Enzyme composite material with osteogenic antibacterial activity as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and discloses an enzyme composite material with osteogenic antibacterial activity as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing an imidazole-2-formaldehyde solution with an alkaline phosphatase storage mother solution, then adding a zinc nitrate aqueous solution, and synthesizing the enzyme composite material in situ. The composite material disclosed by the invention has good tolerance and cycling stability, and also has osteogenic antibacterial activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to an enzyme composite material with osteogenic and antibacterial activities, a preparation method thereof, and an application thereof. Background Art

[0002] Alkaline Phosphatase (ALP) is a dimeric metalloenzyme composed of several isozymes, which can catalyze the hydrolysis of pyrophosphate, an inhibitor of bone mineralization, provide inorganic phosphate ions for the nucleation and formation of hydroxyapatite, and promote mineralization. In recent years, as the role of ALP in bone defect and bone repair has been gradually known to people, many studies have incorporated it into hydrogels or used it as a surface coating material to be loaded onto the surfaces of metals and inert bone implants, taking advantage of the osteogenic property of ALP to enhance the bioactivity of bone implants and assist bone repair treatment. For example, in the prior art, an enzyme / CaGP mineralized hydrogel was prepared in 2012 by incorporating ALP into the hydrogel biomaterial to induce calcium phosphate (CaP) mineralization; the prior art also reported grafting ALP onto the surface of metal bone implants through the activation of trichloride to endow the material with enzyme bioactivity; and covalently fixing ALP onto two bio-inert ceramics through aldehyde groups to enhance the hydrophilicity and cell adhesion of the ceramic implants.

[0003] Although many important application progresses have been made in the modification of bone implants by ALP, there are still many challenges. For example, only when the action conditions are mild can the enzyme exhibit excellent specificity and significant catalytic activity, and the enzyme is not heat-resistant and is prone to denaturation during storage and operation. In addition, during the production process, residual enzymes may become contaminants in the product, requiring expensive separation and purification processes, which seriously hinder the large-scale and industrial application of enzymes.

[0004] The tolerance and cyclic stability of the enzyme catalysts in the prior art are poor.

[0005] Therefore, there is an urgent need to provide an enzyme catalyst with good tolerance and cyclic stability. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above prior art. For this purpose, the present invention provides an enzyme composite material with osteogenic and antibacterial activities, a preparation method thereof, and an application thereof. The composite material of the present invention has good tolerance and cyclic stability, and also has osteogenic and antibacterial activities.

[0007] The present invention uses ZIF-90 as an "armor" to load the ALP enzyme to improve its temperature tolerance and cyclic stability, and further modifies the "armored enzyme" onto the surfaces of inert implants such as Ti and PEEK (polyetheretherketone) with the help of silk fibroin, endowing it with enzyme catalytic activity, osteogenic activity, and antibacterial property.

[0008] Metal-Organic Frameworks (MOF) have highly tunable structures and pore properties and are good matrices for enzyme immobilization. Imidazole-2-carboxaldehyde (2-ICA) can react with Zn 2+ to synthesize Zn-MOF with a porous structure (denoted as ZIF-90). In the present invention, it is used as a carrier for immobilized enzyme. On the one hand, it can provide physical protection for the enzyme, enabling the enzyme to stably exhibit catalytic activity for a long time; on the other hand, the degradation of Zn-MOF releases Zn 2+ , which can play a role in slow-release antibacterial action.

[0009] The first aspect of the present invention provides a method for preparing an enzyme composite material with both osteogenic and antibacterial activities.

[0010] A method for preparing an enzyme composite material with both osteogenic and antibacterial activities, comprising the following steps:

[0011] Mix the imidazole-2-carboxaldehyde solution with the alkaline phosphatase (ALP) stock solution, and then add an aqueous zinc nitrate solution to in-situ synthesize the enzyme composite material (the enzyme composite material can also be referred to as "armored enzyme").

[0012] Preferably, the concentration of the imidazole-2-carboxaldehyde solution is 0.05 - 0.2 M, and more preferably 0.1 M.

