Fibroin-calcium carbonate composite material as well as preparation method and application thereof

Abstract

The invention discloses a fibroin-calcium carbonate composite material and a preparation method and application thereof, and the preparation method comprises the following steps: step 1, adding degummed fibroin into a calcium chloride solution, and carrying out boiling water bath to obtain a fibroin-calcium chloride solution; step 2, adding the fibroin-calcium chloride solution obtained in the step 1 into a DES solvent, and uniformly stirring to obtain a solution A; adding the calcium carbonate suspension into a DES solvent, and uniformly stirring to obtain a suspension B; step 3, adding the turbid liquid B into the solution A, and stirring for reaction to obtain milk white turbid liquid; 4, after the milk white turbid liquid obtained in the step 3 is centrifuged or filtered and separated, the solid part is washed with deionized water and then dried, and the faint yellow fibroin-calcium carbonate composite material is obtained. According to the prepared fibroin-calcium carbonate composite material, on one hand, due to the existence of fibroin, the biocompatibility of the material is higher, so that the product has the potential of becoming an implanted filling material and a whitening cosmetic; on the other hand, due to the existence of amino groups on the surface of the carrier, further modification is facilitated, for example, after glutaraldehyde or genipin is used as a cross-linking agent for treatment, the material has the potential of becoming an immobilized enzyme carrier.

Description

Technical Field

[0001] The invention relates to the technical field of new materials, and in particular to a silk fibroin-calcium carbonate composite material and a preparation method and application thereof. Background Art

[0002] As a natural protein fiber, silk has long been used in the production of high-end textiles due to its unique biocompatibility and excellent mechanical properties. In recent years, with the development of biomaterials science, the use of silk has far exceeded the textile industry. Silk fibroin, the main component of silk fiber, has become one of the research hotspots in the field of biomedical materials due to its good biocompatibility, degradability and mechanical properties. Therefore, silk fibroin can not only be used to make textiles, but also as a basic raw material for biomedical materials, such as tissue engineering scaffolds, drug sustained-release carriers, wound dressings, etc.

[0003] Calcium carbonate (CaCO 3 ) is an inorganic compound that has been widely used in many industrial fields due to its good chemical stability, non-toxicity and relatively low price. In particular, nano-scale calcium carbonate, due to its high specific surface area and special physical and chemical properties, has shown superior performance in the fields of medicine, food, chemical industry, cosmetics, etc.

[0004] The present invention utilizes silk fibroin derived from silk to prepare a silk fibroin-calcium carbonate composite material, which can achieve modification of calcium carbonate particles to make the calcium carbonate particles have better biocompatibility and functionality. Summary of the invention

[0005] The purpose of the present invention is to provide a silk fibroin-calcium carbonate composite material and its preparation method and application, so as to solve the problem of poor biocompatibility and poor functionality of the above-mentioned calcium carbonate particles. The silk fibroin of the present invention can be composited with calcium carbonate particles to give them better dispersibility and stability, and can also be combined with active groups on the surface of calcium carbonate through covalent bonds or non-covalent interactions, thereby enhancing its performance in specific applications.

[0006] To achieve the above object, the present invention provides a method for preparing a silk fibroin-calcium carbonate composite material in a first aspect, comprising the following steps:

[0007] Step 1: Add the degummed silk into a calcium chloride solution and place in a boiling water bath to obtain a silk-calcium chloride solution;

[0008] Step 2: Add the silk fibroin-calcium chloride solution obtained in step 1 to the DES solvent, stir evenly, and obtain solution A; add the calcium carbonate suspension to the DES solvent, stir evenly, and obtain suspension B;

[0009] Step 3: Add suspension B to solution A, stir and react to obtain a milky white turbid solution;

[0010] Step 4: After the milky white turbid liquid obtained in step 3 is separated by centrifugation or filtration, the solid part is washed with deionized water and then dried to obtain a light yellow silk fibroin-calcium carbonate composite material.

[0011] Preferably, the concentration of the calcium chloride solution in step 1 is 5-6 M, and the mass volume ratio of the degummed silk fibroin to the calcium chloride solution is 0.5-1 g: 5-10 mL.

