells, further destroying the tumor's resistance to oxidative stress. This synergistic photodynamic / starvation therapy / ferroptosis multi-treatment strategy can reinforce each other, not only showing effective anti-tumor effects in vitro and in vivo, but also having good biosafety and good prospects for clinical transformation.

[0086] According to the embodiment, the iron MOF nanoparticles of the present invention that relieve tumor hypoxia and have both photodynamic and ferroptosis therapeutic effects need near-infrared light irradiation to exert photodynamic therapy, and the irradiation wavelength is 500-800nm; preferably 600-700nm; more preferably 660nm, and the irradiation power is 100mW·cm -2 The irradiation time is 5 minutes.

[0087] Preparation method of iron MOF nanoparticles

[0088] The present invention provides a method for preparing iron MOF nanoparticles which can be carried out using conventional synthesis steps and parameters in the art. Typically, the method for preparing iron MOF nanoparticles of the present invention comprises the following steps:

[0089] (1) Synthesis of MOF: FeCl 3 6H 2 O solution (100 mg dissolved in 4 mL of deionized water) was slowly added to dithiodiacetic acid solution (100 mg dissolved in 16 mL of water). Stir magnetically for about 30 minutes. When the liquid turned into a creamy yellow color, centrifuge at 8000-10000 rpm for 10 minutes to remove the supernatant.

[0090] (2) Ce 6Synthesis of HGMOF: GOX was dissolved in water, mixed with the MOF synthesized above, and mixed with magnet for 24 hours. The centrifugal speed was 10,000 rpm, and the samples were washed three times for collection. At the same time, a Ce 6 -HCPT liposomes. 200mg egg yolk lecithin, 50mg cholesterol, 4mg DSPE, 5mg Ce 6 and 5 mg HCPT were thoroughly mixed in 20 mL of ethanol / dichloromethane (1:1) solution, and the solvent was removed by rotary evaporation at 40 °C. The membrane liposomes were then hydrated with deionized water and completely dispersed by contact ultrasonic treatment for 20 min. The drug-loaded liposome solution was mixed with the drug-loaded MOF and the Ce 6 @HGMOF nanoparticles. Finally, the nanoparticles were centrifuged (3000 rpm) for 10 minutes, the supernatant was removed, and the nanoparticles were washed three times with deionized water and collected.

[0091] Compared with the prior art, the main advantages of the present invention include:

[0092] (1) The present invention is based on the self-oxygenation of peroxidase nanoparticles, the peroxidase-like activity of the nanoparticles themselves and the intracellular H 2 O 2 In vitro and in vivo experiments have shown that the photodynamic therapy is very effective by reversing the hypoxic microenvironment of the tumor. The microenvironment can well enhance the effect of photodynamic therapy. At the same time, drug efflux and the expression of angiogenesis-related proteins are also improved. The expression of angiogenesis-related proteins in tumor cells can be reduced, thereby enhancing the anti-tumor effect.

[0093] (2) The "starvation therapy" of the present invention consumes glucose, thereby cutting off the nutrient supply to the tumor and enhancing the acidic microenvironment of the tumor, which is beneficial to improving the peroxidase-like activity of the nanoparticles. 2 O 2 is recycled and reacts with the nanoparticles to produce O again 2 , thus making up for the O consumed during the "hunger therapy" 2 The amount of O consumed during the "starvation therapy" 2 , and form a virtuous circle.

[0094] (3) The present invention introduces a therapeutic strategy of ferroptosis. As an iron-dependent, non-apoptotic form of cell death, a therapeutic strategy of ferroptosis is introduced to overcome resistance to apoptosis. Overcome the resistance of tumor cells to apoptosis. In addition, ferroptosis consumes intracellular GSH, induces inactivation of GPX4 and SLC7A11, destroys the anti-oxidative stress system in the tumor, and enhances the therapeutic effect of photodynamic therapy. Ce6@HGMOF has great potential in reversing the hypoxic inhibitory microenvironment of tumors. It has great potential in the microenvironment and can greatly improve the efficiency of photodynamic therapy. Although there have been many cases of enhancing photodynamic therapy for tumors by relieving hypoxia, there are few reports on relieving hypoxia while relieving hypoxia and synergizing photodynamic therapy with multiple treatment methods. This synergistic strategy is not only synergistic, but also complementary. In addition, Ce6@HGMOF can effectively accumulate in the tumor site, which greatly reduces the dosage of the drug, helps to reduce its side effects, and demonstrates exciting clinical applications.

