A nano metal organic framework compound and a preparation method and application thereof

Abstract

The application discloses a kind of nano metal organic framework compounds and preparation method and application thereof.The nano metal organic framework compound includes the core from inside to outside in turn, the first shell layer being covered in the core surface, and the second shell layer being chemically modified and covered in the first shell layer surface, the core includes copper-cysteamine nanoparticles, the first shell layer includes 2-methyl imidazole zinc salt, and the second shell layer includes hyaluronic acid.The nano metal organic framework compound material provided by the application has photosensitive characteristics and enzyme catalytic activity, and can effectively target tumor cells.The whole preparation process is simple, environmentally friendly, and conducive to large-scale production, providing a new idea and method for the treatment of cancer.

Description

Technical Field

[0001] This invention relates to a nano-metal-organic framework compound, specifically, to a core-shell-shell structured nano-metal-organic framework compound, its preparation method, and its applications, belonging to the field of nanobiomedical technology. Background Technology

[0002] Cancer remains a major disease posing a serious threat to human health worldwide. According to data from the International Agency for Research on Cancer (IARC), the number of new cancer cases globally is projected to reach 29.5 million by 2040. Data from the National Cancer Center of China in 2022 reported approximately 4.06 million new cancer cases annually in China, with a trend towards younger onset, posing a serious threat to public health. Treatment methods mainly include surgery, chemotherapy, radiotherapy, and immunotherapy, but these have drawbacks such as patient intolerance, significant trauma, severe systemic side effects, damage to surrounding normal tissues, and immune-related adverse reactions. Therefore, developing new and effective methods for cancer treatment is essential.

[0003] With the rapid development of nanotechnology, nanomaterials for tumor treatment have entered the field of scientific research. Nanomaterials can serve as multifunctional platforms, combining chemotherapy, photothermal therapy, and photodynamic therapy to achieve synergistic therapeutic effects. They can also be modified to connect specific targeting molecules, enabling precise targeting of tumor cells, increasing local drug concentration, and thus enhancing therapeutic efficacy. Furthermore, they can reduce damage to normal tissues by targeting tumor tissue. Metal-organic frameworks (MOFs) are compounds containing both inorganic and organic functions, possessing good biocompatibility, high porosity, structural tunability, large surface area, and excellent catalytic performance, showing broad application prospects in ion exchange, small molecule storage, heterogeneous catalysis, luminescence, and magnetism. Among them, ZIF-8 metal-organic frameworks are widely used due to their simple preparation methods and large specific surface area; however, existing compounds suffer from limited crystallinity and poor electrical conductivity. Summary of the Invention

[0004] The main objective of this invention is to provide a nano-metal-organic framework compound, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0006] This invention provides a nano-metal-organic framework compound comprising, from the inside out, a core, a first shell layer covering the surface of the core, and a second shell layer chemically modified and covering the surface of the first shell layer. The core comprises copper-cysteine ​​nanoparticles, the first shell layer comprises 2-methylimidazolium zinc salt, and the second shell layer comprises hyaluronic acid.

[0007] This invention also provides a method for preparing a nano-metal-organic framework compound, comprising:

[0008] Copper-cysteine ​​nanoparticles were prepared by reacting copper salt with cysteine ​​hydrochloride.

[0009] 2-Methylimidazole, polyvinylpyrrolidone, and water were mixed and then mixed with a mixture containing zinc salt and copper-cysteine ​​nanoparticles to obtain copper-cysteine ​​nanoparticles coated with 2-methylimidazole zinc salt.

[0010] Hyaluronic acid was chemically modified and coated onto the surface of the 2-methylimidazolium zinc salt of the copper-cysteine ​​nanoparticles coated with 2-methylimidazolium zinc salt to prepare a nano-metal-organic framework compound.

[0011] This invention also provides nano-metal-organic framework compounds prepared by the aforementioned method.

[0012] Accordingly, embodiments of the present invention also provide the application of the aforementioned nano-metal-organic framework compounds in the preparation of oxidase mimics.

[0013] Compared with the prior art, the nano-metal-organic framework compounds of the present invention have the following advantages and significant progress:

[0014] 1) The nano-metal framework compound provided by the present invention has a core-shell-shell structure. This structure is unique and novel, and has both photosensitizing properties and good catalase and oxidase catalytic activities. Its enzyme activity is significantly increased, and it has high stability.

[0015] 2) The preparation method of the nano-metal-organic framework compound composite nanomaterials with core-shell-shell structure provided by the present invention is simple and easy to implement, low in cost, and conducive to large-scale production, thereby obtaining more types of composite nanomaterials with wider applications. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram illustrating the preparation process principle of the nano-metal-organic framework compound in Example 1 of the present invention;

[0018] Figure 2aThis is a SEM image of the ZIF-8 nanoparticles prepared in Example 1 of this invention. Figure 2b This is a TEM image of Cu-cy Nps prepared in Example 1 of the present invention. Figure 2c This is a TEM image of Cu-cyNps@ZIF-8 nanoparticles prepared in Example 1 of this invention;

[0019] Figure 3 EDS mapping image of CNZH, a nano-metal-organic framework compound prepared in Example 1 of this invention;

[0020] Figure 4 The XRD pattern of CNZH, a nano-metal-organic framework compound prepared in Example 1 of this invention;

[0021] Figure 5a This is the total peak diagram of the XPS spectrum of CNZH, a nano-metal-organic framework compound prepared in Example 1 of this invention. Figures 5b-5f The peak profiles are for Cu2p, Zn2p, C1s, O1s, and N1s, respectively.

[0022] Figure 6a , Figure 6b The images are macroscopic images of the nano-metal-organic framework compound CNZH prepared in Example 1 of this invention under natural light and 365nm ultraviolet light irradiation conditions, respectively.

[0023] Figure 7a , Figure 7b , Figure 7c The figures show the enzyme catalytic activity of the nano-metal-organic framework compound CNZH prepared in Example 1 of this invention at pH 5.0, at different concentrations, and at different pH values ​​(5.0, 6.5, 7.4).