[0012] Preferably, the boiling water bath time in step 1 is 8 to 12 minutes.

[0013] Preferably, in step 2, the volume ratio of the silk fibroin-calcium chloride solution to the DES solvent is 1:(1-1.2), the concentration of the calcium carbonate suspension is 2.5-3.5 M, and the volume ratio of the calcium carbonate suspension to the DES solvent is 1:(2-2.2).

[0014] The configuration method of the above calcium carbonate suspension is as follows:

[0015] Taking 100mL suspension as an example, calculate the amount of solid calcium carbonate powder to be added to water according to the above concentration (2.5-3.5M), then weigh the calculated amount of calcium carbonate powder and add it to 80mL of water, stir evenly, then add water to 100mL, continue to stir evenly, and obtain a calcium carbonate suspension.

[0016] Preferably, in step 3, the volume ratio of solution A to suspension B is 1:(2-2.2).

[0017] Preferably, the DES solvent is prepared by mixing urea and betaine in a molar ratio of 2:1 or glucose and choline chloride in a molar ratio of 1:1 and then heating the mixture.

[0018] Preferably, the heating temperature is 100-150° C. and the heating time is 10-30 min.

[0019] Preferably, the centrifugal speed is 5000-8000 rpm and the time is 5-15 min.

[0020] The second aspect of the present invention provides a silk fibroin-calcium carbonate composite material prepared by the above preparation method.

[0021] The third aspect of the present invention provides an application of a silk fibroin-calcium carbonate composite material, and the application of the silk fibroin-calcium carbonate composite material in filling materials, immobilized enzyme carriers and whitening cosmetics.

[0022] Although there are many types of existing implant filling materials, they generally have some problems, such as immune rejection reaction, difficulty in controlling the biodegradation rate, etc. The silk fibroin-calcium carbonate composite material prepared by the present invention is used in implant filling materials. By compounding silk fibroin with calcium carbonate, a filling material with good biocompatibility can be prepared to reduce the occurrence of immune response.

[0023] Enzyme immobilization technology is an effective means to improve enzyme catalytic efficiency, but in practical applications, how to maintain the activity and stability of the enzyme is a challenge. The silk fibroin-calcium carbonate composite material prepared by the present invention is used in an immobilized enzyme carrier. The silk fibroin and calcium carbonate composite material is used as a carrier of the immobilized enzyme to provide a stable microenvironment to protect the enzyme from external factors. At the same time, the structure of calcium carbonate is used to increase the chance of the enzyme contacting the substrate, thereby improving the catalytic efficiency.

[0024] Many whitening cosmetics contain chemically synthesized whitening agents, which may cause irritation or allergic reactions to human skin. The silk fibroin-calcium carbonate composite material prepared by the present invention is used in whitening cosmetics. The silk fibroin has good moisture retention and biocompatibility. After being compounded with calcium carbonate, it can be used as a natural whitening ingredient, which can not only reduce the generation of melanin, but also moisturize the skin and reduce the occurrence of side effects. In addition, the silk fibroin also has certain antioxidant properties, which can help resist the damage of free radicals to the skin.

[0025] Therefore, the present invention adopts the above-mentioned silk fibroin-calcium carbonate composite material and its preparation method and application, which has the following beneficial effects:

[0026] (1) The raw material used in the present invention is natural protein, which is harmless to biological individuals. The product produced can directly contact with the organism, and the production process is green and environmentally friendly.

[0027] (2) The reaction medium for preparing calcium carbonate particles in the method of the present invention is a low eutectic solvent, and the non-toxic and harmless reaction environment is conducive to dispersion, separation and subsequent utilization.

[0028] (3) In the process of preparing calcium carbonate particles, the present invention dissolves degummed silk in a high concentration calcium chloride solution, so that the calcium carbonate particles with a single surface group are transformed into a silk-calcium carbonate composite material with amino groups. On the one hand, the presence of silk makes the material more biocompatible, so that the product has the potential to become an implant filling material and a whitening cosmetic; on the other hand, the presence of amino groups on the surface of the carrier facilitates further modification, such as using glutaraldehyde or genipin as a cross-linking agent to treat the material, so that the material has the potential to become an immobilized enzyme carrier.

