CN110665050B

CN110665050B - Biological adhesive and preparation method thereof

Info

Publication number: CN110665050B
Application number: CN201911087311.5A
Authority: CN
Inventors: 赖佳惠; 周娟; 陈敬华; 方诗莹; 杨陈; 王攀琛; 曾以妍; 张羲和
Original assignee: Jiangnan University
Current assignee: Shanghai Zechong Biotechnology Co ltd
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2021-02-26
Anticipated expiration: 2039-11-08
Also published as: CN110665050A

Abstract

The invention discloses a biological adhesive and a preparation method thereof, belonging to the technical field of biological adhesives. The adhesive is prepared by carrying out Michael addition reaction on structural proteins (collagen, gelatin and the like) and tannic acid under an oxidation condition, carrying out subsequent treatment, and carrying out cross-linking by Transglutaminase (TG). The raw materials used by the biological adhesive provided by the invention have good biocompatibility, and toxic chemical components used in the traditional biological adhesive are replaced; meanwhile, the synergistic effect among the raw materials can ensure that the product is stably adhered to the wound in a gel state for a longer time, and the effects of resisting bacteria and repairing skin damage of the tannic acid can be more durably acted on the wound, thereby better promoting the wound to be repaired and healed; the biological adhesive can be accurately positioned by an injector, can firmly adhere to a combined part, and has important application value in the field of biological medicines.

Description

Biological adhesive and preparation method thereof

Technical Field

The invention relates to a biological adhesive and a preparation method thereof, belonging to the technical field of biological adhesives.

Background

The traditional surgical suture can provide good tensile strength, relatively low accident rate and the like, but has the defects of complexity, time consumption, secondary mechanical injury to wounds caused by suture, generation of cell rejection immunity, granulation tumor caused by long residual time in vivo, certain discomfort and pain caused by suture removal and the like. The biological adhesive is a biomedical material which is prepared from a material with excellent biocompatibility, is sterile, has no immunogenicity, and can inhibit bacteria within a certain period of time. The components of the adhesive are free of solvent or the solvent is water, and the adhesive can still generate a certain strength adhesion with body tissues under a wet environment (namely the existence of water, interstitial fluid, blood and the like). The dissolved matters of the biological adhesive and the like do not generate toxicity to organism cells, do not influence the wound healing of organism tissues, are degradable, can be absorbed by the human tissues after meeting the use requirements, and are finally metabolized out of the human body.

Currently, fibrin adhesives and mussel mucin adhesives are commonly used in clinical practice, but these adhesives still have many drawbacks. For example, fibrin adhesives have the disadvantages of low adhesive strength, long preparation time, high price and potential risk of viral infection; mussel mucin adhesives are expensive and sold in the U.S. at $ 115/mg.

Therefore, researchers have been investigating alternatives to these two types of bioadhesives. In recent years, gelatin-based adhesives become an important biological adhesive, and have high adhesive strength, low cost and good biocompatibility. For example, patent CN105879109A discloses a low-cost, strong-adhesion, surgical bio-adhesive, which is formed by mixing gelatin aqueous solution and glutaraldehyde aqueous solution. Although the adhesives disclosed in this patent can meet the requirements of adhesion and gel time, glutaraldehyde is prone to cytotoxic reactions and is extremely unsafe for application to the wound surface of a patient, and thus, bioadhesives that meet the ideal requirements have not yet been developed.

Disclosure of Invention

The invention aims to solve the defects of high price, poor biocompatibility and degradability, no antibacterial property and the like of the existing biological adhesive, and provides an injectable antibacterial biological adhesive and a preparation method thereof.

A first object of the present invention is to provide a method for preparing an injectable antibacterial bio-adhesive, comprising the steps of:

1): carrying out Michael addition reaction on the structural protein and tannic acid under an oxidation condition to obtain a tannic acid modified structural protein;

2): dissolving the tannin modified structural protein prepared in the step 1) in a buffer solution, adding transglutaminase, and uniformly mixing to obtain the biological adhesive.

