Application of crystal face adjustable and controllable organic framework material capable of enhancing thrombin activity in preparation of hemostatic material

Abstract

The invention relates to application of a crystal face adjustable and controllable organic framework material capable of enhancing thrombin activity in preparation of a hemostatic material, and belongs to the technical field. According to the application of the crystal face adjustable and controllable organic framework material capable of enhancing the thrombin activity in preparation of the hemostatic material, the surfaces of metal-organic framework materials (metal-organic framework, MOF) with different exposed crystal faces can adsorb thrombin and adjust the catalytic activity of the attached thrombin, and the material has excellent blood coagulation capacity and can be used for preparing the hemostatic material. The compound can be used as a hemostatic active molecule or loaded on a carrier to prepare a hemostatic material applied to surgical hemostasis. More importantly, the thrombin activity is enhanced by regulating and controlling the MOF morphology and the exposed surface.

Description

Technical Field

[0001] The invention belongs to the technical field of application of organic framework materials, and relates to the application of a crystal face-regulatable organic framework material capable of enhancing thrombin activity in the preparation of hemostatic materials. Background Art

[0002] Deaths caused by severe trauma account for 8% of the world's total annual deaths, of which 30% to 40% are caused by failure to promptly control acute bleeding, and about half of the patients die before arriving at the hospital. Rapid use of hemostatic materials to effectively stop bleeding when trauma occurs can save the patient's life to the greatest extent.

[0003] Surgical hemostatic materials are usually composed of hemostatic active molecules with procoagulant ability and a substrate that provides mechanical support. The procoagulant ability of the hemostatic active molecules determines the hemostatic effect of the hemostatic material. Zeolite and kaolin of aluminosilicate are the most representative traditional inorganic hemostatic active molecules, which achieve hemostatic effects by absorbing water to release calcium ions and activating coagulation factors in plasma, respectively. Hemostatic gauze (QuikClotACS+ and QuikClotCombatGauze) with them as hemostatic active molecules have been widely used by the US military in pre-hospital emergency care during the Iraq War. However, the side effects caused by the exothermic reaction of zeolite and the hydrophilic expansion of kaolin limit its hemostatic effect to a certain extent, and the lack of an efficient procoagulant mechanism makes it difficult to effectively stop bleeding for massive bleeding caused by arterial damage.

[0004] In order to obtain a high-performance hemostatic active molecule with excellent procoagulant ability and few side effects, its procoagulant mechanism should focus on the blood coagulation process. Blood coagulation is a complex cascade reaction, in which the conversion of prothrombin to thrombin is a key reaction in the coagulation cascade. The speed and activity of thrombin production will directly affect the formation of fibrin, and then affect the final formation of blood clots. Improving thrombin activity provides a new way to design new hemostatic active molecules.

[0005] Metal-organic framework (MOF) is an emerging multifunctional porous nanomaterial. Inspired by the structure of molecular sieves, a zeolitic imidazolate framework (ZIF-8) with a zeolite-like structure was derived. Compared with traditional porous nanomaterials such as zeolites and carbon, ZIF-8 has a large specific surface area, many active sites, and adjustable crystal faces. Its surface can be used as a substrate for enzyme immobilization to adsorb enzymes and regulate the catalytic activity of attached enzymes. In recent years, it has shown good application potential in the field of enzyme engineering.

[0006] Therefore, utilizing the adjustable properties of metal-organic framework (MOF) crystal surfaces to prepare a MOF with high biocatalytic activity for thrombin to accelerate blood coagulation is of great significance for the research and development of new hemostatic active molecules. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a method for preparing a crystal face-regulatable organic framework material that can enhance the activity of thrombin; a second object of the present invention is to provide a crystal face-regulatable organic framework material that can enhance the activity of thrombin; a fourth object of the present invention is to provide an application of a crystal face-regulatable organic framework material that can enhance the activity of thrombin in the preparation of hemostatic materials.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] 1. A method for preparing a surface-adjustable organic framework material capable of enhancing thrombin activity, the preparation method being as follows:

[0010] Using zinc salt as a metal source, 2-methylimidazole as an organic ligand, and hexadecyltrimethylammonium bromide as a structure directing agent, the zinc salt is dissolved in solvent I to form a B solution, 2-methylimidazole and hexadecyltrimethylammonium bromide are dissolved in solvent I together to form an A solution, the A solution is added to the B solution, mixed evenly, and then allowed to react at room temperature for 2 to 24 hours, and then the mixture after the reaction is completed is centrifuged, washed and vacuum dried to obtain a crystal face-regulatable organic framework material that can enhance thrombin activity.

