Nano metal organic framework compound as well as preparation method and application thereof

Abstract

The invention discloses a nano metal organic framework compound as well as a preparation method and application thereof. The nano metal organic framework compound comprises an inner core, a first shell layer and a second shell layer which are sequentially arranged from inside to outside, the surface of the inner core is coated with the first shell layer, the surface of the first shell layer is chemically modified and coated with the second shell layer, the inner core comprises copper-cysteamine nano-particles, the first shell layer comprises 2-methylimidazole zinc salt, and the second shell layer comprises copper-cysteamine nano-particles. The second shell layer comprises hyaluronic acid. The nano metal organic framework compound material provided by the invention has a photosensitive characteristic and enzyme catalytic activity, and can effectively target tumor cells. And the whole preparation process is simple and environment-friendly, large-scale production is facilitated, and a new thought and method are provided for cancer treatment.

Description

Technical Field

[0001] The present invention relates to a nano metal organic framework compound, in particular to a nano metal organic framework compound with a core-shell-shell structure and a preparation method and application thereof, belonging to the technical field of nano biomedicine. Background Art

[0002] Tumors are still a major disease that seriously threatens human health worldwide. According to data from the International Agency for Research on Cancer (IARC), it is estimated that by 2040, the number of new cancer cases worldwide will reach 29.5 million. According to data from the National Cancer Center in 2022, there are about 4.06 million new cancer cases in China each year, and the incidence trend is getting younger, posing a serious threat to the health of the people. Treatment methods mainly include surgery, chemotherapy, radiotherapy, and immunotherapy, but there are disadvantages such as intolerance by patients, large trauma, serious systemic side effects, damage to surrounding normal tissues, and immune-related adverse reactions. Therefore, it is very necessary to develop new and effective means to treat tumors.

[0003] With the vigorous development of nanotechnology, the use of nanomaterials in tumor treatment has also entered the field of vision of scientific researchers. Nanomaterials can be used as a multifunctional platform to combine chemotherapy, photothermal therapy and photodynamic therapy to play a synergistic therapeutic role. They can also be connected to specific targeting molecules through surface modification to achieve precise attack on tumor cells and increase local drug concentration, thereby enhancing the therapeutic effect; in addition, they can also reduce damage to normal tissues by targeting tumor tissues. Metal organic framework compounds, namely MOFs, are compounds that contain both inorganic and organic functions. They have good biocompatibility, high porosity, structural adjustability, large surface area and excellent catalytic performance. They have broad application prospects in the fields of ion exchange, small molecule storage, heterogeneous catalysis, luminescence, and magnetism. Among them, ZIF-8 metal organic framework compounds have the advantages of simple preparation method and large specific surface area and are widely used, but the disadvantages of existing compounds are limited crystallization ability and poor conductivity. Summary of the invention

[0004] The main purpose of the present invention is to provide a nano metal organic framework compound and a preparation method and application thereof, so as to overcome the deficiencies in the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0006] An embodiment of the present invention provides a nano metal organic framework compound, which includes an inner core, a first shell layer coated on the surface of the inner core, and a second shell layer chemically modified and coated on the surface of the first shell layer, wherein the inner core includes copper-cysteamine nanoparticles, the first shell layer includes 2-methylimidazole zinc salt, and the second shell layer includes hyaluronic acid.

[0007] The present invention also provides a method for preparing a nano-metal organic framework compound, which comprises:

[0008] reacting a copper salt with cysteamine hydrochloride to prepare copper-cysteamine nanoparticles;

[0009] Mixing 2-methylimidazole, polyvinyl pyrrolidone and water, and mixing the mixture with a mixed solution containing zinc salt and copper-cysteamine nanoparticles to prepare copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt;

[0010] The hyaluronic acid is chemically modified and coated on the 2-methylimidazole zinc salt surface of the copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt to prepare a nano metal organic framework compound.

[0011] The embodiment of the present invention also provides a nano metal organic framework compound prepared by the above preparation method.

[0012] Correspondingly, the embodiments of the present invention also provide the use of the aforementioned nano metal organic framework compound in the preparation of oxidase mimics.

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

[0014] 1) The nanometal framework compound provided by the present invention has a core-shell-shell structure, which is unique and novel, has both photosensitivity and good catalase and oxidase catalytic activity, its enzyme activity is significantly increased, and has the characteristics of high stability;

[0015] 2) The preparation method of the nano-metal organic framework compound composite nanomaterial with a 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 broader applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is a schematic diagram of 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 the present invention. Figure 2b TEM image of Cu-cy Nps prepared in Example 1 of the present invention, Figure 2c TEM image of Cu-cyNps@ZIF-8 nanoparticles prepared in Example 1 of the present invention;

[0019] Figure 3 This is an EDS mapping image of the nano-metal organic framework compound CNZH prepared in Example 1 of the present invention;

[0020] Figure 4 The XRD spectrum of the nano metal organic framework compound CNZH prepared in Example 1 of the present invention;

[0021] Figure 5a This is the total peak diagram of the XPS spectrum of the nano metal organic framework compound CNZH prepared in Example 1 of the present invention, Figure 5b-5f They are the peak separation diagrams of Cu2p, Zn2p, C1s, O1s and N1s respectively;

