CN106668845B - Preparation method of thrombin-immobilized chitosan/silk fibroin microspheres - Google Patents
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Abstract
The invention discloses a preparation method of chitosan/silk fibroin microspheres for fixing thrombin, which comprises the following steps: s1, adding degummed silk fibroin fibers into an ethanol solution containing calcium chloride for dissolving, dialyzing, and filtering to obtain a silk fibroin solution; s2, dissolving chitosan in an acetic acid solution, and adding the fibroin solution and CuSO of S14Obtaining a mixed solution; s3, adding the mixed solution in the S2 into an alkaline ethanol solution to prepare blank microspheres, adding EDTA, stirring to remove the complexed Cu2+Filtering, dewatering, cleaning and drying to obtain chitosan/fibroin microspheres; s4, swelling the chitosan/silk fibroin microspheres in the S3, adding a thrombin solution and a buffer solution, and adding a glutaraldehyde solution for an immobilized enzyme reaction; the invention adopts chitosan and fibroin as carriers of immobilized enzymes, can improve the stability of the enzymes and ensure the activity recovery efficiency of immobilization, and has great practical significance.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a preparation method of thrombin-immobilized chitosan/silk fibroin microspheres.
Background
Thrombin is widely used in medical research at present, is a good hemostatic, but has the problems of reduced effectiveness and even inactivation due to poor stability. Some researchers have been working on studies to improve the immobilization effect of thrombin, and they have desired to find suitable carriers for enzymes and storage methods to prolong their storage time. In some literatures and reports, the thrombin is fixed by adding stabilizing agents glycine and gelatin; the method is characterized by exploring the immobilization carriers of enzyme drugs, such as complex coagulation sponge, water-soluble chitosan, sodium alginate microcapsules and other immobilization methods.
Chitosan is often used as a carrier, and has great advantages as a carrier of drugs and has groups capable of being combined with ligands. Meanwhile, the chitosan has good biocompatibility and biodegradability, and is widely applied to the aspects of biological medicine, food, environmental protection, agricultural production and the like.
The silk fibroin is obtained by processing silk left after silkworm pupa dragging, is a natural fibrin, is insoluble in water, has great imbibition property, and is always used as a beauty product due to the porosity and the super-strong water holding capacity. However, silk fibroin has poor mechanical ratio and cannot achieve the expected effect when being used as a carrier alone.
Disclosure of Invention
The invention aims to provide a preparation method of thrombin-immobilized chitosan/silk fibroin microspheres according to the defects in the prior art.
The invention firstly prepares the chitosan/fibroin microspheres as a carrier, and then fixes the thrombin, thereby ensuring the activity and prolonging the stability of the thrombin.
The invention combines chitosan and silk fibroin to make up for the defects of each other. The silk fibroin contains abundant acylamino and a small amount of hydroxyl, and forms hydrogen bonds with chitosan, so that the hydrogen bonds among chitosan chains are destroyed, and a part of amino in the chitosan is free. Amino in the chitosan has positive charge, and can neutralize negative charge of silk fibroin after being blended with silk fibroin, and has special effect on immobilization of charged negative enzyme.
The purpose of the invention is realized by the following technical scheme:
a preparation method of chitosan/silk fibroin microspheres for fixing thrombin comprises the following steps:
s1, adding degummed silk fibroin fibers into an ethanol solution containing calcium chloride for dissolving, dialyzing, and filtering to obtain a silk fibroin solution;
s2, dissolving chitosan in an acetic acid solution, and adding the fibroin solution and CuSO of S14Obtaining a mixed solution;
s3, adding the mixed solution in the S2 into an alkaline ethanol solution to prepare blank microspheres, adding EDTA, stirring to remove the complexed Cu2+Filtering, dewatering, cleaning and drying to obtain chitosan/fibroin microspheres;
s4, swelling the chitosan/silk fibroin microspheres in the S3, adding a thrombin solution and a buffer solution, and adding a glutaraldehyde solution for an immobilized enzyme reaction;
the pH value of the buffer solution in the thrombin solution in S4 is 70-80U/mL/L, S4, the concentration of the glutaraldehyde solution is 0.1-0.4%, and the reaction time of the immobilized enzyme is 1.5-2.5 h.
Preferably, the amount of the added enzyme in the thrombin solution is 70U/mL, the pH of the buffer solution in S4 is 6, the concentration of the glutaraldehyde solution is 0.3%, and the reaction time of the immobilized enzyme is 2 h.
Preferably, in S2, chitosan is dissolved in acetic acid solution to prepare 1.5-3% chitosan solution, the volume ratio of the fibroin solution to the chitosan solution is 1 (3-6), and CuSO4The mass fraction of the solution is 3-7%.
Preferably, in S3, the alkaline ethanol solution is NaOH or C2H5OH and H2Mixed solution of O, NaOH, C2H5OH:H2The volume ratio of O is 2:3: 5.