[0029] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a SEM image of the composite material prepared in Example 1;

[0031] Figure 2 This is a SEM image of the composite material prepared in Example 2;

[0032] Figure 3 It is a magnified SEM image of the composite material prepared in Example 1 and pure calcium carbonate;

[0033] Figure 4 The cell proliferation test results of the materials in Example 1-2;

[0034] Figure 5 The material biocompatibility test results of Examples 1-2;

[0035] Figure 6 These are the test results of the immobilized enzyme activity of the materials of Example 1-2. DETAILED DESCRIPTION

[0036] The present invention will be further described below. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and a specific operation process, but the present invention is not limited to this embodiment.

[0037] Example 1

[0038] A method for preparing a silk fibroin-calcium carbonate composite material comprises the following steps:

[0039] (1) 23.4 g of betaine and 24.0 g of urea were heated at 150° C. for 30 minutes and mixed to obtain a clear eutectic solvent (DES solvent);

[0040] (2) adding 0.7 g of degummed silk powder into 7 mL of 5.8 M calcium chloride solution and boiling it in a boiling water bath for 10 minutes to obtain a silk fibroin-calcium chloride solution;

[0041] (3) Add the silk fibroin-calcium chloride solution and 14 mL of 3.0 M calcium carbonate suspension obtained in the previous step to 14 mL of DES solvent and 28 mL of DES solvent, respectively, and stir evenly; add the DES solvent containing calcium carbonate suspension (suspension B) to the evenly mixed DES solvent containing silk fibroin-calcium chloride (solution A) to obtain a milky turbid solution;

[0042] (4) The milky white turbid liquid obtained in the previous step was centrifuged at 6000 rpm for 10 minutes and washed three times with deionized water to obtain a light yellow silk fibroin-calcium carbonate composite material.

[0043] Example 2

[0044] A method for preparing a silk fibroin-calcium carbonate composite material comprises the following steps:

[0045] (1) 35.0 g of choline chloride and 45.0 g of glucose were heated at 100° C. for 10 minutes and mixed to obtain a clear deep eutectic solvent (DES solvent);

[0046] (2) adding 0.7 g of degummed silk powder into 7 mL of 5.8 M calcium chloride solution and boiling it in a boiling water bath for 10 minutes to obtain a silk fibroin-calcium chloride solution;

[0047] (3) Add the silk fibroin-calcium chloride solution and 14 mL of 3.0 M calcium carbonate suspension obtained in the previous step to 14 mL of DES solvent and 28 mL of DES solvent, respectively, and stir evenly; add the DES solvent containing calcium carbonate suspension (suspension B) to the evenly mixed DES solvent containing silk fibroin-calcium chloride (solution A) to obtain a milky turbid solution;

[0048] (4) The milky white turbid liquid obtained in the previous step was centrifuged at 6000 rpm for 10 minutes and washed three times with deionized water to obtain a light yellow silk fibroin-calcium carbonate composite material.

[0049] Comparative Example 1

[0050] The difference between this comparative example and Example 1 is that the DES solvent is replaced by deionized water.

[0051] Comparative Example 2

[0052] The difference between this comparative example and Example 1 is that 24.0 g of urea is dissolved in 23.4 g of water to form a solvent.

[0053] Comparative Example 3

[0054] The difference between this comparative example and Example 1 is that 23.4 g of betaine is dissolved in 24.0 g of water to form a solvent.

[0055] Comparative Example 4

[0056] The difference between this comparative example and Example 2 is that 45.0 g of glucose is dissolved in 35.0 g of water to form a solvent.

[0057] Comparative Example 5

[0058] The difference between this comparative example and Example 2 is that 35.0 g of choline chloride is dissolved in 45.0 g of water to form a solvent.