[0013] Preferably, the solvent in the imidazole-2-carboxaldehyde solution is an ethanol aqueous solution, and more preferably an ethanol aqueous solution with a volume fraction of 45 - 50%.

[0014] Preferably, the concentration of the aqueous zinc nitrate solution is 0.05 - 0.2 M, and more preferably 0.05 M.

[0015] Preferably, the aqueous zinc nitrate solution can be an aqueous zinc nitrate hexahydrate solution.

[0016] Preferably, the concentration of the alkaline phosphatase stock solution is 3 - 4 kU / mL, and more preferably 4 kU / mL.

[0017] Preferably, the volume ratio of the imidazole-2-carboxaldehyde solution, the alkaline phosphatase stock solution, and the aqueous zinc nitrate solution is 15 - 20:1:5 - 10, and more preferably 20:1:10.

[0018] Preferably, the preparation method comprises the following steps:

[0019] (1) Pipette and mix the imidazole-2-carboxaldehyde solution with the alkaline phosphatase stock solution in proportion, then add the aqueous zinc nitrate solution, seal, and react on a shaker at 100 - 150 rpm for 0.5 - 1 h to obtain a mixture;

[0020] (2) Centrifuge the mixture obtained in step (1) at 5000 - 10000 rpm for 10 - 15 min, take the precipitate, wash it with water 1 - 3 times, and lyophilize it to obtain the enzyme composite material.

[0021] The second aspect of the present invention provides an enzyme composite material with both osteogenic and antibacterial activities.

[0022] An enzyme composite material with both osteogenic and antibacterial activities, which is prepared by the above preparation method.

[0023] The third aspect of the present invention provides an application of an enzyme composite material with both osteogenic and antibacterial activities.

[0024] The application of the enzyme composite material with both osteogenic and antibacterial activities prepared by the above preparation method in the field of medical device preparation.

[0025] A preparation method of a bone implant, comprising the following steps:

[0026] (1) Prepare the enzyme composite material into an enzyme composite material solution, and then add a silk fibroin solution and mix to obtain a mixed solution;

[0027] (2) Surface - modify the implant material with dopamine and protocatechuic aldehyde, after air - drying, coat the mixed solution in step (1) on the surface of the implant material, add pure water, seal it, dialyze it on a shaker, and air - dry it to obtain the bone implant.

[0028] Preferably, the concentration of the enzyme composite material solution is 20 - 40 U / mL, and more preferably 30 - 40 U / mL.

[0029] Preferably, the concentration of the silk fibroin solution is 25 - 28.57 mg / mL, and more preferably 26 - 28.57 mg / mL.

[0030] Preferably, the volume ratio of the enzyme composite material solution to the silk fibroin solution is 3:(5 - 7), and more preferably 3:7.

[0031] Preferably, the implant material includes Ti or PEEK (polyetheretherketone).

[0032] Preferably, the shaker dialysis is carried out at 50 - 60 rpm for 10 - 12 h.

[0033] The bone implant prepared by the above method uses the implant material as the substrate and the enzyme composite material as the surface coating component.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] (1) The enzyme composite material of the present invention has the structural feature of encapsulating ALP with ZIF-90, which significantly improves the temperature tolerance and cyclic stability of ALP.

[0036] (2) The enzyme composite material coating of the present invention can significantly improve the cell adhesion and biocompatibility of inert bone implant materials, and play a certain role in promoting osteogenesis and antibacterial. Description of the Drawings

[0037] Figure 1 is a schematic diagram of the synthesis of the enzyme composite material in Example 1;

[0038] Figure 2 is a schematic diagram of the antibacterial and osteogenesis-promoting principles of the enzyme composite material in Example 1;

[0039] Figure 3 is a scanning electron micrograph of the Zn-MOF obtained in Comparative Example 1 and the ALP@Zn-MOF material obtained in Example 1;

[0040] Figure 4 is the activity of "armored enzyme" in three groups of bone implant samples (ALP@MOF-Silk-dPEEK, Zn / ALP-Silk-dPEEK, ALP-dPEEK) under cyclic treatment at different temperatures;

