CN112813007A - Method for repairing biological material film by biological template method - Google Patents
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Abstract
The invention discloses a method for repairing a biological material film by a biological template method, which belongs to the field of biochemical engineering and biological materials and comprises the following steps: step 1: preparing a biological template material: uniformly mixing the template substrate and a corresponding solvent, adding a modifier or a reaction substrate corresponding to the template substrate, and carrying out post-treatment after complete reaction to obtain a biological template material; step 2: repairing the biological membrane, namely placing the damaged membrane in a container, and adding a biological template material to ensure that the damaged membrane is fully contacted with the biological template material; adding an ionic liquid cross-linking agent until the reaction is complete; filtering; adding an ionic liquid detergent for washing; carrying out oscillation treatment; and (4) performing post-treatment to obtain the repaired biological membrane. The invention utilizes the principles of self-assembly, material in-situ growth and biological self-repair, adopts a biological template method to repair the biological material film, solves the problem of damage repair of the biological material film, and has better application potential in the aspects of film material repair, biological material repair and tissue and organ film repair.
Description
Technical Field
The invention belongs to the field of biochemical engineering and biological materials, and relates to a method for repairing a biological material film by a biological template method.
Background
The biological material film refers to a film composed of biological materials, and comprises film materials such as biological films, cellulose films, chitosan films and the like. The biological membrane refers to a membrane constituting each "member" of a cell of a living body, and cellular membranes, nuclear membranes, endoplasmic reticulum, golgi apparatus, mitochondria and other organelles in the cell, which are functionally related "components" of this "machine", and they are all constituted by membranes, and these membranes have similar chemical compositions and substantially the same basic structure, and are collectively called biological membranes. The protein biological membrane material with bioactivity and good compatibility is used as a field, the protein has the composition similar to human epithelial tissue amino acid, and the studied animal extraction of collagen, silk fibroin, keratin and the like is favorable for cell attachment and spreading and is very favorable for proliferation. The biological membrane material has the characteristics of excellent biocompatibility, easy biodegradation and the like, and meanwhile, the degradation product of the biological membrane material has no toxic or side effect on tissues and can provide nutrition and repair effects on tissues, organs and the like.
The research on repairing the biological material film is less, the research on repairing the biological material film by adopting a biological template method is not available, and the invention has better application potential in the aspects of film material repair, biological material repair and tissue and organ film repair.
The biological template method is a new technology for preparing nano materials which is developed in recent years internationally. This technique utilizes the naturally occurring nanostructures of organisms as templates. The controllable synthesis of nano materials with different shapes and sizes can be realized by selecting a proper template; meanwhile, when the nano material with ordered arrangement and complex structure is prepared, the template method has more advantages than other synthesis methods. The biological tissue template mainly comprises a biological membrane, a solid biological tissue and a liquid biological tissue, and is now a research hotspot. The biological template has a specific spatial structure, can be well combined with nanoparticles, and has the advantages of degradability and environmental friendliness.
In nature, many natural biomasses contain inorganic nano materials or organic nano materials with complex structures, and the biological nano materials have the characteristics of high uniformity in geometric dimension and structural diversity. Certain biological macromolecules can also form a supramolecular structure through self-assembly, which greatly expands the diversity of natural biomasses as templates. In addition, the natural biomass contains various active functional groups, has higher chemical reaction activity, and can generate coordination reaction with various metal ions, thereby laying the chemical foundation for synthesizing the nano material by using the natural biomass as a template. Therefore, the natural biomass is an ideal template for preparing the nano material with a complex structure and monodisperse particle size distribution.
The controllable synthesis of nano materials with different shapes and sizes can be realized by selecting a proper template; meanwhile, when the nano material with ordered arrangement and complex structure is prepared, the template method has more advantages than other synthesis methods. The template used in the template method has wide sources, and at present, the natural biomass used as the template for synthesizing the nano material mainly comprises cellulose, chitosan, protein, microorganisms and the like.