Preferably, the ethanol solution containing calcium chloride in S1 is CaCl2、H2O and C2H5Mixed solution of OH in which CaCl is present2︰H2O︰C2H5The molar ratio of OH is 1: 8: 2.
Preferably, the degummed silk fibroin fiber in S1 is prepared by: adding cocoon sheets into 0.5% sodium carbonate solution, heating for degumming, washing, repeatedly degumming and washing, and drying to obtain degummed silk fibroin fibers.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention adopts chitosan and fibroin as carriers of immobilized enzymes, can improve the stability of the enzymes and ensure the activity recovery efficiency of immobilization, and has great practical significance.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1:
1. preparation of fibroin solution
Putting a certain amount of cocoon sheets into 0.5% sodium carbonate solution at 98 + -2 deg.C at a bath ratio of 1: 50 for 30min, taking out the degumming silk, and washing with tap water. And then repeating the operation once again, taking out the degummed silk, washing the degummed silk with tap water, washing the degummed silk twice with deionized water, and drying the degummed silk in a drying oven at 40 ℃ to obtain the degummed silk fibroin fiber.
Adding the obtained silk fibroin fiber into CaCl at a bath ratio of 1: 102︰H2O︰C2H5Dissolving in mixed solution of OH = 1: 8: 2 (molar ratio) at 70 deg.C, and cooling to obtain brown yellow fibroin solution. The mixture is put into a dialysis bag with the carrying capacity of 8000-14000, and is dialyzed for two days by tap water and then by deionized water for one day. Then filtering to remove the solventAfter the impurities are removed, the reagent bottles are stored and put into a refrigerator for standby.
2. Preparation of chitosan/fibroin blank micro-beads
Weighing 2g of chitosan, dissolving the chitosan in 2% acetic acid solution to prepare 100mL of chitosan solution with concentration of 2%, adding 20mL of fibroin solution, mixing and stirring for 30 min; 10mL of prepared CuSO with the mass fraction of 5%4Adding the solution into the chitosan/fibroin mixed solution, and continuously stirring and uniformly mixing.
The solution was aspirated by a 5mL syringe, and added dropwise to an alkaline solution NaOH-C2H5OH-H2O (20: 30: 50), preparing blank microbeads, filtering by using a screen, and repeatedly washing residual alkali liquor by using distilled water. Putting the washed microbeads into a beaker, adding distilled water and a proper amount of 0.5mol/LEDTA, stirring to remove the complexed Cu2+Filtering, adding acetone for dehydration, washing with ethanol, and drying at 50 deg.C to obtain chitosan/fibroin microsphere.
3. Absorbing the surface moisture of the swelled microspheres, and weighing 0.5g of the microspheres in a conical flask; respectively adding 5.0mL of prepared 75U/mL thrombin solution, adding 3mL of phosphoric acid buffer solution with adjusted fixed pH, and adsorbing for 30 minutes; adding a certain amount of glutaraldehyde solution and distilled water into each conical flask by a mass fraction method, and adjusting the concentration of glutaraldehyde in the whole system; covering the opening of the conical flask with tinfoil at 4 ℃; after reacting for 2h, taking out, washing with 0.9% physiological saline for 2-3 times, and removing surface water.
The reaction conditions in examples 1 to 6 are shown in table 1:
TABLE 1 Experimental conditions for examples 1 to 6
Example 7:
adding 5mL of 100U/mL heparin sodium solution into the systems of the embodiments 1-6 respectively, adsorbing for 30 minutes, and centrifuging at 20 ℃ by a centrifuge and r 3000; taking supernatant and fixing the volume to 100 mL; respectively taking 5.0mL of solution, putting the solution into a test tube, and then adding 1.0mL of barbital solution and 1.0mL of azure dye solution; the blank group is 5.0mL of distilled water, 1.0mL of barbital solution and 1.0mL of azure dye solution; the absorbance was measured at a wavelength of 505nm, and the heparin sodium titer was measured to determine the effect of immobilized enzyme.
The method for measuring the titer of the heparin sodium comprises the following steps:
(1) preparation of test reagent
Preparation of Barbituo buffer solution
1g of sodium hydroxide is dissolved in 50mL of distilled water heated to boiling to obtain a 0.5mol/LNaOH solution. Barbiturate (diethylbarbituric acid) 5.52g was weighed out and dissolved in the above 0.5mol/LNa0H solution, and after complete dissolution, it was cooled, diluted to 500mL with distilled water and corrected with a pH meter.
Preparation of azure solution
Weighing 0.5g of azure (biological dye), completely dissolving with a small amount of distilled water, diluting to 500mL, filtering, storing the filtrate (namely stock solution) in a refrigerator, sucking 5mL of stock solution when in use, adding 25mL of distilled water, and mixing uniformly to obtain the final product.