[0095] (4) The preparation method of the present invention is simple, has mild conditions, low cost, and is easy to promote and apply.

[0096] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0097] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0098] Example 1: Synthesis of Camptothecin Nanomedicine

[0099] Step (1): Synthesis of MOF. Use a peristaltic pump to transfer FeCl 3 6H 2 O solution (100 mg dissolved in 4 mL of deionized water) was slowly added with dithiodiacetic acid solution (100 mg dissolved in 16 mL of water). The mixture was stirred magnetically for about 30 minutes. When the liquid turned milky yellow, it was centrifuged at 8000-10000 rpm for 10 minutes to remove the supernatant to obtain MOF.

[0100] Step (2): Preparation of Ce6@HGMOF by thin lipid film hydration and sequential extrusion method.

[0101] GOX (glucose oxidase) was dissolved in water, mixed with the MOF synthesized above, and stirred with a magnetic force for 24 hours. The mixture was centrifuged at 10,000 rpm and washed three times to obtain GMOF.

[0102] 200 mg egg yolk phosphatidylcholine, 50 mg cholesterol, 4 mg DSPE, 5 mg Ce6 (photosensitizer) and 5 mg HCPT (hydroxycamptothecin) were fully mixed in 20 mL solution (ethanol / dichloromethane, 1:1), and the solvent was removed by rotary evaporation at 40 °C. The membrane liposomes were then hydrated with deionized water and completely dispersed by contact ultrasonic treatment for 20 min. The drug-loaded liposome solution was mixed with the drug-loaded GMOF and pressed repeatedly to obtain Ce6@HGMOF nanoparticles. Finally, the above nanoparticles were centrifuged (3000 rpm) for 10 min, the supernatant was removed, and the Ce6@HGMOF nanoparticles were collected by washing 3 times with deionized water.

[0103] Similarly, Ce6@MOF and Ce6@GMOF nanoparticles were obtained using the above method, except that glucose oxidase and hydroxycamptothecin (Ce6@MOF) and hydroxycamptothecin (Ce6@GMOF) were not added.

[0104] Example 2: Characterization of Nanoparticles

[0105] The size and morphology of Ce6@HGMOF were analyzed by transmission electron microscopy (TEM, FEI Tecnai G2 F20) and scanning electron microscopy (SEM, Hitachi Rcgulus 8100). The zeta potential was measured on a Malvern Zetasizernano. The absorbance was recorded on a Weiss spectrophotometer (Thermo Fisher Scientific). The physical adsorption apparatus (Micromeritics ASAP 2020) was used to analyze the Ce6@HGMOF under N 2 Surface area and pore distribution were obtained on adsorption. Fourier transform infrared spectrometer (BRUKER) was used to determine the chemical structure. Dissolved oxygen analyzer (JPB-@7A) was purchased from Shanghai Yidian Scientific Instrument Co., Ltd (Shanghai, China). Anaerobic culture tanks and anaerobic bags were purchased from Mitsubishi Chemical Co., Ltd. (Tokyo, Japan). 660nm near-infrared laser was from Xi'an Lei Ze Electronic Technology Co., Ltd (Xi'an, China). Flow cytometry was analyzed on a BD BeckmanCoulter flow cytometer (Brea, CA). CLSM images were recorded on a Zeiss LSM 780 microscope. ( Figure 1 A and 1B). After loading the drug, the surface was covered with liposome extrudates. TEM and magnified images revealed that a coating appeared on the surface of the MOF, indicating that the liposomes could encapsulate it ( Figure 2 C).

[0106] In order to verify the main element composition of MOF, we observed through mapping and found that MOF has the most Fe elements, which verifies that MOF is mainly composed of Fe ions ( Figure 2 D).

[0107] Next, we observed the changes in particle size and zeta potential before and after drug synthesis. The average particle size of MOF was 124.58nm, and the average particle size of Ce6@HGMOF was 148.45nm ( Figure 2 E), indicating that the particle size of MOF increases after liposome encapsulation.

[0108] The average zeta potential of MOF is 5.84 mV, the potential of the loaded GOX increases to 9.45 mV, the average potential of Ce6 is -47.3 mV, and the potential of the synthesized Ce6@HGMOF is 2.59 mV ( Figure 2 F).

[0109] The synthesis of nanoparticles was further confirmed by Fourier transform infrared spectroscopy (FTIR). The characteristic peak of GOX is 1407.04 cm -1 ; HCPT characteristic peak is 2913.6cm -1 ; Ce6 characteristic peak is 3286.1cm -1 ; MOF characteristic peak is 579.28cm -1 ; Compared with the above components, Ce6@HGMOF shows similar characteristic peaks, proving that each component is successfully loaded ( Figure 2 G).