Disclosure of Invention
The invention aims to solve the problems that: the invention solves the problem of repairing the damage of the biological material film, and adopts the principles of self-assembly, material in-situ growth and biological self-repair and adopts a biological template method to repair the biological material film.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for repairing a biofilm by a biological template method comprises the following steps:
step 1: preparing a biological template material: uniformly mixing the template substrate and a corresponding solvent, adding a modifier or a reaction substrate corresponding to the template substrate, and carrying out post-treatment after complete reaction to obtain a biological template material;
step 2: repairing biofilms
Step 2.1: placing the damaged film in a container, adding a biological template material, and fully contacting the damaged film with the biological template material;
step 2.2: adding an ionic liquid cross-linking agent until the reaction is complete;
step 2.3: filtering;
step 2.4: adding an ionic liquid detergent for washing;
step 2.5: carrying out oscillation treatment;
step 2.6: and (4) performing post-treatment to obtain the repaired biological membrane.
Preferably, the template matrix in the step 1 adopts chitosan, 0.2g of chitosan is mixed in 50ml of 1-ethyl-3-methylimidazole acetate ionic liquid, 0.05g of chloroacetic acid is added, and the mixture is washed and filtered; adding 20g of 4-ethyl-4-methylmorpholine bromide, dropwise adding ammonia water until the pH value reaches 11-13, adding 1ml of glutaraldehyde solution (2%, v%), stirring for 10min, washing until the pH value reaches about 7, and drying to obtain the chitosan template.
Preferably, the template matrix in the step 1 is made of cellulose, 0.5g and 15g of 1-ethyl-3-methylimidazol diethyl phosphate ionic liquid are respectively added into an iodine measuring flask, the mixture is placed into an ultrasonic cleaning tank and pretreated by ultrasonic waves of 40kHz and 300W, then the iodine measuring flask is placed into a magnetic heating stirrer at a certain temperature for reaction, after mulberry fibers are completely dissolved, 0.15g of maleic anhydride is added into the dissolved solution in parts for several times, the reaction is carried out at a constant temperature, after the reaction is finished, the product is cooled to room temperature, filtered and washed for 3-5 times, ammonia water (25% -28%) is added dropwise until the pH value reaches 11-13, the reaction is carried out at 50 ℃ for 3 hours, the obtained product is washed until the pH value is 7, and dried at the constant temperature for 24 hours, and the cellulose template powder is obtained.
Preferably, silk fibroin mixed solution is adopted as the template matrix in the step 1, 10ml of keratin solution with the mass fraction of 5% is mixed with 10ml of silk fibroin with the mass fraction of 0.5%, 10ml of chitosan with the mass fraction of 5% is added with 85ml of phospholipid and is uniformly mixed.
Preferably, the template matrix in the step 1 adopts a yeast culture solution or a acetobacter xylinum culture solution, the yeast culture solution is 50ml of saccharomyces cerevisiae fermentation culture solution, 0.2g of nano-cellulose is added, and the mixture is uniformly shaken and mixed at the temperature of 30-35 ℃ for 2 d; the culture solution contains Acetobacter xylinum OD 50ml600=0.6-0.8, glucose 0.2g, yeast extract 0.03 g, peptone 0.03 g.
Preferably, the template matrix is prepared in step 2.1 as a solution with a concentration of 30-55% (wt%) using an ionic liquid; ultrasonic treatment is carried out for 30-50s in the step 2.5 until the biological membrane grows in a self-assembly way; the work-up in step 2.6 comprises drying and washing 2 to 3 times.
Preferably, the silk fibroin mixed solution is mixed and cultured with the damaged biological membrane in the step 2.1 until the biological membrane is grown; washing with 5-20 wt% concentration ionic liquid detergent in step 2.4; and (5) carrying out ultrasonic treatment for 10-20s in the step 2.5 to obtain the repaired biological membrane.
Preferably, step 2.1 adds a yeast culture or acetobacter xylinum culture to the membrane to be damaged, and shake-cultures until the biofilm is "grown".