In one embodiment, the buffer is Tris-HCl buffer.

In one embodiment, the structural protein comprises gelatin or collagen.

In one embodiment, the mass ratio (w/w) of the structural protein to the deionized water in the step 1) is (0.005-0.1): 1, the mass ratio (w/w) of the tannic acid to the structural protein is (0.01-0.1): 1.

in one embodiment, the mass ratio (w/w) of tannic acid to structural protein in step 1) is (0.01-0.1): 1.

in one embodiment, the michael addition reaction in step 1) is specifically performed by: mixing structural protein with deionized water, stirring and dissolving, reacting for 0.5-3.0 h, and adjusting the pH of the solution to be alkaline, wherein the pH is 8.0-9.0; adding tannic acid, continuously stirring, introducing oxygen to react, keeping the pH of the solution consistent with the pH, reacting at 50-70 ℃ for 1.0-4.0 h, and adjusting the pH of the solution to be neutral and slightly alkaline after the reaction is finished, wherein the pH is 7.0-7.8.

In one embodiment, the michael addition reaction in step 1) is followed by dialysis and drying; the dialysis refers to the deionized water dialysis of the solution by using a dialysis tube with a molecular weight cut-off value of 3500 Da; the drying refers to freeze drying.

In one embodiment, the buffer in step 2) is Tris-HCl buffer, and the pH is 7.5 to 9.5.

In one embodiment, the tannin modified structural protein is dissolved in Tris-HCl buffer solution with the concentration of 1-15 g/100 mL; the final concentration of the transglutaminase in the Tris-HCl buffer solution is 0.1-5 g/100mL, and within the range, the product has more stable structure, shorter gel forming time and better appearance.

In one embodiment, the concentration of the Tris-HCl buffer is 7.5g/100mL, and each 100mL of the Tris-HCl buffer contains 1-15 g of the tannin modified structural protein.

In one embodiment, the transglutaminase is added in an amount of 3g/100mL, and the enzyme activity of the transglutaminase is 200U/g).

A second object of the invention is to protect a transglutaminase crosslinked structural protein and tannin based bioadhesive prepared using any of the above methods.

In one embodiment, the structural protein includes, but is not limited to, gelatin, collagen.

The invention also claims the application of the method or the biological adhesive in the preparation of the adhesion between active physiological tissues, the plugging and leak repairing of physiological pipelines, the medicine for sealing and stopping bleeding or repairing injured wounds, or medical devices.

Has the advantages that: the invention provides an injectable antibacterial biological adhesive and a preparation method thereof, compared with the prior art:

(1) the invention adopts structural protein, tannic acid, transglutaminase and the like as raw materials, has good biocompatibility and good bacteriostatic effect (on 10)⁶CFU bacteria has 100% of bacteriostasis rate) to replace toxic chemical components used in the traditional biological adhesive, and the synergistic effect of the three components can ensure that the product can be stably adhered to wounds for a long time in a gel state so as to better promote wound repair and healing.

(2) The invention has the function of injection, can fill and adhere wounds in any shapes, and leads the adhesive to be tightly attached to the wounds, thereby better promoting the healing of the wounds.

(3) The invention is beneficial to promoting the wound healing in humid environment and has quite strong wet tissue adhesion strength.

(4) The invention has good biological activity and small stimulation to biological tissues.

(5) The invention has good mechanical property, the bonding strength can reach 5.528KPa, and the colloid can not be broken due to the deformation of the wound.

(6) The invention is biodegradable, and can effectively avoid secondary damage caused by removing the medicine.

(7) The production process of the invention has no pollution, and the final product has no pollution to the environment and human body, thus being green and environment-friendly.

(8) The preparation method is simple, has low requirements on equipment, easily available raw materials and low cost, and is suitable for large-scale production.

