Protein-delivering mof nanoparticles with low metal content and methods of making and using the same

Abstract

The application relates to the technical field of biological macromolecule delivery, and discloses a protein delivery MOF nanoparticle with low metal content and a preparation method and application thereof. The protein delivery MOF nanoparticle with low metal content comprises a protein and a MOF carrier loaded with the protein; the MOF carrier comprises 2-methyl imidazole and zinc ions, and the zinc ions are respectively coordinated with histidine residues of the protein and 2-methyl imidazole. The preparation method comprises the following steps: dissolving the protein in deionized water, adding a low-zinc-ion-content aqueous solution, stirring to obtain a first solution; adding 2-methyl imidazole into the first solution, stirring to obtain a reaction mixture, centrifuging the mixture, and collecting a precipitate; and washing the precipitate for multiple times, and drying to obtain the product. The nanoparticle has ultrahigh loading capacity, extremely low metal content and low biological toxicity; has good pH responsiveness, can be dissociated under acidic conditions, releases the protein and restores the protein activity.

Description

Technical Field

[0001] This invention relates to the field of biomacromolecule delivery technology, specifically to a low-metal-content protein delivery MOF nanoparticle, its preparation method, and its applications. Background Technology

[0002] Metal-organic frameworks (MOFs) are porous organic-inorganic hybrid materials assembled from metal ions and organic ligands, offering numerous advantages. The diversity of metal ion and organic ligand structures allows MOFs to possess a wider range of morphologies and tunable chemical and size properties, making them more suitable for loading drug molecules with varying physicochemical properties. The high porosity and specific surface area of ​​MOFs result in high loading capacity, enabling the effective loading of both small molecule drugs and biomacromolecules. Organic ligands facilitate covalent modification of MOFs. Weak coordination bonds contribute to the good biodegradability of MOFs, facilitating controlled drug release. Based on these advantages, MOFs are widely used in drug delivery.

[0003] However, existing MOF drug delivery systems still have some problems. First, the loading efficiency of macromolecular drugs such as proteins and nucleic acids in MOFs is low, which seriously inhibits the clinical therapeutic effect of macromolecular drugs. Second, traditional MOF drug delivery systems contain a high content of metal ions. When these metal ions accumulate in some organs and tissues in vivo, the release of metal ions as the MOF degrades may lead to an increase in local metal ion concentration and produce serious side effects. In addition, the preparation of MOFs often involves organic solutions such as DMSO and DMF. These organic solvents have significant biotoxicity and may adversely affect the structure and function of some biomolecular drugs. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention aims to provide a low-metal-content MOF nanoparticle for protein delivery, its preparation method, and its applications. This nanoparticle possesses ultra-high protein loading capacity, extremely low metal content, and good biocompatibility. In a neutral pH environment, the MOF structure of the nanoparticle remains intact, protecting the protein while simultaneously reducing its activity in neutral pH conditions. In an acidic environment, the MOF structure undergoes responsive degradation, releasing the protein and restoring its activity. The MOF nanoparticle can also synergistically deliver other drugs.

[0005] To achieve the above objectives, the present invention employs the following technical solutions.

[0006] The present invention provides a low-metal-content protein delivery MOF nanoparticle, comprising a protein and an MOF carrier loading the protein; the MOF carrier contains 2-methylimidazole and zinc ions, wherein the zinc ions coordinate with histidine residues and 2-methylimidazole residues of the protein, respectively.

[0007] Preferably, the protein includes one or more of bovine serum albumin, collagenase, hyaluronidase, catalase, horseradish peroxidase, and superoxide dismutase.

[0008] Further preferred options are bovine serum albumin or collagenase.

[0009] Preferably, in the low-metal-content protein delivery MOF nanoparticles, proteins, 2-methylimidazole, and zinc ions form a three-dimensional spatial network structure through coordination self-assembly, and the proteins are encapsulated inside the nanoparticles.

[0010] This invention provides a method for preparing protein-delivered MOF nanoparticles with low metal content, comprising the following steps:

[0011] S1, Dissolve the protein in deionized water, add zinc ion aqueous solution, and stir to obtain the first solution;

[0012] When a small amount of zinc ions is added to a protein solution, the zinc ions coordinate with the histidine residues of the protein to form nodes.

[0013] S2, add 2-methylimidazole to the first solution and stir to obtain a mixture; centrifuge the mixture and collect the precipitate;

[0014] After the addition of 2-methylimidazole, zinc ions on the nodes coordinate with 2-methylimidazole and undergo coordination self-assembly to form a three-dimensional network structure, in which the protein is encapsulated inside the nanoparticles.

[0015] S3 is obtained by washing the precipitate multiple times and drying it.