Preferably, the ionic liquid is: any one or combination of 1-ethyl-3-methylimidazole diethyl phosphate, 1-ethyl-3-methylimidazole acetate, 1-butyl-3-methylimidazole acetate, 1-amido-3-methylimidazole chloride, 1-methyl-3-methylimidazole dimethyl phosphate, 1-methyl-3-methylimidazole diethyl phosphate, 1-ethyl-3-methylimidazole dimethyl phosphate, 1-butyl-3-methylimidazole chloride or 1-butyl-3-methylpyridine chloride;
the ionic liquid cross-linking agent is a mixture of ionic liquid and a cross-linking agent, and the mixing ratio is 90-95:5-10 (wt/wt); the ionic liquid detergent is 5-55% (wt%) ionic liquid water solution, cetyl trimethyl ammonium bromide is added to make the final concentration of the ionic liquid detergent be 2-10% (wt%), and polyvinylpyrrolidone is added to make the final concentration of the ionic liquid detergent be 0.1-0.2 mol/L; the mass ratio of the ionic liquid to 0.15 percent (wt%) of glutaraldehyde is 9: 1.
The invention has the advantages and positive effects that:
(1) the invention utilizes the principles of self-assembly, material in-situ growth and biological self-repair, adopts a biological template method to repair the biological material film, solves the problem of damage repair of the biological material film, and has better application potential in the aspects of film material repair, biological material repair and tissue and organ film repair.
(2) According to the invention, according to the principle of self-assembly of the micro-nano material, the repaired biological material film has good stability, and the function can be completely recovered by attaching the repair process to the film material.
(3) The invention adopts the ionic liquid detergent, the main component of the ionic liquid is green salt ionic liquid, the ionic liquid can be recycled through suction filtration, rotary evaporation and drying, and the ionic liquid is green and environment-friendly.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a repair of cellulose membrane obtained in example.
Detailed Description
The technical solution of the present invention is described in detail below, but the scope of the present invention is not limited to the embodiments.
Preparing an ionic liquid:
preparing an ionic liquid detergent, adding Cetyl Trimethyl Ammonium Bromide (CTAB) into 55% (wt%) 1-ethyl-3-methylimidazolium dimethyl phosphate saline solution to enable the final concentration of CTAB to be 2% (wt%), and then adding polyvinylpyrrolidone to enable the final concentration to be 0.1 mol/L;
preparing an ionic liquid cross-linking agent: 90g of ionic liquid and 10g of 0.15% (wt%) glutaraldehyde were mixed;
example 1
Repairing the biological membrane by adopting a chitosan template:
1: preparation of chitosan template material
1.1 mixing 0.2g of chitosan in 50ml of ionic liquid [ EMIM ] [ Ac ], adding 0.05g of chloroacetic acid, washing and filtering;
1.2 adding 20g of ionic liquid 4-ethyl-4-methyl morpholine bromide;
1.3 adding ammonia water drop by drop until the pH value reaches 11-13;
1.4 adding 1ml of glutaraldehyde solution (2%, v%), stirring for 10min, washing until the pH value reaches about 7, and drying for later use;
2: repairing biofilms
2.1: placing the damaged film in a container, preparing the chitosan template material obtained in the step 1 into a solution with the concentration of 30-55% (wt%), and adding the solution into the container;
2.2: adding an ionic liquid cross-linking agent;
2.3: filtering;
2.4: adding an ionic liquid detergent for washing;
2.5: ultrasonic processing for 30-50s until the biological membrane is self-assembled and grows; step 2.6: post-treating to obtain a repaired biological membrane;
2.6: drying and washing for 2 to 3 times.
Example 2
Repairing the biological membrane by adopting a cellulose template:
1: preparation of cellulose formwork Material
1.1 Add cellulose 0.5g and 15g ionic liquid [ EMIM ] [ DEP ] into a 20ml iodine flask, respectively;
1.2 placing an iodine measuring flask in an ultrasonic cleaning tank for pretreatment by ultrasonic treatment at 40kHz and 300W;
1.3 placing an iodine measuring flask in a magnetic heating stirrer for reaction, adding 0.15g of maleic anhydride into the dissolved solution for several times after the mulberry fibers are completely dissolved, and continuing to react for a period of time at constant temperature;
1.4 after the reaction is finished, cooling the product to room temperature, filtering and washing for 3-5 times;
1.5, dropwise adding ammonia water (25-28%) until the pH value reaches 11-13, stirring at 50 ℃ for reaction for 3 hours, washing the obtained product until the pH value is 7, and drying at constant temperature for 24 hours to obtain cellulose template powder;
2: repairing biofilms
2.1: placing the damaged film in a container, preparing the cellulose template material obtained in step 1 into a solution with a concentration of 30-55% (wt%), and adding the solution into the container;
2.2: adding an ionic liquid cross-linking agent;
2.3: filtering;
2.4: adding an ionic liquid detergent for washing;
2.5: ultrasonic processing for 30-50s until the biological membrane is self-assembled and grows; step 2.6: post-treating to obtain a repaired biological membrane;
2.6: drying and washing for 2 to 3 times.