Drawings

FIG. 1 is a technical scheme of the present invention;

FIG. 2 is a graph showing the effect of the antibacterial test of the bioadhesive prepared by the present invention;

FIG. 3 is a schematic structural view of the adhesion strength test of the present invention;

FIG. 4 is a result of an adhesive strength test of the bioadhesive prepared by the present invention;

FIG. 5 shows the results of cytotoxicity tests of the bioadhesive prepared according to the present invention;

FIG. 6 shows the results of in vitro degradation experiments for bioadhesives prepared according to the present invention;

fig. 7 is an SEM image of the bioadhesive prepared in example 3 of the present invention.

Detailed Description

The invention firstly dissolves the structural protein in the deionized water and then adjusts the pH value to be alkaline. The source of the structural protein is not particularly limited in the present invention, and commercially available products or laboratory preparations known to those skilled in the art may be used.

Adhesive strength test method: thawing the treated pigskin in a 37 deg.C water bath, and cutting into 1 × 4cm pieces²The sample, surface moisture was fully absorbed with absorbent paper and kept at 37 ℃. The obtained biological adhesive is coated on the surface of pig skin (1 × 3 cm)²) Then, the two pigskins were bonded, gently pressed with a finger for 30 seconds, kept warm in a water bath, and after the sample gelled, the sample was subjected to uniaxial tensile stress-strain test on a universal mechanical tester with a 50N weighing cell at a crosshead speed of 5 mm/min. Each stretch to break was taken as the final maximum adhesion and the corresponding bond strength was calculated.

To further illustrate the present invention, the following examples are provided for illustration.

Example 1

(1) Weighing 1g of gelatin, adding 100mL of deionized water, and dissolving the gelatin for 2.0h at 60 ℃ under the condition of heating and stirring to finally obtain a gelatin solution with the mass fraction of 1%.

(2) Adjusting the pH of the gelatin solution prepared in step (1) to 8.5 with 12mol/L sodium hydroxide solution, weighing 34mg tannic acid and sufficiently dissolving in a small amount of deionized water, slowly dropwise adding the above tannic acid solution into the gelatin solution with adjusted pH, adjusting the pH to 8.5 with 1mol/L sodium hydroxide solution again, heating at 60 deg.C under stirring for 3.0h, adjusting the pH with 1mol/L sodium hydroxide solution to maintain 8.5, and continuously introducing air.

(3) After the reaction in the step (2) is finished, adjusting the pH to 7.4 by using 0.5mol/L dilute hydrochloric acid, cooling the reactant to room temperature, dialyzing the reactant by using deionized water for 3 times by using a dialysis bag with a molecular weight cut-off value of 3500Da, and finally freeze-drying the dialyzed substance.

(4) And (4) taking 100mg of the freeze-dried sample obtained in the step (3), uniformly mixing the freeze-dried sample with 1ml of Tris-HCL buffer solution to obtain a solution, adding 10mg of transglutaminase, and uniformly mixing to obtain the adhesive.

Example 2

(4) And (4) taking 100mg of the freeze-dried sample obtained in the step (3), uniformly mixing the freeze-dried sample with 1ml of Tris-HCL buffer solution to obtain a solution, adding 20mg of transglutaminase, and uniformly mixing to obtain the adhesive.

Example 3

(4) And (4) taking 100mg of the freeze-dried sample obtained in the step (3), uniformly mixing the freeze-dried sample with 1ml of Tris-HCL buffer solution to obtain a solution, adding 30mg of transglutaminase, and uniformly mixing to obtain the adhesive.

Example 4 gel formation time test

The gel forming time after transglutaminase was added in step (4) of the production processes of examples 1 to 3 was measured, and the gel forming time of the bioadhesive was measured at room temperature (25 ℃) and body temperature (37 ℃), respectively. The results are shown in Table 1. The gel time can be as low as 9min at 25 ℃.

TABLE 1 gel time results for adhesives obtained from different example preparations

Examples	Example 1	Example 2	Example 3
				Enzyme concentration (%, w/v)	1	2	3
Gel formation time (minutes) at 25 ℃	30	20	9
				Gel formation time (minutes) at 37 ℃	＞60	50	30

Example 5 bacteriostatic test

The bacteriostatic effect of the biological adhesive on escherichia coli ATCC 8739 was determined by colony counting.

