Multifunctional metal-organic framework nano-drug delivery system and preparation method and application thereof

Abstract

The application discloses a multifunctional metal organic framework nano drug delivery system and a preparation method and application thereof, and belongs to the technical field of medicines. The system is composed of two-dimensional metal organic framework nanosheets with light-heat conversion capacity, natural enzymes and modifiers capable of charge flipping under a weak acid environment. The two-dimensional metal organic framework nanosheets with light-heat conversion capacity are coordination polymers formed by coordination of porphyrin photosensitizers and metal ions. The preparation method of the system comprises the following steps: 1) reacting porphyrin photosensitizers, trifluoroacetic acid, polyvinylpyrrolidone and metal salts to prepare two-dimensional metal organic framework nanosheets; 2) loading natural enzymes on the two-dimensional metal organic framework nanosheets through an adsorption reaction to obtain drug-loaded two-dimensional metal organic framework nanosheets; and 3) modifying the drug-loaded two-dimensional metal organic framework nanosheets with the modifiers capable of charge flipping under a weak acid condition through electrostatic adsorption to obtain the multifunctional metal organic framework nano drug delivery system.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a multifunctional metal-organic framework drug delivery system and its preparation method, as well as its synergistic application in tumor photothermal therapy, photodynamic therapy and chemokinetic therapy. Background Technology

[0002] Cancer is one of the major public health problems worldwide. Although traditional methods such as surgery, chemotherapy, and radiotherapy are well-established in clinical practice, they still face challenges such as poor selectivity, significant toxic side effects, and the easy development of drug resistance. To develop safer and more effective anti-tumor methods, emerging therapies such as photothermal therapy (PTT), photodynamic therapy (PDT), and chemodynamic therapy (CDT) have attracted widespread attention due to their non-invasiveness and high controllability. To maximize treatment efficacy and reduce toxic side effects, many researchers have begun to combine multiple therapies to achieve synergistic anti-tumor effects.

[0003] Photothermal therapy (PTT) is a non-invasive treatment strategy that utilizes photothermal agents with high near-infrared light absorption to convert light energy into heat energy under external near-infrared laser irradiation, thereby locally ablating tumors. Its mechanism of action involves the photothermal agent absorbing photon energy and generating localized high temperatures through non-radiative relaxation, leading to protein denaturation, membrane structure disruption, and organelle damage in tumor cells, ultimately inducing apoptosis or necrosis. PTT offers advantages such as strong spatiotemporal controllability and minimal damage to normal tissues. An ideal photothermal agent requires strong absorption within the near-infrared biological window, high photothermal conversion efficiency, and good biocompatibility. Based on morphology, photothermal agents can be classified into zero-dimensional, one-dimensional, two-dimensional, and three-dimensional nanomaterials. While zero-dimensional nanomaterials are simple to synthesize, easy to functionalize, and biocompatible, their symmetrical structure limits the upper limits of light absorption and conversion efficiency. One-dimensional nanomaterials achieve efficient utilization of near-infrared light through tunable optical anisotropy, but their high aspect ratio poses challenges to synthetic control and in vivo behavior. Three-dimensional nanomaterials achieve extremely high photothermal conversion efficiency through their ingenious three-dimensional structures, but their synthetic complexity also increases significantly. Two-dimensional nanomaterials, with their extremely high specific surface area, enhanced light absorption, and ideal platform for multifunctional integration, are gradually becoming the research focus for constructing efficient nanotherapeutic systems.

[0004] PDT is a treatment method that relies on the combined effects of a photosensitizer, appropriate wavelength light exposure, and oxygen in the tissue. Its core mechanism involves the photosensitizer absorbing light energy and transitioning to an excited state, then converting ground-state oxygen into highly oxidizing singlet oxygen via energy transfer. 1O2) attacks intracellular lipids, proteins, and nucleic acids, leading to cell death. PDT has advantages such as high selectivity and the ability to induce immunogenic cell death, but its efficacy is limited by the depth of light penetration. Tetra(4-carboxyphenyl)porphyrin (TCPP) is a commonly used photosensitizer for PDT, but it is prone to strong phototoxicity under sunlight, which limits its clinical application.

[0005] CDT is a technology that utilizes transition metal ions (such as Fe) 2+ Cu + This therapeutic strategy catalyzes a Fenton-like reaction in excess hydrogen peroxide (H₂O₂) within the tumor microenvironment, generating highly toxic hydroxyl radicals (•OH). •OH can trigger lipid peroxidation, DNA damage, and protein inactivation, leading to tumor cell death. CDT is photosensitive, suitable for deep tumors, and utilizes the weak acidity of the tumor microenvironment to enhance the reaction rate. Copper ions (Cu) + / Cu 2+ As an effective Fenton-like catalyst, copper-based materials can cycle through their valence state within the tumor microenvironment, continuously catalyzing the production of •OH from H2O2. Furthermore, copper-based materials typically have a wider pH application window than iron-based materials. However, their therapeutic efficacy is limited by insufficient H2O2 concentration within tumor cells. Therefore, enzymes capable of catalyzing H2O2 production in the tumor microenvironment (TME) are increasingly being used in therapeutic processes.