The method for repairing the cellulose membrane comprises the following steps: can solve the problems that natural cellulose has non-uniformity and the prepared biological membrane has poor uniformity. The cellulose solvent and the cellulose are used for preparing the biological template, the high-grade space structure of the cellulose can be destroyed by heating the cellulose solvent, the cellulose with different sizes is dissolved, and finally the cellulose is homogenized.
Example 3
Adopting an acetobacter xylinum template to repair the biological membrane:
1: preparation of Acetobacter xylinum template material
1.1 preparation of a culture solution of Acetobacter xylinum
2: repairing biofilms
2.1 placing the damaged membrane in a container, adding 50ml of acetobacter xylinum nutrient solution, and performing shake culture for 5 days until a cellulose membrane grows;
2.2 pouring out the liquid, and adding 5ml of ionic liquid cross-linking agent for treatment for 2 h;
2.3 pouring out the liquid, adding 30ml of ionic liquid detergent, shaking for 30min, freeze-drying, washing and drying to obtain the repaired biological material film.
Example 4
Repairing the biological membrane by adopting a yeast template:
1: preparation of Yeast template Material
1.1 adding 0.2g of nano-cellulose into 50ml of saccharomyces cerevisiae fermentation culture liquid, shaking and uniformly mixing, wherein the temperature is 30-35 ℃, and the culture time is 2 days.
Example 5
Repairing a biological membrane by adopting silk fibroin:
1: preparing a silk fibroin template material, mixing 10ml of a keratin solution with the mass fraction of 5% with 10ml of silk fibroin with the mass fraction of 0.5%, adding 10ml of 5% chitosan, adding 85ml of phospholipid, and uniformly mixing.
2: repairing the biological membrane:
2.1 placing the damaged biological membrane in a container, adding 20ml of silk fibroin mixed solution, carrying out ultrasonic treatment for 1min, and placing on a vibration table concentrator to vibrate and uniformly mix for 1 h;
2.2 pouring out the liquid, adding 5ml of ionic liquid cross-linking agent for treatment for 10 min;
2.3 pouring out the liquid, adding 30ml of ionic liquid detergent, and shaking for 30 min;
and 2.4 washing with deionized water for 2 times, and freeze-drying to obtain the repaired biological membrane.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A method for repairing a biofilm by a biological template method is characterized by comprising the following steps:
step 1: preparing a biological template material: uniformly mixing the template substrate and a corresponding solvent, adding a modifier or a reaction substrate corresponding to the template substrate, and carrying out post-treatment after complete reaction to obtain a biological template material;
step 2: repairing biofilms
Step 2.1: placing the damaged film in a container, adding a biological template material, and fully contacting the damaged film with the biological template material;
step 2.2: adding an ionic liquid cross-linking agent until the reaction is complete;
step 2.3: filtering;
step 2.4: adding an ionic liquid detergent for washing;
step 2.5: carrying out oscillation treatment;
step 2.6: and (4) performing post-treatment to obtain the repaired biological membrane.
2. The method for repairing a biofilm by using a biological template method as claimed in claim 1, wherein chitosan is adopted as the template matrix in the step 1, 0.2g of chitosan is mixed in 50ml of 1-ethyl-3-methylimidazolium acetate ionic liquid, 0.05g of chloroacetic acid is added, and the mixture is washed and filtered; adding 20g of 4-ethyl-4-methylmorpholine bromide, dropwise adding ammonia water until the pH value reaches 11-13, adding 1ml of glutaraldehyde solution (2%, v%), stirring for 10min, washing until the pH value reaches about 7, and drying to obtain the chitosan template.