Activating strains: 1mL of the bacterial solution was put in 20mL of liquid medium and shake-cultured for 12 hours (37 ℃ C., 100 r/min).

Transferring 50uL of the activated bacterial suspension to 20mL of liquid medium, culturing for 2h (37 ℃, 100rpm) by shaking, and collecting 20uL of bacterial suspension (about 1X 10)⁶CFU/mL) was added to a well plate containing 100uL of the bioadhesive sample prepared in examples 1, 2 and 3 (3 parallel and 3 blank controls), and the broth was diluted 10 h (37 ℃ C., 100r) after 2h of shake culture⁴And (3) taking 100uL of the diluted bacterial liquid, smearing the diluted bacterial liquid on a solid culture medium, putting the solid culture medium into a constant-temperature incubator (37 ℃) to culture for 12 hours, and calculating the total number of colonies.

Bacteriostatic ratio (%) - (total number of blank colonies-total number of bioadhesive colonies) ÷ total number of blank colonies × 100

The experimental results are as follows: the results of the biological adhesive of the present invention on the inhibition of escherichia coli are shown in table 2 and fig. 2, and the inhibition rates of the biological adhesives prepared by the methods of examples 1 to 3 were all 100%.

TABLE 2 bacteriostatic effect of the adhesives obtained in the different examples

Examples	Example 1	Example 2	Example 3
				TA/gelatin(％，w/w)	3.4	3.4	3.4
Enzyme concentration (%, w/v)	1	2	3
				Bacteriostatic ratio (%)	100	100	100

Example 6 adhesion Strength test

Adhesive strength test method: thawing the treated pigskin in a 37 deg.C water bath, and cutting into 1 × 4cm pieces²The sample, surface moisture was fully absorbed with absorbent paper and kept at 37 ℃. The bioadhesive prepared in examples 1, 2 and 3 was applied to the surface of the pigskin (1X 3 cm)²) Then, the two pigskins were bonded, gently pressed with a finger for 30 seconds, kept warm in a water bath, and after the sample gelled, the sample was subjected to uniaxial tensile stress-strain test on a universal mechanical tester with a 50N weighing cell at a crosshead speed of 5 mm/min. Each stretch to break was taken as the final maximum adhesion and the corresponding bond strength was calculated. The results are shown in table 3 and fig. 4.

Table 3 results of adhesive strengths of adhesives obtained from different examples

Examples	Example 1	Example 2	Example 3
				Enzyme concentration (%, w/v)	1	2	3
Adhesive Strength (KPa)	2.857	3.773	5.528

Example 7 cytotoxicity assay

Cytotoxicity test methods: the cytotoxicity of the bioadhesive against 3T3 fibroblasts was determined using the MTT method.

The specific operation is as follows: freeze-drying the biological adhesive prepared in example 3, and soaking the freeze-dried adhesive in a cell culture medium at a concentration of 10mg/mL after ultraviolet sterilization for 0.5h, wherein the extract is used for culturing cells; spreading 3T3 cells with good growth state in 96-well plate at 3000/well at 37 deg.C and 5% CO₂Culturing in an incubator for 24 hours; after the cells were attached to the wall, 100. mu.L of different concentrations of cement extract (0.25, 0.5, 1, 5mg/mL) were added, 6 replicates per concentration were set, and a set of blank controls was set per plate. After the cells were cultured for 24, 48 and 72 hours, the medium was discarded, 100. mu.L of 0.5mg/ml MTT was added thereto, and the mixture was placed in a cell culture chamber and cultured for 4 hours to terminate the culture. The supernatant in the well plate was aspirated, 100. mu.L of dimethyl sulfoxide was added to each well, the mixture was allowed to stand for 15 minutes, and the absorbance at 570nm was measured with a microplate reader.

The experimental results are as follows: the results of the cytotoxicity test of the bioadhesive of the present invention are shown in fig. 5. The bioadhesive prepared in example 3 had no inhibitory effect on cell growth at concentrations of 0.25, 0.5mg/mL, and had no significant effect on cell growth at concentrations of 1, 2 mg/mL.