[0059] Performance Testing

[0060] (1) Characterization of the particle size and dispersion of silk fibroin-calcium carbonate composites. Figure 1 and Figure 2It can be seen from the figure that it shows the material morphology after calcium carbonate is connected to silk fibroin, with uniform particle size and good dispersibility. Figure 3 From the enlarged picture, it can be found that the composite material connected with silk (a) is significantly different from the pure calcium carbonate (b). It can be seen intuitively that silk increases the specific surface area of ​​the calcium carbonate material. The introduction of silk will also increase biocompatibility, which is beneficial to the subsequent implant filling or enzyme immobilization application needs.

[0061] (2) Testing the cell proliferation effect of the materials prepared in Examples 1 and 2

[0062] Proliferation of human fibroblasts and detection of collagen content: After cell recovery, the cells were inoculated in a 96-well plate and cultured in a cell culture incubator for 24 hours. 100 μL of supernatant solution with concentrations of 0.05, 0.1, 0.15, and 0.2 mg / mL prepared with 2.5% serum culture medium was added to each well. No drug was added to the blank control group, and 6 parallels were set up in each group. After incubation for 24 hours, 20 μL of 5 mg / mL MTT solution was added to each well, mixed and incubated in a cell culture incubator for 4 hours. The liquid in the culture plate was aspirated, 100 μL of DMSO solution was added to each well and mixed for 15 minutes, and the absorbance was measured at 570 nm on a microplate reader. Relative cell viability = (OD 实验组 -OD 交白 / OD 对照组 -OD 交白 )×100%, the collagen content was detected by the kit method.

[0063] Test results such as Figure 4 As shown, it is shown that Example 1 and Example 2 can promote the proliferation of human fibroblasts at a concentration of less than 0.15 mg / mL, have no cytotoxicity, and have collagen inducing activity.

[0064] (3) Evaluation of the biocompatibility of the materials prepared in Example 1-2

[0065] Blood compatibility evaluation: Place the centrifuge tubes containing samples (Examples 1 and 2) in a 37°C water bath for 30 minutes, add 1 mL of diluted fresh rabbit blood to each tube (200 μL rabbit blood is diluted with 10 mL of 9 g / L NaCl solution); rabbit blood diluted with 9 g / L NaCl solution is used as a negative control, and 200 μL rabbit blood is diluted with 10 mL of distilled water as a positive control; place each group of centrifuge tubes in a 37°C water bath again for 1 hour, take the supernatant and transfer it to a colorimetric cup, use a spectrophotometer to measure the OD545nm value, and calculate the hemolysis rate of each sample. Hemolysis rate = (OD 样品 -OD 阴性对照 ) / (OD 阳性对照 -OD 阴性对照 )×100%.

[0066] Test results such as Figure 5 As shown, the hemolysis rates of the materials prepared in Example 1 and Example 2 are approximately 0.65% and 0.73%, respectively, and the materials with a hemolysis rate of less than 2% are non-hemolytic biomedical materials, indicating that the samples in Example 1 and Example 2 of the present invention have a low hemolysis rate, little destructiveness to red blood cells, good blood compatibility, and are suitable for non-hemolytic biomedical materials.

[0067] (4) Effect of Example 1-2 on Immobilized Enzyme

[0068] The materials obtained in Example 1 and Example 2 were cross-linked with 10 mL of glutaraldehyde at room temperature for 3 hours, and then the excess glutaraldehyde was washed away with 0.1 M PBS buffer (pH 7.0), and 2 mL of papain solution (1 mg / mL) was added. After fixing at room temperature for 10 hours, the free enzyme was washed away with PBS buffer. The silk material of the immobilized enzyme was taken, 1 mL of activator was added, 5 mg / mL of substrate casein was added after 37°C water bath, and after reacting at 37°C for 10 minutes, 5% trichloroacetic acid solution was immediately added to terminate the reaction. After standing for 30 minutes, the supernatant was taken and the absorbance value A was measured at a wavelength of 275 nm to calculate the enzyme activity. After the enzyme was fixed under the optimal immobilization conditions, its enzyme activity was determined to be 100%, and other reaction conditions were constant. The reaction temperatures were set to 40°C, 50°C, 60°C, 70°C and 80°C, respectively, and the relative enzyme activities of the free papain in the control group and the immobilized enzymes of the two examples were compared.

