PH / GSH dual-response type Fe-based MOF drug carrier as well as synthesis method and application of pH / GSH dual-response type Fe-based MOF drug carrier

Abstract

The invention provides a pH / GSH dual-response type Fe-based MOF drug carrier as well as a synthesis method and application thereof, and belongs to the technical field of drug carrier synthesis. Fe-based MOF is synthesized from MOF containing GSH sensitive bonds, then an anti-cancer drug model DOX is entrapped in channels of the Fe-based MOF, a functional material is modified on the surface of the Fe-based MOF, and the pH / GSH double-response type Fe-based MOF drug carrier is obtained. The Fe-based MOF drug carrier achieves the purpose of inhibiting or killing tumor cells through mutual cooperation of ferroptosis and chemotherapy, has good application in cancer resistance, and has good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug carrier synthesis, and relates to a pH / GSH dual-responsive Fe-based MOF drug carrier, a synthesis method and application thereof. Background Art

[0002] For decades, cancer has been threatening human health, and researchers have been constantly innovating cancer treatments. The characteristics of the tumor cell microenvironment, such as slightly acidic, overexpression of glutathione (GSH), and high reactive oxygen species (ROS), provide new treatment ideas for environmentally responsive nanodrug delivery systems. In addition, ferroptosis is a type of programmed death in cells. It accumulates iron, causing lipids inside the cells to be continuously oxidized into cytotoxic lipid peroxides, which eventually cause cell rupture and death after damage to the cell membrane. Based on the above mechanism, the researchers introduced different sensitive groups to respond to different environmental stimuli, and combined the mechanism of ferroptosis to design and prepare the required nanodrugs to inhibit or kill tumor cells.

[0003] Currently, researchers have developed many Fe-based MOFs nanomaterials to induce ferroptosis by indirectly inactivating the GPX4 enzyme by inhibiting or depleting overexpressed GSH (up to 10 mmol / L in cancer cells). For example, some MnO2-based nanomaterials have also been reported to have the ability to deplete intracellular antioxidant GSH to enhance ROS-induced tumor therapy. On the other hand, the lack of GSH also enhances ROS-related therapies such as radiotherapy, chemotherapy, and photodynamic therapy (PDT), which consumes cytotoxic ROS to maintain homeostasis in the body. Meng et al. developed a Ce6-loaded glutathione-depleting MOF that can lead to intracellular GSH depletion through disulfide-thiol exchange reactions, thereby synergizing with the antitumor effect of PDT therapy. Single treatment methods often do not have very good efficacy and have certain limitations. At the same time, the efficacy of ferroptosis therapy may still be limited by the insufficient supply of endogenous H2O2 in tumor tissues. Combining programmed cell death and apoptosis provides a promising treatment approach that can significantly improve the therapeutic effect of anticancer therapy. Therefore, it is necessary to develop a new nanoplatform that can amplify ferroptosis therapy by reducing GSH and increasing ROS content, forming a triple synergistic treatment strategy of ferroptosis, chemotherapy, and starvation therapy. Summary of the invention

[0004] In view of some deficiencies in the prior art, the present invention provides a pH / GSH dual-responsive Fe-based MOF drug carrier and a synthesis method and application thereof; the present invention synthesizes an Fe-based MOF using a MOF containing a GSH-sensitive bond, then encapsulates an anticancer drug model DOX in the pores of the Fe-based MOF, and modifies the surface of the Fe-based MOF with a functional material to obtain the pH / GSH dual-responsive Fe-based MOF drug carrier; the Fe-based MOF drug carrier achieves the purpose of inhibiting or killing tumor cells through the synergy of ferroptosis and chemotherapy, has a good application in anti-cancer and has good practicality.

[0005] In order to achieve the above technical purpose, the present invention adopts the following technical means.

[0006] The present invention first provides a pH / GSH dual-responsive Fe-based MOF drug carrier, wherein the Fe-based MOF drug carrier has a rectangular sheet-shaped Fe-based MOF as the main structure, and the outer surface of the Fe-based MOF is modified with a functional material; there are pores inside the Fe-based MOF, and the anticancer drugs are loaded in the pores; The Fe-based MOF is composed of Fe 3+ As the active center, MOF containing GSH sensitive bonds is used as the organic ligand; The functional material includes mPEG-NH2 or mPEG-GOx.

[0007] Preferably, the MOF containing GSH sensitive bonds includes HOOC-(CH2)2-SS-(CH2)2-COOH or RSSR; The anticancer drug includes DOX.

[0008] The present invention also provides a method for synthesizing the pH / GSH dual-responsive Fe-based MOF drug carrier, the method comprising: (1) Synthesis of Fe-based MOF: Dissolving FeCl3 and MOF containing GSH sensitive bonds in solvents respectively to obtain FeCl3 solution and organic ligand solution; Under ultrasonic conditions, the FeCl3 solution is slowly added to the organic ligand solution, TEA is added thereto, and then stirred for reaction. After the reaction is completed, blank nanoparticle Fe-based MOF is obtained, which is washed, dried, and collected for later use; (2) Synthesis of pH / GSH dual-responsive Fe-based MOF drug carrier: The anticancer drug aqueous solution is slowly dripped into the Fe-based MOF aqueous solution under ultrasonic conditions, and the reaction 1 is carried out under stirring conditions. After the reaction is completed, the Fe-based MOF loaded with the anticancer drug is obtained by centrifugation and washing. An excess amount of functional material is added to an aqueous solution of Fe-based MOF loaded with anticancer drugs, and reaction 2 is carried out under stirring conditions. After the reaction, the solution is dialyzed with distilled water overnight and dried to obtain the pH / GSH dual-responsive Fe-based MOF drug carrier.