[0011] Preferably, the molar ratio of zinc ion, 2-methylimidazole and hexadecyltrimethylammonium bromide in the zinc salt is 1:4-57:0-0.073.

[0012] Preferably, the zinc salt is zinc nitrate hexahydrate or zinc acetate dihydrate;

[0013] The solvent is methanol or deionized water;

[0014] The vacuum drying is performed at 60° C. for 12 to 24 hours.

[0015] 2. The surface-adjustable organic framework material that can enhance thrombin activity is prepared according to the above preparation method.

[0016] Preferably, the particle size of the face-adjustable organic framework material capable of enhancing thrombin activity is 100 to 200 nm.

[0017] Preferably, the face-adjustable organic framework material capable of enhancing thrombin activity is a polyhedron exposing any one or both of the {100} face or the {110} face;

[0018] The surface area of ​​the {100} crystal plane or the {110} crystal plane in the crystal plane-adjustable organic framework material capable of enhancing thrombin activity accounts for 0 to 100%.

[0019] Preferably, the face-adjustable organic framework material capable of enhancing thrombin activity is a cubic hexahedron exposing a {100} face, a truncated octahedron exposing {100} and {110} faces, or a rhombic dodecahedron exposing a {110} face.

[0020] Preferably, the face-adjustable organic framework material capable of enhancing thrombin activity is a cubic hexahedron in which the surface area of ​​the {100} face accounts for 100% and the surface area of ​​the {110} face accounts for 0%;

[0021] The crystal face adjustable organic framework material capable of enhancing thrombin activity is a truncated octahedron in which the surface area of ​​the {100} crystal face accounts for 40% and the surface area of ​​the {110} crystal face accounts for 60%;

[0022] The crystal face adjustable organic framework material capable of enhancing thrombin activity is a rhombic dodecahedron in which the surface area of ​​the {100} crystal face accounts for 0% and the surface area of ​​the {110} crystal face accounts for 100%.

[0023] 3. Application of the above-mentioned face-regulatable organic framework material that can enhance thrombin activity in the preparation of hemostatic materials.

[0024] Preferably, in the application, the material is loaded on a carrier, and the carrier is a protein material, a polypeptide-type silk fibroin, gelatin or its derivatives, fibrin, a polysaccharide material or a polymer material;

[0025] The polysaccharide material is any one or more of cellulose, starch, agarose, dextran, chitosan, alginate or hyaluronic acid;

[0026] The polymer material is any one or more of polyethylene glycol, polyvinyl alcohol, polyurethane, polycaprolactone, polylactic acid-glycolic acid, and polyethylene oxide.

[0027] The beneficial effects of the present invention are as follows: the present invention discloses an application of a crystal face adjustable organic framework material capable of enhancing thrombin activity in the preparation of a hemostatic material, and the metal-organic framework (MOF) material surface with different crystal face exposures can adsorb thrombin and regulate the catalytic activity of the attached thrombin, and has excellent coagulation ability, and can be used as a hemostatic active molecule or as a hemostatic active molecule loaded on a carrier to prepare a hemostatic material for surgical hemostasis. More importantly, the present invention achieves the enhancement of thrombin activity by regulating the MOF morphology and exposed surface.

[0028] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0030] Figure 1 Schematic diagram of the face-adjustable organic framework material (MOF) prepared in Example 1, Example 2 and Example 3 that can enhance thrombin activity (a), and schematic diagram of the {100} face and the {110} face (b);

[0031] Figure 2 The crystal diffraction pattern (a) and the enlarged pattern (b) in the range of 2θ 6 to 14 of the crystal face tunable organic framework material (MOF) that can enhance thrombin activity prepared in Example 1, Example 2 and Example 3;

[0032] Figure 3 Transmission electron microscope images of the surface-adjustable organic framework materials (MOFs) that can enhance thrombin activity prepared in Examples 1, 2, and 3, with a scale bar of 50 nm;

[0033] Figure 4 The images of thrombin adsorbed on the surface of the surface-adjustable organic framework material (MOF) capable of enhancing thrombin activity prepared in Examples 1, 2 and 3, the scale bar is 100 μm;