[0016] Preferably, the number of zinc ions required per 1g of protein is 0.026-1.8 mmol.

[0017] Preferably, the molar ratio of zinc ions to 2-methylimidazole is 1:(1750-14000).

[0018] Preferably, the centrifugation parameters are: rotation speed 14000 rpm, centrifugation temperature 4℃, and centrifugation time 30 min.

[0019] Preferably, the drying method is freeze drying; the freeze drying temperature is -80℃, the pressure is 3-5 Pa, and the time is 2-3 days.

[0020] Applications of the aforementioned low-metal-content protein-delivered MOF nanoparticles or the low-metal-content protein-delivered MOF nanoparticles prepared by the aforementioned preparation method in loading photosensitizers, contrast agents, chemotherapeutic drugs, antibacterial drugs, or immunomodulatory drugs.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1) The protein delivery MOF nanoparticles of the present invention have ultra-high loading capacity and extremely low metal content, and have lower biotoxicity than traditional MOF mineralized loaded protein nanoparticles.

[0023] 2) Protein delivery MOF nanoparticles have good pH responsiveness and can dissociate under acidic conditions, releasing proteins and restoring protein activity.

[0024] 3) The preparation method is simple, green and safe, without the potential toxicity of organic solvents or the adverse effects of organic solvents on protein structure and function. Attached Figure Description

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0026] Figure 1 This is a schematic diagram illustrating the synthesis principle of low-metal-content protein delivery MOF nanoparticles according to the present invention.

[0027] Figure 2 For different Zn 2+ MOF prepared under / 2-MIM ratio conditions Col Transmission electron microscopy (TEM) results;

[0028] Figure 3a For different Zn 2+ MOF prepared under / 2-MIM ratio conditions Col Dynamic light scattering detection results;

[0029] Figure 3b Dynamic light scattering detection results of MOF nanoparticles prepared from normal collagenase and MOF nanoparticles prepared from DEPC alkylated collagenase. Figure 4 For different Zn 2+ MOF prepared under / 2-MIM ratio conditions Col Encapsulation efficiency results graph;

[0030] Figure 5 For MOF Col Thermogravimetric analysis curves of ZIF-2 and ZIF-8;

[0031] Figure 6 MOF Col-2. X-ray diffraction analysis spectra of Col@ZIF-8 and simulated ZIF-8;

[0032] Figure 7 Free collagenase and collagenase + Zn in aqueous solution as detected by DLS 2+ and MOF Col Surface potential diagram at -2;

[0033] Figure 8 MOF Col Stability test results of aqueous solution of -2;

[0034] Figure 9 MOF at a type I collagenase concentration of 1 mg / mL Col State diagrams of -2 and Col@ZIF-8 solutions;

[0035] Figure 10 MOF Col -2 Collagenase responsive release curve in an acidic environment;

[0036] Figure 11a MOF Col -2 Collagenase Activity Test Results Graph;

[0037] Figure 11b MOF Col -2 Results of gelatin degradation volume in each group during collagenase activity test;

[0038] Figure 12 MOF Col -2 biotoxicity test results graph;

[0039] Figure 13a MOF Col -2 Diagram of collagen degradation in tumor tissue after treatment;

[0040] Figure 13b MOF Col -2. Results of collagen area ratio in tumor tissue after treatment;

[0041] Figure 14 MOF Col / FD Transmission electron microscope image;

[0042] Figure 15 For FD1080 and MOF Col / FD Visible-near-infrared absorption spectrum of DMSO solution;

[0043] Figure 16a To achieve a power density of 1.0 W / cm² 2 Under 1064 nm (NIR-II) laser irradiation, 0.4 mg / mL MOFCol / FD Solution temperature change curve;

[0044] Figure 16b To achieve a power density of 1.0 W / cm² 2 Under 1064 nm laser irradiation, 0.4 mg / mL MOF Col / FD Temperature change curves of the aqueous solution after four heating / cooling cycles. Detailed Implementation

[0045] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0046] Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means familiar to those skilled in the art.

[0047] Traditional MOF-mineralized collagenase encapsulated nanoparticles have a high metal ion content, with metal ions and 2-MIM forming bulky MOF structures that encapsulate the protein. When these encapsulate proteins in organs and tissues, the degradation of the MOF releases metal ions, leading to increased local metal ion concentrations and potentially causing serious side effects. Furthermore, the high metal ion content and bulky MOF structure result in low loading efficiency for traditional MOF-mineralized collagenase encapsulated nanoparticles. The applicant has discovered that, through a specific method, protein delivery MOF nanoparticles prepared with minimal metal ions possess a unique structure, exhibiting low metal ion content, high loading efficiency, and low side effects and biotoxicity.