[0110] Example 3: Encapsulation efficiency

[0111] To determine the amount of loaded drug, Ce6@HGMOF was placed in a thermostatic refrigerator at 4°C for 2 h for exposure to ultrasound followed by centrifugation (13,000 rpm, 20 min). The effective loading of HCPT and Ce6 was obtained using a UV-visible spectrophotometer. The loading of GOX was determined by BCA protein assay.

[0112] The results showed that the drug encapsulation efficiencies of HCPT and GOX were approximately 27.8% and 22.4%, respectively, and the loading rate of Ce6 was approximately 30–40%.

[0113] Example 4: HCPT drug / Fe ion release from Ce6@HGMOF

[0114] To evaluate drug release from HCPT, free HCPT and Ce6@HGMOF were sealed in dialysis bags with a MWCO of 1000Da, after which the bags were placed in different beakers. An appropriate amount of PBS at pH 5.4 was added to each beaker with or without 10 mM GSH, and all dialysis bags were shaken at 300 rpm, and a portion of the supernatant buffer was collected at predetermined time intervals. Drug release was observed by measuring the HCPT content with a UV-Vis spectrophotometer.

[0115] A similar approach was used for the Fe ion release experiment, where Ce6@HGMOF was sealed in a dialysis bag with a MWCO of 1000Da, which was then placed in different beakers. An appropriate amount of PBS with a pH of 7.4 or 5.4 was added to each beaker with or without 10mM GSH, all the dialysis bags were shaken at 300 rpm, and a portion of the supernatant buffer was collected at a predetermined interval. Iron ions were measured by using ICPMS. We also tested the BET specific surface area of ​​the MOF for nitrogen adsorption and found that the average specific surface area of ​​the MOF was 19.8842m 2 / g. In contrast, the average specific surface area of ​​Ce6@HGMOF is only 1.8562m 2 / g, demonstrating the success of liposome encapsulation ( Figure 3 A).

[0116] The decomposition of MOF and the release of iron ions in an acidic environment are key factors for the nanoparticles to function. We tested the drug / iron ion release of Ce6@HGMOF in different environments. We found that the nanoparticles slowed down the release of HCPT through liposome encapsulation, which may prolong the half-life of HCPT in vivo. At the same time, when Ce6@HGMOF enters tumor cells and encounters GSH, targeted release can be achieved ( Figure 3 B).

[0117] We observed the release of ferric iron at different pH values ​​and found that the release of ferric iron in an acidic environment of pH 5.4 was significantly greater than that in a normal environment of pH 7.4, and the release increased significantly after the addition of GSH. This is because MOF is composed of disulfide bonds and ferric iron, and both disulfide bonds and ferric iron can react with GSH, which will accelerate the disintegration of MOF ( Figure 3 C).

[0118] Example 5: Assessment of extracellular and intracellular GSH

[0119] GSH is a naturally occurring antioxidant in cells, and GSH depletion is an important indicator of ferroptosis.

[0120] First, the GSH consumption capacity of MOF was examined extracellularly. A 10mM GSH solution was mixed and stirred with MOF (10mg / mL), and the GSH content in the solution was measured at different time points (0, 2, 4, 12 and 24h), and a GSH solution without MOF was used as a control group. The GSH consumption capacity of MOF was then evaluated immediately in the cell. EC109 cells were seeded in a 10cm cell culture dish and cultured for 24 hours before use. After various treatments, the cells were harvested, washed twice with PBS, and then exposed to ultrasonic treatment at 150W for 30 seconds and repeated 10 times, followed by centrifugation at 8000 rpm for 10 minutes. The supernatant was collected and placed in a 4°C low-temperature ice bath box, and the changes in intracellular GSH content were calculated according to the absorbance value of the enzyme marker in the GSH kit instructions.

[0121] To further demonstrate the ability of MOF to consume GSH, we co-incubated MOF with GSH for 24 h and found that the content of GSH gradually decreased after co-incubation with MOF compared with GSH alone, indicating that MOF can react with GSH and consume GSH ( Figure 3 D). At the same time, we used the GSH kit Ce6@HGMOF to test. GSH in esophageal cancer cells can be consumed by Ce6@HGMOF nanomedicine ( Figure 5 I).

