CN100467680C - Antibacterial silk composite nanofiber material and preparation method thereof - Google Patents

Abstract

This invention discloses a compound antibacterial silk nanometer fibrous material, accounting for weight its component proportion are: Sericin protein 58-90%; chitosan or chitin 0-40%, antibacterial agent 0-5%, electric conduction 1-4%,crossing linking 1-4%. Its process is: dissolve the pure sericin protein, antibiotics separately in polar solvent, join electric conduction medicinal preparation and crosslinking agent, using the static electricity spinning technique, making the average aperture for 0.5-5.0 micron, factor of porosity 60-90%, the mean diameter is 40-100nm and the material is nanometer textile fiber adhesive-bonded membrane. This invention has good organization compatibility with human body, Antibiotics and sericin insoluble is in homogeneous solvent, massive holes among the nanometer textile fiber adhesive-bonded membrane, which has outstanding aqueous percolation permeability, antibacterial agent distributes evenly in the nanometer textile fiber, and the medicine release effect is perfect, so it can be a kind of ideal resist bacteria injured medical material.

Description

Antibacterial silk composite nanofiber material and preparation method thereof

technical field

The invention relates to a medical biological material and a preparation method thereof, in particular to a nanofiber antibacterial material composed of silk fibroin and an antibacterial agent and a preparation method thereof, belonging to the technical field of polymer materials.

Background technique

Before the present invention was made, there were reports on the use of natural or synthetic high polymers as main raw materials to make nanofiber non-woven membranes through electrospinning, such as: raw materials are polyacrylonitrile, polyamide, polyvinyl alcohol, cellulose And its derivatives, etc., but some of the above materials have poor compatibility with the human body, and some spun fibers are relatively thick, with an average diameter of several hundred to several thousand nanometers, which are not suitable for medical materials.

Silk protein is a fairly pure natural protein, which is composed of 18 kinds of amino acids. It has a great similarity with human collagen. It is non-toxic, non-irritating, and has good biocompatibility. Therefore, silk protein is the preparation Ideal basic raw material for medical biomaterials. There is also a nanofiber non-woven film made of silk protein by electrospinning, but it has no antibacterial properties, and its application range is limited to a certain extent.

The Chinese invention patent with the publication number CN1348820A "An Antibacterial Silk Fibroin Wound Protective Film and Its Preparation Method" discloses that silk is used as the main film-forming raw material, antibacterial agents and additives are added, and the salivation method is applied through layered coating and hot air drying. The prepared antibacterial silk fibroin wound protective film is a dense film formed by hot air drying. Although it has a good antibacterial effect, the flexibility of the silk fibroin wound protective film will be affected by hot air drying; Pores affect its water permeability and air permeability, which is not conducive to wound healing, and the effect is affected to a certain extent.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a medical antibacterial wound covering material with good compatibility with the human body, excellent antibacterial properties, water permeability and air permeability, and a preparation method thereof.

An antibacterial silk composite nanofiber material according to the present invention is characterized in that it is a nanofiber non-woven membrane structure with an average pore diameter of 0.5-5.0 μm, a porosity of 60-90%, and an average fiber diameter of 40-5.0 μm. 100nm; its components and the proportions by weight of each component are: silk fibroin 58-90%, chitosan or chitin 0-40%, antibacterial agent 0-5%, conductive agent 1-4%, 1-4% crosslinking agent; the antibacterial agent is one or more of quaternary ammonium salts, guanidines, polyguanidines, and salts containing Ag ⁺ , Zn ²⁺ metal ions; the conductive agent It is one or more of borates, acetates and silicates; the crosslinking agent is one or more of tannic acid, citric acid, pentaerythritol and boric acid.