[0048] This invention provides low-metal-content protein delivery MOF nanoparticles, comprising a protein and an MOF support loading the protein; the MOF support contains 2-methylimidazole and zinc ions, wherein the zinc ions coordinate with histidine residues and 2-methylimidazole residues of the protein, respectively. In the low-metal-content protein delivery MOF nanoparticles of this invention, zinc ions self-assemble in three dimensions with histidine residues and 2-methylimidazole residues of the protein, forming a three-dimensional network structure, significantly reducing the zinc ion content in the nanoparticles. Figure 1 The diagram shown illustrates the principle of synthesizing protein-delivered MOF nanoparticles with low metal content.

[0049] The low-metal-content protein delivery MOF nanoparticles of this invention contain few zinc ions, forming a thin mineralized layer on the protein surface for encapsulation. Histidine residues of the protein participate in this process, and the proteins bind together through this mineralized layer to form nanoparticles. The nanoparticles are spherical, with the protein encapsulated inside. The surface of the nanoparticles is a MOF formed by zinc ions and 2-methylimidazole. In a neutral pH environment, the MOF structure of the nanoparticles remains intact, protecting the protein and reducing its activity in a neutral pH environment. In an acidic environment, the MOF structure undergoes responsive degradation, releasing the protein and restoring its activity.

[0050] Specifically, the protein includes one or more of bovine serum albumin, collagenase, hyaluronidase, catalase, horseradish peroxidase, and superoxide dismutase, with bovine serum albumin or collagenase being preferred. Zinc ions can be an aqueous solution of zinc nitrate or its hydrate, zinc chloride or its hydrate, or zinc sulfate or its hydrate.

[0051] The low-metal-content protein-delivered MOF nanoparticles of the present invention have ultra-high loading capacity and can load photosensitizers, contrast agents, chemotherapeutic drugs, antibacterial drugs or immunomodulatory drugs.

[0052] Example 1 Preparation of Zn molar ratio 2+ MOF nanoparticles encapsulating collagenase with a low metal content of / 2-MIM=1:14000 are denoted as MOF. Col -1

[0053] 1.1 Dissolve 5 mg of type I collagenase in 2 mL of deionized water, then add 1.13 μmol of zinc nitrate aqueous solution, stir at room temperature for 10 min to obtain the first solution;

[0054] 1.2 Add 15.75 mmol of 2-methylimidazole solution to the first solution and continue stirring the reaction at room temperature for 4 h to obtain a mixture; transfer the mixture to a 1.5 mL centrifuge tube, set the centrifuge conditions to 14000 rpm and 4℃, centrifuge for 30 min, remove the supernatant and collect the precipitate.

[0055] 1.3 After rinsing the precipitate with deionized water, centrifuge for 30 min at 14000 rpm and 4℃. Remove the supernatant and collect the product. Repeat this washing process three times. Freeze the product at -80℃ for 4 h, then transfer it to a freeze dryer. Set the freeze-drying temperature to -90℃, the pressure to 3-5 Pa, and the time to 2-3 days. The final product is stored at 4℃ for later use.

[0056] Example 2 Preparation of Zn molar ratio 2+MOF nanoparticles encapsulating collagenase with a low metal content of / 2-MIM=1:7000 are denoted as MOF. Col -2

[0057] The difference between Example 2 and Example 1 is that the amount of zinc nitrate used is 2.25 μmol.

[0058] Example 3 Preparation of Zn molar ratio 2+ MOF nanoparticles encapsulating collagenase with a low metal content of / 2-MIM=1:3500 are denoted as MOF. Col -3

[0059] The difference between Example 3 and Example 1 is that the amount of zinc nitrate used is 4.50 μmol.

[0060] Example 4 Preparation of Zn molar ratio 2+ MOF nanoparticles with low metal content encapsulating collagenase ( / 2-MIM=1:1750) are denoted as MOF. Col -4.

[0061] The difference between Example 4 and Example 1 is that the amount of zinc nitrate used is 9.0 μmol.

[0062] Example 5 Preparation of low-metal-content encapsulated collagenase MOF nanoparticles synergistically loaded with photosensitizer FD1080, denoted as MOF Col / FD

[0063] The difference between Example 5 and Example 2 is that 250 μg FD1080 was added simultaneously when 2.25 μmol zinc nitrate was added.