[0034] Figure 5 The in vitro coagulation time of the crystal face-adjustable organic framework material (MOF) that can enhance thrombin activity prepared in Example 1, Example 2 and Example 3, the results are expressed as mean ± standard error (n = 3), *p < 0.05;

[0035] Figure 6 The endogenous thrombin potential of the facet-tunable organic framework materials (MOFs) that can enhance thrombin activity prepared in Examples 1, 2 and 3 is shown. The results are expressed as mean ± standard error (n=3), *p<0.05. DETAILED DESCRIPTION

[0036] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0037] Example 1

[0038] A crystal face tunable organic framework material (MOF) that can enhance thrombin activity, the specific preparation method is as follows:

[0039] (1) Dissolve 11.35 g of 2-methylimidazole and 65 mg of hexadecyltrimethylammonium bromide in 225 mL of deionized water to obtain solution A.

[0040] (2) Dissolve 0.725 g of zinc nitrate hexahydrate in 25 mL of deionized water to obtain solution B.

[0041] (3) Solution A was added to solution B under stirring conditions (250 rpm / min). After complete mixing, stirring was continued for 5 min, and then the reaction was allowed to stand for 3 h.

[0042] (4) After the reaction is completed, the product is centrifuged and washed with methanol for 3 to 5 times, and finally placed in a vacuum dryer at 60°C for 12 h to obtain a crystal face-regulatable organic framework material (MOF, i.e., ZIF-8 / C) that can enhance thrombin activity.

[0043] Example 2

[0044] A crystal face tunable organic framework material (MOF) that can enhance thrombin activity, the specific preparation method is as follows:

[0045] (1) Dissolve 1.115 g of 2-methylimidazole and 0.984 mg of hexadecyltrimethylammonium bromide in 5 mL of deionized water to obtain solution A.

[0046] (2) Dissolve 300 mg of zinc acetate dihydrate in 5 mL of deionized water to obtain solution B.

[0047] (3) Add solution B to solution A under stirring conditions (250 rpm / min). Continue stirring for 15 seconds after complete mixing, and then let it react for 2 hours.

[0048] (4) After the reaction is completed, the product is centrifuged and washed with methanol for 3 to 5 times, and finally placed in a vacuum dryer at 60°C for 18 h to obtain a crystal face-tunable organic framework material (MOF, i.e., ZIF-8 / TRD) that can enhance thrombin activity.

[0049] Example 3

[0050] A crystal face tunable organic framework material (MOF) that can enhance thrombin activity, the specific preparation method is as follows:

[0051] (1) 810.6 mg of 2-methylimidazole was dissolved in 50 mL of methanol to obtain solution A.

[0052] (2) 734.4 mg of zinc nitrate hexahydrate was dissolved in 50 mL of methanol to obtain solution B.

[0053] (3) Add solution A to solution B under stirring conditions (800 rpm / min). Stop stirring immediately after complete mixing, and then let the reaction stand for 24 hours.

[0054] (4) After the reaction is completed, the product is centrifuged and washed with methanol for 3 to 5 times, and finally placed in a vacuum dryer at 60°C for 24 hours to obtain a crystal face-regulatable organic framework material (MOF, i.e., ZIF-8 / RD) that can enhance thrombin activity.

[0055] Performance Testing

[0056] The crystal face adjustable organic framework materials (MOF) prepared in the above embodiments that can enhance the activity of thrombin are respectively cubic hexahedron (ZIF-8 / C) with exposed {100} crystal face, truncated octahedron (ZIF-8 / TRD) with exposed {100} and {110} crystal face, and rhombic dodecahedron (ZIF-8 / RD) with exposed {110} crystal face. The particle size of MOF is about 100 to 200 nm. In the in vitro blood coagulation promotion experiment, blood coagulation is achieved in about 35 to 85 seconds. In the in vitro thrombin generation experiment, the endogenous thrombin potential is about 35 to 60 NIH U min.

[0057] Figure 1 Schematic diagram (a) of the facet-tunable organic framework material (MOF) prepared in Example 1, Example 2 and Example 3 that can enhance thrombin activity, and schematic diagram (b) of the {100} facet and the {110} facet. Figure 1 It can be seen that Example 1 is a cubic hexahedron (ZIF-8 / C) exposing the {100} crystal face, Example 2 is a truncated octahedron (ZIF-8 / TRD) exposing the {100} and {110} crystal faces, and Example 3 is a rhombic dodecahedron (ZIF-8 / RD) exposing the {110} crystal face.