[0041] Figure 5 is to soak three groups of bone implant samples (ALP@MOF-Silk-dPEEK, MOF-Silk-dPEEK, PEEK) in an aqueous solution of calcium glycerophosphate, mineralize for 3 days (days), wash and dry, and use SEM to observe the deposition of inorganic calcium salts on the material surface;

[0042] Figure 6 is the image of the number of surviving colonies on the agar plate after co-culturing the composite material solution of Example 1 with different concentrations and Staphylococcus aureus for 8 h at different pH values and the statistical chart of the number of colonies of some concentrations;

[0043] Figure 7 is the image of the number of surviving colonies on the agar plate after co-culturing different groups of samples with bacteria for 6 h and the statistical chart of the number of colonies, and the image of the number of colonies of the bacteria adhered to different groups of samples on the agar plate and the statistical chart of the number of colonies. Detailed Embodiments

[0044] In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0045] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0046] Example 1

[0047] A preparation method of an enzyme composite material with both osteogenic and antibacterial activities, comprising the following steps:

[0048] Under ultrasonic treatment and heating conditions, 2-ICA (imidazole-2-carboxaldehyde) was dissolved in an aqueous ethanol solution with a volume fraction of 50% to obtain a 0.1 M 2-ICA solution; to the cooled 2-ICA solution, a 4 kU / mL ALP (alkaline phosphatase) stock solution was added so that the concentration of ALP in the final reaction system was 40 U / mL; an aqueous solution of Zn(NO3)2·6H2O (0.05 M) was added to the mixed solution of 2-ICA and ALP, and the clear yellow solution immediately became turbid;

[0049] After sealing, at room temperature, the reaction was carried out on a shaker at 150 rpm for 1 h, centrifuged (10000 rpm, 15 min), the precipitate was taken, washed with water 3 times, freeze-dried, to obtain an enzyme composite material (denoted as ALP@MOF or ALP@Zn-MOF).

[0050] A preparation method of a bone implant, comprising the following steps:

[0051] (1) Silkworm cocoon → Silk: Take 2 L of pure water and boil it in a beaker; weigh Na2CO3 (4.24 g) and silkworm cocoon (5 g, cut into 1 cm 2 size); after the water boils, add Na2CO3 (slowly add to avoid generating a large amount of bubbles and causing bumping), and then add the silkworm cocoon and boil for 30 min (during which it is necessary to stir irregularly to promote fiber dispersion, and the liquid inside the silk can be squeezed out with a glass rod in a timely manner to promote the boiling out of sericin); take out the silk, rinse and cool it with pure water, and rub it in water until there is no slippery feeling; place the silk in pure water (1 L) and wash for 20 min, repeat 3 times; take out the silk, squeeze out the water, and air-dry overnight;

[0052] (2) Cut the dialysis bag into small sections of 10 - 20 cm; boil the dialysis bag in a large volume of 2% (w / v, 2 g / mL) NaHCO3 and 1 mM EDTA (ethylenediaminetetraacetic acid) for 10 min, and then wash it with distilled water; then, boil it in 1 mM EDTA for 10 min and also wash it with distilled water; after cooling, place it in 75% alcohol and put it in a 4°C refrigerator; wash the dialysis bag with distilled water before use;

[0053] (3) Weigh 0.5 g of the silk prepared in step (1) and 2.424 g of LiBr, and sterilize them under ultraviolet light. Under sterile conditions, add sterile water to LiBr to prepare a 3 mL solution. Place the silk in a sterile glass bottle, and add 2 mL of the LiBr solution (9.3 M) from the top. After sealing, place it in an oven and keep it at 60 °C for 4 h. The silk fibroin dissolves to obtain a silk fibroin solution, which is a pale yellow viscous liquid.

[0054] (4) Load the silk fibroin solution from step (3) into the dialysis bag (cut-off molecular weight MWCO: 3500) treated in step (2). Add pure water at a ratio of 1 L of pure water / 12 mL of silk fibroin solution, and dialyze for 48 h, changing the water every 12 h. Collect the liquid in the dialysis bag, centrifuge at 10000 rpm for 15 min, collect the supernatant to obtain the final silk fibroin solution, and store it in a refrigerator at 4 °C. Weigh the "aluminum foil bowl", take 1 mL of the final silk fibroin solution into the "aluminum foil bowl", dry it in the oven and then measure the total weight to calculate the concentration of silk fibroin in the solution.