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

[0023] Figure 7a , Figure 7b , Figure 7c The enzymatic activity of the nano-metal organic framework compound CNZH prepared in Example 1 of the present invention at pH 5.0, the enzymatic activity at different concentrations, and the enzymatic activity at different pH values ​​(5.0, 6.5, 7.4) are shown respectively;

[0024] Figure 8 This is a Western blot image of the nano metal organic framework compound CNZH prepared in Example 1 of the present invention;

[0025] Fig. 9 This is a graph showing the biosafety results of the nano-metal organic framework compound CNZH prepared in Example 1 tested by the CCK-8 method;

[0026] Fig.10 This is a diagram showing the therapeutic effect of the nano-metal organic framework compound CNZH prepared in Example 1 by live / dead cell staining experiments;

[0027] Fig.11 is the SEM image of ZIF-8 nanoparticles obtained in Comparative Example 1;

[0028] Fig.12 is the SEM image of ZIF-8 nanoparticles obtained in Comparative Example 2;

[0029] Fig.13 is the SEM image of ZIF-8 nanoparticles obtained in Comparative Example 3;

[0030] Fig.14 This is the TEM image of Cu-cy Nps obtained in Comparative Example 4. DETAILED DESCRIPTION

[0031] In view of the defects of the prior art, the inventors of this case have proposed the technical solution of the present invention after long-term research and extensive practice. The main purpose is to provide a nano metal organic framework compound, which uses ZIF-8 (2-methylimidazole zinc salt) as a skeleton material to integrate nanomaterials into an efficient nano platform, providing a new idea and method for the treatment of tumors.

[0032] For ease of understanding of the present application, the present application will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0033] Specifically, as one aspect of the technical solution of the present invention, a new type of nanomaterial, namely a nano metal organic framework compound, is presenting a core-shell-shell structure, specifically including an inner core arranged in sequence from the inside to the outside, a first shell layer coated on the surface of the inner core, and a second shell layer chemically modified and coated on the surface of the first shell layer, the inner core includes copper-cysteamine nanoparticles, the first shell layer includes 2-methylimidazole zinc salt, and the second shell layer includes hyaluronic acid.

[0034] In some embodiments, the inner core is formed by aggregation of a plurality of copper-cysteamine nanoparticles.

[0035] Furthermore, the particle size of the copper-cysteamine nanoparticles is 140-170 nm.

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

[0037] In some embodiments, the nano metal organic framework compound includes the following components calculated by weight: 3 to 6 parts of copper-cysteamine nanoparticles, 1 to 3 parts of 2-methylimidazole zinc salt, and 2 to 7 parts of hyaluronic acid.

[0038] In some embodiments, the nano metal organic framework compound has photosensitivity and enzyme catalytic activity, and has the function of targeting 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 is distributed inside the nanomaterial, ZIF-8 is used as a carrier material to constitute the inner shell of the nanomaterial (i.e., the aforementioned first shell), and the outermost layer is coated with HA as an outer shell (i.e., the aforementioned second shell) through chemical modification, wherein Cu-cy Nps is copper-cysteamine nanoparticles, ZIF-8 is 2-methylimidazole zinc salt, and HA is hyaluronic acid.

[0040] Furthermore, ZIF-8 as a skeleton material is pH responsive, which can overcome the defect of extremely short diffusion distance (<50nm) of ROS generated by photosensitizers in cells and tissues; in addition, ZIF-8 also has the advantages of excellent biocompatibility, high porosity, structural adjustability, large surface area, excellent thermal stability and excellent catalytic performance. It can effectively encapsulate Cu-cy Nps in it, and HA can also be modified on its surface to give the nanomaterial a better targeting effect.

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

[0042] In summary, the nano metal organic framework compound provided by the present invention has a core-shell-shell structure, which is unique and novel, has both photosensitivity and good catalase and oxidase catalytic activity, its enzyme activity is significantly increased, and has the characteristics of high stability, etc. It can effectively target tumor cells, providing a new idea and method for the treatment of cancer.

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

[0044] reacting a copper salt with cysteamine hydrochloride to prepare copper-cysteamine nanoparticles;

[0045] Mixing 2-methylimidazole, polyvinyl pyrrolidone and water, and mixing the mixture with a mixed solution containing zinc salt and copper-cysteamine nanoparticles to prepare copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt;

[0046] The hyaluronic acid is chemically modified and coated on the 2-methylimidazole zinc salt surface of the copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt to prepare a nano metal organic framework compound.

[0047] In some embodiments, the preparation method comprises: uniformly mixing copper salt, cysteamine hydrochloride and water, adjusting the pH value to 7-9, and heating to boiling for reaction to obtain copper-cysteamine 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 comprises copper (II) chloride dihydrate (CuCl 2 ·2H 2 O), but not limited to this.

[0050] In some preferred embodiments, the preparation method specifically comprises: 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 cysteamine hydrochloride is 1-3:1-4. That is, in other words, in terms of weight, the raw materials of Cu-cy Nps include: copper (II) chloride dihydrate (CuCl 2 ·2H 2 O) 1-3 parts, cysteamine hydrochloride (HSCH 2 CH 2 NH 2 ·HCl) 1 to 4 parts.