[0058] The crystal structures of the face-adjustable organic framework materials (MOFs) that can enhance thrombin activity prepared in Examples 1, 2, and 3 were measured by X-ray diffraction, and the results are as follows: Figure 2 As shown, a is the crystal diffraction pattern, and b is the enlarged view of 2θ in the range of 6 to 14. Figure 2 It can be seen from a that the characteristic peaks of the face-adjustable organic framework materials (MOF) that can enhance the activity of thrombin in Examples 1, 2 and 3 are consistent with the simulation data and the peak values ​​are very sharp, indicating that the method of the present invention successfully prepared a face-adjustable organic framework material that can enhance the activity of thrombin with high crystallinity; Figure 2 As can be seen from b, from Example 1 to Example 3, the intensity of the (110) diffraction peak gradually increases, indicating that the exposure rate of the {110} crystal plane increases, while the intensity of the (200) diffraction peak gradually decreases, indicating that the exposure rate of the {100} crystal plane decreases.

[0059] The crystal face tunable organic framework materials (MOF) prepared in Example 1, Example 2 and Example 3 that can enhance thrombin activity were observed by transmission electron microscopy, and the results are as follows Figure 3 As shown. Figure 3 It can be seen that Example 1 is a cubic hexahedron (ZIF-8 / C), Example 2 is a truncated octahedron (ZIF-8 / TRD), and Example 3 is a rhombic dodecahedron (ZIF-8 / RD). The particle sizes of the crystal face-adjustable organic framework materials (MOFs) that can enhance thrombin activity prepared in Examples 1, 2, and 3 are all in the range of 100 to 200 nm.

[0060] The adsorption of thrombin on the surface of the crystal face-adjustable organic framework (MOF) material capable of enhancing thrombin activity prepared in Examples 1, 2 and 3 was observed by laser scanning confocal microscopy (Bright is the bright field image of MOF, FITC is the fluorescence of thrombin labeled with fluorescein isothiocyanate, and Merged is the overlap of the bright field image and the fluorescence image). The results are as follows: Figure 4 As shown. Figure 4 It can be seen that the bright field image of MOF and the fluorescence image of FITC-labeled thrombin completely overlap, indicating that thrombin can be successfully adsorbed on the surface of the crystal face-adjustable organic framework material (MOF) that can enhance thrombin activity prepared in Examples 1, 2 and 3.

[0061] The in vitro coagulation-promoting effect of the crystal face-adjustable organic framework material (MOF) that can enhance thrombin activity prepared in Example 1, Example 2 and Example 3 was measured by an in vitro coagulation experiment. The results are as follows: Figure 5 The results are shown as mean ± standard error (n = 3), *p < 0.05. Figure 5It can be seen that the natural coagulation process takes about 425 seconds, while after adding the crystal face adjustable organic framework material (MOF) that can enhance thrombin activity prepared in Example 1, Example 2 and Example 3, blood coagulation can be achieved in about 35 to 85 seconds, indicating that the crystal face adjustable organic framework material (MOF) that can enhance thrombin activity prepared by the present invention has a good in vitro coagulation-promoting effect.

[0062] The total amount of free thrombin generated on the surface of the MOF material (MOF) with adjustable crystal surface that can enhance thrombin activity prepared in Example 1, Example 2 and Example 3 was measured by an in vitro thrombin generation experiment. The results are as follows: Figure 6 The results are shown as mean ± standard error (n = 3), *p < 0.05. Figure 6 It can be seen that the total amount of free thrombin generated on the surface of the facet-controlled organic framework material (MOF) that can enhance thrombin activity prepared in Examples 1, 2 and 3 is higher than that in the negative control platelet-poor plasma group, among which the total amount of free thrombin generated on the surface of Example 2 is as high as 3 times that of the positive control platelet-rich plasma group.