[0006] Metal-organic frameworks (MOFs) are crystalline porous materials formed by the self-assembly of metal ions and organic ligands through coordination bonds. MOFs possess advantages such as high specific surface area, tunable pore size and structure, and ease of functionalization, showing great potential in drug delivery, catalysis, and bioimaging. Two-dimensional MOF nanosheets (Cu-TCPP) formed by the coordination of copper ions and TCPP not only effectively reduce the phototoxicity of TCPP, but also possess high loading capacity as a nanomedicine carrier, photothermal conversion performance as a photothermal agent, and the ability to achieve drug delivery therapy (CDT) through copper ion-mediated Fenton-like reactions. Therefore, Cu-TCPP MOFs provide an ideal platform for constructing intelligent nanodelivery systems integrating multiple therapeutic modalities. Summary of the Invention

[0007] The purpose of this invention is to construct a multifunctional metal-organic framework nanomedicine delivery system that promotes the accumulation of the nanomedicine delivery system in tumor cells by the weak acidity of the tumor microenvironment, increases the accumulation of reactive oxygen species (ROS) at the tumor site, and achieves synergistic anti-tumor effects of PTT / PDT / CDT.

[0008] The technical solution adopted in this invention is as follows: This invention provides a multifunctional metal-organic framework nanoparticle drug delivery system, which consists of two-dimensional metal-organic framework nanosheets with photothermal conversion capabilities, natural enzymes, and modifiers that can be charge-reversed under weakly acidic conditions.

[0009] Preferably, the two-dimensional metal-organic framework nanosheets with photothermal conversion capability are coordination polymers formed by coordinating porphyrin-based photosensitizers with metal ions. The porphyrin-based photosensitizer is selected from one of tetrakis(4-carboxyphenyl)porphyrin, hematoporphyrin derivatives, verteporfen, talaporfen, and temopofen; the metal ion is selected from one of copper ions, iron ions, manganese ions, and zinc ions.

[0010] Preferably, the natural enzyme is selected from one of D-amino acid oxidase, glucose oxidase, xanthine oxidase, and monoamine oxidase.

[0011] Preferably, the charge-reversible modifier in a weakly acidic environment is selected from one of aminated poly(2-ethyl-2-oxazoline), chitosan, carboxymethyl chitosan, and chitosan quaternary ammonium salt.

[0012] The method for preparing two-dimensional metal-organic framework nanosheets of the multifunctional metal-organic framework nanocarrier system of the present invention includes the following steps:

[0013] (1) Dissolve metal salts and polyvinylpyrrolidone in N,N-dimethylformamide (DMF) and anhydrous ethanol. Add a mixed solution of DMF and anhydrous ethanol containing porphyrin photosensitizer under stirring. Add trifluoroacetic acid dropwise. React in a water bath. Centrifuge and wash to obtain two-dimensional metal-organic framework nanosheets.

[0014] (2) The two-dimensional metal-organic framework nanosheets obtained in step (1) are dispersed in a buffer solution, a natural enzyme solution is added, the mixture is stirred in the dark, and then centrifuged and washed to obtain drug-loaded two-dimensional metal-organic framework nanosheets.

[0015] (3) Disperse the drug-loaded two-dimensional metal-organic framework nanosheets obtained in step (2) in a buffer solution, add a charge-reversible modifier, stir the reaction, centrifuge and wash to obtain the multifunctional metal-organic framework nano-drug delivery system.

[0016] Preferably, in step (1), the volume ratio of DMF to anhydrous ethanol is 1:1 to 4:1, the molar ratio of porphyrin photosensitizer to metal salt is 1:2 to 1:6, the amount of polyvinylpyrrolidone is 20 mg to 50 mg, the amount of trifluoroacetic acid is 20 μL to 50 μL, and the reaction time is 2 h to 6 h.

[0017] Preferably, in step (2), the buffer solution is a phosphate buffer salt used to adjust the reaction pH, with a pH range of 5.5 to 8.0, the mass ratio of the natural enzyme to the two-dimensional metal-organic framework nanosheets is 1:1 to 1:4, and the reaction time is 12 h to 24 h.

[0018] Preferably, in step (3), the mass ratio of the charge-reversed modifier to the drug-loaded two-dimensional metal-organic framework nanosheets under a weakly acidic environment is 1:2 to 2:1, and the reaction time is 4 h to 12 h.