[0064] Comparative Example 1: Preparation of metal-organic framework material ZIF-8

[0065] 1.1 Dissolve 15.75 mmol 2-MIM in 4.5 mL of water;

[0066] 1.2 Add 0.23 mmol of zinc nitrate aqueous solution to the solution in 1.1 and stir the reaction at room temperature for 10 min; transfer the milky white mixture to a 1.5 mL centrifuge tube and centrifuge at 5000 rpm and 4℃ for 10 min, and collect the precipitate;

[0067] 1.3 Wash the precipitate twice with anhydrous ethanol, and then once with 5% SDS (w / v) solution to obtain the intermediate product. Mix the intermediate product with 5 mL of 3% PVP (w / v) aqueous solution at room temperature and stir for 30 min. Centrifuge at 5000 rpm and 4℃ for 10 min. After removing the supernatant, resuspend in ultrapure water. Freeze the suspension at -80℃ for 4 h, then transfer it to a lyophilizer. Set the lyophilization temperature to -90℃, the pressure to 3-5 Pa, and the time to 2-3 days to obtain the final product. Store the final product at 4℃ for later use.

[0068] Comparative Example 2: Preparation of conventional MOF mineralization and encapsulation of collagenase nanoparticles, denoted as Col@ZIF-8

[0069] 2.1 Add 5 mg of type I collagenase to 4.5 ml of 2-methylimidazole solution, wherein the 2-methylimidazole content is 15.75 mmol, and stir at room temperature for 10 min to obtain the first solution;

[0070] 2.2 Add 0.5 ml of zinc nitrate solution to the first solution, wherein the zinc nitrate content is 0.23 mmol; stir the reaction at room temperature for 10 min, transfer the mixture to a 1.5 mL centrifuge tube, set the centrifuge conditions to 14000 rpm and 4℃, centrifuge for 30 min, remove the supernatant, and collect the precipitate.

[0071] 2.3 The precipitate was washed twice with anhydrous ethanol, and the product was washed once with 5% SDS (w / v) solution to obtain the intermediate product. The intermediate product was mixed with 5 mL of 3% PVP (w / v) aqueous solution at room temperature and stirred for 30 min, then centrifuged at 5000 rpm and 4℃ for 10 min. After removing the supernatant, the product was resuspended in ultrapure water, and then the suspension was frozen at -80℃ for 4 h. It was then transferred to a lyophilizer and the freeze-drying temperature was set to -90℃, the pressure to 3-5 Pa, and the time to 2-3 days. The final product was stored at 4℃ for later use.

[0072] Comparative Example 3: Preparation of histidine alkylation modified collagenase MOF nanoparticles, denoted as MOF. Col-DEPC

[0073] 3.1 Prepare a 10 mmol / L diethyl pyrocarbonate (DEPC) solution using 0.01 mol / L PBS buffer;

[0074] 3.2 Add 5 mg of type I collagenase to 5 mL of DEPC solution and stir the mixture at room temperature for 1 h to obtain the reaction solution;

[0075] 3.3 The reaction solution was transferred to an ultrafiltration tube with a molecular weight cutoff of 10 kDa and ultrafiltered at 1000 rpm and 4℃ to remove residual DEPC, yielding alkylated collagenase. DEPC ;

[0076] 3.4 Collagenase was confirmed by detecting UV absorption at 227 nm. DEPC Alkylation of histidine residues resulted in a gradual increase in the protein's UV absorption at 227 nm as DEPC alkylation modification progressed.

[0077] 3.5 Following the steps of Example 2, use Collagenase DEPC Nanoparticles were prepared by replacing normal type I collagenase.

[0078] The low-metal-content encapsulated collagenase MOF nanoparticles prepared in Examples 1-4 Col -1, MOF Col -2, MOF Col -3, MOF Col -4, Example 5: Low-metal-content encapsulated collagenase MOF nanoparticles with synergistic photosensitizer FD1080 prepared. Col / FD And ZIF-8 (Comparative Example 1), MOF-mineralized collagenase nanoparticles Col@ZIF-8 (Comparative Example 2), and MOF-mineralized collagenase nanoparticles (Comparative Example 3) Col-DEPC A series of tests were conducted. The testing instruments were: transmission electron microscope (model: JEM-2100 Plus), JEOL Ltd.; thermogravimetric analyzer (model: TG 209 F1 Libra), NETZSCH, Germany; dynamic light scattering particle size analyzer (model: ZS 90), Malvern Instruments Ltd., UK; X-ray diffractometer (model: SmartLab™ 9 kW Cu), Rigaku Ltd., Japan; X-ray photoelectron spectroscopy (model: AXIS SUPRA+), Kratos, UK; microplate reader (SpectraMaxiD5), Miko Molecular Instruments Ltd.; BCA protein quantification kit (catalog number: 23225), Thermo Fisher Scientific Ltd.