[0122] Implementation:6: Measurement of OH levels in solution

[0123] Methylene blue was used to detect the production of ·OH. Whether MOF has peroxidase-like activity is the key to its ability to alleviate tumor hypoxia. Methylene blue (MB) is a biological dye and redox indicator used to detect ·OH. 10mM H 2 O 2 Methylene blue (MB) solution (64 μg / mL) was prepared by mixing or not mixing with 5 mM glucose into different groups. MOF was added to the resulting mixed solution and incubated. Finally, the samples were centrifuged and the absorbance change of the supernatant at 664 nm was recorded by UV-Vis spectroscopy. Dye and redox indicator for the detection of ·OH.

[0124] To verify whether MOF is capable of Fenton reaction, we co-cultured MB with MOF and measured the amount of ·OH produced by detecting the absorption peak of the sample at 664 nm. Figure 4 As shown, it was found that the MB group and MB+H 2 O 2 Compared with the group, after adding MOF, the absorption peak in the above solution was significantly reduced, which may be due to the interaction between MOF and H 2 O 2 The ·OH produced by the Fenton reaction between them effectively consumes MB.

[0125] Example 7: Peroxidase-like activity of MOF and pH effect

[0126] This example tests the peroxidase-like activity of the prepared MOF on TMB oxidation: 10 mg / mL MOF aqueous dispersion was added to 0.1 M acetate buffer (pH 4.0), and then incubated with 0.1 mL, 10 mM TMB solution at room temperature for 20 minutes. The resulting solution was measured by UV-vis spectrometer at a wavelength of 652 nm.

[0127] Different buffer solutions with pH values ​​of 3-8 (3-6 AcOH buffer, 7-8 phosphate buffer, concentration 0.2 M) were used to study the effect of pH on the cascade reaction activity. 10 mg / mL MOF and 2 mM TMB were then used at 25°C for 40 minutes. Then, a UV Weiss spectrophotometer was operated to measure the absorbance at 285 nm.

[0128] TMB is a peroxidase substrate that can be oxidized by H in the presence of peroxidase. 2 O 2 Oxidation produces a bright blue substance. This colored substance is known to have maximum absorption peaks at wavelengths of 370 and 652 nm. Based on the depth of color, the absorbance of the reaction is measured using a spectrophotometer and then converted into the enzyme activity or enzyme concentration of the sample to be tested. We tested the catalase activity of MOF through TMB experiments and found that MOF and H 2 O 2 When incubated with TMB, the absorbance value increased significantly, indicating that MOF has catalase activity ( Figure 5 E). Studies have shown that the lower the intracellular pH, the stronger the catalase-like activity of MOF. Therefore, we simulated the tumor microenvironment and tested the ability of GOX to react with glucose to produce gluconic acid and lower the pH.

[0129] 5 mg of GOX was incubated with 0, 5, 10, 15, and 20 mmol of glucose, and the solution was incubated at ambient temperature for 2 hours with gentle shaking, and the pH change was monitored with pH test paper. It was found that after incubation with GOX and glucose, the pH value of the solution decreased significantly from 7 to more than 4 points ( Figure 3 F). Subsequently, we verified the changes in pH after incubation with GOX and different concentrations of glucose, and the results showed that the pH of the solution continued to decrease with the increase in glucose concentration. When glucose was 20 mM, the pH value decreased to more than 3 points ( Figure 3 G).

[0130] Prior art reports that the peroxidase-like activity of MOF is pH-dependent. To verify whether the peroxidase-like activity of MOF increases with decreasing pH, we again studied the correlation between MOF and pH using TMB. The results showed that the absorbance value near 300nm increased with decreasing pH, indicating that MOF exhibited more peroxidase-like activity at lower pH (3-4). Figure 3 H).

[0131] GOX can react with glucose to produce gluconic acid, thereby lowering the pH of the acidic tumor microenvironment, confirming the speculation that this may further enhance the peroxidase-like activity of MOF.

[0132] Example 8: Oxygen Production

[0133] 10 mg / mL Ce6@HGMOF was added into 10% H 2 O 2 In the solution, use ddH 2 O was used as a control. Then, a portable dissolved oxygen meter was used to detect O 2 How to alleviate hypoxia and the oxygen production capacity of nanomedicines is an important issue we are concerned about. Therefore, we simulated the tumor microenvironment outside the cell and used Ce6@HGMOF with H 2 O 2 After incubation with glucose, the changes in oxygen content in the liquid were detected using a dissolved oxygen analyzer.