[0019] Preferably, step (1) specifically involves dissolving the metal salt and polyvinylpyrrolidone in DMF and anhydrous ethanol (V) respectively. DMF V 乙醇 Mix thoroughly with water bath sonication (ratio 1:1~4:1). Then, while stirring, add a mixture of DMF containing a porphyrin-based photosensitizer and anhydrous ethanol (V...). DMF V 乙醇 The mixture was prepared by adding trifluoroacetic acid dropwise (ratio 1:1 to 4:1), mixing thoroughly in a water bath using ultrasonication, and then reacting in a water bath. After the reaction was complete, the sample was centrifuged at 8000 rpm to 12000 rpm for 10 to 30 minutes, and the supernatant was discarded. The precipitate was washed twice with deionized water and twice with anhydrous ethanol, and then dried under vacuum to obtain the two-dimensional metal-organic framework support.

[0020] Preferably, step (2) is as follows: after dissolving the natural enzyme, add phosphate buffer solution to adjust the pH of the system, and add the two-dimensional metal-organic framework carrier dropwise. After stirring at room temperature, centrifuge at 8000 rpm to 12000 rpm for 10 min to 30 min, discard the supernatant, and wash the precipitate twice with deionized water to obtain the drug-loaded two-dimensional metal-organic framework.

[0021] Preferably, step (3) specifically involves: modifying the carrier with charge-reversing properties under weakly acidic conditions using electrostatic adsorption, dispersing the carrier in phosphate buffer, and adjusting its pH value. The dissolved charge-reversing material is added dropwise, stirred at room temperature, and then centrifuged at 6000 rpm to 10000 rpm for 10 to 30 minutes. The supernatant is discarded, and the precipitate is washed three times with deionized water and dried under vacuum to obtain the nano-drug delivery system.

[0022] This invention also provides the application of the aforementioned multifunctional metal-organic framework nanoparticle drug delivery system in the preparation of antitumor drugs. The antitumor drug dosage forms include injections, tablets, and implantable delivery systems.

[0023] The present invention also provides the application of the aforementioned multifunctional metal-organic framework nanocarrier system in the preparation of drugs for the synergistic treatment of tumors using photothermal therapy, photodynamic therapy, and chemokinetics.

[0024] The weakly acidic environment of the tumor microenvironment allows the zeta potential on the surface of the nanoparticles to change from negative to positive. At the same time, it consumes GSH and provides H2O2, which promotes the uptake of the drug delivery system by tumor cells and increases the generation of ROS to kill tumor cells.

[0025] Preferably, the present invention uses metal-organic framework nanosheets as a carrier, loads glucose oxidase on the surface of the nanosheets and modifies the surface with carboxymethyl chitosan that can reverse charge in the weakly acidic environment of the tumor to form a multifunctional metal-organic framework nano-drug delivery system.

[0026] Among them, Cu is used 2+ A metal-organic framework (MOF) with a size of approximately 240 nm was constructed by coordinating with the porphyrin-based photosensitizer TCPP. This framework effectively stabilized the photosensitizer, reducing its toxicity under sunlight irradiation and minimizing damage to normal tissues. The framework formation endowed the material with excellent near-infrared photothermal conversion capabilities. This coordination structure altered the electronic energy levels of TCPP, causing a redshift and broadening of its absorption spectrum, resulting in enhanced light absorption in the near-infrared region. During the return of electrons from the excited state to the ground state, energy was not released in the form of fluorescence, but rather primarily converted into lattice thermal vibrations via non-radiative relaxation, thus efficiently converting light energy into heat energy and achieving localized heating. The material can degrade to Cu in tumor microenvironments with high glutathione expression. 2+ Together with TCPP, this not only disrupts the antioxidant defense system in tumor cells but also provides substrates for CDT and PDT. Furthermore, glucose oxidase, loaded onto the surface of metal-organic framework nanosheets via π-π adsorption, can catalyze the production of H₂O₂ from glucose, providing a substrate for Fenton-like reactions. Simultaneously, surface modification with carboxymethyl chitosan via electrostatic interactions leads to protonation of the amino groups under weakly acidic conditions, changing the surface charge from negative to positive, thereby significantly enhancing the uptake efficiency by tumor cells.

[0027] This nano-drug delivery system achieves a charge reversal from negative to positive in the tumor microenvironment, enhancing the uptake of the drug by tumor cells. The G-CuP@C nano-drug delivery system can exert a cascade synergistic anti-tumor effect in the tumor microenvironment. After the system is efficiently internalized by tumor cells through the charge reversal effect, the glucose oxidase it carries catalyzes the overexpressed glucose within the cells, continuously generating H2O2 and exacerbating microenvironment acidification. This provides substrates for CDT on one hand, and promotes the degradation of metal-organic frameworks on the other. Under 808 nm near-infrared laser irradiation, CuP nanosheets, acting as a photothermal conversion agent, can convert light energy into heat energy, achieving PTT; simultaneously, the released TCPP ligands can generate [something] under 660 nm laser excitation. 1 O2 enables photodynamic therapy. Furthermore, Cu released from the degraded scaffold... +It can undergo a Fenton-like reaction with H2O2 produced by glucose oxidase catalysis, generating highly toxic hydroxyl radicals (•OH), thus achieving CDT; while Cu + / Cu 2+ The cycle can simultaneously consume high concentrations of glutathione within cells, disrupting the redox balance of tumor cells and thus significantly enhancing the killing effect of reactive oxygen species. This nano-drug delivery system achieves enrichment and degradation at the tumor site through the weak acidity of the tumor microenvironment and the high expression of glutathione, and realizes synergistic effects of PTT, PDT, and CDT on tumors, exhibiting excellent anti-tumor efficacy.