[0024] Figure 8 This is a Western blot image of CNZH, a nano-metal-organic framework compound prepared in Example 1 of this invention.

[0025] Figure 9 The results of the CCK-8 assay for the biosafety of CNZH, a nano-metal-organic framework compound prepared in Example 1, are shown in the figure.

[0026] Figure 10 A diagram showing the therapeutic effect of the nano-metal-organic framework compound CNZH prepared in Example 1, as detected by a live / dead cell staining assay.

[0027] Figure 11 SEM images of the ZIF-8 nanoparticles obtained in Comparative Example 1;

[0028] Figure 12 SEM images of the ZIF-8 nanoparticles obtained in Comparative Example 2;

[0029] Figure 13 SEM images of the ZIF-8 nanoparticles obtained in Comparative Example 3;

[0030] Figure 14 This is a TEM image of Cu-cy Nps obtained in Comparative Example 4. Detailed Implementation

[0031] In view of the deficiencies of the prior art, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention, which mainly provides a nano-metal-organic framework compound that uses ZIF-8 (2-methylimidazolium zinc salt) as a framework material to integrate nanomaterials into a highly efficient nano-platform, providing a new idea and method for the treatment of tumors.

[0032] To facilitate understanding of this application, it will be described in more detail below. However, it should be understood that the invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. Rather, these embodiments or examples are provided to provide a thorough and complete understanding of the disclosure of this invention.

[0033] Specifically, as one aspect of the technical solution of the present invention, a novel nanomaterial, namely a nano-metal-organic framework compound, is involved, which has a core-shell-shell structure. Specifically, it includes a core arranged from the inside out, a first shell layer covering the surface of the core, and a second shell layer that is chemically modified and covered on the surface of the first shell layer. The core includes copper-cysteine ​​nanoparticles, the first shell layer includes 2-methylimidazolium zinc salt, and the second shell layer includes hyaluronic acid.

[0034] In some implementations, the core is formed by the aggregation of multiple copper-cysteine ​​nanoparticles.

[0035] Furthermore, the copper-cysteine ​​nanoparticles have a particle size of 140–170 nm.

[0036] In some implementations, the thickness of the first shell layer is 3–8 nm.

[0037] In some embodiments, the nanometal-organic framework compound comprises the following components in parts by weight: 3 to 6 parts of copper-cysteine ​​nanoparticles, 1 to 3 parts of 2-methylimidazolium zinc salt, and 2 to 7 parts of hyaluronic acid.

[0038] In some embodiments, the nanometal-organic framework compound has photosensitizing properties and enzyme catalytic activity, and has the ability to target tumor cells.

[0039] In some more specific embodiments, in the nano-metal-organic framework compound Cu-cy Nps@ZIF-8@HA (abbreviated as CNZH), Cu-cy Nps are distributed inside the nanomaterial, ZIF-8 serves as a carrier material to form the inner shell of the nanomaterial (i.e., the aforementioned first outer shell), and the outermost layer is chemically modified and coated with HA as the outer shell (i.e., the aforementioned second outer shell). Cu-cy Nps are copper-cysteine ​​nanoparticles, ZIF-8 is 2-methylimidazolium zinc salt, and HA is hyaluronic acid.

[0040] Furthermore, ZIF-8, as a framework material, exhibits pH responsiveness, which can overcome the defect of extremely short diffusion distance (<50nm) of ROS generated by photosensitizers within cells and tissues. In addition, ZIF-8 also has advantages such as excellent biocompatibility, high porosity, structural tunability, large surface area, excellent thermal stability, and superior catalytic performance. It can effectively encapsulate Cu-cy Nps within it, and its surface can also be modified with HA to give the nanomaterials better targeting capabilities.

[0041] Furthermore, the zinc salt in the nano-metal-organic framework compound is zinc nitrate.

[0042] In summary, the metal-organic framework compounds provided by this invention possess a core-shell-shell structure, which is unique and novel. This structure combines photosensitivity with excellent catalase and oxidase catalytic activities, exhibiting significantly increased enzyme activity and high stability. Furthermore, it can effectively target tumor cells, providing a new approach and method for cancer treatment.

[0043] As another aspect of the technical solution of the present invention, a method for preparing a nano-metal-organic framework compound includes:

[0044] Copper-cysteine ​​nanoparticles were prepared by reacting copper salt with cysteine ​​hydrochloride.

[0045] 2-Methylimidazole, polyvinylpyrrolidone, and water were mixed and then mixed with a mixture containing zinc salt and copper-cysteine ​​nanoparticles to obtain copper-cysteine ​​nanoparticles coated with 2-methylimidazole zinc salt.

[0046] Hyaluronic acid was chemically modified and coated onto the surface of the 2-methylimidazolium zinc salt of the copper-cysteine ​​nanoparticles coated with 2-methylimidazolium zinc salt to prepare a nano-metal-organic framework compound.

[0047] In some embodiments, the preparation method includes: mixing copper salt, cysteine ​​hydrochloride and water evenly, adjusting the pH value to 7-9, and heating to boiling to carry out the reaction, thereby obtaining copper-cysteine ​​nanoparticles (Cu-cy Nps).

[0048] In some preferred embodiments, the reaction temperature is 100–150°C and the reaction time is 30–50 min.

[0049] Furthermore, the copper salt includes, but is not limited to, copper(II) chloride dihydrate (CuCl2·2H2O).

[0050] In some preferred embodiments, the preparation method specifically includes: adjusting the pH value to 7-9 using an alkaline reagent.

[0051] In some preferred embodiments, the mass ratio of copper(II) chloride dihydrate to cysteine ​​hydrochloride is 1-3:1-4. That is, in other words, the raw materials for Cu-cy Nps, by weight, include: 1-3 parts of copper(II) chloride dihydrate (CuCl2·2H2O) and 1-4 parts of cysteine ​​hydrochloride (HSCH2CH2NH2·HCl).

[0052] In some preferred embodiments, the preparation method further includes: centrifuging and washing the prepared copper-cysteine ​​nanoparticles 1 to 4 times with a mixed solution of water and ethanol. The volume ratio of water to ethanol is 4 to 5: 3 to 4.