Example 8 in vitro degradation experiments

The in vitro biodegradability of the bioadhesive was investigated with collagenase i in PBS.

The specific operation is as follows: the bioadhesives obtained in examples 1, 2 and 3 were freeze-dried, and the freeze-dried adhesive was soaked in collagenase I (1U/ml) in PBS (pH7.4) and shaken at 37 ℃ and 100 rpm/min. The solution was centrifuged and the unreacted enzyme was removed periodically (1d, 3d, 7d, 14d), and the pellet was freeze-dried and weighed. In addition, a control group without enzyme was provided.

Biodegradation rate (%) - (W0-W1)/W0 × 100, wherein W0 is the weight of the original freeze-dried adhesive and W1 is the weight of the sample after freeze-drying at a predetermined time.

The experimental results are as follows: the results of the in vitro degradation experiments of the bioadhesive of the present invention are shown in fig. 6, and the degradation rates of examples 1, 2 and 3 reached 60.2%, 48.9% and 21.3%, respectively, after seven days; 78.7%, 67.5% and 40.7% were reached after fourteen days, respectively.

Example 9 apparent morphology detection

The apparent morphology detection method comprises the following steps: the pore morphology of the bioadhesive was observed by Scanning Electron Microscopy (SEM).

The specific operation is as follows: (1) detection of an attachment surface: thawing the treated pigskin in a 37 deg.C water bath, cutting into 1 × 4cm2 samples, absorbing surface water with absorbent paper, and keeping the temperature at 37 deg.C. After the bioadhesive prepared in example 3 was applied to the surface of the pigskin (1X 3cm2), the two pigskins were glued together, gently pressed with a finger for 30s, incubated in a water bath, and the sample was freeze-dried after gelling. And tearing the two frozen and dried pigskins apart, and using the sample for detecting the attachment surface.

(2) Non-adhesive surface detection: the bioadhesive prepared in example 3 was freeze-dried and subjected to non-adherent surface examination directly on its surface.

(3) Internal cross section detection: the bioadhesive prepared in example 3 was freeze-dried and fractured to obtain internal cross-sections for internal morphology studies.

The experimental results are as follows: the result of the detection of the apparent morphology of the biological adhesive is shown in fig. 7, and the biological adhesive has a good pore structure and high porosity, is beneficial to the absorption of wound exudate, can be moisture permeable and breathable, and creates a space for cell proliferation.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for preparing an injectable antibacterial biological adhesive, which is characterized by comprising the following steps:

(1) weighing 1g of gelatin, adding 100mL of deionized water, and dissolving the gelatin for 2.0h at 60 ℃ under the condition of heating and stirring to finally obtain a gelatin solution with the mass fraction of 1%;

(2) adjusting the pH of the gelatin solution prepared in the step (1) to 8.5 by using 12mol/L sodium hydroxide solution, weighing 34mg of tannic acid to be fully dissolved in a small amount of deionized water, slowly dropwise adding the tannic acid solution into the gelatin solution with the adjusted pH, adjusting the pH to 8.5 by using 1mol/L sodium hydroxide solution again, heating for 3.0h under the condition of stirring at 60 ℃, adjusting the pH to keep 8.5 by using 1mol/L sodium hydroxide solution, and continuously introducing air;

(3) after the reaction in the step (2) is finished, adjusting the pH to 7.4 by using 0.5mol/L dilute hydrochloric acid, cooling the reactant to room temperature, dialyzing the reactant by using deionized water for 3 times by using a dialysis bag with a molecular weight cut-off value of 3500Da, and finally freeze-drying the dialyzed substance;

2. A bioadhesive prepared by the method of claim 1.

3. Use of the method of claim 1 or the bioadhesive of claim 2 for the preparation of a medicament for adhesion between living physiological tissues, tamponade and leakage repair of physiological tracts, sealing of wounds for hemostasis or repair, or medical devices.