[0028] The beneficial effects of this invention are:

[0029] (1) The multifunctional metal-organic framework constructed in this invention achieves tumor enrichment and reactive oxygen species generation in a slightly acidic and high-glutathione environment at the tumor site, and effectively consumes glutathione, thereby realizing Cu + / Cu 2+ It promotes the circulation of hormones and enhances the effectiveness of tumor treatment.

[0030] (2) This invention significantly improves the anti-tumor effect by accumulating in the tumor, generating a photothermal effect through dual near-infrared light irradiation, and inducing a large amount of reactive oxygen species to kill tumor cells; at the same time, it consumes a large amount of glutathione overexpressed in the tumor, which breaks the tumor cell's antioxidant defense system; in vivo pharmacodynamic studies revealed that under the multiple effects of PTT, PDT and CDT, tumor growth is severely inhibited, realizing the synergistic application of multiple treatments. Attached Figure Description

[0031] Figure 1 Transmission electron microscope images of the CuP support prepared in Example 1 (A) with a scale bar of 500 nm and (B) with a scale bar of 500 nm.

[0032] Figure 2 The waveforms of CuP and TCPP prepared in Example 1 under ultraviolet-visible-near-infrared wavelengths are shown.

[0033] Figure 3 The Fourier transform infrared spectrum of CuP prepared in Example 1 is shown.

[0034] Figure 4 The X-ray photoelectron spectra of CuP prepared in Example 1 are (A) full spectrum and (B) high-resolution Cu 2p spectrum.

[0035] Figure 5The diagram shows the TCPP release of CuP prepared in Example 1 under (A) pH 7.4 PBS, (B) pH 6.5 PBS, (C) pH 7.4 PBS + GSH and (D) pH 6.5 PBS + GSH.

[0036] Figure 6 CuP prepared in Example 1 was subjected to Cu in pH 7.4 PBS, pH 6.5 PBS, pH 7.4 PBS + GSH, and pH 6.5 PBS + GSH. + / Cu 2+ The release status diagram.

[0037] Figure 7 Transmission electron micrographs of CuP prepared in Example 1 after 1 day and 7 days in pH 7.4 PBS, pH 6.5 PBS and pH 6.5 PBS+GSH.

[0038] Figure 8 The graph shows the reaction of G-CuP prepared in Example 2 with glucose (A) to generate H2O2 and (B) pH value changes.

[0039] Figure 9 The graph shows the zeta potential changes of G-CuP@C prepared in Example 2 at different pH values.

[0040] Figure 10 The following are the photothermal temperature rise diagrams of the G-CuP@C drug-loaded system prepared in Example 2: (A) temperature rise diagrams at different concentrations, (B) temperature rise diagrams at different irradiation powers, and (C) photothermal conversion efficiency diagram of G-CuP@C.

[0041] Figure 11 The graph shows the ability of G-CuP@C prepared in Example 2 to consume GSH at different concentrations.

[0042] Figure 12 The G-CuP@C drug delivery system prepared in Example 2 produced at different times after GSH treatment. 1 Diagram showing the situation with O2.

[0043] Figure 13 The diagram shows the generation of •OH in the G-CuP@C drug delivery system prepared in Example 2 after different treatments.

[0044] Figure 14 The cytotoxicity of CuP, G-CuP and G-CuP@C prepared in Examples 1 and 2 under dual NIR irradiation in L929 cells (A) in darkness, (B) under light, and (C) in 4T1 cells.

[0045] Figure 15The graph shows the intracellular ROS generation of CuP and G-CuP@C prepared in Examples 1 and 2 under different treatments. The scale bar is 50 μm.

[0046] Figure 16 The NDA fluorescence signal diagrams of CuP and G-CuP@C prepared in Examples 1 and 2 under different treatments were observed and detected by (A) CLSM and (B) FCM, and (C) average fluorescence intensity bar graphs were also shown. The scale bar is 50 μm.

[0047] Figure 17 CuP and G-CuP@C prepared in Examples 1 and 2 were observed by (A) CLSM under different treatments. 2+ The intracellular production status and (B) the relative fluorescence intensity of rhodamine B hydrazide in each group, with a scale bar of 50 μm.

