Mussel-mucin-sodium alginate composite microspheres and preparation method thereof

Abstract

The application discloses a kind of mussel mucin-sodium alginate composite microspheres and preparation method thereof, and sugar and mussel mucin aqueous solution are prepared into mixed solution 1, then mixed solution 1 is mixed with sodium alginate solution, and free radical initiator is added, and the system pH is adjusted to 3-11, under the stirring state, after 4-12 hours of reaction, organic alcohol solution is added to terminate the reaction, finally, divalent ion solution is added, and mussel mucin-sodium alginate composite microspheres are precipitated.The composite microspheres prepared by the application solve the defects that mussel mucin is easy to oxidize and unstable in the prior art solution, at the same time, the composite microspheres prepared by the application are good in dispersibility, and the reaction system is not limited by pH condition.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to a mussel adhesive protein-sodium alginate composite microsphere and a preparation method thereof. BACKGROUND

[0002] Mussel adhesive protein is a kind of mucous protein of glycoprotein type, which has super adhesion performance. It has the characteristics of wide adhesion range, water resistance, corrosion resistance, good biocompatibility, no immune response, etc. In addition, it also has the functions of antibiosis, promotion of cell tissue growth, hemostasis, etc. It has important applications in the field of biomedicine, such as the adhesion of surgical incisions and wounds of mucosal tissue, skin tissue, bone, soft tissue, etc.; postoperative treatment and nursing of chronic wounds such as diabetes and bedsores, etc.

[0003] The excellent adhesion of mussel adhesive protein mainly depends on the large amount of DOPA groups contained therein. The functional residue has strong wet adhesion, but the DOPA group is very easy to oxidize in solution state. Some phenolic hydroxyl groups in DOPA are oxidized into quinone, which leads to the crosslinking of oxidized DOPA and unoxidized DOPA, forming high molecular polymers precipitated from the protein solution, thereby reducing the content of mussel adhesive protein in the solution.

[0004] In the prior art, the stability of mussel adhesive protein in solution state is usually improved by preparing it into the form of microspheres. However, the prepared microspheres have the problems of poor dispersibility and easy agglomeration. In addition, the existing methods for preparing mussel adhesive protein microspheres are mainly based on the self-polymerization of mussel adhesive protein under alkaline conditions, so the reaction conditions of the system are required to be high. SUMMARY

[0005] The present application aims to provide a mussel adhesive protein-sodium alginate composite microsphere and a preparation method thereof. The mussel adhesive protein-sodium alginate composite microsphere is prepared by using mussel adhesive protein and sodium alginate as raw materials, so as to solve the problems of easy oxidation and instability of mussel adhesive protein solution in the prior art. At the same time, the problems of poor dispersibility of mussel adhesive protein microspheres and the fact that the preparation of microspheres can only be carried out under alkaline conditions are solved.

[0006] To achieve the above-mentioned purpose, the present application provides a preparation method of a mussel adhesive protein-sodium alginate composite microsphere, which comprises the following steps:

[0007] (1) uniformly mixing a sugar solution and a mussel adhesive protein aqueous solution to obtain a mixed solution 1, wherein the mixed solution 1 contains 0.5-5 wt% of the sugar substance and 1-20 wt% of the mussel adhesive protein;

[0008] (2) 0.1-20 wt% of sodium alginate aqueous solution is mixed with the mixed solution 1 to form a mixed solution 2;

[0009] (3) 1-20 wt% of radical initiator is added to the mixed solution 2, the pH range of the solution is adjusted to 3-11, and the solution is continuously stirred at 200-800 r / min for 4-12 hours to form a mixed solution 3;

[0010] (4) 0.05-2 wt% of organic alcohol is added to the mixed solution 3 to form a mixed solution 4;

[0011] (5) The mixed solution 4 is added dropwise to 1-5 wt% of divalent ion solution at a stirring speed of 200-800 r / min, and the cross-linking reaction is carried out for 2-4 hours, and then the product is precipitated to obtain mussel adhesive protein-sodium alginate composite microspheres.

[0012] Preferably, the saccharide in the step (1) is selected from one of trehalose, beta-glucan, lactose, glucose and sucrose.