[0079] 1. Dynamic light scattering particle size analyzer and transmission electron microscopy:

[0080] The nanoparticles prepared in Examples 1-4 were analyzed using a dynamic light scattering particle size analyzer and a transmission electron microscope to compare different Zn content. 2+ The particle size of nanoparticles prepared under / 2-MIM ratio conditions. The detection medium was aqueous phase, and the detection temperature was 25℃.

[0081] Figure 2For different Zn 2+ MOF prepared under / 2-MIM ratio conditions Col Transmission electron microscopy (TEM) images, from Figure 2 It can be seen that the nanoparticles in Examples 1-4 all exhibit a complete spherical shape.

[0082] Figure 3a For different Zn 2+ MOF prepared under / 2-MIM ratio conditions Col Dynamic light scattering detection results show that when Zn 2+ When the / 2-MIM ratios were 1:14000, 1:7000, 1:3500, and 1:1750, the prepared nanoparticles had particle sizes of 144.6 nm, 88.5 nm, 213.4 nm, and 230.1 nm, respectively, and the particle size distribution of the nanoparticles in each combination was relatively narrow.

[0083] Figure 3b Dynamic light scattering assays were performed on MOF nanoparticles prepared from normal collagenase and MOF nanoparticles prepared from DEPC-alkylated collagenase. The results showed that when histidine in the collagenase protein was alkylated, it could not coordinate with zinc ions, thus blocking the formation of nanoparticles.

[0084] 2. Encapsulation efficiency test:

[0085] The encapsulation efficiency of the nanoparticles prepared in Examples 1-4 was tested using the BCA method. Col -1, MOF Col -2, MOF Col -3, MOF Col -4, MOF Col The collagenase content in the -5 samples was compared and the encapsulation efficiency of the nanoparticles was measured. The encapsulation efficiency was calculated as M1 / M2*100%, where M1 is the mass of collagenase encapsulated by the nanoparticles as measured by the BCA method, and M2 is the total mass of collagenase fed into the corresponding system.

[0086] Figure 4 For different Zn 2+ MOF prepared under / 2-MIM ratio conditions Col Encapsulation efficiency. The results show that when Zn 2+ When the feed content is small, it follows the Zn 2+ As the Zn content increases, the encapsulation efficiency gradually rises. 2+ When the / 2-MIM ratio reaches 1:3500, the encapsulation efficiency reaches its maximum of 90.1%. Afterwards, it follows Zn... 2+ As concentration increases, encapsulation efficiency begins to decrease.

[0087] 3. Surface potential, protein loading, stability, and X-ray diffraction characterization tests:

[0088] MOF detection using dynamic light scattering particle size analyzer Col -2. Collagenase and Zn 2+ The surface potential of collagenase after incubation was measured in an aqueous medium at a temperature of 25°C.

[0089] MOF was obtained by thermogravimetric analysis. Col The weight change curves of ZIF-2 and ZIF-8 with temperature were used to calculate the protein loading after excluding the weight loss caused by changes in ZIF-8 crystals.

[0090] MOF Col -2 was dispersed in an aqueous solution and left for 9 days. The particle size and polydispersity index were measured using a dynamic light scattering particle size analyzer on days 0, 1, 3, 5, 7 and 9.

[0091] MOF detection using X-ray diffraction Col MOF lattice states in -2 and Col@ZIF-8 were compared with simulated ZIF-8 X-ray diffraction patterns.

[0092] Figure 5 MOF Col Thermogravimetric analysis curves of ZIF-2 and ZIF-8. The analysis results for the ZIF-8 group show that, due to the oxidation of the organic imidazole, the main weight loss of ZIF-8 occurs after approximately 620℃. Correspondingly, MOF... Col -2 The weight loss of the metal-organic framework in the thermogravimetric analysis should be completed after 620℃. Therefore, the weight loss in the 100℃ to 623℃ range is attributed to the weight of collagenase, and the results show that the collagenase content is 80.3%.

[0093] Figure 6 MOF Col -2. X-ray diffraction analysis spectra of Col@ZIF-8 and simulated ZIF-8. The results show that Col@ZIF-8 and MOF... Col -2 exhibits multiple peaks between 5° and 35°, and matches well with the simulated X-ray diffraction pattern of ZIF-8, indicating that Col@ZIF-8 and MOF... Col ZIF-8 crystalline structures are present in both -2 and MOF. Col -2 both exhibit broad peak shapes, indicating a semi-crystalline structure. This is because MOF Col The low Zn(II) content in -2 leads to the loss of long-range ordered structures, and the protein surface structure interferes with the formation of crystal structures.

[0094] Figure 7This shows the free collagenase and collagenase + Zn in aqueous solution as detected by DLS. 2+ and MOF Col The surface potential of -2 indicates the MOF Col -2 has a large negative potential.