[0048] Figure 18 The graph shows the damage to mitochondria in tumor cells caused by CuP and G-CuP@C prepared in Examples 1 and 2 under different treatments, as observed by CLSM. The scale bar is 50 μm.

[0049] Figure 19 (A) Mouse tumor growth curve and (B) Mouse body weight change curve for CuP and G-CuP@C prepared in Examples 1 and 2. Detailed Implementation

[0050] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0051] Example 1

[0052] Preparation, characterization, and degradation of CuP supports:

[0053] (1) Preparation of CuP support

[0054] 10.2 mg Cu(NO3)2·3H2O and 50 mg polyvinylpyrrolidone (PVP K30) were dissolved in a mixed solvent of 12 mL DMF and 4 mL anhydrous ethanol, and stirred for 10 minutes. Separately, 8.0 mg TCPP was dissolved in 8 mL of a DMF / anhydrous ethanol mixed solution (V:V = 3:1). The TCPP solution was slowly added dropwise to the Cu(NO3)2·3H2O / PVP solution with stirring. After the addition was complete, 20 μL of trifluoroacetic acid was added. The mixture was placed in an 80°C water bath and stirred for 3 hours. After the reaction was complete, the mixture was allowed to cool naturally to room temperature, centrifuged at 11000 rpm for 15 minutes, and the precipitate was collected. The precipitate was washed twice each with deionized water and anhydrous ethanol, and finally dried in a vacuum drying oven to obtain a dark purple solid powder, which is the CuP nanosheet.

[0055] (2) Characterization of CuP support

[0056] The morphology of CuP was observed using transmission electron microscopy, and the results are shown in the attached figure. Figure 1 As shown, CuP exhibits an irregular two-dimensional sheet structure with a lateral dimension of approximately 240 nm, demonstrating the successful preparation of the CuP support.

[0057] The full-wavelength waveforms of CuP and TCPP were measured using a UV spectrophotometer, and the results are shown in the attached figure. Figure 2 As shown, compared with the sharp Soret absorption band of free TCPP at 418 nm, the absorption peak of CuP at 418 nm is red-shifted and broadened, and it has strong absorption in the near-infrared region of 600 nm-1000 nm, which confirms the formation of CuP coordination structure and its potential photothermal properties.

[0058] Fourier transform infrared spectroscopy was used to investigate Cu 2+ The coordination details are shown in the attached figure. Figure 3 As shown, this illustrates Cu 2+ Successful coordination led to the formation of a CuP metal-organic framework.

[0059] The surface elemental composition and valence state of CuP were characterized by X-ray photoelectron spectroscopy, and the results are shown in the attached figure. Figure 4 As shown, copper ions in CuP are in the form of Cu 2+ and Cu + It exists in the form of.

[0060] (3) Degradation of CuP support

[0061] The release of TCPP from CuP in different media was investigated using a UV-spectrum spectrophotometer. The results are shown in the attached figure. Figure 5 As shown, TCPP releases are highest in a pH 6.5 PBS+GSH environment.

[0062] The release of Cu from CuP in different media was investigated using an atomic absorption spectrophotometer. + / Cu 2+ The situation and results are as follows. Figure 6 As shown, Cu + / Cu 2+ It releases the most in a pH 6.5 PBS+GSH environment.

[0063] The degradation of CuP in different media was investigated using transmission electron microscopy, and the results are shown in the attached figure. Figure 7 As shown, CuP is most completely degraded in a pH 6.5 PBS+GSH environment.

[0064] Example 2

[0065] Preparation of G-CuP@C nanocarrier system:

[0066] (1) Preparation of G-CuP drug-loaded nanosheets

[0067] 10 mg of CuP carrier was dispersed in pH 7.4 PBS, and 5.0 mg of glucose oxidase was dissolved in pH 7.4 PBS. The dissolved glucose oxidase solution was added dropwise, and the mixture was reacted at room temperature with stirring for 12 hours. After centrifugation at 11,000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with deionized water to obtain G-CuP drug-loaded nanosheets.

[0068] The loading of glucose oxidase was investigated by measuring the amount of H2O2 generated and the pH value after reaction with glucose. The results are shown in the attached figure. Figure 8 As shown, the H2O2 level gradually increased and the pH value gradually decreased, proving the successful loading of glucose oxidase and the fact that it maintained good enzyme activity after loading.

[0069] (2) Preparation of G-CuP@C nanocarrier system

[0070] 10 mg of G-CuP was dispersed in pH 5.0 PBS, and 10 mg of carboxymethyl chitosan was dissolved in pH 5.0 PBS. The dissolved carboxymethyl chitosan solution was added dropwise, and the mixture was reacted at room temperature with stirring for 6 hours. After centrifugation at 8000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed three times with deionized water to obtain the G-CuP@C nanoparticle drug delivery system.