[0053] In some embodiments, the preparation method specifically includes:

[0054] Zinc salt, water and copper-cysteine ​​nanoparticles are mixed to form the first mixture;

[0055] 2-Methylimidazole, polyvinylpyrrolidone, and water were mixed evenly, and then the first mixture was added dropwise. After stirring evenly and standing, the mixture was centrifuged and washed with water to obtain copper-cysteine ​​nanoparticles (Cu-cy Nps@ZIF-8) coated with 2-methylimidazole zinc salt.

[0056] This invention uses ZIF-8 as the inner shell. Firstly, because nanomaterials diffuse over extremely short distances within tumor cells, effectively facilitating their entry into these cells is crucial. ZIF-8, being pH-responsive, reacts with the acidic environment of tumor cells, enabling the effective decomposition of nanomaterials within them. Secondly, ZIF-8 is easily chemically modified, allowing for further surface modification with HA to enhance the nanomaterial's targeting ability and biocompatibility.

[0057] Furthermore, the settling time is 1 to 5 hours.

[0058] Furthermore, the number of centrifugal washing cycles is 1 to 4, with each centrifugation lasting 8 to 20 minutes and the centrifugation speed being 8000 to 11000 rpm.

[0059] Furthermore, the zinc salt includes, but is not limited to, zinc nitrate hexahydrate.

[0060] In some preferred embodiments, the mass ratio of 2-methylimidazole, zinc nitrate hexahydrate, and polyvinylpyrrolidone is (18-21):(1-3):(2-6). That is, in other words, by weight, the raw materials for ZIF-8 include: 18-21 parts of 2-methylimidazole (C4H6N2), 1-3 parts of zinc nitrate hexahydrate (Zn(NO3)2·6H2O), and 2-6 parts of polyvinylpyrrolidone (C6H9NO).

[0061] In some embodiments, the preparation method specifically includes: dissolving the copper-cysteine ​​nanoparticles coated with 2-methylimidazolium zinc salt in water, adding hyaluronic acid, and stirring the reaction at room temperature for 2-6 hours to obtain the nano-metal-organic framework compound.

[0062] In some embodiments, the mass ratio of the copper-cysteine ​​nanoparticles, 2-methylimidazolium zinc salt, and hyaluronic acid is (3-6):(1-3):(2-7). That is, in other words, the nano-metal-organic framework compound comprises, by weight: 3-6 parts of copper-cysteine ​​nanoparticles (Cu-cy Nps), 1-3 parts of 2-methylimidazolium zinc salt (ZIF-8), and 2-7 parts of hyaluronic acid (HA).

[0063] In some more specific implementation examples, the preparation method of the nano-metal-organic framework compound includes the following steps:

[0064] (1) Dissolve copper(II) dihydrate (CuCl2·2H2O) in deionized water, then add cysteine ​​hydrochloride (HSCH2CH2NH2·HCl) and stir until homogeneous. Adjust the pH value to 7-9, and finally heat the solution to boiling. Centrifuge and wash 1-4 times with a mixture of water and ethanol. The resulting solution is Cu-cy Nps solution.

[0065] (2) Dissolve zinc nitrate in deionized water, then add Cu-cy Nps solution, and set the solution aside. The volume of deionized water added is 10 mL.

[0066] (3) Dissolve 2-methylimidazolium and polyvinylpyrrolidone in deionized water, then slowly add the solution prepared in step (2), stir evenly and let stand for 1 to 5 hours, centrifuge at 8000 to 11000 rpm and wash with water 1 to 4 times, each centrifugation time is 8 to 20 minutes, the precipitate obtained is Cu-cy Nps@ZIF-8, for later use, add 10 mL of deionized water;

[0067] (4) Add the Cu-cy Nps@ZIF-8 obtained in step (3) to deionized water, dissolve it completely, add HA, stir at room temperature for 2-6 hours, and obtain the nano-metal-organic framework compound after the reaction is completed.

[0068] Furthermore, in step (1), the pH value is adjusted to 7-9, and the reagent used is sodium hydroxide (NaOH).

[0069] In summary, the preparation method of the core-shell-shell structured metal-organic framework compound composite nanomaterials provided by this invention mainly utilizes the novel nanomaterial ZIF-8 loaded in Cu-cy Nps, and uses HA to chemically modify the ZIF-8 nanoparticles to impart targeting properties. The preparation method is simple, environmentally friendly, easy to implement, low in cost, and conducive to large-scale production, thereby obtaining more types of composite nanomaterials with wider applications.

[0070] As another aspect of the technical solution of the present invention, it also relates to nano-metal-organic framework compounds prepared by the aforementioned preparation method.

[0071] Accordingly, as another aspect of the technical solution of the present invention, it also relates to the application of the nano-metal-organic framework compound in the preparation of oxidase mimics.

[0072] Furthermore, the oxidase includes catalase.

[0073] Furthermore, the metal-organic framework compounds provided by this invention were characterized by SEM, TEM, and XRD, showing that the prepared nanomaterials were successfully prepared with a particle size of approximately 250 nm and exhibited photosensitizing properties. TMB experiments demonstrated the enzymatic catalytic activity of the nanomaterials. CCK-8 assays proved the good biocompatibility of the nanomaterials. Western blotting experiments verified the targeting ability of the nanomaterials, meeting the standards for biomedical applications.

[0074] The present invention is further illustrated by the following embodiments: The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the present invention as described in detail in the claims.

[0075] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment and testing methods used in the following embodiments are all known in the art.

[0076] Example 1

[0077] like Figure 1As shown, the nanomaterial provided in this embodiment has a core-shell-shell structure. Specifically, the structure of the nanomaterial includes: Cu-cy Nps distributed inside the nanomaterial, ZIF-8 as a carrier material forming the inner shell of the nanomaterial, and the outermost layer coated with HA through chemical modification as the outer shell, wherein Cu-cy Nps are copper-cysteine ​​nanoparticles, ZIF-8 is 2-methylimidazolium zinc salt, and HA is the nanomaterial metal-organic framework compound hyaluronic acid.