[0095] Figure 8 MOF is shown Col The stability of -2 after 9 days in aqueous solution, during which MOF Col The particle size and polydispersity index of -2 showed no significant fluctuations, and the MOF Col -2 exhibits high stability.

[0096] Figure 9 The MOF is shown when the concentration of type I collagenase is 1 mg / mL. Col Photographs of -2 and Col@ZIF-8 solutions, in which MOF Col -2 appears colorless and transparent in water, while Col@ZIF-8 is a milky white colloidal suspension.

[0097] 4. MOF Col -2 acid response release detection

[0098] MOF Col -2 was dispersed in PBS buffer solutions at pH 7.4 and pH 6.5, placed in dialysis tubes for incubation, and external sample solutions were collected at different time points. The cumulative amount of collagenase released was detected and recorded by BCA method.

[0099] The specific method is as follows:

[0100] (1) MOF Col -2 was pre-dissolved in PBS buffer solutions at pH 7.4 and pH 6.5 (0.4 mg / mL for type I collagenase) and incubated for 12 h to pre-release collagenase.

[0101] (2) Dissolve 5 g of gelatin in 12.5 mL of ultrapure water and incubate at 50°C for 2 h.

[0102] (3) Use a pipette to add 0.3 mL of gelatin solution into a 1 mL syringe while it is still hot, and let it cool and solidify at room temperature to build a gel model.

[0103] (4) Using a pipette, transfer 0.5 mL of MOF incubated at different pH environments. Col -2 solution, untreated pure collagenase solution, and control group PBS buffer solution were added to a gelatin gel syringe and incubated at 37°C for 48 h.

[0104] (5) After incubation, allow the syringe to cool to room temperature, remove the degraded gelatin from the supernatant, and calculate the volume of degraded gelatin by subtracting the remaining volume from the original volume. This is used to detect MOF. Col -2 In vitro pH-responsive dissociation characteristics.

[0105] Figure 10 MOF Col -2 Collagenase responsive release curve in an acidic environment. The results show that when MOF Col When incubated with PBS buffer at pH 7.4, MOF Col -2 inhibition remained stable, and collagenase was encapsulated in the particles, so even after 24 hours of incubation, the amount of collagenase released was still relatively small; while when MOF Col When incubated with PBS buffer at pH 6.5, the nanoparticles dissociate, releasing over 80% of the collagenase after 24 hours. MOF Col -2 exhibits good pH responsiveness.

[0106] Figure 11 shows MOF Col -2 Collagenase activity test diagram. Figure 11a As shown, MOFs pretreated with PBS buffer, free collagenase solution, and PBS buffer solutions at pH 7.4 and pH 6.5, respectively, were... Col The gel was incubated with the -2 solution at 37°C for 48 h. After cooling to room temperature, the supernatant was removed for comparison. The results showed that MOF pretreated with PBS buffer solution at pH 7.4... Col In a solution of -2, MOF Col -2 remains stable; the MOF structure inhibits collagenase activity; MOF Col -2 exhibited lower enzyme activity. Meanwhile, MOF pretreated with PBS buffer at pH 6.5 showed... Col In the -2 solution, collagenase is released, regains its activity, and causes significant gel degradation.

[0107] Figure 11b for Figure 11a Gelatin degradation volume in each group. MOF Col -2 In a neutral environment, collagenase activity is inhibited; in an acidic environment, MOF Col -2 degradation, in response to release collagenase and restore collagenase activity.

[0108] 5. MOF Col MOF -2 Col -2 Cytotoxicity Detection

[0109] The testing method is as follows:

[0110] (1) Panc02 cells and L929 cells were seeded in 96-well plates at a density of 5000 cells per well and cultured in a carbon dioxide incubator for 24 h;

[0111] (2) Remove the old culture medium and add 100 μL of MOF containing different collagenase concentrations to each well. Col -2 or Col@ZIF-8 medium, with collagenase concentration gradients of 0 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL, with 5 replicates per group.

[0112] (3) After culturing the cells in a carbon dioxide incubator for 24-48 h, add 100 μL of complete culture medium containing MTT (1 mg / mL) to each well and incubate in the incubator for 4 h.

[0113] (4) Remove the supernatant culture medium, add 100 μL DMSO to each well, and shake for 10 min in the dark at room temperature.

[0114] (5) The absorption of the solution in each well at 490 nm was detected by an ELISA reader to analyze the MOF. Col Cytotoxicity of -2 or Col@ZIF-8.