[0009] Preferably, in step (1), the MOF containing a GSH-sensitive bond comprises HOOC-(CH2)2-SS-(CH2)2-COOH or RSSR; When the MOF containing GSH sensitive bonds is HOOC-(CH2)2-SS-(CH2)2-COOH, the mass ratio of FeCl3 and the GSH sensitive MOF is 1:1, and the sum of the mass of FeCl3 and the GSH sensitive MOF to the amount of TEA is 1~2g:1mL; When the MOF containing a GSH sensitive bond is RSSR, the mass ratio of FeCl3 to the GSH sensitive MOF is 1:1, and the sum of the masses of FeCl3 and the GSH sensitive MOF to the TEA dosage ratio is 1-2 g:1 mL. Preferably, the synthesis method of the RSSR comprises: 4,4'-dithiodibutyric acid and N,N'-carbonyldiimidazole are dissolved in anhydrous dichloromethane, evaporated under nitrogen protection, and then the residue is dissolved in dichloromethane, repeatedly washed with ultrapure water and sodium carbonate solution, the organic phase is collected and dried in anhydrous sodium sulfate, the dichloromethane is removed by rotary evaporation, and then dried to obtain the RSSR.

[0010] Preferably, in step (1), when the MOF containing the GSH sensitive bond is HOOC-(CH2)2-SS-(CH2)2-COOH, the stirring reaction conditions are: reacting at 120°C for 24h; When the MOF containing the GSH sensitive bond is RSSR, the stirring reaction conditions are: stirring at 40° C. under nitrogen protection for 8 hours.

[0011] Preferably, in step (2), the dosage ratio of the anticancer drug to the Fe-based MOF is 1-3:1; The anticancer drug includes DOX; The conditions of the reaction 1 are: reacting at room temperature in the dark for 24 hours.

[0012] Preferably, in step (2), the functional material includes mPEG-NH2 or mPEG-GOx; The conditions of reaction 2 are: reacting at room temperature for 24 hours.

[0013] Preferably, the synthesis method of PEG-GOx comprises: GOx is dissolved in ultrapure water, EDC and NHS are added thereto, and the mixture is uniformly mixed under stirring. After the reaction is completed, mPEG-NH2 is added and the mixture is reacted under stirring. After the reaction is completed, the mixture is dialyzed with distilled water overnight and dried to obtain the PEG-GOx.

[0014] The present invention also provides the use of the pH / GSH dual-responsive Fe-based MOF drug carrier in loading anticancer drugs.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention prepares pH / GSH dual-responsive Fe-based MOF drug carriers Fe(III)-(3,3'-SS-)@DOX@PEG and Fe(III)-RSSR@DOX@PEG-GOx. The Fe(III)-(3,3'-SS-)@DOX@PEG consumes the high concentration of GSH in tumor cells through the disulfide bonds contained in the organic skeleton, and causes the MOF structure to collapse by breaking the bonds, thereby releasing the internally encapsulated DOX to form chemical therapy; then the Fe reduced by GSH 2+ It rapidly reacts with the high content of endogenous H2O2 in the tumor to produce a Fenton reaction, resulting in excessive accumulation of ROS, which ultimately stimulates efficient ferroptosis therapy, thus forming a therapeutic strategy of ferroptosis and chemotherapy synergistic with each other. Under high GSH acidic release conditions, the release of DOX in 72 h reached more than 90%, indicating that the polymer has good pH / GSH responsiveness. The Fe generated during the release process was regulated by the chemical system. 2+ Monitoring with •OH demonstrated that excessive ROS can be produced in tumors and rapidly induce ferroptosis.

[0016] The Fe(III)-RSSR@DOX@PEG-GOx of the present invention consumes the high concentration of GSH in tumor cells through the disulfide bonds contained in the organic framework, and causes the MOF structure to collapse by breaking the bonds, thereby releasing the internally encapsulated DOX to form chemical therapy; after the surface-modified GOx enters the cell, it catalyzes glucose to generate H2O2, amplifies the ROS content in the tumor site, and reacts with the Fe(III)-RSSR@DOX@PEG-GOx reduced by GSH. 2+ The Fenton reaction occurs rapidly, leading to excessive accumulation of ROS, which eventually leads to cascade amplification of ferroptosis therapy; GOx consumes a large amount of glucose, blocking the energy delivery of tumor cells, thus forming a triple synergistic treatment strategy of ferroptosis, chemotherapy, and starvation therapy. The in vitro simulated release experiment showed an outstanding sustained release effect, and by monitoring Fe 2+The generation of and •OH proved the occurrence of cascade reaction. Under high GSH acidic release conditions, the release of DOX in 48 h reached more than 82.10%, indicating that the polymer has good pH / GSH responsiveness. Cell experiments proved that Fe(Ⅲ)-RSSR@DOX@PEG-GOx has good cytotoxicity and biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the release process of pH / GSH dual-responsive Fe-based MOF drug carrier; in the figure, a is Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG; b is Fe(Ⅲ)-RSSR@DOX@PEG-GOx.

[0018] Figure 2 This is a scanning electron microscope image of Fe-based MOF; in the figure, a is Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG; b is Fe(Ⅲ)-RSSR@DOX@PEG-GOx.

[0019] Figure 3 Figure 2 shows the N2 adsorption-desorption curves (a, c) and pore size distribution diagrams (b, d) of Fe-based MOFs; in the figure, a and b are Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG; c and d are Fe(Ⅲ)-RSSR@DOX@PEG-GOx.

[0020] Figure 4 XPS spectrum (a), infrared spectrum (b), UV absorption spectrum (c) and XRD pattern (d) of Fe(Ⅲ)-(3,3'-SS-)@DOX.