[0052] In some preferred embodiments, the preparation method further comprises: washing the prepared copper-cysteamine nanoparticles by centrifugation for 1 to 4 times with a mixed solution of water and ethanol, wherein the volume ratio of water to ethanol is 4 to 5:3 to 4.

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

[0054] mixing zinc salt, water and copper-cysteamine nanoparticles to form a first mixed solution;

[0055] 2-methylimidazole, polyvinyl pyrrolidone and water were mixed evenly, and then the first mixed solution was added dropwise, stirred evenly and allowed to stand, centrifuged and washed with water to obtain copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt (Cu-cy Nps@ZIF-8).

[0056] The present invention uses ZIF-8 as the inner shell. On the one hand, since the diffusion distance of nanomaterials in tumor cells is extremely short, it is very important to make nanoparticles effectively enter tumor cells. Because ZIF-8 has pH responsiveness, it can react with the acidic environment of tumor cells to effectively decompose nanomaterials in tumor cells. On the other hand, ZIF-8 has the advantage of being easy to chemically modify, so the nanomaterial can be further modified with HA on its surface to improve the targeting and biosafety of the nanomaterial.

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

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

[0059] Further, the zinc salt includes zinc nitrate hexahydrate, but is not limited thereto.

[0060] In some preferred embodiments, the mass ratio of 2-methylimidazole, zinc nitrate hexahydrate and polyvinyl pyrrolidone is (18-21): (1-3): (2-6). In other words, in terms of weight, the raw materials of ZIF-8 include: 2-methylimidazole (C 4 H 6 N 2 ) 18-21 parts, zinc nitrate hexahydrate (Zn(NO 3 ) 2 6H 2 O) 1-3 parts, polyvinyl pyrrolidone (C 6 H 9 NO) 2 to 6 parts.

[0061] In some embodiments, the preparation method specifically includes: adding the copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt into water to fully dissolve, then adding hyaluronic acid, and reacting at room temperature with stirring for 2 to 6 hours to obtain the nano metal organic framework compound.

[0062] In some embodiments, the mass ratio of the copper-cysteamine nanoparticles, 2-methylimidazole zinc salt and hyaluronic acid is (3-6): (1-3): (2-7). That is, from another perspective, the nano metal organic framework compound includes, by weight: 3-6 parts of copper-cysteamine nanoparticles (Cu-cy Nps), 1-3 parts of 2-methylimidazole zinc salt (ZIF-8), and 2-7 parts of hyaluronic acid (HA).

[0063] Among them, in some more specific implementation cases, the preparation method of the nano metal organic framework compound includes the following steps:

[0064] (1) Copper (II) chloride dihydrate (CuCl 2 ·2H 2 O) was dissolved in deionized water, and then cysteamine hydrochloride (HSCH 2 CH 2 NH 2 ·HCl) and stirred evenly, adjusting the pH value to 7-9, and finally heating the solution to boiling, and washing it 1-4 times by centrifugation with a mixed solution of water and ethanol, and the obtained solution is a Cu-cy Nps solution;

[0065] (2) Dissolve zinc nitrate in deionized water, then add Cu-cy Nps solution, and keep the resulting solution for later use. The volume of deionized water added is 10 mL;

[0066] (3) 2-methylimidazole and polyvinyl pyrrolidone were dissolved in deionized water, and then the solution prepared in step (2) was slowly added dropwise, stirred evenly, and then allowed to stand for 1 to 5 hours. The mixture was centrifuged at 8000 to 11000 rpm and washed with water for 1 to 4 times, each centrifugation time being 8 to 20 minutes. The resulting precipitate was Cu-cy Nps@ZIF-8, which was set aside. The volume of deionized water added was 10 mL;

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

[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 nano-metal organic framework compound composite nanomaterial provided by the present invention mainly utilizes the new nanomaterial ZIF-8 to be loaded on Cu-cy Nps, and chemically modifies the ZIF-8 nanoparticles with HA to impart targeting. The preparation is simple, environmentally friendly, easy to implement, low-cost, and conducive to large-scale production, thereby obtaining more types of composite nanomaterials with broader applications.

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

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

[0072] Furthermore, the oxidase comprises catalase.

[0073] Furthermore, the nano metal organic framework compound provided by the present invention was characterized by SEM, TEM and XRD, and the results showed that the prepared nano material was successfully prepared, with a particle size of about 250nm and photosensitivity. The enzyme catalytic activity of the nano material was shown by TMB experiment. The CCK-8 experiment proved that the nano material had good biosafety. The targeting of the nano material was verified by Western Blot experiment, which met the standards for biomedical applications.

[0074] The present invention is further described by the following examples: The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention 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 examples are all well known in the art.

[0076] Example 1

[0077] like Figure 1 As shown, a nano metal organic framework compound (hereinafter referred to as "nanomaterial") provided in this embodiment has a core-shell-shell structure. Specifically, the structure of the nanomaterial includes: Cu-cy Nps is distributed inside the nanomaterial, ZIF-8 is used as a carrier material to form the inner shell of the nanomaterial, and the outermost layer is coated with HA as an outer shell through chemical modification, wherein Cu-cy Nps is copper-cysteamine nanoparticles, ZIF-8 is 2-methylimidazole zinc salt, and HA is a nano metal organic framework compound hyaluronic acid.