[0115] Test results are as follows Figure 12 As shown. Figure 12 The relative cell viability of each group of cells after incubation with different concentrations of MOFCol-2 or Col@ZIF-8 collagenase for different times. Among them, a is PancO2, 24 h; b is PancO2, 48 h; c is L929 cells, 24 h; d is L929 cells, 48 ​​h.

[0116] The test results show that, compared to the traditional MOF biomineralization encapsulation protein structure Col@ZIF-8, MOF… Col -2 has lower biotoxicity.

[0117] MOFs with different protein concentrations Col Panc02 and L929 cells were incubated with -2 or Col@ZIF-8 for 24 or 48 h, and cell viability was detected using the MTT assay. Results showed that MOF... Col-2 exhibits good biocompatibility, showing low cytotoxicity even when the collagenase concentration is increased to 50 μg / mL during cell incubation. However, when Col@ZIF-8 cells were incubated with Panc02 cells for 24 h, cell viability significantly decreased at a collagenase concentration of 10 μg / mL; and at a collagenase concentration of 50 μg / mL, cell viability was completely inhibited in all Col@ZIF-8 experimental groups.

[0118] 6. MOF Col -2 Detection of type I collagen degradation in a pancreatic cancer tumor model

[0119] The mice used in the experiment were 6-8 weeks old, C57BL / 6 female mice, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0120] Panc02 tumor-bearing mice were intravenously injected with MOF Col -2 nanoparticles were processed twice, with an interval of 12 h, including MOF Col The dosage of -2 nanoparticles was 10 mg / kg. After 48 h, mice were euthanized by cervical fracture, and tumor tissue was extracted, paraffin-embedded, sectioned, and then dewaxed for Masson's trichrome staining to observe collagen degradation in the tumor tissue.

[0121] The steps for Mason tricolor staining are as follows:

[0122] (1) After the Panc02 tumor-bearing mice were euthanized, the tumor tissue was removed and fixed with 4% paraformaldehyde.

[0123] (2) The tumor tissue was dehydrated and then embedded in paraffin.

[0124] (3) The paraffin sections from step (2) are then transferred to a glass slide for dewaxing.

[0125] (4) Place the dewaxed sections into Bouin solution and mordant at 37°C for 2 h.

[0126] (5) Rinse with pure water until the yellow color on the slices from step (4) disappears.

[0127] (6) Add the azurite blue staining solution to the slice from step (5), incubate for 2-3 minutes, and wash with water to remove residual staining solution.

[0128] (7) Add Mayer hematoxylin staining solution to the slices from step (6), incubate for 2-3 min, and wash with water to remove residual staining solution.

[0129] (8) Immerse the slices from step (7) in acidic ethanol differentiation solution for 5-10 seconds, then rinse with running water for 10 minutes.

[0130] (9) Add the Ponceau S and Fuchsia staining solution to the slice from step (8), incubate for 10 min, and rinse with running water.

[0131] (10) Add the phosphomolybdic acid solution to the slice from step (9) and incubate for about 10 min.

[0132] (11) After discarding the phosphomolybdic acid solution, directly add the aniline blue staining solution to the slice from step (10) and stain for 5 minutes.

[0133] (12) Rinse with a weak acid solution for 2 min.

[0134] (13) Soak the slices from step (12) in 95% ethanol for 5-10 seconds to rapidly dehydrate them.

[0135] (14) Immerse the slices from step (13) in anhydrous ethanol for 5-10 seconds, repeating 3 times.

[0136] (15) Soak the slices from step (14) in xylene for 1-2 min, repeating 3 times.

[0137] (16) After drying, seal with neutral resin.

[0138] Figure 13a MOF Col -2 Diagram of collagen degradation in tumor tissue after treatment. Results showed a large amount of blue collagen signal in the tumor sections of Xiao Hu in the PBS-treated group. MOF... Col In group -2, collagen signal was significantly reduced, with approximately 90% of collagen being absorbed by MOF. Col -2 degradation. That is, tumor-bearing mice injected with MOF Col -2 formulation can degrade collagen in tumor tissue. Figure 13b for Figure 13a The proportion of collagen area in tumor tissues of each group, MOF Col In group -2, the proportion of collagen area was significantly smaller than that in the PBS-treated group, MOF Col -2 is very effective at degrading collagen.

[0139] 7. Low-metal-content MOF nanoparticles encapsulated with synergistic photosensitizer FD1080 for collagenase Col / FD Photothermal performance characterization

[0140] Free FD1080 at a concentration of 20 μg / mL and MOF Col / FD The DMSO solutions were placed in a UV spectrophotometer to detect their absorption in the NIR-II region, and the effect of nanoparticle encapsulation on the performance of FD1080 was observed.