[0021] Figure 5 The drug loading capacity and drug loading efficiency of Fe(Ⅲ)-(3,3'-SS-)@DOX under different dosage ratios.

[0022] Figure 6 The infrared spectrum (a), Zeta potential change diagram (b) and continuous stability diagram (c) of Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG.

[0023] Figure 7 XPS spectrum (a), IR spectrum (b) and XRD pattern (c) of Fe(Ⅲ)-RSSR@DOX.

[0024] Figure 8 The drug loading capacity and drug loading efficiency of Fe(Ⅲ)-RSSR@DOX under different dosing ratios.

[0025] Fig. 9The infrared spectrum (a), Zeta potential change diagram (b) and continuous stability diagram (c) of Fe(Ⅲ)-RSSR@DOX@PEG-GOx.

[0026] Fig.10 The in vitro release curves of DOX from the pH / GSH dual-responsive Fe-based MOF drug carrier under different release conditions; in the figure, a is Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG; b is Fe(Ⅲ)-RSSR@DOX@PEG-GOx.

[0027] Fig.11 Fe release under different conditions for pH / GSH dual-responsive Fe-based MOF drug carriers 2+ In vitro release curve; in the figure, a is Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG; b is Fe(Ⅲ)-RSSR@DOX@PEG-GOx.

[0028] Fig.12 The in vitro release curve of •OH from the pH / GSH dual-responsive Fe-based MOF drug carrier under different release conditions; in the figure, a is Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG; b is Fe(Ⅲ)-RSSR@DOX@PEG-GOx.

[0029] Fig.13 The cytotoxicity graphs of Fe(Ⅲ)-(3,3'-SS-) MOF in L929 cells (A) and 4T1 cells (C) and the cytotoxicity graph of Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG in L929 cells (B).

[0030] Fig.14 The cytotoxicity graphs of Fe(Ⅲ)-RSSR MOF in L929 cells (A) and 4T1 cells (C) and the cytotoxicity graph of Fe(Ⅲ)-RSSR@DOX@PEG-GOx in L929 cells (B).

[0031] Fig.15 Confocal microscopy image of Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG (30 μg / mL) co-incubated with 4T1 cells (scale bar: 20 μm).

[0032] Fig.16 Confocal microscopy image of Fe(Ⅲ)-RSSR@DOX@PEG-GOx (30 μg / mL) co-incubated with 4T1 cells (Scale bar: 20 μm). DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are followed. The reagents or instruments used are not indicated by the manufacturer and are conventional products that can be purchased on the market.

[0034] In the following examples, normal mouse L929 cells were purchased from China Center for Type Culture Collection (Accession No.: GDC0034); 4T1 cells were purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0035] Example 1: Synthesis of pH / GSH dual-responsive Fe-based MOF drug carrier In this example, Fe(III)-(3,3'-SS-)@DOX@PEG was synthesized, and the specific steps are as follows: (1) Synthesis of Fe(Ⅲ)-(3,3'-SS-) MOF: 1.00 g FeCl3·6 H2O and 1.00 g 3,3'-dithiodipropionic acid were accurately weighed and dissolved in a beaker containing 50 mL DMF and 1 mL TEA. Ultrasonication was continued for 5 minutes, and then the mixture in the beaker was transferred to a 100 mL hydrothermal autoclave and reacted continuously at 120°C for 24 hours. After the reaction was completed and the mixed system returned to room temperature, it was centrifuged at 12,000 rpm and washed three times with DMF and ultrapure water respectively. After vacuum drying, the final blank nanoparticles Fe(Ⅲ)-(3,3'-SS-) MOF were obtained and collected for later use.

[0036] Figure 2 The SEM image of Fe(Ⅲ)-(3,3'-SS-) MOF shows that the synthesized nanoparticles have a rectangular sheet structure with a uniform size of about 200 nm in length and 50 nm in width. The average particle size of the nanoparticle aqueous solution was determined by dynamic light scattering to be 143.30 nm (PDI=0.278), indicating that the synthesized Fe(Ⅲ)-(3,3'-SS-)MOF nanoparticles have a nanometer size that is more suitable as a drug carrier.

[0037] Figure 3 The N2 adsorption-desorption curve and particle size distribution of Fe-based MOF are shown in the figure. It can be seen from the figure that the specific surface area, pore volume and average pore size of Fe(Ⅲ)-(3,3'-SS-)MOF are 24.93 m 2 g -1 , 0.106 cm 3 g-1 and 17.61 nm. Therefore, Fe(Ⅲ)-(3,3'-SS-)MOF with mesopores as the main pore structure has a certain drug loading capacity and can be used as a carrier of anticancer drugs.

[0038] (2) Synthesis of Fe(Ⅲ)-(3,3'-SS-)@DOX: In order to load the drug into the voids of Fe(Ⅲ)-(3,3'-SS-) MOF, the DOX aqueous solution (1 mg / mL) was slowly dripped into the Fe(Ⅲ)-(3,3'-SS-) MOF aqueous solution under ultrasound, and stirred continuously for 24 h at room temperature in the dark. The mixed solution was centrifuged (12 000 rpm, 15 min), and the unloaded DOX was repeatedly washed with distilled water. After drying, the drug-loaded nano-Fe(Ⅲ)-(3,3'-SS-)@DOX was obtained. All the supernatants after centrifugation were collected, and their ultraviolet absorbance was measured at 480 nm. The drug loading capacity (DLC) and encapsulation efficiency (EE) of DOX were calculated using the UV-visible spectroscopy standard curve method.