[0078] Specifically, the synthesis method of the nanometal organic framework compound will be described and characterized as follows.

[0079] 1. Preparation of raw materials (all commercially available)

[0080] Copper(II) chloride dihydrate (CuCl 2 ·2H 2 O), cysteamine hydrochloride (HSCH 2 CH 2 NH 2 ·HCl), sodium hydroxide (NaOH), 2-methylimidazole (C 4 H 6 N 2 ), zinc nitrate hexahydrate (Zn(NO 3 ) 2 6H 2 O), polyvinyl pyrrolidone (C 6 H 9 NO), hyaluronic acid, anhydrous ethanol and deionized water.

[0081] 2. Preparation methods of nanomaterials

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

[0083] Weigh copper (II) chloride dihydrate (CuCl 2 ·2H 2O) 0.460g in a beaker, add 20ml of deionized water and place on a magnetic stirrer (500rpm / min), add 0.636g of cysteamine hydrochloride after fully dissolving, wait for the above solution to be mixed evenly, then adjust the pH value of the solution to 8 (add NaOH solution) and the color of the solution gradually turns black, stir at room temperature for 1.5h, then heat to boiling, react at 120℃ for 40min, and stop the reaction when crystalline substances are seen at the bottom of the beaker. Discard the solution and add a solution with a volume ratio of deionized water and ethanol of 5:4, fully ultrasonicate, then centrifuge and wash 3 times (11000rpm×10min), finally add the above deionized water and ethanol mixture to obtain Cu-cy Nps solution, and store in a refrigerator at 4℃.

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

[0085] An electronic balance was used to weigh 1.94 g of 2-methylimidazole (2-MIM) and 375.2 mg of polyvinylpyrrolidone (PVP) into a flask, and 10 ml of deionized water was added and stirred to fully dissolve the mixture to obtain solution A. Then zinc nitrate hexahydrate (Zn(NO 3 ) 2 6H 2 O) 100 mg was placed in a centrifuge tube, and 10 ml of deionized water was added for ultrasonic dispersion to obtain solution B. Solution B was slowly added dropwise to solution A during stirring. After 10 min, the round-bottom flask was placed in a cool and dry place for 3 h, and washed with water three times in a centrifuge (11000 rpm×10 min) to obtain the nanomaterial carrier ZIF-8, which was stored in a refrigerator at 4°C.

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

[0087] First, obtain solution A by the method in step 2.2. Then weigh Zn(NO 3 ) 2 6H 2 O) 100 mg was placed in a centrifuge tube, and then 10 ml of deionized water was added. The sample was ultrasonically treated until the solution became transparent, and 4 ml of Cu-cy Nps solution was added and ultrasonicated for 5 min to obtain solution C. Finally, solution C was slowly added dropwise to solution A while stirring, and after the addition was completed, it was stirred for another 10 min. The flask was transferred to a cool and dry place and allowed to stand for 3 h. Finally, the solution was collected and washed with water by centrifugation 3 times (11000 rpm×10 min), and Cu-cy Nps@ZIF-8 was obtained by ultrasonic mixing, which was stored in a refrigerator at 4°C.

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

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

[0090] 3. Characterization

[0091] 3.1 SEM characterization of ZIF-8

[0092] Take 1 ml of ZIF-8 aqueous solution, dilute it 10 times with deionized water, and disperse it by ultrasonic for 1 hour. Drop the fully dispersed ZIF-8 suspension onto a round glass slide, dry it in an oven at 60°C, and then test the 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 respectively, dilute it 10 times with deionized water, ultrasonically disperse it for 1 hour, drop it onto the copper grid, dry it naturally, and then test the morphology by TEM.

[0095] 3.3 EDS mapping image detection of nanomaterials CNZH

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

[0097] 3.4 XRD detection of nanomaterial CNZH

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

[0099] 3.5 XPS spectrum detection of nanomaterial CNZH

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

[0101] 3.6 Photosensitivity of nanomaterial CNZH

[0102] Take 32.5 ml of deionized water and CNZH solution respectively and put them into centrifuge tubes of uniform specifications, and then take photos and record them under natural light and 365 nm ultraviolet light conditions respectively.

[0103] 3.7 Detection of enzyme-like activity of TMB catalyzed by 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, resulting in color change and a significant enhancement of the absorption peak at 652nm.

[0105] 3.7.1 Enzymatic 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, H 2 O 2 , 50 mM, UV-visible absorption spectrum recorded the characteristic absorption spectrum of ox TMB.

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

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

[0109] 3.7.3 Enzymatic activity of Cu-cy Nps@ZIF-8 towards TMB at different pH

[0110] Take three portions of Cu-cy Nps@ZIF-8 with a concentration of 200 μg / mL, and then adjust the pH to 5.0, 6.5, and 7.4 respectively, and record the characteristic absorption spectrum of ox TMB by UV-visible absorption spectroscopy.