[0134] The results showed that the oxygen production capacity of Ce6@HGMOF gradually increased with the extension of time. After 300 seconds, the generated oxygen reached 4 mg / L, indicating that it has excellent self-oxygen supply capacity ( Figure 5 C).

[0135] Example 9: Singlet oxygen detection

[0136] Measurement using DPBF (singlet oxygen indicating fluorescent probe, 1,3-Diphenylisobenzofuran) probe 1 O 2 Under laser irradiation (660nm, 100mW / cm -2, 20 minutes), 200 μL DPBF solution (10 mM in DMSO) was added to 2 mL Ce6@HGMOF solution (0.4 mg / mL, calculated by Ce6). The absorbance of DPBF at 420 nm was recorded every 2 minutes. Ce6@HGMOF nanomedicine can play a photodynamic role, and we tested the ability of Ce6@HGMOF nanomedicine to produce singlet oxygen through DPBF. It is a fluorescent probe suitable for intracellular singlet oxygen detection. Singlet oxygen can undergo epoxidation reaction with DPBF, reducing its absorption spectrum by about 420 nm, which is positively correlated with the singlet oxygen level.

[0137] like Figure 3 I. The results show that the DPBF content in the DPBF+Ce6@HGMOF nanoparticles+laser group was more significantly reduced compared with the DPBF+laser group. After 20 minutes of irradiation, the DPEF intensity of the DPBF+laser group only decreased from 100% to about 90%, while the DPBF intensity of the DPBF+Ce6@HGMOF nanoparticles+laser group decreased from 100% to about 50%. This indicates that Ce6@HGMOF nanoparticles have a photodynamic effect and can produce singlet oxygen and consume DPBF under 660nm laser irradiation.

[0138] Example 10: ATPlite assay

[0139] For cell proliferation assay, EC109 cells (3 × 10 3 Cells (cells / well) were seeded in complete medium in 96-well plates overnight and then cultured under the following different conditions: (1) control, (2) MOF, (3) Ce6 + laser, (4) Ce6@MOF + laser, (5) Ce6@GMOF + laser, and (6) Ce6@HGMOF + laser (660 nm, 100 mW / cm -2 After 48 hours, the culture medium was removed, and then ATPlite solution was added and shaken for 8 minutes in the dark, and the luminescence value of each well was detected using an enzyme marker to calculate the proliferation inhibition effect.

[0140] like Figure 7 A, The proliferation ability of EC109 esophageal cancer cells treated with different groups was evaluated by ATPlite experiment, and it was found that the esophageal cancer cells treated with Ce6@HGMOF nanoparticles were concentration-dependent. The MOF group had no obvious inhibitory effect on cell proliferation at low concentrations, while the Ce6+laser group had a satisfactory inhibitory effect on cell proliferation, proving the powerful anti-tumor effect of photodynamic therapy.

[0141] In particular, the Ce6@GMOF+laser group and Ce6@HGMOF+laser group had the most significant effects, demonstrating the best synergistic effect of nanoparticle therapy and that the cells were almost dead at high concentrations.

[0142] Example 11: Live / dead staining assay

[0143] EC109 cells were treated with (1) control, (2) MOF, (3) Ce6 + laser, (4) Ce6@MOF + laser, (5) Ce6@GMOF + laser, and (6) Ce6@HGMOF + laser (calculated as MOF, 50 μg / mL; calculated as Ce6, 5 μg / mL) (laser parameters: 660 nm, 100 mW / cm -2 , 5 minutes) and treated with the above conditions for 24 hours. Cell death was analyzed by live / dead staining. Figure 7 B, comparing the red fluorescence representing living cells and the red fluorescence representing dead cells, it was found that the red fluorescence of cells treated with Ce6@HGMOF+laser group far exceeded that of other groups, proving that the nanoparticles have a strong killing ability against EC109 esophageal cancer cells.

[0144] Example 12: Apoptosis analysis by FACS

[0145] For apoptosis analysis, EC109 cells were seeded in 6-well plates and treated with the following different conditions: (1) control, (2) MOF, (3) Ce6 + laser, (4) Ce6@MOF + laser, (5) Ce6@GMOF + laser, and (6) Ce6@HGMOF + laser (calculated as MOF, 50 μg / mL; calculated as Ce6, 5 μg / mL) (660 nm, 100 mW / cm -2 After culturing for 24 h, cells were collected and stained with Annexin VFITC / PI apoptosis detection kit. Flow cytometry was used for analysis. We evaluated the ability to induce tumor cell apoptosis under different groups by flow cytometry. Figure 7 C and Figure 7 D. It was also found that the degree of apoptosis of cells treated with Ce6@GMOF+laser group and Ce6@HGMOF+laser group was much higher than that of other groups, especially the late apoptosis of cells was dominant, which proved that the nanoparticles could effectively induce apoptosis of EC109 cells and exert effective antitumor activity.