[0039] from Figure 4 It can be seen that the XPS full scan spectrum of Fe(Ⅲ)-(3,3'-SS-)@DOX shows five main peaks, which are 163, 285, 400, 531 and 712 eV, corresponding to S 2p, C 1s, N 1s, O 1s and Fe 2p. Compared with the XPS full spectrum of blank Fe(Ⅲ)-(3,3'-SS-) MOF, the N element contained in DOX appears for the first time in the full spectrum of Fe(Ⅲ)-(3,3'-SS-)@DOX, which preliminarily confirms the successful loading of DOX ( Figure 4 a). At the same time, in the infrared contrast spectrum ( Figure 4 b) The characteristic peak of DOX appears, and the UV contrast spectrum ( Figure 4 c) An absorption peak representing DOX also appeared at 480 nm, further proving the successful loading of DOX. Figure 4 d The overall peak shape of Fe(Ⅲ)-(3,3'-SS-)@DOX did not change much within the overall angle range, indicating that the loading process of drug DOX would not destroy the crystal structure of the drug-loaded material.

[0040] This step also explored the drug loading capacity of Fe(Ⅲ)-(3,3'-SS-) MOF materials. Experiments were designed with different MOF:DOX dosing ratios. The supernatant after centrifugation of Fe(Ⅲ)-(3,3'-SS-)@DOX prepared under different conditions was collected. The drug loading capacity (DLC) and drug loading efficiency (EE) at different dosing ratios were determined and calculated using an ultraviolet standard curve to explore the optimal dosing ratio of the synthesized material. The results are shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that after increasing the DOX input content, DLC gradually increases, but EE is on a downward trend after exceeding 1:1. Combined with EE data and considering the economic cost of synthesis, the drug loading conditions are optimal when the MOF:DOX ratio is around 1:1. Therefore, Fe(Ⅲ)-(3,3'-SS-)@DOX with MOF:DOX=1:1 was selected to complete the remaining experiments. The DLC of Fe(Ⅲ)-(3,3'-SS-)@DOX is 28.08% and the EE is 39.15%.

[0041] (3) Synthesis of Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG: In this step, Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG nanoparticles were synthesized by self-assembly method, which specifically included: adding excess mPEG-NH2 to an aqueous solution of Fe(Ⅲ)-(3,3'-SS-), stirring the mixture continuously at 25°C for 24 h, dialyzing the reaction mixture with distilled water overnight, and freeze-drying to obtain Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG nanoparticles.

[0042] from Figure 6 a As can be seen from the figure, the appearance of the characteristic peak of PEG proves the successful modification of PEG; at the same time, the numerical change of Zeta potential can also prove the successful modification of PEG, and obtain a more stable Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG nano-drug delivery composite material (6b). In addition, the changes in the particle size and PDI of the Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG nanoparticle aqueous solution for seven consecutive days show that the particle size and PDI values ​​of the nano-drug delivery remain almost unchanged for a week, which indicates that the nano-drug delivery material has good stability (6c).

[0043] Example 2: Synthesis of pH / GSH dual-responsive Fe-based MOF drug carrier In this example, Fe(III)-RSSR@DOX@PEG-GOx was synthesized, and the specific steps are as follows: (1) Synthesis of mPEG-GOx: GOx (20.0 mg, 0.12 mmol) was dissolved in 10 mL ultrapure water, EDC (10.0 mg, 0.06 mmol) and NHS (7.0 mg, 0.06 mmol) were added thereto, and the mixture was stirred at 25 °C for 2 h. After the stirring, mPEG-NH2 (100 mg, 0.02 mmol) was added thereto, and then the mixture was stirred at 25 °C for 20 h. After the reaction, the reactant was transferred to a dialysis bag (MWCO 6000-8000 Da), dialyzed with distilled water overnight, and then freeze-dried for later use.

[0044] (2) Synthesis of RSSR: 4,4'-dithiodibutyric acid (0.3778 g, 1.585 mmol) and N,N'-carbonyldiimidazole (0.6333 g, 3.906 mmol) were dissolved in 40 mL of anhydrous dichloromethane, and then evaporated under nitrogen protection at 40°C for 8 hours to obtain the residue. The residue was dissolved in 40 mL of dichloromethane, washed repeatedly with ultrapure water and sodium carbonate (NaCO3) solution (0.10 mol / L), and then the organic phase was collected and dried in anhydrous sodium sulfate (Na2SO4). After drying, the dichloromethane was treated by vacuum rotary evaporation, and the precipitate phase RSSR was obtained after vacuum drying as the MOF organic ligand RSSR containing disulfide sensitive bonds.

[0045] (3) Synthesis of Fe(III)-RSSR MOF: Accurately weigh FeCl3 (80.0 mg) and dissolve it in 10 mL DMF to form solution I, and dissolve RSSR (100.0 mg) in 20 mL DMF to form solution II. Under ultrasonic conditions, solution I was slowly dripped into a beaker containing solution II and 1 mL TEA, and ultrasonication was continued for 5 minutes. Then the beaker was placed on a magnetic stirrer and stirred at 350 rpm at room temperature for 24 hours. The final blank nanoparticle Fe(Ⅲ)-RSSR MOF was obtained by centrifugation at 12000 rpm, washed 3 times with DMF and ethanol, respectively, vacuum dried, and collected for later use.

[0046] Figure 2 The SEM image of Fe(Ⅲ)-(3,3'-SS-) MOF shows that the synthesized nanoparticles have a rectangular flake structure, good dispersibility, and a uniform particle size of about 100 nm in length and 50 nm in width. The average particle size of the nanoparticle aqueous solution was determined by dynamic light scattering to be 131.00 nm (PDI=0.121), indicating that the Fe(Ⅲ)-RSSRMOF nanoparticles have a relatively suitable nanometer size as a drug carrier and have good stability.