[0055] (5) Surface polishing and cleaning of PEEK: Take a PEEK sheet and polish it smoothly according to the sides (#400, indicating the grit number of the sandpaper is 400), front and back (#400, #2000, indicating the grit number of the sandpaper is 400, 2000), and ultrasonically clean it in acetone, ethanol, and water for 15 min each.

[0056] (6) DA / PA modification of the PEEK surface

[0057] 1. Treatment with dopamine hydrochloride (DA)

[0058] Prepare Tris (tris(hydroxymethyl)aminomethane) buffer solution (25 mM, pH = 8.5); dissolve DA in Tris buffer at a concentration of 2 mg / mL to obtain a DA solution. Place the PEEK sheet treated in step (5) into a 24-well plate, with the front side facing up, and add 2 mL of the DA solution to each well. Shake it on a shaker for 24 h (37 °C, 60 rpm, protected from light). Take out the PEEK sheet, ultrasonically clean it for 10 min, and air dry it for later use.

[0059] 2. Treatment with protocatechuic aldehyde (PA) / DA

[0060] Dissolve 2 mg of DA and 1.14 mg of PA in 1 mL of Tris buffer to obtain a DA / PA solution. Place the PEEK sheet obtained from "1. Treatment with dopamine hydrochloride (DA)" in step (6) into a 24-well plate, with the front side facing up, and add 2 mL of the above-prepared DA / PA solution to each sheet. Shake it on a shaker at 37 °C, 60 rpm, and protect it from light for 8 h. After rinsing, ultrasonically clean it for 10 min and air dry it at room temperature.

[0061] (7) Construction of the "armored enzyme" coating on the PEEK surface

[0062] a. Prepare 0.1 M 2-ICA solution and 0.05 M Zn(NO3)2·6H2O solution, sterilize them with a 0.22 μm microporous filter membrane and reserve for use.

[0063] b. Dilute the final silk fibroin solution (denoted as Silk solution) prepared in step (4) to 28.57 mg / mL with sterile pure water and reserve for use. Thaw the ALP stock solution in advance and reserve for use.

[0064] c. Prepare 4 groups of material solutions according to Tables 1, 2, 3, and 4 (first add the component solutions of the first three groups from left to right in the order in the table, seal, react on a shaker at 150 rpm for 1 h, and then add Silk solution / pure water).

[0065] Table 1: Components of ALP@MOF-Silk solution

[0066]

[0067] Table 2: Components of Zn / ALP-Silk solution

[0068]

[0069] Table 3: Components of ALP solution

[0070]

[0071] Table 4: Components of MOF-Silk solution

[0072]

[0073]

[0074] Put the PEEK sheets processed in step (6) into 24-well plates, and coat 50 μL of the material solutions in Tables 1 to 4 on the surface of each PEEK sheet respectively (that is, only coat one of the material solutions in Tables 1 to 4 on the surface of one PEEK sheet), and air dry for later use.

[0075] Add pure water according to the ratio of 20 mL / sheet, seal, dialyze on a shaker (60 rpm) for 12 h, and then air dry in a laminar flow hood for later use. Respectively prepare bone implants ALP@MOF-Silk-dPEEK (treatment corresponding to the material solution in Table 1), Zn / ALP-Silk-dPEEK (treatment corresponding to the material solution in Table 2), ALP-dPEEK (treatment corresponding to the material solution in Table 3), and MOF-Silk-dPEEK (treatment corresponding to the material solution in Table 4).

[0076] Comparative Example 1

[0077] Compared with Example 1, the composite material prepared in Comparative Example 1 is an enzyme-free composite material, and the corresponding preparation method includes the following steps:

[0078] Under ultrasonic treatment and heating conditions, dissolve 2-ICA (imidazole-2-carboxaldehyde) in an ethanol aqueous solution with a volume fraction of 50% to obtain a 0.1 M 2-ICA solution; add an aqueous solution of Zn(NO3)2·6H2O (0.05 M) to the 2-ICA solution;

[0079] After sealing, react at room temperature on a shaker at 150 rpm for 1 h, centrifuge (10,000 rpm, 15 min), take the precipitate, wash it with water 3 times, and freeze-dry to obtain a composite material (denoted as Zn-MOF).