[0078] Specifically, the synthesis method and characterization of nano-metal-organic framework compounds will be described below.

[0079] I. Raw materials for preparation (all commercially available)

[0080] Copper(II) chloride dihydrate (CuCl2·2H2O), cysteine ​​hydrochloride (HSCH2CH2NH2·HCl), sodium hydroxide (NaOH), 2-methylimidazolium (C4H6N2), zinc nitrate hexahydrate (Zn(NO3)2·6H2O), polyvinylpyrrolidone (C6H9NO), hyaluronic acid, anhydrous ethanol, and deionized water.

[0081] II. Preparation methods of nanomaterials

[0082] 2.1 The preparation method of Cu-cy Nps includes the following steps:

[0083] Weigh 0.460 g of copper(II) chloride dihydrate (CuCl2·2H2O) into a beaker, add 20 ml of deionized water, and place the beaker on a magnetic stirrer (500 rpm / min). After dissolving completely, add 0.636 g of cysteine ​​hydrochloride. Once the solution is thoroughly mixed, adjust the pH to 8 (by adding NaOH solution). The solution will gradually turn black. Stir at room temperature for 1.5 h, then heat to boiling and react at 120 °C for 40 min. Stop the reaction when crystalline substances are seen at the bottom of the beaker. Discard the solution and add a solution of deionized water and ethanol in a 5:4 volume ratio. Sonicate thoroughly, then centrifuge and wash three times (11000 rpm × 10 min). Finally, add the above mixture of deionized water and ethanol to obtain the Cu-cy Nps solution, which should be stored at 4 °C.

[0084] 2.2 The preparation method of ZIF-8 includes the following steps:

[0085] 1.94 g of 2-methylimidazole (2-MIM) and 375.2 mg of polyvinylpyrrolidone (PVP) were weighed into a flask using an electronic balance. 10 ml of deionized water was added, and the mixture was stirred until fully dissolved to obtain solution A. 100 mg of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was then weighed into a centrifuge tube, and 10 ml of deionized water was added. The mixture was ultrasonically dispersed to obtain solution B. Solution B was slowly added dropwise to solution A while stirring. After 10 minutes, the round-bottom flask was placed in a cool, dry place and allowed to stand for 3 hours. The flask was then centrifuged and washed three times with water (11000 rpm × 10 min) to obtain the nanomaterial carrier ZIF-8, which was stored at 4℃.

[0086] 2.3 The preparation method of Cu-cy Nps@ZIF-8(CNZ) includes the following steps:

[0087] First, obtain solution A using the method in step 2.2. Then, weigh 100 mg of Zn(NO3)2·6H2O and place it in a centrifuge tube, add 10 ml of deionized water, and sonicate the sample until the solution becomes transparent. Add 4 ml of Cu-cy Nps solution and sonicate for 5 min to obtain solution C. Finally, slowly add solution C dropwise to solution A while stirring. After the addition is complete, stir for another 10 min. Transfer the flask to a cool, dry place and let it stand for 3 h. Finally, collect the solution, centrifuge and wash with water 3 times (11000 rpm × 10 min), and sonicate to mix to obtain Cu-cy Nps@ZIF-8. Store at 4℃.

[0088] The preparation method of 2.4Cu-cy Nps@ZIF-8@HA(CNZH) includes the following steps:

[0089] Weigh 4 mg of hyaluronic acid (HA) into a round-bottom flask, add 9 ml of deionized water, and stir with a magnetic stirrer (500 rpm / min) until HA is completely dissolved. Then add 1 ml of CNZ solution and continue stirring for 3 h. Collect the liquid, centrifuge, and then wash once with water (11000 rpm × 10 min) to obtain the nanomaterial CNZH. Store at 4℃.

[0090] III. Characterization

[0091] 3.1 Characterization of ZIF-8 by SEM

[0092] Take 1 ml of ZIF-8 aqueous solution, dilute it 10 times with deionized water, and sonicate it for 1 hour. Drop the fully dispersed ZIF-8 suspension onto a circular glass slide, dry it in an oven at 60°C, and then test its morphology.

[0093] 3.2 TEM characterization of Cu-cy Nps and Cu-cy Nps@ZIF-8

[0094] Take 1 ml of Cu-cy Nps and Cu-cy Nps@ZIF-8 aqueous solution, dilute with deionized water 10 times, disperse by ultrasonication for 1 h, drop onto copper grid and air dry naturally, and then test the morphology by TEM.

[0095] 3.3 Nanomaterials CNZH for EDS mapping image detection

[0096] CNZH was freeze-dried, and 1 ml of the powder was taken for testing.

[0097] 3.4 XRD Detection of Nanomaterials by CNZH

[0098] The sample preparation method is the same as in 3.3.

[0099] XPS spectral analysis of 3.5 nm materials using CNZH.

[0100] The sample preparation method is the same as in 3.3.

[0101] Photosensitivity of 3.6 nm CNZH material

[0102] Take 32.5 ml of deionized water and CNZH solution respectively and put them into centrifuge tubes of the same specification. Then take pictures and record them under natural light and 365 nm ultraviolet light irradiation conditions respectively.

[0103] Detection of enzyme-like activity of TMB catalyzed by 3.7 nanomaterials

[0104] Using 3,3′,5,5′-tetramethylbenzidine as an indicator, when the nano-metal-organic framework compound CNZH has peroxidase activity, it can promote the oxidation of 3,3′,5,5′-tetramethylbenzidine, thereby causing a color change and a significant enhancement of the absorption peak at 652 nm.

[0105] 3.7.1 Enzymatic catalytic activity of TMB at pH 5.0

[0106] Take 200 μg / mL of Cu-cy Nps@ZIF-8, ZIF-8 and Cu-cy Nps respectively, add TMB 0.1M and H2O2 50mM, and record the characteristic absorption spectra of ox TMB using UV-Vis absorption spectroscopy.

[0107] 3.7.2 Enzyme catalytic activity of different concentrations of Cu-cy Nps@ZIF-8

[0108] The characteristic absorption spectra of ox TMB were recorded by UV-Vis absorption spectroscopy using Cu-cyNps@ZIF-8 at concentrations of 0, 10, 25, 50, 100, 200, 300, 400, and 500 μg / mL.