[0146] Example 13: Detection of Tumor Cell Hypoxia and Reactive Oxygen Species

[0147] EC109 cells were treated differently according to the groups and cultured for 24 hours. Pyocyanin (200 μM, 30 minutes) was added to the cells as a positive control for ROS induction, while deferoxamine (200 μM) was a hypoxia inducer. According to the manufacturer's instructions, the assay kit mixture was added to the tumor cells in sequence. After incubation for 30 minutes, the cells were washed thoroughly, and ROS and hypoxia signals were monitored by fluorescence microscopy. Secondly, in order to observe the effect of hypoxia relief on photodynamics, we used the ROS-ID Hypoxia / Oxidative Stress Detection Kit to detect the relationship between hypoxia relief and ROS production in tumors. Secondly, in order to observe the effect of hypoxia relief on photodynamics, we used the ROS-ID Hypoxia / Oxidative Stress Detection Kit to detect the relationship between hypoxia relief and ROS production in tumors. As Figure 5 D and 5E, indicating that alleviating hypoxia can improve photodynamics.

[0148] Example 14: Detection of LPO in tumor cells

[0149] EC109 cells were seeded in 12-well plates and cultured overnight. The concentrations of the Ce6@MOF+laser group and Ce6@HGMOF+laser group (calculated according to MOF, 50 μg / mL) (660 nm, 100 mW / cm -2 , 5 minutes) experimental conditions for treating cells for 24h. C11-BODIPY (LPO probe) was used to stain cells, and fluorescent images of cells were obtained using a fluorescence microscope. C11-BODIPY is a highly selective and sensitive LPO fluorescent probe that can selectively capture LPO on intracellular membrane structures. Therefore, we used this probe to stain tumor cells after different treatments and observed them under a fluorescence microscope.

[0150] like Figure 6 Compared with the control group, when MOF and Ce6@HGMOF were added, obvious green fluorescence could be observed in tumor cells, indicating the accumulation of LPO in the cells. This proves that MOF and Ce6@HGMOF can induce ferroptosis in tumor cells. And this phenomenon is more obvious after irradiation, which may be that photodynamics promotes LPO in tumor cells to some extent.

[0151] Example 15: In vivo anticancer treatment

[0152] All experimental procedures involving animals were performed in accordance with the guidelines approved by the Southern Model Animal Care and Use Committee (IACUC) Shanghai Research Center (IACUC No. 2023-0015). EC109 tumor-bearing BALB / c nude mice were randomly divided into 7 groups (n = 6 in each group): (1) control group, (2) MOF group, (3) Ce6 + laser group, (4) Ce6@MOF + laser group, (5) Ce6@GMOF + laser group, (6) Ce6@HGMOF + laser group (calculated as MOF, 4 mg / kg; calculated as Ce6, 0.4 mg / kg) (660 nm, 100 mW / cm -2 , 5 minutes). Tumor size and body weight were measured every other day after treatment for 18 days (tumor volume = W2×L / 2, W = width, L = length). The relative tumor volume of each mouse was calculated as V / V 0 (V 0 is the tumor volume at the start of treatment). To verify the antitumor effect of Ce6@HGMOF in mice, we conducted EC109 tumor-bearing mice experiments.

[0153] like Fig. 9 As shown in the animal experiment flow chart of A, mice were first injected subcutaneously. After the mice were injected subcutaneously, tumors were formed in the mice after 7 days, and then the mice were injected with drugs once every two days. The drugs were administered by intravenous injection and irradiated on the day after administration. The irradiated group received 660nm near-infrared irradiation within 24 hours after administration. After administration, multiple treatment cycles were repeated, and the mice were killed after 18 days. Changes in mouse weight and tumor volume were closely monitored during the treatment.

[0154] Compared with the control group, the tumor size and weight in the MOF group decreased to some extent, but the changes were small, indicating that the anti-tumor effect of ferritin alone may be weak. The tumor inhibition effect of the Ce6@MOF+laser group was enhanced compared with the Ce6+laser group. The enhanced tumor inhibition effect of the Ce6@MOF+laser group was due to the enhanced efficacy of photodynamic therapy after hypoxia relief.