[0013] The saccharide solution and the mussel adhesive protein aqueous solution are mixed at the ratio, the saccharide solution can not only improve the hydrogen bonding and hydration of the mussel adhesive protein, but also protect the natural structure of the mussel adhesive protein molecules, and at the same time, the mussel adhesive protein is more dispersed in the solution state and is not easy to aggregate, which is beneficial to obtain microspheres with good dispersity in the later stage.

[0014] Preferably, the volume ratio of the sodium alginate solution to the mixed solution 1 in the step (2) is (0.5-20):1.

[0015] The sodium alginate solution and the mixed solution 1 are mixed at the mass percentage and the volume ratio, the mussel adhesive protein in the mixed solution 1 contains a large number of positively charged amino groups, which can form a self-assembly polymer system through hydrogen bonding and electrostatic interaction with the negatively charged carboxyl groups in the sodium alginate; this step utilizes the viscosity of sodium alginate to hinder the self-polymerization of the mussel adhesive protein, which is beneficial to obtain microspheres with good dispersity in the later stage.

[0016] Preferably, the volume ratio of the radical initiator to the mixed solution 2 in the step (3) is (2-20):1.

[0017] Preferably, the radical initiator in the step (3) is selected from one or more of oxygen, copper sulfate, sodium periodate, ammonium persulfate, potassium dichromate, perchloric acid, hydrogen peroxide and peroxydisulfuric acid.

[0018] The free radical initiator is mixed with the mixed solution 2 at the volume ratio, when the reaction system is in an acidic condition, the free radical initiator activates the active sites of the mussel adhesive protein in the mixed solution 2, and then enables the mussel adhesive protein to also occur in the oxidation self-polymerization reaction under the acidic condition, overcoming the limitation that the mussel adhesive protein can only occur in the self-polymerization reaction under the alkaline condition; and when the system is in an alkaline condition, the self-polymerization rate of the mussel adhesive protein in the mixed solution 2 is also faster under the action of the free radical initiator, the reaction time is shortened, and the preparation process is optimized.

[0019] Preferably, the volume ratio of the organic alcohol solution to the mixed solution 3 in the step (4) is (1-20): 1.

[0020] Preferably, the organic alcohol solution in the step (4) is selected from one or more of methanol, ethanol, propanol and isopropanol.

[0021] When the organic alcohol solution is added to the polymer obtained in the step (3), strong hydrogen bonding can be formed between the hydroxyl groups on the organic alcohol and the catechol groups on the mussel adhesive protein in the mixed solution 3, thereby hindering the active sites of the mussel adhesive protein from occurring in the oxidation self-polymerization reaction, so as to terminate the self-polymerization reaction of the mussel adhesive protein, which avoids the phenomenon of excessive self-polymerization and agglomeration of the mussel adhesive protein, and thus makes the dispersion of the subsequent prepared microspheres better.

[0022] Preferably, in the step (5), the volume ratio of the divalent ion solution to the mixed solution 4 is (2-20): 1, wherein the divalent ion can be selected from one of Ca 2+ , Co 2+ and Zn 2+ .

[0023] When the volume ratio between the divalent ion solution and the mixed solution 4 is too low, the exchange speed between the sodium ions and the divalent ions in the mixed solution 4 system is slow, thereby causing the crosslinking degree of the divalent ions and the sodium alginate to be low, and the coagulation ability to be weak; and when the volume ratio between the divalent ion solution and the mixed solution 4 is too high, a dense crosslinked structure is rapidly formed on the surface of the microspheres, thereby causing the finally prepared product to be unable to form an integrated microsphere structure.

[0024] Preferably, the method further comprises: after the composite microspheres are filtered, the composite microspheres are washed repeatedly with purified water for more than 2 times, and the washed composite microspheres are placed in a refrigerator below-20℃ until completely frozen, and then the frozen sample is placed in a freeze dryer for drying.

[0025] The application also discloses a mussel adhesive protein-sodium alginate composite microsphere prepared by the above preparation method.