The method for preparing antibacterial silk composite nanofiber material, first degumming, dissolving, dialysis, purifying and drying the silk to obtain pure silk fibroin, which is characterized in that the processing is carried out in the following steps:

a. Dissolve pure silk fibroin, chitosan or chitin or antibacterial agents in polar solvents respectively, and then fully mix them to make a uniform solution. The polar solvents are glacial acetic acid, dimethyl One or more of formamide, anhydrous formic acid, and trifluoroethanol;

b. Add a conductive agent and a cross-linking agent to the above solution to prepare a spinning stock solution with a mass fraction of 8-20%. The components in the solute and the proportions by weight of each component are: silk fibroin 58-90% , chitosan or chitin 0-40%, antibacterial agent 0-5%, conductive agent 1-4%, cross-linking agent 1-4%;

c. Using the electrospinning process for the above spinning stock solution, an antibacterial silk composite nanofiber nonwoven membrane with an average pore size of 0.5-5.0 μm, a porosity of 60-90%, and an average fiber diameter of 40-100 nm is obtained on the receiving plate .

The technological conditions of the above electrospinning are: voltage 15-35KV, spinning distance 7-20cm, feeding amount of spinning solution 0.1-3.0ml/h.

The antibacterial silk composite nanofiber nonwoven membrane prepared above is irradiated with gamma rays.

Compared with the prior art, the advantages of the antibacterial silk composite nanofiber material provided by the present invention are:

1. It uses silk as the main raw material, and silk is a relatively pure natural protein, which has good tissue compatibility with the human body, is non-toxic, non-irritating and sensitizing. Therefore, the wound medical material made is compatible with The wound surface has good adhesion.

2. Chitosan or chitin and antibacterial agents are compounded in silk protein, and chitosan or chitin is a natural high polymer with antibacterial properties; the antibacterial agents used are non-antibiotics and have a wide antibacterial spectrum , has excellent killing effect on Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, has little side effects on the human body, low toxicity, no irritation to the skin, good light and heat stability, and they are soluble in the same species as silk fibroin In the solvent, a uniform spinning solution is formed, and in the composite nanofiber nonwoven membrane formed by electrospinning, chitosan, antibacterial agent, etc. can be evenly compounded and blended with silk fibroin in the nanofiber nonwoven membrane. The specific surface area of the fiber is large, and chitosan acts according to the bactericidal mechanism of contact barriers, showing excellent bactericidal properties; because the antibacterial agent is evenly distributed in the nanofibers, the fibers are thin and the specific surface area is large, so the initial release concentration is large, making the drug release The concentration exceeds the minimum bactericidal concentration of the drug, so it has excellent bactericidal effect, and because the non-woven film is composed of many nanofibers, the drug needs to be released gradually from the inside of the fiber, so the antibacterial agent in the film can be released continuously , while maintaining the persistence of the drug effect.

3. Since the nanofiber non-woven membrane formed by electrospinning has a large number of pores that penetrate up and down, it has excellent water permeability and air permeability, which is beneficial to wound treatment. Also, because the electrospinning is carried out at room temperature, the antibacterial agent will not be affected by the temperature and cause changes in the composition of the drug, ensuring a good use effect.

4. The antibacterial silk composite nanofiber material is used as a wound medical material after being irradiated and sterilized. Its appearance is white, uniform in thickness, smooth, flat, soft, not easy to break, and very convenient to use.

Description of drawings

Fig. 1 is the scanning electron microscope (SEM) figure of the antibacterial silk composite nanofiber material that the technical scheme that provides by the embodiment of the present invention 1 makes;

Fig. 2 is a SEM image of the antibacterial silk composite nanofiber material prepared according to the technical solution provided in Example 3 of the present invention.

Detailed ways

The present invention will be further elaborated below in conjunction with embodiment.