[0109] 3.7.3 Enzymatic catalytic activity of Cu-cy Nps@ZIF-8 at different pH values ​​for TMB

[0110] Three portions of Cu-cy Nps@ZIF-8 with a concentration of 200 μg / mL were taken, and the pH was adjusted to 5.0, 6.5 and 7.4 respectively. The characteristic absorption spectra of ox TMB were recorded by UV-Vis absorption spectroscopy.

[0111] 3.8 Detection of the targeting of CNZH nanomaterials

[0112] Western blot analysis was performed to detect the targeting of CNZH (100 μg / mL) to A-431 cells. First, cells were lysed using whole-cell lysate, and all proteins were separated by SDS-PAGE (10% gel). The lysate was then transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with rapid blocking buffer for 20 min, washed once with TBST buffer containing Tween 20, and then incubated overnight with the primary polyclonal antibody CD44, followed by incubation with a secondary antibody at room temperature for 2 h. Finally, antibody binding was detected using an infrared imaging system (Odyssey CLX).

[0113] 3.9 CCK-8 assay for the biocompatibility of CNZH in nanomaterials

[0114] First, CNZH was diluted to specific concentrations (0, 25, 50, 100, 150, 200 μg / mL) using DMEM medium containing 10% FBS. Mouse fibroblasts (L-929) and normal human lung epithelial cells (BEAS) were then cultured at a concentration of 1×10⁻⁶. 4 Cells were initially seeded at a density of cells / mL in 96-well plates and cultured in an incubator. After cell attachment, the cells were incubated with different concentrations of fresh DMEM samples for 24 h. Then, fresh cell counting kit (CCK-8) solution was added and incubated for 3 h. Cell viability was measured at 450 nm absorbance.

[0115] 3.10 Live / dead cell staining assay to detect the therapeutic effect of CNZH nanomaterial.

[0116] Human epidermal cancer cell A-431 (1×10⁻⁶) 4Cells / mL were seeded into 96-well plates and incubated overnight. In different groups, after co-culturing with 100 μg / mL CNZ and CNZH for 3 h, the light group was irradiated with 365 nm UV light for 10 min, and then cultured for another 3 h. The supernatant in the 96-well plates was replaced with 100 μL of calcein-AM / PI (KeyGen) solution for staining live / dead cells. After co-incubation for 45 min, the experiment was observed under an inverted fluorescence microscope (Olympus IX 73) using different colors.

[0117] IV. Analysis of Characterization Results

[0118] 4.1 Preparation and Characterization of Nanomaterials

[0119] Figures 2a-2c These are the morphological characteristics of nanoparticles. Among them, Figure 2a ZIF-8 SEM image, Figure 2b TEM image of Cu-cyNps Figure 2c TEM image of Cu-cy Nps@ZIF-8. (e.g.) Figure 2a As shown, scanning electron microscopy revealed that the average size of the ZIF-8 nanoparticles was approximately 250 nm, and they were uniformly distributed. Transmission electron microscopy was used to further analyze the nanoparticles. Figure 2b It can be seen that the average size of Cu-cy Nps is approximately 150 nm, which are uniform spherical shapes. Figure 2c The displayed Cu-cy Nps@ZIF-8 nanoparticles have an average size of approximately 250 nm. For example... Figure 3 As shown, the EDS mapping image of the nanomaterial shows the uniform distribution of various elements through different colors (carbon red, nitrogen orange, oxygen green, zinc blue and copper violet), indicating that ZIF-8 was successfully loaded in Cu-cy Np. Finally, HA was modified on the surface of the framework to make the nanomaterial CNZ targeted, thus obtaining the nanomaterial CNZH. Figures 5a-5f The XPS spectrum of CNZH nanomaterials is shown. Figure 5a In the overall peak diagram, Cu2p, Zn2p, N1s, and C1s atoms can be observed. Figure 5b In the figure, Cu2p has two pairs of peaks, among which 932.73 eV (Cu2p) has the highest peak. 3 / 2 ) and 952.33eV (Cu 2p 1 / 2 ) represents Cu + Of which 934.97 eV (Cu 2p 3 / 2 ) and 953.68eV (Cu 2p 1 / 2 ) represents Cu 2+ This indicates that Cu in CNZH exists in two oxidation states, +1 and +2. Figure 5cAs can be seen, there are two peaks in Zn 2p, at 1021.7 eV and 1044.7 eV, representing Zn 2p. 3 / 2 and Zn 2p 1 / 2 .exist Figures 5d-5f Characteristic peaks for C, O, and N can be observed in the image. Figure 4 The XRD pattern of the nanomaterial CNZH shows diffraction angles of 10.38°, 12.71°, 18.01°, 24.51°, and 29.6°, corresponding to crystal structures (002), (112), (022), (222), (233), and (044), which is consistent with the Zif-8 standard card. In summary, this invention successfully prepared the nanomaterial CNZH.

[0120] 4.2 Photosensitive properties of nanomaterials

[0121] Figure 6a and Figure 6b This is a macroscopic image of the nanomaterial CNZH. (Example) Figure 6a As shown, under natural light conditions, the solution of the nanomaterial CNZH is gray, while under 365nm ultraviolet light irradiation (such as...), the solution is gray. Figure 6b If the fluorescence is bright orange-yellow, it indicates that CNZH has photosensitizing properties, and the coating of HA does not affect its performance.