[0141] With a power density of 1.0 W / cm² 2 MOF particles with a concentration of 0.4 mg / mL were irradiated with a 1064 nm laser. Col / FD The solution was left to stand for 10 minutes, and temperature changes were recorded every 5 seconds using a digital infrared camera to assess its photothermal conversion performance. Repeated irradiation heating / natural cooling was then performed to assess its photothermal stability.

[0142] Figure 14 The molecular structure of FD1080 and MOF are shown. Col / FD Solution photography and MOF Col / FD Transmission electron microscopy images of nanoparticles, MOF Col / FD With MOF Col Similar spherical morphology and particle size.

[0143] Figure 15 This shows the free FD1080 and MOF at a concentration of 20 μg / mL. Col / FD The visible-near-infrared absorption spectra of DMSO solution showed that FD1080 in MOF Col / FD The material still exhibits strong NIR-II band light absorption, similar to the performance of free FD1080, indicating that the encapsulation of nanoparticles did not significantly affect the performance of FD1080.

[0144] Figure 16 shows MOF Col / FD It exhibits excellent photothermal conversion performance and photothermal stability. Figure 16a To achieve a power density of 1.0 W / cm² 2 MOF at 0.4 mg / mL under 1064 nm (NIR-II) laser irradiation Col / FD Solution temperature change curve; Figure 16b To achieve a power density of 1.0 W / cm² 2 Under 1064 nm laser irradiation, 0.4 mg / mL MOF Col / FD Temperature change curves of the aqueous solution after four heating / cooling cycles.

[0145] With a power density of 1.0 W / cm² 2 MOF irradiated with a concentration of 0.4 mg / mL by a 1064 nm laser Col / FD The solution temperature rose by 30°C within 10 minutes, comparable to the free FD1080 group. Through four heating / cooling cycles, MOF... Col / FD It still exhibits high photothermal conversion performance and demonstrates good photothermal stability.

[0146] In summary, the low-metal-content encapsulated collagenase MOF nanoparticles provided by this invention not only have extremely low metal content and lower biotoxicity than traditional MOF mineralized protein-loaded nanoparticles, but also exhibit good pH responsiveness, allowing them to dissociate under acidic conditions, release proteins, and restore protein activity. Furthermore, they possess ultra-high loading capacity and good photothermal stability.

[0147] The method for preparing low-metal-content encapsulated collagenase MOF nanoparticles provided by this invention is simple, green, and safe, without the potential toxicity of organic solvents or the adverse effects of organic solvents on protein structure and function.

[0148] Although the present invention has been described in detail in this specification with general description and specific embodiments, some modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A protein delivery MOF nanoparticle with low metal content, characterized in that, The invention includes a protein and an MOF carrier loaded with the protein; the MOF carrier contains 2-methylimidazole and zinc ions, wherein the zinc ions are coordinated to histidine residues and 2-methylimidazole residues of the protein, respectively. The protein is a collagenase, and the protein is encapsulated within the MOF carrier; the number of zinc ions required per 1 g of protein is 0.026-1.8 mmol. The molar ratio of zinc ions to 2-methylimidazole is 1:3500.

2. The protein-delivered MOF nanoparticles according to claim 1, characterized in that, In the low-metal-content protein delivery MOF nanoparticles, proteins, 2-methylimidazole, and zinc ions form a three-dimensional spatial network structure through coordination self-assembly, and the proteins are encapsulated inside the nanoparticles.

3. The method for preparing protein-delivered MOF nanoparticles with low metal content according to claim 1, characterized in that, Includes the following steps: S1, Dissolve the protein in deionized water, add zinc ion aqueous solution, and stir to obtain the first solution; S2, add 2-methylimidazole to the first solution and stir to obtain a mixture; centrifuge the mixture and collect the precipitate; S3 is obtained by washing the precipitate multiple times and drying it.

4. The method for preparing protein-delivered MOF nanoparticles with low metal content according to claim 3, wherein the centrifugation parameters are: rotation speed 14000 rpm, centrifugation temperature 4℃, and centrifugation time 30 min.

5. The method for preparing protein-delivered MOF nanoparticles with low metal content according to claim 3, characterized in that, The drying process is freeze drying; the freeze drying temperature is -80℃, the drying pressure is 3-5 Pa, and the drying time is 2-3 days.

6. The use of the low-metal-content protein-delivered MOF nanoparticles according to any one of claims 1 to 2 or the low-metal-content protein-delivered MOF nanoparticles prepared by the preparation method according to any one of claims 3 to 5 in the preparation of carriers for loading photosensitizers, contrast agents, chemotherapeutic drugs, antibacterial drugs or immunomodulatory drugs.

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