[0080] Comparative Example 2

[0081] Comparative Example 2 is untreated PEEK.

[0082] Product effect

[0083] Figure 1 It is a schematic diagram of the synthesis of the enzyme composite material in Example 1. Among them, "Rt" represents room temperature, and "Stirring" represents stirring.

[0084] Figure 2 It is a schematic diagram of the antibacterial and osteogenic induction principle of the enzyme composite material in Example 1; among them, R-OH represents a substance containing a hydroxyl group, and HAP represents hydroxyapatite.

[0085] Figure 3 It is the scanning electron microscope image of the Zn-MOF obtained in Comparative Example 1 (corresponding to Figure 3 Figure a therein) and the ALP@Zn-MOF obtained in Example 1 (corresponding to Figure 3 Figure b therein) materials. The surface of ALP@MOF is rougher and the proportion of regular dodecahedrons is less. It is speculated that the possible reason is that Zn-MOF is interfered by the enzyme during the assembly process, resulting in more crystal defects and changing the surface morphology of the product.

[0086] Figure 4 It is the activity of "armored enzyme" in three groups of bone implant samples (ALP@MOF-Silk-dPEEK, Zn / ALP-Silk-dPEEK, ALP-dPEEK) under cyclic treatment at different temperatures; among them, Cycle times represents the number of cycles, and OD(405nm) represents the absorbance at 405 nm.

[0087] From Figure 4It can be seen that under the same number of cycles, the ALP activities of the three groups of samples are as follows: ALP@MOF-Silk-dPEEK > Zn / ALP-Silk-dPEEK > ALP-dPEEK. Analyzing the reasons, on the one hand, because MOF has a special porous structure, it can load more ALP than pure Silk solution and aqueous solution. On the other hand, thanks to the protection of the MOF armor, relatively less ALP is washed away during the cycle, enabling the ALP activity of the ALP@MOF-Silk-dPEEK group of samples to remain at a relatively high level in the later stage of the cycle. At the same time, by comparing the ALP activities under three temperature cycles, it is found that the attenuation rate of the 50 °C cycle is basically the same as that of the other two groups, indicating good temperature tolerance.

[0088] Figure 5 Three groups of bone implant samples (ALP@MOF-Silk-dPEEK, MOF-Silk-dPEEK, PEEK) were immersed in an aqueous solution of calcium glycerophosphate, mineralized for 3 days (d), washed and dried, and SEM was used to observe the deposition of inorganic calcium salts on the material surface. Among them, a corresponds to PEEK in Comparative Example 2; b corresponds to MOF-Silk-dPEEK; c corresponds to ALP@MOF-Silk-dPEEK.

[0089] From Figure 5 It can be seen that there are almost no large particulate deposits on the surface of PEEK; flaky substances accumulate on MOF-Silk-dPEEK, presumably calcium glycerophosphate precipitated due to the increase in temperature during incubation; while a large number of obvious inorganic calcium salt particles can be seen deposited on the surface of ALP@MOF-Silk-dPEEK.

[0090] This proves that ALP@MOF can promote the hydrolysis of organic calcium phosphate salts in vitro and the deposition of inorganic calcium salts such as hydroxyapatite.

[0091] Figure 6 Images of the number of surviving colonies on the agar plate after co-culturing the composite material solution of Example 1 with different concentrations at different pH values for 8 h (corresponding to Figure 6 Figure a in Figure 6 and the statistical chart of the number of colonies at some concentrations (corresponding to

[0092] From Figure 6It can be seen that under the same acidic / basic conditions, the number of surviving colonies decreases with the increase in the concentration of the composite material solution, indicating that all three groups of composite material solutions in the experiment have obvious antibacterial effects, and the antibacterial ability of their solutions is positively correlated with the concentration. In addition, by comparing the number of surviving colonies in two groups with different acid / base properties at the same composite material concentration, it was found that the number of colonies in the pH = 5.5 group was less than that in the pH = 7.4 group. It is speculated that the possible reason is that under acidic environmental conditions, Zn-MOF is more easily degraded, generating more Zn 2+ , and the antibacterial effect is better. However, considering that ALP is more active under slightly alkaline conditions and that inorganic calcium salts such as deposited hydroxyapatite may dissolve under slightly acidic conditions, in order to better exert the osteogenic effect of ALP and simulate the extracellular environment after material implantation, subsequent antibacterial experiments on the material surface were all carried out under the condition of pH = 7.4.