[0026] The application further discloses a mussel adhesive protein-sodium alginate composite microsphere. Advantages

[0027] (1) The mussel adhesive protein-sodium alginate composite microsphere disclosed by the application has a simple preparation method, and the overall reaction system is not limited by pH conditions, and effective polymerization can be achieved in a wide range of pH, and the reaction system has strong adaptability, and the process is simplified; on the other hand, the mussel adhesive protein-sodium alginate microsphere preparation method provided by the application preliminarily controls the agglomeration of mussel adhesive protein by selecting a sugar from the beginning of the whole preparation method, provides a uniform system for the next reaction, and then the appropriate addition of sodium alginate and a free radical initiator and parameter control are combined, so that the mussel adhesive protein-sodium alginate composite microsphere with stable properties and good dispersity is prepared.

[0028] (2) The mussel adhesive protein-sodium alginate composite microsphere preparation method designed by the application first performs a free radical polymerization reaction through the electrostatic interaction between sodium alginate and mussel adhesive protein to form a self-assembled polymer, and this step can hinder the self-polymerization of mussel adhesive protein, so that the prepared microsphere has better dispersity.

[0029] (3) When the free radical initiator is introduced into the self-assembled polymer system, if the reaction system is acidic or weakly acidic, the free radical initiator can excite the active sites of mussel adhesive protein in the self-assembled polymer, so that the mussel adhesive protein occurs oxidation self-polymerization under acidic conditions; if the reaction system is alkaline or weakly alkaline, the free radical initiator can accelerate the self-polymerization rate of mussel adhesive protein in the self-assembled polymer, therefore, the introduction of the free radical initiator overcomes the limitation that mussel adhesive protein is difficult to occur self-polymerization under acidic or weakly acidic conditions and improves the overall reaction rate and optimizes the preparation process.

[0030] (4) The introduction of a proper content of sugar solution into the mussel adhesive protein solution can effectively improve the hydrogen bonding and hydration of mussel adhesive protein, protect the natural structure of the mussel adhesive protein molecule, make the mussel adhesive protein more dispersed in the solution, avoid the occurrence of the aggregation and precipitation phenomenon, and be more conducive to obtaining the composite microsphere with good dispersity. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical scheme of the application, the drawings needed in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0032] Figure 1 Scanning electron microscope images of the present application Example 1, 2, 3, 4 and Comparative Example 1, 2.

[0033] Figure 2 Scanning electron microscope images of the present application Example 1 and Comparative Example 3, 4.

[0034] Figure 3 Scanning electron microscope images of the present application Example 1, 2 and Comparative Example 5, 6.

[0035] Figure 4 Electron microscope images of the present application Example 1 and Comparative Example 7, 8.

[0036] Figure 5 Zeta potential images of the present application Example 1, 2, 3, 4 and Comparative Example 7, 8.

[0037] Figure 6 The present application Example 1 and mussels mucin solution at 40℃ for 4 hours of protein content rate of change of line contrast chart.

[0038] Figure 7 The present application Example 1 and positive control group, negative control group cell morphology contrast chart. DETAILED DESCRIPTION

[0039] The application will be further described below in conjunction with specific examples. It should be understood that the following examples are intended to illustrate but not limit the scope of the present application.

[0040] Example 1:

[0041] The mussel mucin-sodium alginate composite microspheres were prepared according to the following steps:

[0042] A 1 wt% trehalose solution and a 5 wt% mytilus edulis mucin solution were mixed uniformly in a beaker to prepare a mixed solution 1; a 5 wt% sodium alginate solution and the mixed solution 1 were mixed uniformly in a beaker at a volume ratio of 3:1 to obtain a mixed solution 2; the mixed solution 2 and a 3 wt% hydrogen peroxide solution were mixed in a beaker at a volume ratio of 1:10, 1% sodium hydroxide solution was added to adjust the pH of the reaction system to 8, and the reaction was carried out under the condition of continuous stirring at 400 r / min for 8h to obtain a mixed solution 3; a 0.5 wt% ethanol solution and the mixed solution 3 were mixed uniformly at a volume ratio of 10:1 to obtain a mixed solution 4; the mixed solution 4 was added dropwise into a 3 wt% calcium chloride solution under the condition of continuous stirring at 400 r / min, the mixed solution 4 and the 3 wt% calcium chloride solution were mixed at a volume ratio of 1:5 and crosslinked for 2.5h, and then the mytilus edulis mucin-sodium alginate composite microspheres were obtained by standing and precipitation; the composite microspheres were filtered, washed repeatedly with purified water for 5 times to remove the residual calcium chloride on the surface, and placed in a refrigerator below-20℃ until completely frozen, then the frozen sample was placed in a freeze dryer, and the finished product was obtained after drying.