[0122] 4.3 Enzyme catalytic activity of nanomaterials

[0123] like Figures 7a-7c As shown, TMB was used as an indicator to study its peroxidase-like activity. Figure 7a The enzyme catalytic activity at pH 5.0 is [value missing]. Figure 7a As can be seen, the absorbance of Cu-cy Nps and CNZ at 652 nm and 895 nm are consistent, and are 1.5 times that of ZIF-8, indicating that Cu-cy Nps has good POD enzyme activity. Furthermore, encapsulating Cu-cy Nps in ZIF-8 significantly improves its POD enzyme catalytic activity compared to ZIF-8 alone. Figure 7b To represent the catalytic activity of enzymes at different concentrations, in Figure 7b As the concentration of CNZ decreases, the absorbance of ox TMB at 652 nm and 895 nm gradually decreases. Even when the concentration of CNZ is 10 μg / mL, the absorbance of ox TMB can still be observed, indicating that CNZ has excellent POD enzyme activity. Figure 7cTo measure the enzyme catalytic activity at different pH values ​​(5.0, 6.5, 7.4), it can be seen that as the pH value increases, the absorbance of ox TMB gradually decreases at 652 nm and 895 nm, indicating that the POD enzyme activity is strongest in an acidic environment. This is consistent with the tumor microenvironment and is conducive to CNZ exerting catalytic activity in tumor cells.

[0124] Targeting of 4.4 nanometer materials

[0125] Figure 8 This is a Western blot image of the nanomaterial CNZH. The targeting effect of HA was verified by immunoblotting. Figure 8 As can be seen, CD44+ expression gradually decreases with increasing CNZH concentration, indicating that the CD44 antibody in the HA modified on the CNZH surface effectively binds to the CD44 receptor on the surface of tumor cells. This provides a guarantee for nanomaterials to reach the tumor site more precisely and endows the nanomaterial CNZH with targeting properties.

[0126] Biosafety of 4.5 nanometer materials

[0127] The biosafety of CNZH was verified using the CCK-8 assay, such as... Figure 9 As shown, after co-incubating the cells with different concentrations of CNZ and CNZH for 24 hours, even at a relatively high concentration (200 μg / mL), the survival rate of BEAS and L-929 cells could still reach over 90%. The nanomaterial CNZH has good biosafety and is suitable for application in the biomedical field.

[0128] The therapeutic effects of 4.6 nanometer materials

[0129] like Figure 10 The image shows the therapeutic effect of the CNZH nanomaterial detected by a live / dead cell staining assay. The Calcein-AM / PI double staining kit was used to visually represent the live / dead cell status. Cell death was negligible in the control group (Ctrl) and the 365nm UV irradiation group (L365). The CNZ group showed a slight increase in red signal (dead cells) due to the production of a small amount of ·OH caused by CDT. The CNZ+365nm UV irradiation group (LCNZ group) showed a significant increase in red signal, which is attributed to the PDT effect triggered by the photosensitizer under 365nm UV irradiation. Because the CNZ nanomaterial is modified with HA, it can effectively target the CD44 receptor on the surface of tumor cells; therefore, relatively obvious dead cells (red signal) were also observed in the CNZH group. Most notably, the CNZH+365nm UV irradiation group (LCNZH) showed approximately 20% surviving cells (green signal). This indicates that the nanomaterial has good tumor-killing activity and is suitable for tumor treatment.

[0130] Example 2

[0131] Methods for preparing nanomaterials (metal-organic frameworks) include:

[0132] 2.1 The preparation method of Cu-cy Nps includes the following steps:

[0133] Weigh 0.50 g of copper(II) chloride dihydrate (CuCl2·2H2O) into a beaker, add 20 ml of deionized water, and place the beaker on a magnetic stirrer (500 rpm / min). After dissolving completely, add 0.7 g of cysteine ​​hydrochloride. Once the solution is thoroughly mixed, adjust the pH to 7.35 (by adding NaOH solution). The solution will gradually turn black. Stir at room temperature for 1.5 h, then heat to boiling and react at 100 °C for 50 min. Stop the reaction when crystalline substances are seen at the bottom of the beaker. Discard the solution and add a solution of deionized water and ethanol in a 5:4 volume ratio. Sonicate thoroughly, then centrifuge and wash once (10000 rpm × 8 min). Finally, add the above mixture of deionized water and ethanol to obtain the Cu-cy Nps solution, which should be stored at 4 °C.

[0134] 2.2 The preparation method of ZIF-8 includes the following steps:

[0135] 4g of 2-methylimidazole (2-MIM) and 700mg of polyvinylpyrrolidone (PVP) were weighed into a flask using an electronic balance. 15ml of deionized water was added, and the mixture was stirred until fully dissolved to obtain solution A. 200mg of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was then weighed into a centrifuge tube, and 10ml of deionized water was added. The mixture was ultrasonically dispersed to obtain solution B. Solution B was slowly added dropwise to solution A while stirring. After 20 minutes, the round-bottom flask was placed in a cool, dry place and allowed to stand for 2 hours. The flask was then centrifuged and washed four times with water (10000rpm × 8min) to obtain the nanomaterial carrier ZIF-8, which was stored at 4℃.

[0136] 2.3 The preparation method of Cu-cy Nps@ZIF-8(CNZ) includes the following steps:

[0137] First, obtain solution A using the method in step 2.2. Then, weigh 200 mg of Zn(NO3)2·6H2O and place it in a centrifuge tube, add 10 ml of deionized water, and sonicate the sample until the solution becomes transparent. Add 4 ml of Cu-cy Nps solution and sonicate for 10 min to obtain solution C. Finally, slowly add solution C dropwise to solution A while stirring. After the addition is complete, stir for another 20 min. Transfer the flask to a cool, dry place and let it stand for 2 h. Finally, collect the solution, centrifuge and wash with water 4 times (10000 rpm × 8 min), and sonicate to mix thoroughly to obtain Cu-cy Nps@ZIF-8. Store at 4℃.

[0138] The preparation method of 2.4Cu-cy Nps@ZIF-8@HA(CNZH) includes the following steps:

[0139] Weigh 8 mg of hyaluronic acid (HA) into a round-bottom flask, add 9 ml of deionized water, and stir with a magnetic stirrer (500 rpm / min) until HA is completely dissolved. Then add 1 ml of CNZ solution and continue stirring for 2 h. Collect the liquid, centrifuge, and then wash once with water (11000 rpm × 10 min) to obtain the nanomaterial CNZH. Store at 4℃.