[0155] Surprisingly, the Ce6@HGMOF+laser group showed the best antitumor effect compared with the other groups. This demonstrated the superiority of the synergistic effect of multiple treatments in antitumor. The above findings can be confirmed by representative anatomical tumors ( Fig. 9 B) Mouse tumor weight ( Fig. 9 C), tumor weight ( Fig. 9 C) and tumor volume ( Fig. 9 D) were obtained. At the same time, no weight loss occurred in either group ( Fig. 9E), indicating that the above-mentioned nanomaterials and drugs did not affect the body weight of mice and had relatively balanced biosafety.

[0156] To explore the synergistic anti-tumor mechanism of Ce6@HGMOF in mice, we performed fluorescent staining on mouse tumor tissues. Tunel, HIF-1α, CD31, and LPO were used to fluorescently stain mouse tumor tissues. The green fluorescence intensity of apoptosis in tumor tissues gradually increased after various synergistic treatments. After hypoxia was relieved, the fluorescence intensity of HIF-1α, which represents hypoxia, and CD31, which represents angiogenesis, gradually decreased.

[0157] The fluorescence intensity of LPO increased significantly in all groups. Starting from the MOF group, the fluorescence intensity of LPO in all groups increased significantly. This indicates that Ce6@HGMOF can induce the accumulation of LPO and ferroptosis in tumor tissues ( Fig. 9 F). Malondialdehyde (MDA) is the final product of lipid peroxidation, and its concentration can reflect the degree of lipid peroxidation. The MDA content in tumor tissue was detected using this kit, which was consistent with the fluorescence of LPO and the fluorescence intensity of LPO.

[0158] Compared with the control group, the MDA content in the tumor tissues of mice in each drug-treated group increased significantly ( Fig.10 B) Next, we detected the expression of related proteins in tumor tissues by Western blotting. Fig.11 A and Fig.11 B, shows that the expression of hypoxia, iron atrophy, and glycolysis-related proteins is consistent with the fluorescence staining, which once again proves that Ce6@HGMOF plays a synergistic therapeutic role in mice.

[0159] Example 16: In vitro cellular uptake assay

[0160] EC109 cells were cultured at 1.5 × 10 3 Cells were seeded at a density of 10 cells / well in a 20 mm dish and incubated with Ce6@HGMOF (50 μg / mL, calculated as MOF) for 12 h. The culture medium was removed and the cells were rinsed three times with PBS. The nuclei were stained with Hoechst33342 and the lysosomes were stained with LysoTracker Red, then fixed with 5% glutaraldehyde solution and observed under a confocal laser scanning microscope.

[0161] To clarify whether the nanoparticles can enter and exert their effects on tumors, we further evaluated the in vitro cellular uptake of Ce6@HGMOF as well as the in vivo tumor site aggregation and distribution in different tissues. First, the cellular uptake status of Ce6@HGMOF nanoparticles at different times was examined by CLSM. No additional fluorescent dye coating was required because the HCPT loaded by Ce6@HGMOF was autofluorescent in green and Ce6 was autofluorescent in red.

[0162] like Figure 8 A and Figure 8 B, The results show that as the experimental time goes by, the fluorescence of Ce6@HGMOF nanoparticles overlaps with the cell nucleus to a greater extent. At 24 hours, it can be seen that most of the nanoparticles have entered the cell nucleus, indicating that Ce6@HGMOF can be mostly taken up by tumor cells within 24 hours ().

[0163] Example 17: Biodistribution

[0164] Ce6 or Ce6@HGMOF (4 mg / kg calculated as MOF; 0.4 mg / kg calculated as Ce6) was administered to tumor-bearing mice via the tail vein at different time points (2, 6, 12, and 24 h) and measured by in vivo fluorescence imaging. Then, after euthanasia of the mice, tumors and major organs, including heart, liver, spleen, lung, and kidney, were collected. The biodistribution of Ce6@HGMOF in vivo was evaluated using the VISQUE InVivo Elite imaging system.

[0165] Next, we detected the tumor targeting and accumulation ability of Ce6@HGMOF in mice by small animal in vivo imaging system, and as time went on, both free Ce6 and Ce6@HGMOF showed stronger fluorescence intensity at the tumor site. At the same time, Ce6@HGMOF showed stronger fluorescence intensity at the tumor site than free Ce6, proving that free Ce6@HGMOF has better tumor targeting and retention properties than free Ce6.