[0111] 3.8 Detection of the targeting properties of nanomaterial CNZH

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

[0113] 3.9 Detection of biosafety of nanomaterial CNZH by CCK-8 method

[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 human normal lung epithelial cells (BEAS) were cultured at 1×10 4 Cells were seeded in 96-well plates at an initial density of 10 cells / mL and cultured in an incubator. After the cells adhered, they were incubated with fresh DMEM samples of different concentrations for 24 hours. Then, fresh cell counting kit (CCK-8) solution was added and incubated for 3 hours. Cell viability was measured at an absorbance of 450 nm.

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

[0116] Human epidermal cancer cell A-431 cells (1×10 4 cells / mL) were inoculated into 96-well plates and incubated overnight in an incubator. After co-culture with 100μg / mL CNZ and CNZH for 3h in different groups, the illumination group was irradiated with 365nm ultraviolet light for 10min, and then continued to culture for 3h. The supernatant in the 96-well plate was replaced by calcein-AM / PI (KeyGen) solution (100μL) for staining live / dead cells. After co-incubation for 45min, the experiment was observed with different colors under an inverted fluorescence microscope (Olympus IX 73).

[0117] 4. Characterization Results Analysis

[0118] 4.1 Preparation and characterization of nanomaterials

[0119] Figure 2a-2c is the morphological feature of nanoparticles. Figure 2a is the SEM image of ZIF-8. Figure 2b TEM image of Cu-cyNps. Figure 2c TEM image of Cu-cy Nps@ZIF-8. Figure 2a As shown in Figure 2, the average size of ZIF-8 nanoparticles was about 250 nm and they were evenly distributed. Figure 2b It can be seen that the Cu-cy Nps are uniformly spherical with an average size of about 150 nm. Figure 2c The average size of Cu-cy Nps@ZIF-8 nanoparticles is about 250 nm. Figure 3As 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 purple), indicating that ZIF-8 is successfully encapsulated on the Cu-cy Np surface, and finally HA is modified on the surface of the framework to make the nanomaterial CNZ targeted, that is, the nanomaterial CNZH is obtained. Figure 5a-5f This is the XPS spectrum of the nanomaterial CNZH. Figure 5a In the total peak diagram, we can see the presence of Cu2p, Zn2p, N1s, C1s, etc. Figure 5b There are two pairs of peaks in Cu2p, of which 932.73eV (Cu 2p 3 / 2 ) and 952.33 eV (Cu 2p 1 / 2 ) represents Cu + , of which 934.97eV (Cu 2p 3 / 2 ) and 953.68 eV (Cu 2p 1 / 2 ) represents Cu 2+ , indicating that Cu in CNZH exists in two valence states: +1 and +2. Figure 5c It can be seen that there are two peaks in Zn 2p, at 1021.7eV and 1044.7eV, representing Zn 2p 3 / 2 and Zn 2p 1 / 2 .exist Figure 5d-5f The characteristic peaks of C, O, and N can be seen in the figure. Figure 4 The XRD spectrum of the nanomaterial CNZH shows that the diffraction angles are 10.38°, 12.71°, 18.01°, 24.51°, and 29.6°, corresponding to the crystal structures (002), (112), (022), (222), (233) and (044), which are consistent with the standard card of zif-8. In summary, the present invention successfully prepares the nanomaterial CNZH.

[0120] 4.2 Photosensitivity of nanomaterials

[0121] Figure 6a and Figure 6b This is a macroscopic picture of the nanomaterial CNZH. Figure 6a As shown in Figure 2, under natural light conditions, the solution of nanomaterial CNZH is gray, while under 365nm ultraviolet light irradiation conditions (such as Figure 6b ), it showed bright orange fluorescence, indicating that CNZH has photosensitivity and that HA coating does not affect its performance.

[0122] 4.3 Enzyme catalytic activity of nanomaterials

[0123] like Figure 7a-7c As shown, TMB was used as an indicator to study its peroxidase-like activity. Figure 7ais the enzyme catalytic activity at pH 5.0. Figure 7a It can be seen that the absorbance of Cu-cy Nps and CNZ at 652nm and 895nm is consistent, and is 1.5 times that of ZIF-8, indicating that Cu-cy Nps has good POD enzyme activity, and after Cu-cy Nps is encapsulated in ZIF-8, its POD enzyme catalytic activity is greatly improved compared with that of ZIF-8 alone; In addition, Figure 7b is the catalytic activity of the enzyme at different concentrations. Figure 7b It can be seen that as the concentration of CNZ decreases, the absorbance of ox TMB at 652nm and 895nm gradually decreases. Even when the concentration of CNZ is 10μg / mL, the absorbance of ox TMB can still be observed, indicating that CNZ has excellent POD enzyme activity. Figure 7c As for the enzyme catalytic activity at different pH values ​​(5.0, 6.5, and 7.4), it can be seen that with the increase of pH value, the absorbance of ox TMB at 652 nm and 895 nm gradually decreases, indicating that the POD enzyme activity is strongest in an acidic environment, which is also consistent with the tumor microenvironment and is conducive to the catalytic activity of CNZ in tumor cells.