[0047] Figure 3 The N2 adsorption-desorption curve and particle size distribution of Fe-based MOF are shown in the figure. It can be seen from the figure that the specific surface area, pore volume and average pore size of Fe(Ⅲ)-RSSRMOF are 51.34 m 2 g -1 、0.277 cm 3 g -1 and 19.34 nm. Therefore, mesoporous Fe(Ⅲ)-RSSRMOF is expected to have effective drug loading capacity and can be used as a carrier of anticancer drugs.

[0048] (4) Synthesis of Fe(III)-RSSR@DOX: In order to incorporate drugs into Fe(Ⅲ)-RSSR MOF, DOX aqueous solution (1 mg / mL) was slowly dripped into Fe(Ⅲ)-RSSR MOF aqueous solution under ultrasound, stirred for 24 h at room temperature in the dark, and the mixed solution was centrifuged (12 000 rpm, 15 min) and repeatedly washed with distilled water to obtain drug-loaded nano-Fe(Ⅲ)-RSSR@DOX. The supernatant was collected, and the drug loading (DLC) and encapsulation efficiency (EE) of DOX were determined by UV-visible spectroscopy, and the UV absorbance was measured at 480 nm.

[0049] from Figure 7 It can be seen that the XPS full scan spectrum of Fe(Ⅲ)-RSSR@DOX shows 5 main peaks, which are 161, 285, 400, 531 and 710 eV, corresponding to S 2p, C 1s, N 1s, O 1s and Fe 2p. Compared with the XPS full spectrum of blank MOF, the unique N element in DOX appears in the Fe(Ⅲ)-RSSR@DOX full spectrum, which preliminarily confirms the successful loading of DOX (7a). Infrared spectrum ( Figure 7 The characteristic peaks of DOX appeared in b), further proving the successful loading of DOX. In addition, it can be seen from the XRD diagram of Fe(Ⅲ)-RSSR@DOX composite material that the main peak positions can be well matched within the overall angle range, indicating that the drug loading process will basically not destroy the crystal structure of the drug-loaded material ( Figure 7 c).

[0050] This step also explored the drug loading capacity of Fe(Ⅲ)-RSSR MOF materials. Experiments were designed with different MOF:DOX dosing ratios. The supernatant after centrifugation of Fe(Ⅲ)-RSSR@DOX prepared under different conditions was collected, and the drug loading capacity (DLC) and drug loading efficiency (EE) at different dosing ratios were determined and calculated using an ultraviolet standard curve to explore the optimal dosing ratio of the synthesized material. The results are shown in Figure 2. Figure 8 shown.

[0051] from Figure 8 It can be seen that after increasing the DOX input ratio, DLC gradually increases, but due to the fixed pore structure of MOF limiting its drug loading capacity, DLC gradually stabilizes during the increase of DOX input ratio. Combined with EE data and considering the economic cost of synthesis, Fe(Ⅲ)-RSSR@DOX with MOF:DOX=1:3 was selected to complete the remaining test of the experiment. At this time, the DLC of Fe(Ⅲ)-RSSR@DOX was 61.02% and the EE was 52.26%.

[0052] (5) Synthesis of Fe(III)-RSSR@DOX@PEG-GOx: In this step, Fe(Ⅲ)-RSSR@DOX@PEG-GOx nanoparticles were synthesized by self-assembly method, which specifically included: adding excess PEG-GOx to an aqueous solution of Fe(Ⅲ)-RSSR@DOX, stirring the mixture continuously at 25°C for 24 h, dialyzing the reaction mixture with distilled water overnight, and freeze-drying to obtain Fe(Ⅲ)-RSSR@DOX@PEG-GOx nanoparticles.

[0053] from Fig. 9 a As can be seen from the figure, the appearance of the characteristic peak of PEG proves the successful modification of PEG; at the same time, the numerical change of Zeta potential can also prove the successful modification of PEG, and obtain a more stable Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG nano-drug delivery composite material (9b). In addition, the changes in the particle size and PDI of the Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG nanoparticle aqueous solution for seven consecutive days show that the particle size and PDI values ​​of the nano-drug delivery remain almost unchanged for a week, which indicates that the nano-drug delivery material has good stability (9c).

[0054] Example 3: Study on the responsive drug release behavior of pH / GSH dual-responsive Fe-based MOF drug carrier This example investigates the drug release capabilities of Fe(III)-(3,3'-SS-)@DOX@PEG and Fe(III)-RSSR@DOX@PEG-GOx, respectively. The specific steps are as follows: The in vitro release curves of DOX were investigated in the following release media: Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG and Fe(Ⅲ)-RSSR@DOX@PEG-GOx in ABS (0.1 M, pH 5.0), ABS containing 10 mM GSH (0.1 M, pH 5.0), and PBS (0.1 M, pH 7.4), respectively. The results are shown in Figure 2. Fig. 9 shown.

[0055] from Fig.10As can be seen in a, the cumulative release of the drug increased significantly under acidic conditions and the addition of GSH. Under acidic conditions (pH 5.0), the release of DOX was slightly higher than that under neutral conditions (pH 7.4), and the drug release increased from 9.08% to 38.68% within 72 hours, indicating that the drug carrier has good pH responsiveness, which is due to the weakening of the coordination bond interaction of Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG under acidic conditions. In 10 mmol / L GSH solution, the release of DOX under pH 5.0 for 72 hours reached 90.24%, which is much higher than 38.68% when no GSH was added, indicating that the addition of GSH broke the -SS- sensitive bond, and the special skeleton structure made Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG have excellent pH / GSH responsiveness. It can be analyzed from the drug release curve that the overall drug release process is relatively slow and stable, and there is no sudden release of the drug, which can alleviate the side effects of patients. Considering the high GSH and slightly acidic tumor microenvironment, Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG with pH / GSH dual responsiveness can efficiently release DOX at the tumor site, reduce the number of dosing times and achieve the purpose of effectively inhibiting tumor growth.