[0071] The changes in zeta potential of the G-CuP@C nanoparticle drug delivery system at different pH values ​​were measured using dynamic light scattering. The results are shown in the attached figure. Figure 9 As shown, the Zeta potential of G-CuP@C changes from negative to positive under weakly acidic conditions, proving the success of the modification.

[0072] Example 3

[0073] Characterization of the G-CuP@C nano-drug delivery system:

[0074] The photothermal properties of the G-CuP@C nanoparticle drug delivery system were evaluated by irradiating it with a NIR laser. The results are shown in the attached figure. Figure 10 As shown, the photothermal effect of the drug delivery system is concentration- and power-dependent, with a photothermal conversion efficiency of 39.2%, indicating that G-CuP@C has good photothermal conversion capability.

[0075] The ability of the G-CuP@C drug delivery system to consume GSH at different concentrations was investigated using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The results are attached. Figure 11 As shown, the ability of G-CuP@C to consume GSH increases with increasing concentration, exhibiting a concentration-dependent effect on GSH consumption.

[0076] The formation of G-CuP@C was investigated using 1,3-diphenylisobenzofuran (DPBF). 1 The capacity of O2, results are attached. Figure 12 As shown, G-CuP@C treated with GSH exhibits good performance. 1 The O2 generation capacity demonstrates that G-CuP@C can degrade to produce TCPP and ROS under the action of GSH, showing excellent potential for photodynamic therapy.

[0077] The ability of G-CuP@C to generate •OH was investigated using 3,3',5,5'-tetramethylbenzidine (TMB), and the results are attached. Figure 13 As shown, G-CuP@C can react directly with H2O2 to generate •OH. This is because G-CuP@C contains a certain amount of copper as Cu. + In its existing form, after G-CuP@C reacts with GSH, more Cu is produced. + The generation of more •OH indicates that G-CuP@C has excellent potential for chemokinetic therapy.

[0078] Example 4

[0079] In vitro cytotoxicity of the drug delivery system to 4T1 cells:

[0080] L929 and 4T1 cells were seeded at appropriate densities in 96-well plates and cultured to the logarithmic growth phase, after which the culture medium was discarded. TCPP, CuP, and G-CuP@C dispersions (n=5) prepared from blank culture medium were added. For L929 cells, dark and sunlight groups were established; 4 h after drug administration, the light group was exposed to sunlight for 30 min. For 4T1 cells, dark, 808 nm NIR, 660 nm NIR, and 808+660 nm NIR groups were established; 4 h after drug administration, the NIR groups were exposed to the corresponding wavelength for 3 min (808 nm, 1.00 W / cm²). 2 660 nm, 0.20 W / cm 2 After incubation for 24 h, all groups were incubated with 5 mg / mL MTT solution for an additional 4 h. After 4 h of incubation, the supernatant was aspirated, and 150 μL of DMSO was added to each well. After shaking for 15 min, the absorbance at 570 nm was measured using a microplate reader, and cell viability was calculated. Results are attached. Figure 14 As shown, TCPP exhibits significant cytotoxicity under sunlight, while G-CuP@C shows reduced cytotoxicity under sunlight irradiation, indicating that the drug delivery system has a role in reducing sunlight toxicity. In the NIR-irradiated treatment groups, the G-CuP@C group subjected to dual NIR irradiation showed the strongest cell-killing effect, with an IC50 value of [missing value]. 50 The drug delivery system showed the strongest therapeutic effect in the dual NIR group, with a concentration of 2.523 μg / mL.

[0081] Example 5

[0082] Drug delivery systems contribute to the generation of ROS within tumor cells:

[0083] 4T1 was seeded in 24-well plates and cultured to 60% density, then the culture medium was discarded. Control, G-CuP@C, G-CuP@C + 808 nm NIR, CuP + 808 / 660 nm NIR, and G-CuP@C + 808 / 660 nm NIR groups were set up. Each group was prepared with blank medium at a concentration of 25 μg / mL. After incubation for 4 h, the culture medium was discarded, and the samples were washed three times with PBS. The DCFH-DA probe was diluted with blank medium to a final concentration of 10 μM, added to each well, and incubated at 37°C in the dark for 30 min. Simultaneously, the NIR light group was irradiated for 3 min at the corresponding wavelength (808 nm, 1.00 W / cm²). 2 660 nm, 0.20 W / cm 2After incubation for 30 min, the staining solution was discarded, and the cells were washed three times with PBS. Cell nuclei were stained with 10 μg / mL Hoechst staining solution for 20 min. The slide was then removed and inverted onto a glass slide with mounting medium. Intracellular ROS production was observed using CLSM. Results are shown in the attached figure. Figure 15 As shown, the G-CuP@C group exhibits a small amount of green fluorescence without NIR irradiation, which is due to the release of Cu from the formulation. 2+ / Cu + A Fenton-like reaction occurs, generating •OH; G-CuP@C irradiated with 808 nm NIR exhibits a photothermal effect, with increased temperature accelerating the Fenton-like reaction and increasing intracellular ROS levels; G-CuP@C irradiated with 808 nm and 660 nm produces •OH through a photodynamic pathway. 1 O2 further increases the intracellular ROS level; therefore, G-CuP@C (808+660 nm) has a stronger ROS generation capacity and has the characteristics of PTT / PDT / CDT working together.