[0124] 4.4 Targeting of Nanomaterials

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

[0126] 4.5 Biosafety of Nanomaterials

[0127] The biosafety of CNZH was verified by CCK-8 method. Fig. 9 As shown, after the cells were incubated with different concentrations of CNZ and CNZH for 24 hours, the survival rate of BEAS and L-929 cells could still reach more than 90% even at a relatively high concentration (200 μg / mL). The nanomaterial CNZH has good biosafety and is suitable for application in the biomedical field.

[0128] 4.6 Therapeutic effects of nanomaterials

[0129] like Fig.10As shown in the figure, the therapeutic effect diagram of the nanomaterial CNZH was detected by live / dead cell staining experiment. The Calcein-AM / PI double staining kit was used to intuitively display the live / dead cell situation. The cell death in the control group (Ctrl) and the 365nm ultraviolet light irradiation group (L365) was negligible. Due to the effect of CDT, a small amount of ·OH was produced in the CNZ group, which slightly increased the red signal (dead cells). The red signal in the CNZ+365nm ultraviolet light irradiation group (LCNZ group) increased significantly, which was caused by the PDT effect of the photosensitizer triggered by the irradiation of 365nm ultraviolet light. Since HA was modified on the surface of the CNZ nanomaterial, the CD44 receptor on the surface of tumor cells can be effectively targeted, so obvious dead cells (red signals) can also be seen in the CNZH group. The most significant is the CNZH+365nm ultraviolet light irradiation group (LCNZH), where the surviving cells (green signals) are about 20%. It shows that the nanomaterial has a good tumor killing effect and is suitable for the treatment of tumors.

[0130] Example 2

[0131] The preparation method of nano metal organic framework compound (referred to as nano material) includes:

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

[0133] Weigh copper (II) chloride dihydrate (CuCl 2 ·2H 2 O) 0.50g in a beaker, add 20ml of deionized water and place on a magnetic stirrer (500rpm / min), add 0.7g of cysteamine hydrochloride after fully dissolving, wait for the above solution to be mixed evenly, then adjust the pH value of the solution to 7.35 (add NaOH solution) and the color of the solution gradually turns black, stir at room temperature for 1.5h, then heat to boiling, react at 100℃ for 50min, and stop the reaction when crystalline substances are seen at the bottom of the beaker. Discard the solution and add a solution with a volume ratio of deionized water and ethanol of 5:4, fully ultrasonicate, then centrifuge and wash once (10000rpm×8min), finally add the above deionized water and ethanol mixture to obtain Cu-cy Nps solution, and store in a refrigerator at 4℃.

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

[0135] Weigh 4 g of 2-methylimidazole (2-MIM) and 700 mg of polyvinylpyrrolidone (PVP) using an electronic balance and put them into a flask. Add 15 ml of deionized water and stir to fully dissolve them to obtain solution A. Then weigh zinc nitrate hexahydrate (Zn(NO 3 ) 2 6H2 O) 200 mg was placed in a centrifuge tube, and 10 ml of deionized water was added for ultrasonic dispersion to obtain solution B. Solution B was slowly added dropwise to solution A during stirring. After 20 min, the round-bottom flask was placed in a cool and dry place for 2 h, and washed with water 4 times in a centrifuge (10000 rpm×8 min) to obtain the nanomaterial carrier ZIF-8, which was stored in a refrigerator at 4°C.

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

[0137] First, obtain solution A by the method in step 2.2. Then weigh Zn(NO 3 ) 2 6H 2 O) 200 mg was placed in a centrifuge tube, and then 10 ml of deionized water was added. The sample was ultrasonically treated until the solution became transparent, and 4 ml of Cu-cy Nps solution was added and ultrasonicated for 10 min to obtain solution C. Finally, solution C was slowly added dropwise to solution A while stirring, and after the addition was completed, it was stirred for another 20 min. The flask was transferred to a cool and dry place and allowed to stand for 2 h. Finally, the solution was collected and washed with water by centrifugation 4 times (10000 rpm×8 min), and Cu-cy Nps@ZIF-8 was obtained by ultrasonic mixing, which was stored in a refrigerator at 4°C.

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

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

[0140] Example 3

[0141] The preparation method of nano metal organic framework compound (referred to as nano material) includes:

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

[0143] Weigh copper (II) chloride dihydrate (CuCl 2 ·2H 2O) 0.30g in a beaker, add 20ml of deionized water and place it on a magnetic stirrer (500rpm / min), add 0.4g of cysteamine hydrochloride after it is fully dissolved, wait for the above solution to be mixed evenly, then adjust the pH value of the solution to 8.5 (add NaOH solution) and the color of the solution gradually turns black, stir at room temperature for 1.5h, then heat to boiling, react at 150℃ for 30min, and stop the reaction when crystalline substances are seen at the bottom of the beaker. Discard the solution and add a solution with a volume ratio of deionized water and ethanol of 5:4, fully ultrasonicate, then centrifuge and wash 4 times (8000rpm×20min), and finally add the above deionized water and ethanol mixture to obtain Cu-cy Nps solution, which is stored in a refrigerator at 4℃.