[0043] Example 2

[0044] The mytilus edulis mucin-sodium alginate composite microspheres were prepared according to the following steps:

[0045] A 5 wt% lactose solution and a 20 wt% mytilus edulis mucin solution were mixed uniformly in a beaker to prepare a mixed solution 1; a 20 wt% sodium alginate solution and the mixed solution 1 were mixed uniformly in a beaker at a volume ratio of 20:1 to obtain a mixed solution 2; the mixed solution 2 and a 20 wt% perchloric acid solution were mixed in a beaker at a volume ratio of 1:20, 1% sodium hydroxide solution was added to adjust the pH of the reaction system to 11, and the reaction was carried out under the condition of continuous stirring at 800 r / min for 12h to obtain a mixed solution 3; a 2 wt% propanol solution and the mixed solution 3 were mixed uniformly at a volume ratio of 20:1 to obtain a mixed solution 4; the mixed solution 4 was added dropwise into a 5 wt% zinc chloride solution under the condition of continuous stirring at 800 r / min, the mixed solution 4 and the 5 wt% zinc chloride solution were mixed at a volume ratio of 1:20 and crosslinked for 4h, and then the mytilus edulis mucin-sodium alginate composite microspheres were obtained by standing and precipitation; the composite microspheres were filtered, washed repeatedly with purified water for 5 times to remove the residual zinc chloride on the surface, and placed in a refrigerator below-20℃ until completely frozen, then the frozen sample was placed in a freeze dryer, and the finished product was obtained after drying.

[0046] Example 3

[0047] The mussel myoglobin-sodium alginate composite microspheres are prepared by the following steps:

[0048] 0.5 wt% glucose solution and 1 wt% mussel myoglobin solution are mixed uniformly in a beaker to prepare mixed solution 1; 0.1 wt% sodium alginate solution is mixed with mixed solution 1 in a beaker at a volume ratio of 0.5:1 to obtain mixed solution 2; mixed solution 2 is mixed with 1 wt% sodium periodate solution in a beaker at a volume ratio of 1:2, 1% hydrochloric acid solution is added to adjust the pH of the reaction system to 3, and the reaction is carried out under the condition of continuous stirring at 200 r / min for 4h to obtain mixed solution 3; 0.05 wt% isopropyl alcohol solution is mixed with mixed solution 3 at a volume ratio of 1:1 to obtain mixed solution 4; mixed solution 4 is added dropwise into 1 wt% calcium chloride solution under the condition of continuous stirring at 200 r / min, and mixed solution 4 and 1 wt% calcium chloride solution are mixed at a volume ratio of 1:2 for cross-linking reaction for 2h, then the mixture is left to stand and precipitate to obtain mussel myoglobin-sodium alginate composite microspheres; the composite microspheres are filtered and washed repeatedly with purified water for 5 times to remove residual calcium chloride on the surface, and the composite microspheres are placed in a refrigerator below-20℃ until completely frozen, then the frozen sample is placed in a freeze dryer, and the finished product is obtained after drying.

[0049] Example 4

[0050] The mussel myoglobin-sodium alginate composite microspheres are prepared by the following steps:

[0051] Mix 1 wt% sucrose solution and 5 wt% mussel adhesive protein solution in a beaker to prepare mixed solution 1. Mix 5 wt% sodium alginate solution with mixed solution 1 in a beaker at a volume ratio of 3:1 to obtain mixed solution 2. Mix mixed solution 2 with 3 wt% ammonium persulfate solution and 3 wt% perchloric acid aqueous solution in a volume ratio of 1:4:6 in a beaker, then add 1% sodium hydroxide solution to adjust the pH of the reaction system to 8, and react for 8 hours under continuous stirring at 400 r / min to obtain mixed solution 3. Mix 0.2 wt% methanol solution, 0.3 wt% isopropanol solution and mixed solution 3 in a volume ratio of 5:5:1 to obtain mixed solution 4. Continue to add mixed solution 4 dropwise to 3 wt% cobalt chloride solution while stirring at 400 r / min, wherein mixed solution 4 and 3 wt%... A cobalt chloride solution was mixed at a volume ratio of 1:5 and cross-linked for 2.5 hours. After standing and precipitation, mussel adhesive protein-sodium alginate composite microspheres were obtained. The composite microspheres were filtered and washed repeatedly with purified water 5 times to remove residual cobalt chloride on the surface. The composite microspheres were then placed in a refrigerator below -20°C until completely frozen. The frozen sample was then placed in a freeze dryer and dried to obtain the finished product.