3. The method for repairing a biofilm by using a biological template method as claimed in claim 1, wherein the template matrix in step 1 is made of cellulose, 0.5g and 15g of 1-ethyl-3-methylimidazolium phosphate diethyl ester ionic liquid are respectively added into an iodine measuring flask, the mixture is placed into an ultrasonic cleaning tank for pretreatment by ultrasonic at 40kHz and 300W, then the iodine measuring flask is placed into a magnetic heating stirrer at a certain temperature for reaction, after the mulberry fibers are completely dissolved, 0.15g of maleic anhydride is added into the dissolved solution in several times, the reaction is carried out at a constant temperature, after the reaction is finished, the product is cooled to room temperature, the solution is filtered and washed for 3-5 times, ammonia water (25% -28%) is added dropwise until the pH value reaches 11-13, the reaction is carried out at 50 ℃ for 3 hours under stirring, the obtained product is washed until the pH value is 7, drying for 24 h at constant temperature to obtain cellulose template powder.
4. The method for repairing a biological material film by using a biological template method as claimed in claim 1, wherein silk fibroin mixed solution is adopted as the template matrix in the step 1, 10ml of keratin solution with the mass fraction of 5% is mixed with 10ml of silk fibroin with the mass fraction of 0.5%, 10ml of chitosan with the mass fraction of 5% is added with 85ml of phospholipid and mixed uniformly.
5. The method for repairing a biofilm by using a biological template method as claimed in claim 1, wherein a yeast culture solution or a acetobacter xylinum culture solution is adopted as a template matrix in the step 1; the yeast culture solution is 50ml of saccharomyces cerevisiae fermentation culture solution, 0.2g of nano-cellulose is added, the mixture is uniformly shaken and mixed, the temperature is 30-35 ℃, and the culture time is 2 d; the culture solution contains Acetobacter xylinum OD 50ml600=0.6-0.8, glucose 0.2g, yeast extract 0.03 g, peptone 0.03 g.
6. The method for repairing a biofilm according to the biomatex method of claim 2 or 3, wherein in step 2.1 the template matrix is configured as a solution with a concentration of 30-55% (wt%) using an ionic liquid; ultrasonic treatment is carried out for 30-50s in the step 2.5 until the biological membrane grows in a self-assembly way; the work-up in step 2.6 comprises drying and washing 2 to 3 times.
7. The method for repairing a biofilm by using a biological template method as claimed in claim 4, wherein the silk fibroin mixed solution and the damaged biofilm are mixed and cultured in the step 2.1 until the biofilm is grown; washing with 5-20 wt% concentration ionic liquid detergent in step 2.4; and (5) carrying out ultrasonic treatment for 10-20s in the step 2.5 to obtain the repaired biological membrane.
8. The method for repairing a biofilm by using a biological template method as claimed in claim 5, wherein the step 2.1 is to add a yeast culture solution or a acetobacter xylinum culture solution into the damaged biofilm, and culture the damaged biofilm by shaking until the biofilm is grown.
9. The method for repairing biofilm by using the biological template method as claimed in claim 1, wherein the biological template method comprises the following steps: the ionic liquid is as follows: any one or combination of 1-ethyl-3-methylimidazole diethyl phosphate, 1-ethyl-3-methylimidazole acetate, 1-butyl-3-methylimidazole acetate, 1-amido-3-methylimidazole chloride, 1-methyl-3-methylimidazole dimethyl phosphate, 1-methyl-3-methylimidazole diethyl phosphate, 1-ethyl-3-methylimidazole dimethyl phosphate, 1-butyl-3-methylimidazole chloride or 1-butyl-3-methylpyridine chloride;
the ionic liquid cross-linking agent is a mixture of ionic liquid and a cross-linking agent, and the mixing ratio is 90-95:5-10 (wt/wt); the ionic liquid detergent is 5-55% (wt%) ionic liquid water solution, cetyl trimethyl ammonium bromide is added to make the final concentration of the ionic liquid detergent be 2-10% (wt%), and polyvinylpyrrolidone is added to make the final concentration of the ionic liquid detergent be 0.1-0.2 mol/L; the ionic liquid cross-linking agent is prepared from ionic liquid and 0.15 percent (wt%) of glutaraldehyde by mass ratio of 9: 1.
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