[0063] In summary, the crystal face adjustable organic framework material (MOF) prepared by the present invention that can enhance the activity of thrombin is obtained by changing the molar ratio of zinc salt, 2-methylimidazole and hexadecyltrimethylammonium bromide, the type of solvent and the reaction conditions to obtain a cubic hexahedron MOF (ZIF-8 / C) exposing the {100} crystal face, a truncated octahedron MOF (ZIF-8 / TRD) exposing the {100} and {110} crystal faces, and a rhombic dodecahedron MOF (ZIF-8 / RD) exposing the {110} crystal face. The MOF surfaces that expose different crystal faces can adsorb thrombin and regulate the catalytic activity of the attached thrombin, have excellent coagulation ability, can be used as a hemostatic active molecule or as a hemostatic active molecule loaded on a carrier to prepare a high-performance hemostatic material for surgical hemostasis, and have great clinical application potential.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a surface-adjustable organic framework material capable of enhancing thrombin activity, characterized in that: The preparation method is as follows: Using zinc salt as a metal source, 2-methylimidazole as an organic ligand, and hexadecyltrimethylammonium bromide as a structure directing agent, the zinc salt is dissolved in solvent I to form a B solution, 2-methylimidazole and hexadecyltrimethylammonium bromide are dissolved in solvent I together to form an A solution, the A solution is added to the B solution, mixed evenly, and then allowed to react at room temperature for 2 to 24 hours, and then the mixture after the reaction is completed is centrifuged, washed and vacuum dried to obtain a crystal face-regulatable organic framework material that can enhance thrombin activity.

2. The preparation method according to claim 1, characterized in that: The molar ratio of zinc ion, 2-methylimidazole and hexadecyltrimethylammonium bromide in the zinc salt is 1:4-57:0-0.

073.

3. The preparation method according to claim 1, characterized in that: The zinc salt is zinc nitrate hexahydrate or zinc acetate dihydrate; The solvent is methanol or deionized water; The vacuum drying is performed at 60° C. for 12 to 24 hours.

4. A surface-adjustable organic framework material capable of enhancing thrombin activity prepared according to the preparation method according to any one of claims 1 to 3.

5. The surface-adjustable organic framework material capable of enhancing thrombin activity according to claim 4, characterized in that: The particle size of the crystal face-adjustable organic framework material capable of enhancing thrombin activity is 100-200 nm.

6. The surface-adjustable organic framework material capable of enhancing thrombin activity according to claim 4, characterized in that: The crystal face adjustable organic framework material capable of enhancing thrombin activity is a polyhedron that exposes any one or both of the {100} crystal face and the {110} crystal face; The surface area of ​​the {100} crystal plane or the {110} crystal plane in the crystal plane-adjustable organic framework material capable of enhancing thrombin activity accounts for 0 to 100%.

7. The surface-adjustable organic framework material capable of enhancing thrombin activity according to claim 4, characterized in that: The crystal face adjustable organic framework material capable of enhancing thrombin activity is a cubic hexahedron exposing a {100} crystal face, a truncated octahedron exposing a {100} and {110} crystal face, or a rhombic dodecahedron exposing a {110} crystal face.

8. The surface-adjustable organic framework material capable of enhancing thrombin activity according to claim 4, characterized in that: The crystal face adjustable organic framework material capable of enhancing thrombin activity is a cubic hexahedron in which the surface area of ​​the {100} crystal face accounts for 100% and the surface area of ​​the {110} crystal face accounts for 0%; The crystal face adjustable organic framework material capable of enhancing thrombin activity is a truncated octahedron in which the surface area of ​​the {100} crystal face accounts for 40% and the surface area of ​​the {110} crystal face accounts for 60%; The crystal face adjustable organic framework material capable of enhancing thrombin activity is a rhombic dodecahedron in which the surface area of ​​the {100} crystal face accounts for 0% and the surface area of ​​the {110} crystal face accounts for 100%.

9. Use of the face-regulatable organic framework material capable of enhancing thrombin activity according to any one of claims 4 to 8 in the preparation of hemostatic materials.

10. The use according to claim 9, characterized in that: In the application, the carrier is loaded on a carrier, and the carrier is a protein material, a polypeptide silk protein, gelatin or its derivatives, fibrin, a polysaccharide material or a polymer material; The polysaccharide material is any one or more of cellulose, starch, agarose, dextran, chitosan, alginate or hyaluronic acid; The polymer material is any one or more of polyethylene glycol, polyvinyl alcohol, polyurethane, polycaprolactone, polylactic acid-glycolic acid, and polyethylene oxide.

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