[0052] Comparative Example 1

[0053] The reaction conditions were the same as in Example 1 above, except that the mass percentages of mussel adhesive protein and sodium alginate were both adjusted to 0.05 wt%.

[0054] Comparative Example 2

[0055] The reaction conditions were the same as in Example 1 above, except that the mass percentages of mussel adhesive protein and sodium alginate were both adjusted to 30 wt%.

[0056] As attached Figure 1 As shown in the scanning electron microscope (SEM) images, when the mass percentages of mussel adhesive protein and sodium alginate are too low, it is difficult for them to form a self-assembled polymer system through electrostatic interaction in the solution, resulting in the inability to form microspheres. When the mass percentages of mussel adhesive protein and sodium alginate are 30 wt%, the addition of a free radical initiator to the reaction system leads to excessive self-aggregation of the mussel adhesive protein under the action of the free radical initiator, resulting in the aggregation of microspheres.

[0057] Comparative Example 3

[0058] The reaction conditions were the same as in Example 1 above, except that the volume ratio of mixed solution 1 to sodium alginate solution was adjusted to 1:0.1.

[0059] Comparative Example 4

[0060] The reaction conditions were the same as those in Example 1 above, except that the volume ratio of the mixed solution 1 to the sodium alginate solution was adjusted to 1:30.

[0061] As shown in the attached Figure 2 As shown in the attached

[0062] Comparative Example 5

[0063] The reaction conditions were the same as those in Example 1 above, except that the mass percentage of the hydrogen peroxide solution was adjusted to 0.5 wt%.

[0064] Comparative Example 6

[0065] The reaction conditions were the same as those in Example 1 above, except that the mass percentage of the hydrogen peroxide solution was adjusted to 30 wt%.

[0066] As shown in the attached Figure 3 As shown in the attached

[0067] Comparative Example 7

[0068] Compared with Example 1, the following method was used to prepare the mytilin-sodium alginate composite microspheres.

[0069] 1 wt% trehalose solution and 5 wt% mytilus mucin solution were mixed uniformly in a beaker to configure a mixed solution 1; 5 wt% sodium alginate solution was mixed with the mixed solution 1 in a beaker at a volume ratio of 3:1 to obtain a mixed solution 2; the mixed solution 2 was added dropwise into 3 wt% calcium chloride solution at a stirring speed of 400 r / min, and the mixed solution 2 and the 3 wt% calcium chloride solution were mixed at a volume ratio of 1:10 for cross-linking reaction for 2.5 hours, and then the mytilus mucin-sodium alginate composite microspheres were formed by standing and precipitating; the composite microspheres were filtered, washed repeatedly with purified water for 5 times to remove the residual calcium chloride on the surface, and placed in a refrigerator below -20℃ until completely frozen. Then the frozen sample was placed in a freeze dryer, and the finished product was obtained after drying.

[0070] Comparative Example 8

[0071] Comparative Example 8 only prepared sodium alginate microspheres compared with Example 1.

[0072] 5 wt% sodium alginate solution was added dropwise into 3 wt% calcium chloride solution at a stirring speed of 400 r / min, and the sodium alginate solution and the calcium chloride solution were mixed at a volume ratio of 1:5 for cross-linking reaction for 2.5 hours, and then the sodium alginate microspheres were formed by standing and precipitating; the sodium alginate microspheres were filtered, washed repeatedly with purified water for 5 times to remove the residual calcium chloride on the surface, and placed in a refrigerator below -20℃ until completely frozen. Then the frozen sample was placed in a freeze dryer, and the finished product was obtained after drying.

[0073] As shown in the attached Figure 4 Comparative Example 7 is the mytilus mucin-sodium alginate composite microspheres A prepared by using the prior art, and Comparative Example 8 is sodium alginate microspheres. It can be seen from the electron microscope graph that the dispersibility of the composite microspheres prepared by the method is better than that of the composite microspheres A prepared by the prior art, and the morphology of the microspheres is more regular.