[0140] Example 3

[0141] Methods for preparing nanomaterials (metal-organic frameworks) include:

[0142] 2.1 The preparation method of Cu-cy Nps includes the following steps:

[0143] Weigh 0.30 g of copper(II) chloride dihydrate (CuCl2·2H2O) into a beaker, add 20 ml of deionized water, and place the beaker on a magnetic stirrer (500 rpm / min). After dissolving completely, add 0.4 g of cysteine ​​hydrochloride. Once the solution is thoroughly mixed, adjust the pH to 8.5 (by adding NaOH solution). The solution will gradually turn black. Stir at room temperature for 1.5 h, then heat to boiling and react at 150 °C for 30 min. Stop the reaction when crystalline substances are seen at the bottom of the beaker. Discard the solution and add a solution of deionized water and ethanol in a 5:4 volume ratio. Sonicate thoroughly, then centrifuge and wash four times (8000 rpm × 20 min). Finally, add the above mixture of deionized water and ethanol to obtain the Cu-cy Nps solution, which should be stored at 4 °C.

[0144] 2.2 The preparation method of ZIF-8 includes the following steps:

[0145] 2.10 g of 2-methylimidazole (2-MIM) and 400 mg of polyvinylpyrrolidone (PVP) were weighed into a flask using an electronic balance. 10 ml of deionized water was added, and the mixture was stirred until fully dissolved to obtain solution A. 100 mg of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was then weighed into a centrifuge tube, and 10 ml of deionized water was added. The mixture was ultrasonically dispersed to obtain solution B. Solution B was slowly added dropwise to solution A while stirring. After 5 minutes, the round-bottom flask was placed in a cool, dry place and allowed to stand for 4 hours. The flask was then centrifuged and washed twice with water (8000 rpm × 20 min) to obtain the nanomaterial carrier ZIF-8, which was stored at 4℃.

[0146] 2.3 The preparation method of Cu-cy Nps@ZIF-8(CNZ) includes the following steps:

[0147] First, obtain solution A using the method in step 2.2. Then, weigh 100 mg of Zn(NO3)2·6H2O and place it in a centrifuge tube, add 10 ml of deionized water, and sonicate the sample until the solution becomes transparent. Add 4 ml of Cu-cy Nps solution and sonicate for 10 min to obtain solution C. Finally, slowly add solution C dropwise to solution A while stirring. After the addition is complete, stir for another 5 min. Transfer the flask to a cool, dry place and let it stand for 4 h. Finally, collect the solution, centrifuge and wash once with water (8000 rpm × 20 min), and sonicate to mix to obtain Cu-cy Nps@ZIF-8. Store at 4℃.

[0148] The preparation method of 2.4Cu-cy Nps@ZIF-8@HA(CNZH) includes the following steps:

[0149] Weigh 2 mg of hyaluronic acid (HA) into a round-bottom flask, add 9 ml of deionized water, and stir with a magnetic stirrer (500 rpm / min) until HA is completely dissolved. Then add 1 ml of CNZ solution and continue stirring for 4 h. Collect the liquid, centrifuge, and then wash once with water (11000 rpm × 10 min) to obtain the nanomaterial CNZH. Store at 4℃.

[0150] Tests showed that Examples 2 and 3 also yielded nano-metal-organic framework compounds with photosensitizing properties and enzyme catalytic activity, which can effectively target tumor cells.

[0151] Comparative Example 1

[0152] The difference between this comparative example and Example 1 is as follows:

[0153] 2.2 The preparation method of ZIF-8 includes the following steps:

[0154] ZIF-8 was prepared using methanol as a solvent. The specific method is as follows: 218.9 mg of 2-methylimidazole (2-MIM) and 352 mg of polyvinylpyrrolidone (PVP) were placed in a flask, and 10 ml of methanol was added. The mixture was stirred until fully dissolved. Then, 100 mg of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and 10 ml of methanol solution were added. The solution was stirred at 550 rpm / min for 2 hours. Finally, the solution was washed twice by centrifugation (11000 rpm × 10 min) with methanol. The ZIF-8 nanoparticles obtained by this method were few in quantity, hexagonal in shape, varied in size, and heavily aggregated, with a particle size of approximately 150 nm. Figure 11 As shown.

[0155] Comparative Example 2

[0156] The difference between this comparative example and Example 1 is as follows:

[0157] 2.2 The preparation method of ZIF-8 includes the following steps:

[0158] Nanoparticles were prepared by replacing the solution with deionized water. 1.94 g of 2-methylimidazole (2-MIM), 375.2 mg of polyvinylpyrrolidone (PVP), and 100 mg of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) were directly mixed in a flask, and 10 ml of deionized water was added. The mixture was stirred at 500 rpm for 10 min to ensure complete dissolution. The solution was then allowed to stand in a cool, dry place for 3 h, and finally centrifuged (11000 rpm × 10 min) and washed three times with water. Although the yield was abundant, the morphology was irregular, and some aggregation still occurred. The particle size was approximately 150-250 nm. The microstructure of ZIF-8 is as follows: Figure 12 As shown.

[0159] Comparative Example 3

[0160] The difference between this comparative example and Example 1 is as follows:

[0161] 2.2 The preparation method of ZIF-8 includes the following steps:

[0162] To further address the adhesion issue, the content of polyvinylpyrrolidone (PVP) was reduced, and the zinc salt and framework material were thoroughly dissolved separately. 100 mg of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was added to 1 ml of deionized water and sonicated until homogeneous. 1.94 g of 2-methylimidazole (2-MIM) and 375.2 mg of polyvinylpyrrolidone (PVP) were added to 9 ml of deionized water and stirred at 500 rpm / min until fully mixed. The zinc salt solution was then added, and stirring was stopped after 10 minutes. The mixture was allowed to stand in a cool, dry place for 3 hours, and finally centrifuged (11000 rpm × 10 min) and washed three times with water. The resulting ZIF-8 nanoparticles were abundant, exhibiting a regular hexagonal morphology with slight adhesion, and a particle size of approximately 120-180 nm. The microstructure is as follows: Figure 13 As shown. This is because the volume of deionized water added in this comparative example changed. Due to the difference in solution volume, the reaction may be incomplete, causing adhesion.