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

[0145] Weigh 2.10 g of 2-methylimidazole (2-MIM) and 400 mg of polyvinylpyrrolidone (PVP) using an electronic balance and put them into a flask. Add 10 ml of deionized water and stir to fully dissolve them to obtain solution A. Then weigh zinc nitrate hexahydrate (Zn(NO 3 ) 2 6H 2 O) 100 mg was placed in a centrifuge tube, and 10 ml of deionized water was added for ultrasonic dispersion to obtain solution B. Solution B was slowly added dropwise to solution A during stirring. After 5 min, the round-bottom flask was placed in a cool and dry place for 4 h, and washed twice with water in a centrifuge (8000 rpm×20 min) to obtain the nanomaterial carrier ZIF-8, which was stored in a refrigerator at 4°C.

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

[0147] First, obtain solution A by the method in step 2.2. Then weigh Zn(NO 3 ) 2 6H 2 O) 100 mg was placed in a centrifuge tube, and then 10 ml of deionized water was added. The sample was ultrasonically treated until the solution became transparent, and 4 ml of Cu-cy Nps solution was added and ultrasonicated for 10 min to obtain solution C. Finally, solution C was slowly added dropwise to solution A while stirring, and after the addition was completed, it was stirred for another 5 min. The flask was transferred to a cool and dry place and allowed to stand for 4 h. Finally, the solution was collected and washed with water once by centrifugation (8000 rpm×20 min), and Cu-cy Nps@ZIF-8 was obtained by ultrasonic mixing, which was stored in a refrigerator at 4°C.

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

[0149] Weigh 2 mg of hyaluronic acid (HA) and put it into a round-bottom flask. Add 9 ml of deionized water and stir on 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 and centrifuge it. Then wash it with water once (11000 rpm×10 min) to obtain the nanomaterial CNZH, which is stored in a refrigerator at 4°C.

[0150] After testing, Examples 2 and 3 also obtained nano-metal organic framework compounds with photosensitivity and enzyme catalytic activity, and which can effectively target tumor cells.

[0151] Comparative Example 1

[0152] Compared with Example 1, this comparative example has the following differences:

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

[0154] Methanol was used as solvent for the preparation of ZIF-8. The specific method was as follows: 218.9 mg of 2-methylimidazole (2-MIM) and 352 mg of polyvinylpyrrolidone (PVP) were placed in a flask, 10 ml of methanol was added, and stirred to fully dissolve. Zinc nitrate hexahydrate (Zn(NO 3 ) 2 6H 2 O) 100 mg and 10 ml methanol solution; the above solution was stirred at 550 rpm / min for 2 hours, and finally washed twice by methanol centrifugation (11000 rpm×10 min). The ZIF-8 nanoparticles obtained by this method were small in quantity, hexagonal, of different sizes and severely adhered, with a particle size of about 150 nm. Fig.11 shown.

[0155] Comparative Example 2

[0156] Compared with Example 1, this comparative example has the following differences:

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

[0158] The solution was replaced with deionized water as the solution for preparing nanoparticles. 1.94 g of 2-methylimidazole (2-MIM), 375.2 mg of polyvinyl pyrrolidone (PVP) and hexahydrate zinc nitrate (Zn(NO 3 ) 2 6H 2O) 100 mg was directly mixed in a flask, 10 ml of deionized water was added, and it was stirred at 500 rpm / min for 10 min to fully dissolve, and then it was allowed to stand in a cool and dry place for 3 h, and finally centrifuged (11000 rpm × 10 min) and washed with water 3 times. Although the yield was rich, the morphology was irregular and there was still adhesion. The particle size was about 150-250 nm. The microscopic morphology of ZIF-8 was as follows Fig.12 shown.

[0159] Comparative Example 3

[0160] Compared with Example 1, this comparative example has the following differences:

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

[0162] To further solve the adhesion problem, on the one hand, the content of polyvinyl pyrrolidone was reduced, and on the other hand, the zinc salt and the skeleton material were fully dissolved. 3 ) 2 6H 2 O) 100 mg was added to 1 ml of deionized water and ultrasonically homogenized; 1.94 g of 2-methylimidazole (2-MIM) and 375.2 mg of polyvinyl pyrrolidone (PVP) were taken, 9 ml of deionized water were added, and the mixture was stirred at 500 rpm / min to fully mix, and the above zinc salt solution was added. After 10 min, stirring was stopped, and the mixture was allowed to stand in a cool and dry place for 3 h. Finally, the mixture was centrifuged (11000 rpm×10 min) and washed with water 3 times. The obtained ZIF-8 nanoparticles had abundant yield, regular hexagonal morphology, slight adhesion, particle size of about 120-180 nm, and microscopic morphology was as follows Fig.13 This is because the volume of deionized water added in this comparative example has changed. Due to the different volumes of the solutions, the reaction may be incomplete, causing adhesion.

[0163] Comparative Example 4

[0164] Compared with Example 1, this comparative example has the following differences:

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

[0166] Weigh copper (II) chloride dihydrate (CuCl 2 ·2H 2O) 0.460g in a beaker, add 20ml of deionized water and place on a magnetic stirrer (500rpm / min), add 0.636g of cysteamine hydrochloride after it is fully dissolved, wait for the above solutions to be mixed evenly, then adjust the pH value of the solution to 8 (add NaOH solution) and the color of the solution will gradually turn black. Stir for 2-3h at room temperature, then heat to 120℃ until the solution boils for 1h, and stop the reaction when crystalline substances are seen at the bottom of the beaker. Discard the solution and add a solution with a volume ratio of deionized water and ethanol of 5:4, fully ultrasonicate, then centrifuge and wash 3 times (11000rpm×10min), and finally add the above deionized water and ethanol mixture to obtain Cu-cy Nps solution. Please refer to Fig.14 TEM observation showed that the obtained Cu-cyNps nanoparticles were spherical. Due to the long stirring time, the diameter was about 250-300 nm. Too large a 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 is not performed.