[0166] To further verify this, we collected and imaged the heart, liver, spleen, lung, kidney, and tumor of mice, e.g. Figure 8 C and Figure 8 D, The results show that free Ce6 and Ce6@HGMOF accumulated most in the tumor and liver sites of mice, and Ce6@HGMOF accumulated significantly more in the tumor site than free Ce6. This once again proves that nanoparticles can better target the accumulated tumor site to exert their effects.

[0167] Example 18: Safety Evaluation

[0168] After the mice were killed, serum was collected and tested by the Medical Laboratory Department of Longhua Hospital of Shanghai University of Traditional Chinese Medicine for ALT, AST, GGT, ALB, CREA, UN and other related indicators to evaluate the health of the mice. Serum was collected from mice to test liver and kidney function, alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum albumin (ALB), creatinine (CREA), blood urea (UN), and blood uric acid (UA).

[0169] like Fig.12 AF, the results showed that there was no statistical difference among the experimental groups, proving that Ce6@HGMOF did not affect the liver and kidney functions of mice and had excellent biosafety and compatibility.

[0170] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An iron MOF nanoparticle, characterized in that: Including: iron MOF, glucose oxidase GOX, photosensitizer Ce6, hydroxycamptothecin HCPT and egg yolk lecithin; Among them, GOX is loaded in the iron MOF, and the outer layer of the iron MOF is encapsulated by liposomes loaded with photosensitizer Ce6 and hydroxycamptothecin HCPT; Among them, the average particle size of the iron MOF is 123-128nm, and the average Zeta potential is 5.84±0.2mV; the average Zeta potential of the iron MOF loaded with GOX is 9.45±0.2mV; the average particle size of the iron MOF nanoparticles is 148-150nm; the average Zeta potential is 2.59±0.2mV; the average specific surface area is 1.8562m 2 / g.

2. A method for preparing the iron MOF nanoparticles according to claim 1, characterized in that: The steps include: (1) Synthesis of iron MOF: FeCl3·6H2O solution was slowly added to dithiodiacetic acid solution using a peristaltic pump and magnetically stirred for about 20-50 minutes; when the liquid turned into a cream-yellow color, it was centrifuged at a speed of 8000-10000 rpm for 10 minutes, and the supernatant was removed to obtain iron MOF; (2) Synthesis of GMOF: Glucose oxidase GOX was dissolved in water, mixed with the iron MOF obtained in step (1), and mixed by magnetic stirring for 24 hours; centrifuged at a speed of 9000-12000 rpm, and centrifuged three times to obtain GMOF; (3) preparing liposomes encapsulating Ce6-HCPT: mixing egg yolk phosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine (DSPE), photosensitizer Ce6 and hydroxycamptothecin (HCPT) in an organic solution, and removing the solvent by rotary evaporation at 35-50° C.; hydrating the membrane liposomes with deionized water, and completely dispersing them by contact ultrasonic treatment for 20 minutes to obtain a solution containing liposomes encapsulating Ce6-HCPT; (4) The solution containing liposomes encapsulating Ce6-HCPT obtained in step (3) is mixed with the GMOF obtained in step (2), and the mixture is pressed repeatedly to obtain Ce6@HGMOF nanoparticles.

3. The method according to claim 2, characterized in that The preparation method of the Ce6-HCPT-encapsulated liposome comprises: thoroughly mixing 200 mg egg yolk lecithin, 50 mg cholesterol, 4 mg DSPE, 5 mg Ce6 and 5 mg HCPT (ethanol / dichloromethane, 1:1) in 20 mL of dichloromethane solution, and removing the solvent by rotary evaporation at 40°C to obtain.

4. The method according to claim 2, characterized in that In step (3), the organic solvent is selected from the following group: C1-C6 alcohol solvents, C1-C6 hydrocarbon solvents, or a combination thereof.

5. The method according to claim 2, characterized in that The concentration of the FeCl3·6H2O solution is 20-40 mg / mL, preferably 25 mg / mL.

6. The method according to claim 2, characterized in that The concentration of the FeCl3·6H2O solution is 4 times that of the dithiodiacetic acid solution.

7. Use of the iron MOF nanoparticles according to claim 1, characterized in that: Used for preparing medicines or preparations for treating cancer.

8. The use according to claim 7, characterized in that The cancer is esophageal cancer.

9. The use according to claim 7, characterized in that The iron MOF nanoparticles are used in combination with near-infrared light to treat cancer.

10. A method for treating esophageal cancer, characterized in that: A therapeutically effective amount of the iron MOF nanoparticles according to claim 1 is administered to a subject in need thereof, and the affected area is irradiated with near-infrared light.

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