Embodiment one:

1. Put 0.1 kg of leftover silk into 3 liters of 0.05% sodium carbonate aqueous solution by mass fraction, boil for 0.5 hour, repeat the treatment three times, remove all the sericin around the silk, and obtain silk fibroin;

2. After drying the obtained silk fibroin, use 0.6 liter of calcium chloride, water and ethanol solution with a molar ratio of 1:8:2 to heat and dissolve at 78±2°C to form a silk protein solution;

3. Pour the above-mentioned silk protein solution into the cellulose semi-permeable membrane, dialyze with tap water first, and then dialyze with deionized water to remove ethanol and calcium chloride small molecules in the solution, and then filter with multi-layer degreasing gauze to prepare Obtain pure silk fibroin solution;

4. Take 100ml of pure silk fibroin solution and pour it into a polystyrene (ABS) dish with an area of 20× ^20cm2 , dry it to form a film at 25°C and constant temperature and humidity of RH 65%, and remove the regenerated silk fibroin Film bagged for use;

5. 3.6 gram chitosan was added in 16 gram glacial acetic acid, stirred 30 minutes and made chitosan acetic acid solution;

6. Add 8.04 grams of the above-mentioned regenerated silk fibroin film to 72 grams of anhydrous formic acid, and stir for 45 minutes;

7. Add the chitosan acetic acid solution obtained in step 5 into the regenerated silk fibroin formic acid solution obtained in step 6, after fully stirring, add 0.18 grams each of sodium borate and tannic acid, continue stirring and dissolving to form a uniform spinning stock solution 100 Gram, its spinning solution mass fraction is 12%;

8. Inject 100 grams of the prepared spinning stock solution into the spinning tube through a micro-injection pump;

9. Adjust the electrostatic voltage of the electrostatic generator to 30kV, the spinning distance to 12cm, and the feeding volume of the spinning solution of the micro-injection pump to 0.5ml/h. The spinning solution is continuously sprayed out through the metal needle in front of the spinning tube, which is extremely The solvent glacial acetic acid and anhydrous formic acid are volatilized in the air, and 12 grams of silk/chitosan composite nanofiber non-woven film can be collected on the receiving plate, and a non-woven film with an area of 15×15 cm ² and a thickness of about 0.015 mm can be obtained. About 25 pieces of medical antibacterial wound mask. According to the principle of electrospinning, the diameter, pore size and pore size of nanofibers can be met by adjusting the mass fraction of spinning solution, static voltage, spinning distance, feeding amount of spinning solution and spinning time, etc. Ratio and the thickness of the non-woven film and other parameters.

10. After the prepared non-woven film was equilibrated in a vacuum drying oven for 12 hours, it was packed in a double-layer plastic bag and sealed on a high-frequency sealing machine, and then sterilized by γ-ray irradiation for use.

Referring to accompanying drawing 1, the antibacterial silk composite nanofiber material that the technical scheme provided by the present embodiment makes, the average diameter of its fiber is about 82nm, as seen from Fig. 1, there is the hole through up and down between the nanofiber nonwoven film, The average pore diameter is about 0.6 μm, and the porosity is about 75%.

Embodiment two:

1. Put 0.2 kg of waste mulberry silk into 6 liters of aqueous sodium carbonate solution with a mass fraction of 0.05%, boil for 0.5 hour, repeat the treatment three times, remove all the sericin around the silk to obtain silk fibroin, and dry at room temperature;

2. Dissolve the dried silk fibroin with 1.2 liters of calcium chloride, water and ethanol solution with a molar ratio of 1:8:2 at 78±2°C to form a silk protein solution;

3. Pour the fibroin solution prepared in step 2 into a cellulose dialysis bag, first dialyze with tap water, then dialyze with deionized water to remove ethanol and calcium chloride in the solution, and then filter with degreasing gauze to obtain Pure silk fibroin solution;

4. Pour 100ml of pure silk fibroin protein solution into a stainless steel plate, freeze at -25°C for 12 hours, and then vacuum-dry it in a freeze dryer for 24 hours to obtain a spongy silk fibroin film, which is bagged for use;

5. 6.7 grams of the spongy silk film obtained in step 4 were added to 73 grams of trifluoroethanol, and stirred for 45 minutes;

6. Weigh 3.0 grams of chitin and add it to 17 grams of trifluoroethanol, and stir for 30 minutes;

7. Add the chitin solution obtained in step 6 into the silk fibroin solution obtained in step 5. After fully stirring, add 0.15 grams each of cerium acetate and pentaerythritol, and continue stirring until 100 grams of uniform spinning solution is formed. The quality score is 10%;