[0163] Comparative Example 4

[0164] The difference between this comparative example and Example 1 is that:

[0165] 2.1 The preparation method of Cu-cy Nps includes the following steps:

[0166] Weigh 0.460 g of copper(II) chloride dihydrate (CuCl2·2H2O) into a beaker, add 20 ml of deionized water, and place the beaker on a magnetic stirrer (500 rpm / min). After complete dissolution, add 0.636 g of cysteine ​​hydrochloride. Once the solution is thoroughly mixed, adjust the pH to 8 (by adding NaOH solution). The solution will gradually turn black. Stir at room temperature for 2-3 hours, then heat to 120°C until boiling for 1 hour. The reaction stops when crystalline substances appear at the bottom of the beaker. Discard the solution and add a solution of deionized water and ethanol in a 5:4 volume ratio. Sonicate thoroughly, then centrifuge and wash three times (11000 rpm × 10 min). Finally, add the above mixture of deionized water and ethanol to obtain the Cu-cy Nps solution. Please refer to [link to relevant documentation]. Figure 14 TEM observation showed that the obtained Cu-cyNps nanoparticles were spherical. Due to the excessive stirring time, the diameter was about 250-300 nm. The excessively large particle size was not conducive to further encapsulation.

[0167] Comparative Example 5

[0168] The difference between this comparative example and Example 1 is that the chemical modification of HA in step 2.4 was not performed.

[0169] The results showed that the targeting ability of unmodified HA nanoparticles was poor. For example... Figure 8As shown, HA modified on the surface of nanomaterials can specifically bind to CD44 receptors in tumor cells, thereby enabling nanoparticles to bind more precisely to tumor cells, improving the targeting of nanomaterials and reducing damage to normal cells during treatment. At the same time, HA, as a widely available natural polysaccharide, improves the biosafety of nanomaterials, making them more suitable for applications in the biomedical field.

[0170] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0171] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. The application of a nano-metal-organic framework compound in the preparation of an oxidase mimic, wherein the oxidase is catalase, and the nano-metal-organic framework compound comprises, from the inside out, a core, a first shell coating the surface of the core, and a chemically modified second shell coating the surface of the first shell. The core is formed by the aggregation of multiple copper-cysteine ​​nanoparticles. The first shell is a 2-methylimidazolium zinc salt shell that responds to the acidic environment of tumor cells, and the second shell is a hyaluronic acid shell for specifically binding to the CD44 receptor on the surface of tumor cells. The nano-metal-organic framework compound comprises, by weight, the following components: 3-6 parts of copper-cysteine ​​nanoparticles, 1-3 parts of 2-methylimidazolium zinc salt, and 2-7 parts of hyaluronic acid. The nano-metal-organic framework compound has photosensitizing properties and enzyme catalytic activity, and has the function of targeting tumor cells. The particle size of the copper-cysteine ​​nanoparticles is 140-170 nm, and the thickness of the first shell is 3-8 nm.

2. The application according to claim 1, characterized in that, The preparation method of the nano-metal-organic framework compound includes: Copper salt, cysteine ​​hydrochloride and water are mixed evenly, the pH value is adjusted to 7-9, and the mixture is heated to boiling to prepare copper-cysteine ​​nanoparticles. The reaction temperature is 100-150℃ and the reaction time is 30-50 min. 2-Methylimidazole, polyvinylpyrrolidone, and water are mixed and then mixed with a mixture containing zinc salt and copper-cysteine ​​nanoparticles to obtain copper-cysteine ​​nanoparticles coated with 2-methylimidazole zinc salt. The 2-methylimidazole zinc salt can be responsively decomposed in the acidic environment of tumor cells. Hyaluronic acid was chemically modified and coated onto the surface of the 2-methylimidazolium zinc salt of the copper-cysteine ​​nanoparticles coated with 2-methylimidazolium zinc salt to prepare a metal-organic framework nanoparticle. The hyaluronic acid was used to specifically bind to the CD44 receptor on the surface of tumor cells. The mass ratio of the copper-cysteine ​​nanoparticles, 2-methylimidazolium zinc salt and hyaluronic acid was (3~6):(1~3):(2~7).

3. The application according to claim 2, characterized in that: The copper salt is copper(II) dihydrate.

4. The application according to claim 2, characterized in that... include: The pH value is adjusted to 7-9 using an alkaline reagent, wherein the alkaline reagent is sodium hydroxide.

5. The application according to claim 3, characterized in that: The mass ratio of copper(II) dihydrate to cysteine ​​hydrochloride is 1~3:1~4.

6. The application according to claim 2, characterized in that... Also includes: The prepared copper-cysteine ​​nanoparticles were centrifuged and washed 1 to 4 times with a mixed solution of water and ethanol, wherein the volume ratio of water to ethanol was 4 to 5: 3 to 4.

7. The application according to claim 2, characterized in that, include: Zinc salt, water and copper-cysteine ​​nanoparticles are mixed to form the first mixture; 2-Methylimidazole, polyvinylpyrrolidone, and water were mixed evenly, and then the first mixture was added dropwise. After stirring evenly and standing, the mixture was centrifuged and washed with water to obtain copper-cysteine ​​nanoparticles coated with 2-methylimidazole zinc salt.

8. The application according to claim 7, characterized in that: The settling time is 1 to 5 hours.

9. The application according to claim 7, characterized in that: The number of centrifugal washes is 1 to 4, each centrifugation lasts 8 to 20 minutes, and the centrifugation speed is 8000 to 11000 rpm.

10. The application according to claim 2, characterized in that: The zinc salt is zinc nitrate hexahydrate.

11. The application according to claim 10, characterized in that: The mass ratio of 2-methylimidazole, zinc nitrate hexahydrate and polyvinylpyrrolidone is (18~21):(1~3):(2~6).

12. The application according to claim 2, characterized in that, include: The copper-cysteine ​​nanoparticles coated with 2-methylimidazolium zinc salt were dissolved in water and then hyaluronic acid was added. The mixture was stirred at room temperature for 2-6 hours to obtain the nano-metal-organic framework compound.

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