[0056] from Fig.10 As can be seen in b, due to the disintegration of the MOF structure, the drug release under acidic conditions and the addition of GSH increased significantly. Under acidic conditions (pH 5.0), the release of DOX was slightly higher than that under neutral conditions (pH 7.4), and the drug release increased from 20.11% to 30.04% within 48 hours, indicating that the drug was pH-responsive, which may be due to the weakening of the coordination effect of Fe(Ⅲ)-RSSR@DOX@PEG-GOx under acidic conditions. In 10 mmol / L GSH solution, the release of DOX under pH 5.0 for 48 hours reached 82.10%, which was much higher than 30.04% without GSH. Considering the redox and acidic tumor microenvironment, the pH / GSH dual-responsive release of DOX by RSSR@DOX@PEG-GOx is the first choice for achieving tumor site-specific release, greatly improving the therapeutic efficiency.

[0057] In summary, the in vitro simulated drug release experiment showed outstanding environmental response release, and under acidic conditions and GSH conditions, the drug can be quickly released at a specific point, improving the drug treatment efficiency.

[0058] Example 4: Verification of the ferroptosis-stimulating effect of pH / GSH dual-responsive Fe-based MOF drug carrier (1) Fe 2+ Generation monitoring: After the pH / GSH dual-responsive Fe-based MOF drug carrier skeleton collapses, in addition to releasing the internally encapsulated drug DOX, it also dissociates Fe as a metal active component to participate in coordination. 3+ At this time, Fe 3+ The role of the tumor is to be reduced to Fe by the high GSH in the tumor 2+ , a large amount of GSH is consumed to destroy the redox homeostasis, and then the Fenton reaction occurs to quickly stimulate ferroptosis.

[0059] O-phenanthroline can react with Fe 2+ This step monitors the Fe generated during the drug release process by measuring the absorbance of the solution after the o-phenanthroline color development. 2+ The monitoring results are as follows Fig.11 shown.

[0060] from Fig.11 As can be seen in a, under acidic conditions (pH 5.0), nearly 169.84 µg of Fe 3+ Converted to Fe 2+ In contrast, in the presence of 10 mM GSH, Fe 2+ The amount of Fe was increased from 169.84 µg to 1007.92 µg. Combined with the analysis of drug release, after adding 10 mmol / L GSH to the pH 5.0 solution, the structure of the drug carrier collapsed, making Fe 3+ Efficiently releases and reacts to generate Fe 2+ These results indicate that GSH can effectively transfer Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG to Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG after responsive dissociation. 3+ Converted to Fe 2+ To stimulate ferroptosis.

[0061] from Fig.11 b It can be seen that under acidic conditions (pH 5.0), 863.52 µg of Fe 2+ Successful release, 12h later Fe 2+ In contrast, in the solution with 10 mM GSH, Fe 2+ The amount of Fe 3+ These results indicate that GSH can effectively release MOF and reduce Fe 3+ Converted to Fe 2+ Play a therapeutic role.

[0062] Therefore, the high GSH content (10 mmol / L) in the tumor can react with the disulfide bonds of MOF, and also needs to react with Fe 3+ The reaction generates Fe 2+ Experiments have shown that at 10 mmol / L GSH, there is sufficient Fe 3+ Can be reduced to Fe 2+ , providing a basis for the subsequent Fenton reaction.

[0063] (2) Monitoring of OH generation: Fe 2+ •OH generated by Fenton reaction with endogenous H2O2 in tumor cells can upregulate the ROS level of tumor cells, promote the accumulation of cytotoxic lipid peroxides and successfully stimulate ferroptosis. As the final product of the Fenton reaction, the generation of •OH in this example can be used to infer the successful occurrence of the Fenton reaction. The measurement results are as follows: Fig.12 shown.

[0064] from Fig.12 As can be seen in a, under acidic conditions (pH 5.0), some •OH was generated within 72 h. In contrast, the addition of 10 mM GSH significantly increased the amount of •OH generated. These results indicate that the synthesized Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG can effectively generate •OH. The generation of a large amount of •OH leads to upregulation of ROS, accumulation of lipid peroxides, ferroptosis, and inhibition of tumor growth.

[0065] from Fig.12 As can be seen in b, the Fe(Ⅲ)-RSSR@DOX@PEG-GOx curve after adding glucose shows a significantly higher TMB absorbance, indicating that a large amount of •OH is indeed produced during the reaction. This is because the glucose oxidase contained in Fe(Ⅲ)-RSSR@DOX@PEG-GOx catalyzes the decomposition of the added glucose and produces H2O2, which eventually reacts with Fe 2+ The reaction generates •OH. Moreover, •OH is the most efficient active oxygen component that stimulates ferroptosis. The generation of a large amount of •OH provides a material basis for the occurrence of ferroptosis, which can effectively achieve the purpose of stimulating ferroptosis and inhibiting tumor growth.

[0066] In summary, monitoring the large amount of •OH generation indicated that the Fenton reaction occurred smoothly.