[0069] Test results from Figure 6 It can be found that the relative enzyme activity of each group increases with increasing temperature and begins to decrease when the optimum temperature is reached. However, compared with the control group, the immobilized enzyme in the example has higher temperature stability, lower sensitivity to catalytic reaction temperature, a wider range of applications, and is not easily inactivated.

[0070] The embodiment was compared with 5 comparative examples by the above test method. It can be seen from Table 1 that there is little difference in particle size between the embodiment and the comparative example, but the dispersion of the embodiment is more uniform, and the contact and connectivity of the silk fibroin are better during the formation of the composite material, which in turn affects the subsequent material in terms of cell proliferation characteristics, biocompatibility and the effect of immobilized enzyme.

[0071] Table 1 Parameter comparison of the embodiment and 5 comparative examples

[0072]

[0073]

[0074] Therefore, the present invention adopts a silk fibroin-calcium carbonate composite material with the above structure, and its preparation method and application. The prepared silk fibroin-calcium carbonate composite material, on the one hand, has stronger biocompatibility due to the presence of silk fibroin, so that the product has the potential to become an implant filling material and a whitening cosmetic; on the other hand, the presence of amino groups on the surface of the carrier facilitates further modification, such as treating with glutaraldehyde or genipin as a cross-linking agent, so that the material has the potential to become an immobilized enzyme carrier.

[0075] 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 they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a silk fibroin-calcium carbonate composite material, characterized in that: The following steps are involved: Step 1: Add the degummed silk into a calcium chloride solution and place in a boiling water bath to obtain a silk-calcium chloride solution; Step 2: Add the silk fibroin-calcium chloride solution obtained in step 1 to the DES solvent, stir evenly, and obtain solution A; add the calcium carbonate suspension to the DES solvent, stir evenly, and obtain suspension B; Step 3: Add suspension B to solution A, stir and react to obtain a milky white turbid solution; Step 4: After the milky white turbid liquid obtained in step 3 is separated by centrifugation or filtration, the solid part is washed with deionized water and then dried to obtain a light yellow silk fibroin-calcium carbonate composite material.

2. The method for preparing a silk fibroin-calcium carbonate composite material according to claim 1, characterized in that: The concentration of the calcium chloride solution in step 1 is 5-6 M, and the mass volume ratio of the degummed silk fibroin to the calcium chloride solution is 0.5-1 g: 5-10 mL.

3. The method for preparing a silk fibroin-calcium carbonate composite material according to claim 1, characterized in that: The boiling water bath time in step 1 is 8 to 12 minutes.

4. The method for preparing a silk fibroin-calcium carbonate composite material according to claim 1, characterized in that: In step 2, the volume ratio of the silk fibroin-calcium chloride solution to the DES solvent is 1:(1-1.2), the concentration of the calcium carbonate suspension is 2.5-3.5 M, and the volume ratio of the calcium carbonate suspension to the DES solvent is 1:(2-2.2).

5. The method for preparing a silk fibroin-calcium carbonate composite material according to claim 1, characterized in that: In step 3, the volumes of solution A and suspension B are 1:(2~2.2).

6. The method for preparing a silk fibroin-calcium carbonate composite material according to claim 1, characterized in that: The DES solvent is prepared by mixing urea and betaine in a molar ratio of 2:1 or glucose and choline chloride in a molar ratio of 1:1 and then heating the mixture.

7. The method for preparing a silk fibroin-calcium carbonate composite material according to claim 6, characterized in that: The heating temperature is 100~150℃ and the time is 10~30 min.

8. The method for preparing a silk fibroin-calcium carbonate composite material according to claim 1, characterized in that: The centrifugal speed is 5000~8000 rpm and the time is 5~15 min.

9. A silk fibroin-calcium carbonate composite material, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. An application of a silk fibroin-calcium carbonate composite material, characterized in that: Application of silk fibroin-calcium carbonate composite materials in filling materials, immobilized enzyme carriers and whitening cosmetics.

Images