[0093] Figure 7 are the images of surviving colonies on the agar plate after co-culturing different groups of samples with bacteria for 6 h (corresponding to Figure 7 Figure a therein) and the quantity statistics (corresponding to Figure 7 Figure c therein), as well as the colony images of the bacteria adhered to different groups of samples on the agar plate (corresponding to Figure 7 Figure b therein) and the quantity statistics (corresponding to Figure 7 Figure d therein), where Counts represents the count.

[0094] As Figure 7 (a, c), there was no significant difference in the number of colonies among the ALP@MOF-Silk-dPEEK, Zn / ALP-Silk-dPEEK, and PEEK groups, but the number of colonies in all three groups of samples was less than that in the ALP-dPEEK group. It is speculated that the reason may be that the hydrophobicity and surface inertness of PEEK are not conducive to bacterial adhesion, and the bacterial adhesion ability of dPEEK after DA / PA treatment is enhanced, which is more conducive to bacterial growth. Therefore, an additional anti-bacterial adhesion experiment was conducted, and the results are as shown in Figure 7 (b, d). By comparing Figure 7 Figure b therein with Figure 7 Figure d therein, it was found that the number of colonies in the ALP-dPEEK group was also more than that in the other three groups.

[0095] This proves that ALP@MOF-Silk-dPEEK has antibacterial and anti-bacterial adhesion effects equivalent to those of PEEK and better than those of ALP-dPEEK.

Claims

1. A preparation method of an enzyme composite material with both osteogenic and antibacterial activities, characterized in that, It includes the following steps: Mix the imidazole-2-carboxaldehyde solution with the alkaline phosphatase stock solution, and then add the zinc nitrate aqueous solution to in-situ synthesize the enzyme composite material.

2. The preparation method according to claim 1, wherein The concentration of the imidazole-2-carboxaldehyde solution is 0.05 - 0.2 M.

3. The preparation method according to claim 1, characterized in that, The concentration of the zinc nitrate aqueous solution is 0.05 - 0.2 M.

4. The preparation method according to claim 1, characterized in that, The concentration of the alkaline phosphatase stock solution is 3 - 4 kU / mL.

5. The preparation method according to any one of claims 1-4, characterized in that, The volume ratio of the imidazole-2-carboxaldehyde solution, the alkaline phosphatase stock solution, and the zinc nitrate aqueous solution is 15 - 20:1:5 - 10.

6. An enzyme composite material with both osteogenic and antibacterial activities, characterized in that, Prepared by the preparation method according to any one of claims 1 - 5.

7. Use of the enzyme composite material with osteogenic and antibacterial activities prepared by the preparation method according to any one of claims 1 - 5 in the field of preparing medical devices.

8. A method for preparing a bone implant, characterized in that, It includes the following steps: (1) Prepare the enzyme composite material solution with the enzyme composite material having osteogenic and antibacterial activities prepared by the preparation method according to any one of claims 1 - 5, and then add the silk fibroin solution and mix to obtain a mixed solution; (2) Modify the surface of the implant material with dopamine and protocatechuic aldehyde. After air-drying, coat the mixed solution in step (1) on the surface of the implant material, add pure water, seal, dialyze on a shaker, and air-dry to obtain the bone implant.

9. The preparation method according to claim 8, characterized in that, The concentration of the enzyme composite material solution is 20 - 40 U / mL; and / or, the concentration of the silk fibroin solution is 25 - 28.57 mg / mL; and / or, the volume ratio of the enzyme composite material solution to the silk fibroin solution is 3:(5 - 7).

10. The preparation method according to claim 8, characterized in that, The implant material includes Ti or polyetheretherketone.

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