[0067] Example 5: Cytotoxicity and endocytosis verification (1) Cytotoxicity verification: In this step, the MTT method was used to detect the cell viability and survival of Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG and Fe(Ⅲ)-RSSR@DOX@PEG-GOx before and after drug loading: the mitochondria of living cells can reduce MTT to crystalline formazan and deposit in the cells, while dead cells cannot. In this way, the cytotoxicity of Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG and Fe(Ⅲ)-RSSR@DOX@PEG-GOx was investigated. The specific steps are as follows: L929 (mouse fibroblasts) and 4T1 (breast cancer cells) were selected and the cell concentration was diluted to 5×10 3 100 μL of 1640 culture medium (containing phenol red, 10% serum and 1% double antibody) were inoculated in 96-well plates, and 100 μL of 1640 culture medium (containing phenol red, 10% serum and 1% double antibody) were added to each well, and cultured in an incubator (5% CO2, 37ºC) for 24 h. The culture medium was carefully removed with an aspirator and replaced with 100 μL of culture medium containing blank nanoparticles or drug-loaded nanoparticles at different concentrations. After incubation for 48 h, the culture medium was removed, washed three times with PBS, and MTT solution (100 μL, volume fraction of 10%) was transferred. The cells were incubated in the dark for 4 h, the liquid was gently aspirated with an aspirator, dissolved with DMSO (150 μL), and the absorbance was quickly measured at a wavelength of 570 nm using an enzyme reader. The cell viability was calculated using the following formula to evaluate the biocompatibility of the carrier and the cytotoxicity of the drug-loaded micelles.

[0068] Relative cell viability (%) = A sample -A0 / A control -A0×100 In this formula, A0 represents the absorbance value of the blank, A control represents the absorbance value of the control group, A sample Indicates the absorbance value of the material. The test results are as follows Fig.13 and 14 shown.

[0069] from Fig.13 It can be seen that when the blank Fe(Ⅲ)-(3,3'-SS-) MOF concentration reaches 125 μg / mL, the normal mouse L929 cells are co-cultured for 48 hours, and the cell survival rate reaches 99.28% ( Fig.13 A). Normal cells still maintain a high activity, which proves that Fe(Ⅲ)-(3,3'-SS-) MOF material has good biocompatibility. Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG has a survival rate of 78.26% for L929 cells at a drug concentration of 125 µg / mL ( Fig.13 (B). The loading of anticancer drug DOX has certain toxicity to normal cells, but the higher cell survival rate still indicates that the nanocarrier has low cytotoxicity and good biocompatibility. Fig.13 C shows that the blank drug carrier Fe(Ⅲ)-(3,3'-SS-) MOF has a certain degree of autotoxicity to 4T1 cells. This is because Fe(Ⅲ)-(3,3'-SS-) MOF breaks bonds and releases Fe under the environment of highly expressed GSH in tumors. 3+ , Fe 3+ Reduced to Fe 2+ , and then the Fenton reaction occurs to stimulate ferroptosis therapy, showing a certain cytotoxicity. However, the survival rate of 4T1 cancer cells of Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG is 6.27%, which is much lower than that of free DOX. Compared with the blank carrier and DOX alone, the drug-loaded carrier Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG shows higher cytotoxicity to 4T1 cells. This is because the intracellular Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG can better release the drug, resulting in a highly efficient therapeutic effect of ferroptosis and DOX synergistically. This result is consistent with the drug release behavior results in vitro.

[0070] from Fig.14 It can be seen that when the blank Fe(III)-RSSR MOF concentration reached 50 μg / mL, the normal mouse L929 cells were co-cultured for 48 hours, and the cell survival rate reached 100.71% ( Fig.14 A). Normal cells still maintain high activity, which proves that Fe(III)-RSSR MOF material has good biocompatibility. At a drug concentration of 50 μg / mL after loading Fe(III)-RSSR@DOX@PEG-GOx, the survival rate of L929 cells was 79.88% ( Fig.14 B). The anticancer drug DOX is toxic to normal cells, but the high cell survival rate still indicates that the nanocarrier has low cytotoxicity. Fig.14C It can be seen that the blank drug carrier Fe(III)-RSSRMOF has a certain autotoxicity to 4T1 cells. This is because Fe(III)-RSSRMOF breaks bonds in the environment of highly expressed GSH in the tumor to produce Fenton reaction, stimulate ferroptosis, and show certain cytotoxicity. However, the cell survival rate of Fe(III)-RSSR@DOX@PEG-GOx is 3.94%, which is much lower than that of free DOX. Compared with the blank carrier and DOX alone, the drug-loaded carrier Fe(III)-RSSR@DOX@PEG-GOx shows higher cytotoxicity to 4T1 cells. This is because the Fe(III)-RSSR@DOX@PEG-GOx in the cell can better release the drug, resulting in a highly effective therapeutic effect of ferroptosis and DOX synergistically. This result is consistent with the drug release behavior results in vitro. At the same time, the GOx modified on the carrier surface consumes glucose in the tumor, produces H2O2, and further promotes the formation of ferritin, forming a triple synergistic treatment situation of ferritin, chemotherapy and starvation therapy.

[0071] In summary, the unloaded blank nanoparticles Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG and Fe(Ⅲ)-RSSR@DOX@PEG-GOx showed good biocompatibility, and the drug-loaded nanoparticles showed better cytotoxicity due to the synergistic treatment of ferroptosis and drug release.

[0072] (2) Cell endocytosis verification: In this step, CLSM was used to study the cellular internalization of Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG and Fe(Ⅲ)-RSSR@DOX@PEG-GOx at different stages. The specific steps were as follows: staining the cell nucleus with DAPI and using DOX as a fluorescent probe. After Fe(Ⅲ)-(3,3'-SS-)@DOX@PEG and Fe(Ⅲ)-RSSR@DOX@PEG-GOx were co-cultured with tumor cells, the cell nucleus had blue fluorescence and DOX was red. DOX was widely internalized into cancer cells and showed strong red fluorescence localization in the cytoplasm and nucleus. The results are shown in Figure 2. Fig.15 and 16 shown.

[0073] from Fig.15 and 16 It can be seen that as the culture time increases from 3 h, 6 h to 12 h, the red fluorescence of DOX increases, while the fluorescence intensity of DAPI remains unchanged. In the confocal images, the merged purple fluorescence is obviously getting stronger and stronger, indicating that more DOX is internalized into the cells.