[0084] Example 6

[0085] Drug delivery systems consume GSH levels within tumor cells:

[0086] Seed 4T1 cells at an appropriate density into 24-well plates containing smears and cultured in an incubator until the logarithmic growth phase. Discard the culture medium and wash twice with sterile PBS. Add 10 μg / mL G-CuP@C and CuP dispersions prepared with blank culture medium to the corresponding wells. G-CuP@C was incubated for 2, 4, and 8 h, respectively, while CuP was incubated for 8 h. After incubation, discard the drug-containing culture medium, wash three times with PBS, and incubate in the dark for 30 min with 50 μM naphthalene-2,3-dicarboxaldehyde (NDA). Stain with 10 μg / mL Hoechst for 20 min, wash twice, remove the smears, and invert them onto slides with mounting medium. Observe intracellular GSH consumption using CLSM. Seed 4T1 cells at an appropriate density into 6-well plates and cultured in an incubator until the logarithmic growth phase. Discard the culture medium and wash twice with sterile PBS. Follow the above steps for setting groups, drug administration, and staining. After staining, discard the staining solution and wash three times with PBS. Add a certain amount of trypsin for digestion and collect the cells. Detect intracellular DNA signal using FCM. Intracellular GSH levels are shown in the attached figure. Figure 16 As shown, both CLSM and FCM showed that NDA fluorescence gradually weakened with time, and compared with CuP, G-CuP@C had the lowest NDA fluorescence intensity after 8 hours of incubation, indicating that GSH was consumed the most. This is beneficial to reduce the consumption of ROS by GSH and kill tumor cells to a greater extent.

[0087] Example 7

[0088] Drug delivery system in tumor cells Cu 2+ The generation of:

[0089] 4T1 cells were seeded at an appropriate density into 24-well plates containing the climbing slides and cultured in an incubator until the logarithmic growth phase. The culture medium was then discarded, and the cells were washed twice with sterile PBS. Groups were set up and incubated with the drug as described in Example 6. After incubation, the drug-containing culture medium was discarded, and the cells were stained according to the Rhodamine B hydrazide instructions. After staining, the cells were washed three times with PBS. The cells were then stained with 10 μg / mL Hoechst for 20 min, followed by two washes. The climbing slides were removed and inverted onto slides containing mounting medium. Intracellular Cu was observed using CLSM. 2+ Release status. Cu in intracellular formulations was detected using CLSM. 2+ Release details and results are attached. Figure 17 As shown. With increasing time, the Cu released by the formulation... 2+ The amount gradually increased. Within the same timeframe, the G-CuP@C group released more Cu compared to the CuP group. 2 + This is mainly due to the modification of CMC, which enhances the uptake of the agent by cells, thereby releasing more Cu into the cells. 2+ The fluorescence intensity was calculated, and the results showed that after 8 hours, the formulation released Cu into the cells. 2+ The amount of Cu increases significantly, and the formulation can release Cu that consumes GSH and further participates in Fenton-like reactions within a certain period of time. 2+ .

[0090] Example 8

[0091] Drug delivery systems cause mitochondrial damage to tumor cells:

[0092] 4T1 cells were seeded at an appropriate density in 24-well plates containing climbing smears and cultured in an incubator until the logarithmic growth phase. The culture medium was then discarded, and the cells were washed twice with sterile PBS. Following Example 5, groups were set up and drug-treated for 4 h, after which the drug-containing culture medium was discarded, and the cells were washed three times with PBS. Simultaneously, NIR light was applied at the appropriate wavelength for 3 min (808 nm, 1.00 W / cm²). 2 660 nm, 0.20 W / cm 2 Continue incubation for 30 min, then wash three times with PBS. Mitochondria were stained according to the JC-1 kit instructions, and washed three times with PBS after staining. The slide was removed and inverted onto a glass slide with mounting medium. Images of JC-1 monomers and aggregates were observed using a CLSM scanner. The results are attached. Figure 18As shown in the figure, bright red fluorescence of JC-1 aggregates with almost no green fluorescence was observed in the control group, indicating that the cell mitochondria were not damaged. In the G-CuP@C group, the red fluorescence weakened and the green fluorescence strengthened, indicating that the surface drug loading system generated ROS, causing damage to the mitochondria. With the intervention of 808 and 660 nm NIR, the green fluorescence gradually increased and the red fluorescence decreased significantly, indicating that the photothermal effect accelerated the generation of ROS and the photodynamic pathway increased the ROS level, suggesting that G-CuP@C+NIR has a stronger ability to damage tumor cell mitochondria.