[0074] Zeta potential test

[0075] The Zeta potential of the microspheres prepared in Examples 1-4 and Comparative Examples 7-8 was determined by a nano DLS dynamic light scattering instrument, so as to directly express the assembly on the surface of the microspheres.

[0076] The microspheres prepared in Examples 1, 2, 3, 4 and Comparative Examples 7, 8 were prepared into a 5% concentration solution, ultrasonically dispersed, and then placed in a nano DLS dynamic light scattering instrument for determination, and the determination was carried out in triplicate. The results are shown in the attached Figure 5It can be seen that the Zeta potential of the composite microspheres prepared in Examples 1, 2, 3, and 4 is -45 mV, the Zeta potential of the mytilus edulis mucin-sodium alginate composite microspheres A prepared in Comparative Example 7 is -18.6 mV, and the Zeta potential of the sodium alginate microspheres prepared in Comparative Example 8 is -24.4 mV, which can indicate that the positively charged mytilus edulis mucin successfully reacts with the negative charge of sodium alginate to form the composite microspheres A by self-assembly. The Zeta potential of the composite microspheres prepared in Examples 1, 2, 3, and 4 is -45 mV, which is due to the fact that the active sites of the mytilus edulis mucin are initiated under the action of the free radical initiator, and the mytilus edulis mucin is oxidized and self-polymerized. Therefore, in Examples 1, 2, 3, and 4, the mytilus edulis mucin first reacts with sodium alginate to form a self-assembled polymer system, and then the mytilus edulis mucin in the self-assembled polymer is oxidized and self-polymerized under the action of the free radical initiator, so that the charge accumulation on the surface of the finally prepared microspheres reduces the Zeta potential. The absolute value of the Zeta potential can also confirm that the stability of the mytilus edulis mucin-sodium alginate composite microspheres prepared in the present scheme is better than that of the mytilus edulis mucin-sodium alginate composite microspheres prepared in Comparative Example 7.

[0077] Comparison of the stability of the mytilus edulis mucin-sodium alginate composite microspheres prepared in the present scheme with the mytilus edulis mucin

[0078] The prepared mytilus edulis mucin-sodium alginate composite microspheres (Example 1) and mytilus edulis mucin were respectively configured into 5% solutions with a pH buffer, and stored at 40°C. At 0 h, 1 h, 2 h, and 4 h, samples were taken and the protein content was determined by spectrophotometry, and a protein content change rate curve was drawn.

[0079] The results are shown in Figure 6 At 40°C, the protein decrease rate of the mytilus edulis mucin in the composite microspheres prepared in the present scheme is much lower than that in the mytilus edulis mucin solution, which shows that the composite microspheres prepared in the present scheme can effectively improve the stability of the mytilus edulis mucin in the solution.

[0080] Evaluation of cytotoxicity

[0081] First, fibroblast L929 cells were inoculated in a 96-well plate, the cell density was 2.5×10 5 cells / well, and MEM medium containing antibiotics and 10% serum was used to culture the cells at 37°C under 5% CO2 conditions for 24 h. Negative and positive control groups were set.

[0082] Then the original culture solution was discarded, the negative control group was added with the culture medium, the positive control group was added with 10% dimethyl sulfoxide, and the extraction solution of each sample was added into the culture plate with cells in the experimental group, and the culture was continued for 24 h. Then the cell solution in the hole was sucked out, 100 μL of culture medium and 10 μL of MTT solution were added into each hole, and the incubation culture was continued for 4 h.

[0083] After the culture ended, the liquid in the hole was discarded, 150 μL of dimethyl sulfoxide was added, and the vibration was shaken on the oscillator for 10 min. The absorbance of each sample at 570 nm was detected by an enzyme-labeled instrument. The cell survival rate was calculated, and the formula was as follows:

[0084] Cell proliferation rate = (average value of absorbance of the experimental group / average value of absorbance of the control group) x 100%

[0085] The cell morphology of each group was observed under an inverted microscope, and the results are shown in FIG. 1. Figure 7 The results show that the cell proliferation rates of Example 1, the negative control group and the positive control group are 101%, 98% and 23% respectively; the cell morphology of the negative control group is similar to that of Example 1, without cell lysis and cell proliferation decrease. In combination with the cell proliferation rate and the cell morphology, it can be determined that the mussel adhesive protein-sodium alginate composite microspheres prepared by the present application have no cytotoxicity, and the biological safety is high.