[0074] In summary, the present invention synthesizes an Fe-based MOF with a MOF containing a GSH-sensitive bond, then encapsulates the anticancer drug model DOX in the pores of the Fe-based MOF, and modifies the surface of the Fe-based MOF with a functional material to obtain the pH / GSH dual-responsive Fe-based MOF drug carrier; the Fe-based MOF drug carrier achieves the purpose of inhibiting or killing tumor cells through the synergy of ferroptosis and chemotherapy, has a good application in anti-cancer, and has good practicality.

[0075] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A pH / GSH dual-responsive Fe-based MOF drug carrier, characterized in that: The Fe-based MOF drug carrier has a rectangular sheet-like Fe-based MOF as the main structure, and the outer surface of the Fe-based MOF is modified with a functional material; there are pores inside the Fe-based MOF, and the anticancer drugs are loaded in the pores; The Fe-based MOF is composed of Fe 3+ As the active center, MOF containing GSH sensitive bonds is used as the organic ligand; The functional material includes mPEG-NH2 or mPEG-GOx.

2. The pH / GSH dual-responsive Fe-based MOF drug carrier according to claim 1, characterized in that: The MOF containing GSH sensitive bonds includes HOOC-(CH2)2-SS-(CH2)2-COOH or RSSR; The anticancer drug includes DOX.

3. The method for synthesizing the pH / GSH dual-responsive Fe-based MOF drug carrier according to claim 1, characterized in that: The synthesis method comprises: (1) Synthesis of Fe-based MOF: Dissolve FeCl3 and MOF containing GSH sensitive bonds in DMF to obtain FeCl3 solution and organic ligand solution respectively; Under ultrasonic conditions, the FeCl3 solution is slowly added to the organic ligand solution, TEA is added thereto, and then stirred for reaction. After the reaction is completed, blank nanoparticle Fe-based MOF is obtained, which is washed, dried, and collected for later use; (2) Synthesis of pH / GSH dual-responsive Fe-based MOF drug carrier: The anticancer drug aqueous solution is slowly dripped into the Fe-based MOF aqueous solution under ultrasonic conditions, and the reaction 1 is carried out under stirring conditions. After the reaction is completed, the Fe-based MOF loaded with the anticancer drug is obtained by centrifugation and washing. An excess amount of functional material is added to an aqueous solution of Fe-based MOF loaded with anticancer drugs, and reaction 2 is carried out under stirring conditions. After the reaction, the solution is dialyzed with distilled water overnight and dried to obtain the pH / GSH dual-responsive Fe-based MOF drug carrier.

4. The method for synthesizing the pH / GSH dual-responsive Fe-based MOF drug carrier according to claim 3, characterized in that: In step (1), the MOF containing a GSH-sensitive bond includes HOOC-(CH2)2-SS-(CH2)2-COOH or RSSR; When the MOF containing GSH sensitive bonds is HOOC-(CH2)2-SS-(CH2)2-COOH, the mass ratio of FeCl3 and the GSH sensitive MOF is 1:1, and the sum of the mass of FeCl3 and the GSH sensitive MOF to the amount of TEA is 1~2g:1mL; When the MOF containing a GSH-sensitive bond is RSSR, the mass ratio of FeCl3 to the GSH-sensitive MOF is 1:1, and the ratio of the sum of the masses of FeCl3 and the GSH-sensitive MOF to the amount of TEA is 1~2g:1mL.

5. The method for synthesizing the pH / GSH dual-responsive Fe-based MOF drug carrier according to claim 4, characterized in that: The synthesis method of the RSSR comprises: 4,4'-dithiodibutyric acid and N,N'-carbonyldiimidazole are dissolved in anhydrous dichloromethane, evaporated under nitrogen protection, and then the residue is dissolved in dichloromethane, repeatedly washed with ultrapure water and sodium carbonate solution, the organic phase is collected and dried in anhydrous sodium sulfate, the dichloromethane is removed by rotary evaporation, and then dried to obtain the RSSR.

6. The method for synthesizing the pH / GSH dual-responsive Fe-based MOF drug carrier according to claim 3, characterized in that: In step (1), when the MOF containing the GSH sensitive bond is HOOC-(CH2)2-SS-(CH2)2-COOH, the stirring reaction conditions are: reacting at 120°C for 24h; When the MOF containing the GSH sensitive bond is RSSR, the stirring reaction conditions are: stirring at 40° C. under nitrogen protection for 8 hours.

7. The method for synthesizing the pH / GSH dual-responsive Fe-based MOF drug carrier according to claim 3, characterized in that: In step (2), the dosage ratio of the anticancer drug to the Fe-based MOF is 1-3:1; The anticancer drug includes DOX; The conditions of the reaction 1 are: reacting at room temperature in the dark for 24 hours.

8. The method for synthesizing the pH / GSH dual-responsive Fe-based MOF drug carrier according to claim 3, characterized in that: In step (2), the functional material includes mPEG-NH2 or mPEG-GOx; The conditions of reaction 2 are: reacting at room temperature for 24 hours.

9. The method for synthesizing the pH / GSH dual-responsive Fe-based MOF drug carrier according to claim 8, characterized in that: The synthesis method of the PEG-GOx comprises: GOx is dissolved in ultrapure water, EDC and NHS are added thereto, and the mixture is uniformly mixed under stirring. After the reaction is completed, mPEG-NH2 is added and the mixture is reacted under stirring. After the reaction is completed, the mixture is dialyzed with distilled water overnight and dried to obtain the PEG-GOx.

10. Use of the pH / GSH dual-responsive Fe-based MOF drug carrier according to claim 1 or 2 in loading anticancer drugs.

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