Preparation of standard heparin solution
Accurately weighing a certain amount of heparin standard, preparing into 150U/mL with distilled water as stock solution, and placing in refrigerator
The sample was stored in a 250mL volumetric flask, and 2.5mL of the stock solution was taken and diluted to the mark with water during the measurement. Thus, 1.5U/mL (l-5U/mL may be prepared).
(2) Drawing of standard curve
After the heparin standard substance is accurately prepared, 1-5U/mL of standard heparin solution is taken and put into a test tube, 1.0mL of barbital buffer solution with the pH value of 8.6 and 1.0mL of azure solution are added, the shaking and shaking are carried out, the optical density value of 505nm is measured by a spectrophotometer, and a standard curve is drawn by taking the titer as the abscissa and the optical density as the ordinate.
(3) Sample assay
And (4) carrying out suction filtration on the washed immobilized microbeads to remove surface water. Then 5.0mL of heparin sodium solution (100U/mL) was added to the system, after adsorption for 30min and centrifugation at r3000, 1mL of supernatant was taken and diluted to 100 mL. And measuring the content of the heparin sodium by using an azure method, and converting the titer of the sample solution according to the following formula.
Pi=
Wherein: pi is the potency (U) of the heparin sodium sample solution after adsorption
Px = number of corresponding units on the standard curve of the optical density values of the measured samples
Total volume of sample (mL) at time of measurement
Volume of sample solution (mL) used for measurement
After the nonspecific adsorption of blank microbeads on heparin sodium is deducted, the adsorption rate of heparin sodium is converted according to the following formula, so that the thrombin fixing effect is indirectly compared.
Adsorption rate w ═ of heparin sodium
Wherein:Poriginal heparin sodium total potency (U) before non-adsorption
The results are shown in table 2:
TABLE 2
Claims (8)
1. A preparation method of chitosan/silk fibroin microspheres for fixing thrombin is characterized by comprising the following steps:
s1, adding degummed silk fibroin fibers into an ethanol solution containing calcium chloride for dissolving, dialyzing, and filtering to obtain a silk fibroin solution;
s2, dissolving chitosan in an acetic acid solution, and adding the fibroin solution and CuSO of S14Obtaining a mixed solution;
s3, dripping the mixed solution in the S2 into alkaline ethanol solution to prepare blank microspheres, adding EDTA, stirring to remove the complexed Cu2+Filtering, dewatering, cleaning and drying to obtain chitosan/fibroin microspheres;
s4, swelling the chitosan/silk fibroin microspheres in the S3, adding a thrombin solution and a buffer solution, and adding a glutaraldehyde solution for an immobilized enzyme reaction;
the enzyme adding amount of the thrombin solution in S4 is 70-80U/mL, the pH of the buffer solution in S4 is 6-7, the concentration of the glutaraldehyde solution is 0.1-0.4%, and the reaction time of the immobilized enzyme is 1.5-2.5 h.
2. The process according to claim 1, wherein the amount of the thrombin solution added is 70U/mL, the pH of the buffer solution in S4 is 6, the concentration of the glutaraldehyde solution is 0.3%, and the immobilized enzyme reaction time is 2 hours.
3. The preparation method of claim 1, wherein in S2, chitosan is dissolved in acetic acid solution to prepare 1.5-3% chitosan solution, the volume ratio of the fibroin solution to the chitosan solution is 1 (3-6), and CuSO is added4The mass fraction of the solution is 3-7%.
4. The method according to claim 1, wherein the alkaline ethanol solution of S3 contains NaOH and C2H5OH and H2Mixed solution of O, NaOH, C2H5OH:H2The volume ratio of O is 2:3: 5.
5. The method according to claim 1, wherein the ethanol solution containing calcium chloride in S1 is CaCl2、H2O and C2H5Mixed solution of OH in which CaCl is present2︰H2O︰C2H5The molar ratio of OH is 1: 8: 2.
6. The method of claim 1, wherein the silk fiber degummed in S1 is prepared by: adding cocoon sheets into 0.5% sodium carbonate solution, heating for degumming, washing, repeatedly degumming and washing, and drying to obtain degummed silk fibroin fibers.
7. Immobilized thrombin microspheres prepared by the process according to any one of claims 1 to 6.
8. Use of the immobilized thrombin microspheres of claim 7 in the preparation of a hemostatic material for medical use.
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| CN112472866B (en) * | 2020-12-16 | 2022-02-08 | 山西省生物研究院有限公司 | Preparation method of starch microsphere and human recombinant tissue factor lipidated compound hemostatic material |
| CN113925997B (en) * | 2021-10-13 | 2022-12-06 | 深圳市华宝生物材料科技有限公司 | Gelatin sponge containing hemostatic microcapsule and preparation method thereof |
| WO2023150616A1 (en) * | 2022-02-02 | 2023-08-10 | Case Western Reserve University | Biosynthetic hemostat and uses thereof |
| CN114539553B (en) * | 2022-04-26 | 2022-09-06 | 深圳湾实验室 | Broad-spectrum antioxidant silk fibroin and preparation method and application thereof |
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