[0086] Hemolysis test

[0087] The extraction medium was sterile physiological saline, the extraction ratio was 0.2 g / mL, the extraction was carried out at 37℃ ± 1℃ for 72 h ± 2 h, and the extraction solution was prepared. The hemolysis test was determined according to the hemolysis test method specified in GB / T 16886.4 and the blood interaction test. The extraction solution of the sample was mixed with the rabbit blood cell suspension according to the ratio of 100 / 2, incubated at 37℃ for 1 h, then the blood cell suspension was centrifuged at 1000 rpm for 5 min, and the absorbance value of the supernatant was determined at 545 nm. Purified water was used as a positive control, and physiological saline was used as a negative control. The hemolysis rate was calculated according to the following formula:

[0088] Hemolysis rate (%) = (sample-negative control) / (positive control-negative control) x 100%

[0089] The specific detection results are shown in Table 1.

[0090] Table 1 Hemolysis rate of mussel adhesive protein-sodium alginate composite microspheres prepared by the present application applied to artificial dura mater

[0091]

[0092] The hemolysis rate of the mussel myo-glycollic acid sodium composite microspheres prepared by the application is not more than 5% according to the hemolysis experiment results in Table 1, so the mussel myo-glycollic acid sodium composite microspheres prepared by the application will not cause hemolysis and have good biocompatibility.

Claims

1. A method for preparing mussel adhesive protein-sodium alginate composite microspheres, characterized in that, Includes the following steps: (1) Mix 0.5~5 wt% sugars with 1~20 wt% mussel adhesive protein aqueous solution to obtain mixed solution 1, wherein the sugars are selected from one of trehalose, β-glucan, lactose, glucose and sucrose; (2) Mix 0.1~20 wt% sodium alginate aqueous solution with the mixed solution 1 at a volume ratio of (0.5~20):1 to obtain mixed solution 2; (3) Add 1~20 wt% of free radical initiator to the mixed solution 2, adjust the pH range of the system to 3~11, and stir continuously for 4~12 hours at 200~800 r / min to obtain mixed solution 3. The free radical initiator is selected from one or more of oxygen, copper sulfate, sodium periodate, ammonium persulfate, potassium dichromate, perchloric acid, hydrogen peroxide, and persulfate. (4) Add 0.05~2 wt% of organic alcohol to the mixed solution 3 to obtain mixed solution 4; (5) Under conditions of 200~800 r / min, the mixed solution 4 was added dropwise to a 1~5 wt% divalent ion solution. After the cross-linking reaction was carried out for 2~4 hours, the mixture was allowed to stand and precipitate to obtain mussel adhesive protein-sodium alginate composite microspheres.

2. The method for preparing mussel adhesive protein-sodium alginate composite microspheres according to claim 1, characterized in that, In step (3), the volume ratio of the free radical initiator to the mixed solution 2 is (2~20):

1.

3. The method for preparing mussel adhesive protein-sodium alginate composite microspheres according to claim 1, characterized in that, In step (4), the volume ratio of the organic alcohol to the mixed solution 3 is (1~20):

1.

4. The method for preparing mussel adhesive protein-sodium alginate composite microspheres according to claim 1, characterized in that, In step (5), the volume ratio of the divalent ion solution to the mixed solution 4 is (2~20):1, and the divalent ion is selected from Ca... 2+ Co 2+ Zn 2+ One of them.

5. The method for preparing mussel adhesive protein-sodium alginate composite microspheres according to claim 1, characterized in that, The organic alcohol in step (4) is selected from one or more of methanol, ethanol, propanol, and isopropanol.

6. The method for preparing mussel adhesive protein-sodium alginate composite microspheres according to claim 1, characterized in that, Also includes: The composite microspheres are filtered, washed, and then placed in a refrigerator at -20°C or below until completely frozen, and then dried in a freeze dryer.

7. A mussel adhesive protein-sodium alginate composite microsphere, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 6.

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