[0169] The results showed that the targeting of nanoparticles without HA chemical modification was poor. Figure 8 As shown, HA modified on the surface of nanomaterials can specifically bind to the CD44 receptors in tumor cells, so that the nanoparticles can bind to tumor cells more accurately, improve the targeting of nanomaterials, and reduce damage to normal cells during treatment; at the same time, HA, as a widely existing natural polysaccharide, improves the biosafety of nanomaterials and makes nanomaterials more suitable for application in the biomedical field.

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

[0171] The above shows and describes 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, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A nano metal organic framework compound, characterized in that: The nano metal organic framework compound includes an inner core, a first shell layer coated on the surface of the inner core, and a second shell layer chemically modified and coated on the surface of the first shell layer, which are arranged in sequence from the inside to the outside. The inner core includes copper-cysteamine nanoparticles, the first shell layer includes 2-methylimidazole zinc salt, and the second shell layer includes hyaluronic acid.

2. The nano metal organic framework compound according to claim 1, characterized in that: The particle size of the copper-cysteamine nanoparticles is 140-170 nm; and / or the inner core is formed by aggregation of a plurality of copper-cysteamine nanoparticles; and / or the thickness of the first shell layer is 3-8 nm.

3. The nano metal organic framework compound according to claim 1, characterized in that: The nano metal organic framework compound comprises the following components calculated by weight: 3 to 6 parts of copper-cysteamine nanoparticles, 1 to 3 parts of 2-methylimidazole zinc salt and 2 to 7 parts of hyaluronic acid; And / or, the nano metal organic framework compound has photosensitivity and enzyme catalytic activity, and has the function of targeting tumor cells.

4. A method for preparing a nano metal organic framework compound, characterized in that: include: reacting a copper salt with cysteamine hydrochloride to prepare copper-cysteamine nanoparticles; Mixing 2-methylimidazole, polyvinyl pyrrolidone and water, and mixing the mixture with a mixed solution containing zinc salt and copper-cysteamine nanoparticles to prepare copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt; The hyaluronic acid is chemically modified and coated on the 2-methylimidazole zinc salt surface of the copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt to prepare a nano metal organic framework compound.

5. The preparation method according to claim 4, characterized in that: include: The copper salt, cysteamine hydrochloride and water are mixed uniformly, the pH value is adjusted to 7-9, and the mixture is heated to boiling for reaction to obtain copper-cysteamine nanoparticles; and / or, the copper salt comprises copper (II) chloride dihydrate; Preferably, the reaction temperature is 100-150°C and the reaction time is 30-50 min; Preferably, the preparation method comprises: adjusting the pH value to 7 to 9 using an alkaline agent; particularly preferably, the alkaline agent comprises sodium hydroxide; Preferably, the mass ratio of copper (II) chloride dihydrate to cysteamine hydrochloride is 1-3:1-4; Preferably, the preparation method further comprises: centrifugally washing the prepared copper-cysteamine nanoparticles with a mixed solution of water and ethanol for 1 to 4 times. Particularly preferably, the volume ratio of water to ethanol is 4 to 5:3 to 4.

6. The preparation method according to claim 4, characterized in that: include: mixing zinc salt, water and copper-cysteamine nanoparticles to form a first mixed solution; 2-methylimidazole, polyvinyl pyrrolidone and water are mixed evenly, and then the first mixed solution is added dropwise, stirred evenly, allowed to stand, centrifuged and washed with water to obtain copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt; Preferably, the standing time is 1 to 5 hours; Preferably, the number of centrifugal washing is 1 to 4 times, the time of each centrifugation is 8 to 20 minutes, and the centrifugal speed is 8000 to 11000 rpm; Preferably, the zinc salt comprises zinc nitrate hexahydrate; And / or, the mass ratio of 2-methylimidazole, zinc nitrate hexahydrate and polyvinyl pyrrolidone is (18-21):(1-3):(2-6).

7. The preparation method according to claim 4, characterized in that: include: The copper-cysteamine nanoparticles coated with 2-methylimidazole zinc salt are added into water and fully dissolved, and then hyaluronic acid is added, and the mixture is stirred at room temperature for 2 to 6 hours to obtain the nano metal organic framework compound; And / or, the mass ratio of the copper-cysteamine nanoparticles, 2-methylimidazole zinc salt and hyaluronic acid is (3-6): (1-3): (2-7).

8. A nano metal organic framework compound prepared by the preparation method according to any one of claims 4 to 7.

9. Use of the nano metal organic framework compound according to any one of claims 1 to 3 and 8 in the preparation of oxidase mimics.

10. The use according to claim 9, characterized in that: The oxidase enzyme includes catalase.

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