[0093] Example 9

[0094] Drug delivery systems for antitumor therapy in mice:

[0095] 4T1 tumor cells were subcutaneously injected into the shaved area on the upper right leg of healthy Balb / c mice. The tumor volume was increased to 100 mm². 3 Mice were randomly divided into 5 groups (n=5): saline group, G-CuP@C group, G-CuP@C+NIR (808 nm) group, CuP+NIR (808 nm+660 nm) group, and G-CuP@C+NIR (808 nm+660 nm) group. Twelve h after tail vein injection of CuP (0.5 mg / mL), the irradiation groups received NIR irradiation at the corresponding wavelength for 5 min (808 nm, 1.00 W / cm²). 2 660nm, 0.20 W / cm 2 The mice were given the drug every 3 days for a total of 4 doses. After the first dose, mouse body weight and tumor length and width were measured every 2 days, and tumor volume was calculated. Results are attached. Figure 19 As shown, compared with the control group, all drug delivery systems exhibited a certain degree of inhibitory effect on mouse tumor growth. After NIR irradiation, the tumor-inhibiting effect of the G-CuP@C group was further enhanced, and the tumor volume showed a significant decreasing trend.

[0096] The above description is only the best specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A multifunctional metal-organic framework nanomedicine delivery system, characterized in that, The nano-drug delivery system consists of two-dimensional metal-organic framework nanosheets with photothermal conversion capabilities, natural enzymes, and modifiers that can undergo charge reversal under weakly acidic conditions.

2. The multifunctional metal-organic framework nanomedicine delivery system according to claim 1, characterized in that, The two-dimensional metal-organic framework nanosheets with photothermal conversion capability are coordination polymers formed by coordination of porphyrin photosensitizers and metal ions; wherein the porphyrin photosensitizers are selected from one of tetra(4-carboxyphenyl)porphyrin, hematoporphyrin derivatives, verteporfen, talaporfen, and temoporfen; and the metal ions are selected from one of copper ions, iron ions, manganese ions, and zinc ions.

3. The multifunctional metal-organic framework nanomedicine delivery system according to claim 1, characterized in that, The natural enzyme is selected from one of D-amino acid oxidase, glucose oxidase, xanthine oxidase, and monoamine oxidase.

4. The multifunctional metal-organic framework nanomedicine delivery system according to claim 1, characterized in that, The charge-reversible modifier under weakly acidic conditions is selected from one of the following: aminated poly(2-ethyl-2-oxazoline), chitosan, carboxymethyl chitosan, and chitosan quaternary ammonium salt.

5. A method for preparing a multifunctional metal-organic framework nanomedicine delivery system according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Dissolve metal salt and polyvinylpyrrolidone in an organic solvent, add porphyrin photosensitizer under stirring, add trifluoroacetic acid dropwise, stir the reaction, and centrifuge and wash after the reaction to obtain two-dimensional metal-organic framework nanosheets. (2) Disperse the two-dimensional metal-organic framework nanosheets obtained in step (1) in a buffer solution, add natural enzymes, stir in the dark, centrifuge and wash to obtain drug-loaded two-dimensional metal-organic framework nanosheets. (3) Disperse the drug-loaded two-dimensional metal-organic framework nanosheets obtained in step (2) in a buffer solution, add a charge-reversible modifier, stir the reaction, centrifuge and wash to obtain the multifunctional metal-organic framework nano-drug delivery system.

6. The preparation method according to claim 5, characterized in that, In step (1), the volume ratio of N,N-dimethylformamide to anhydrous ethanol is 1:1 to 4:1, the molar ratio of porphyrin photosensitizer to metal salt is 1:2 to 1:6, the amount of polyvinylpyrrolidone is 20 mg to 50 mg, the amount of trifluoroacetic acid is 20 μL to 50 μL, and the reaction time is 2 h to 6 h.

7. The preparation method according to claim 5, characterized in that, In step (2), the buffer solution is phosphate buffer, which is used to adjust the pH of the reaction. The pH range is 5.5 to 8.

0. The mass ratio of the natural enzyme to the two-dimensional metal-organic framework nanosheet is 1:1 to 1:

4. The reaction time is 12 h to 24 h.

8. The preparation method according to claim 5, characterized in that, In step (3), the mass ratio of the charge-reversed modifier to the drug-loaded two-dimensional metal-organic framework nanosheets under a weakly acidic environment is 1:2 to 2:1, and the reaction time is 4 h to 12 h.

9. The application of the multifunctional metal-organic framework nanocarrier system according to claim 1 in the preparation of antitumor drugs, characterized in that, The antitumor drug dosage forms include injections, tablets, and implantable delivery systems.

10. The application of the multifunctional metal-organic framework nanocarrier system of claim 1 in the preparation of drugs for the synergistic treatment of tumors using photothermal therapy, photodynamic therapy, and chemokinetics.

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