8. Add 100 grams of spinning solution prepared in step 7 to a micro-injection pump, and inject the spinning solution into the spinning tube by the pump;

9. Fix the anode of the electrostatic generator on the metal needle in front of the spinning tube, connect the cathode to the metal receiving plate, adjust the static voltage of the electrostatic generator to 32kV, and the spinning distance to 15cm, turn on the electrostatic generator and micro-injection Pump, adjust the micro-injection pump so that the feeding amount of the spinning solution is 1.0ml/h, the spinning solution is continuously sprayed out through the metal needle in front of the spinning tube, and the polar solvent trifluoroethanol is volatilized in the air, and on the receiving plate 10 grams of silk/chitin composite nanofiber non-woven membrane can be collected, the average diameter of the fibers is 98nm, there are pores through the fibers, the average pore diameter is 1.0μm, and the porosity is 78%. The area that can be obtained is about About 20 pieces of non-woven film with a size of 15×15cm ² and a thickness of 0.015mm;

10. After the non-woven film prepared in step 9 is balanced in a vacuum drying oven for 24 hours, it is packed in a double-layer plastic bag and sealed on a high-frequency sealing machine, and then sterilized by γ-ray irradiation for use.

The antibacterial silk composite nanofiber materials obtained in the above two examples, after testing, their antibacterial rates to Staphylococcus aureus are respectively 97.8% (6h), 96.2% (6h) and 100% (24h); The antibacterial rates of bacilli were 99.1% (6h), 97.6% (6h) and 100% (24h), respectively, showing excellent antibacterial properties. Because the film has a large number of micropores penetrating up and down and has excellent water permeability and air permeability, it can be used as medical materials such as antibacterial wound medical dressings and antibacterial masks.

Embodiment three:

1. Put 0.2 kg of waste mulberry silk into 6 liters of aqueous sodium carbonate solution with a mass fraction of 0.05%, boil for 0.5 hour, repeat the treatment three times, remove all the sericin around the silk, obtain silk fibroin, and dry at room temperature;

2. Dissolve the dried silk fibroin with 1.2 liters of calcium chloride, water and ethanol solution with a molar ratio of 1:8:2 at 78±2°C to form a silk protein solution;

3. Pour the fibroin solution prepared in step 2 into a cellulose dialysis bag, first dialyze with tap water, then dialyze with deionized water to remove ethanol and calcium chloride in the solution, and then filter with degreasing gauze to obtain Pure silk fibroin solution;

4. Pour 100ml of pure silk fibroin solution into a stainless steel plate, freeze at -18°C for 8 hours, and then vacuum-dry it in a freeze dryer for 18 hours to obtain silk fibroin powder, which is bagged for use;

5. Join 16.2 grams of silk fibroin powder gained in step 4 in the anhydrous formic acid of 73.8 grams, stir for 45 minutes;

6. Select 0.54 grams of antibacterial agent biguanide acetate to join in 8.2 grams of glacial acetic acid, stir and dissolve to obtain antibacterial agent solution;

7. Add the above-mentioned antibacterial agent solution to the solution prepared in step 5. After fully stirring, add 0.63 grams of sodium borate and boric acid respectively, and continue to stir and dissolve until 100 grams of uniform spinning stock solution is formed; the mass fraction of spinning stock solution is 18%;

8. Inject 100 grams of spinning solution prepared in step 6 into the spinning tube through a micro-injection pump;

9. Adjust the electrostatic voltage of the electrostatic generator to 32kV and the spinning distance to 15cm, turn on the electrostatic generator and the micro-injection pump, adjust the micro-injection pump so that the feeding amount of the spinning solution is 0.8ml/h, and the spinning solution passes through the spinning The metal needle in front of the silk tube sprays out continuously, the polar solvents glacial acetic acid and anhydrous formic acid volatilize in the air, and 18 grams of silk/antibacterial drug composite nanofiber non-woven film can be collected on the receiving plate, which can be made into a 15×15cm ^2. About 30 sheets of non-woven film with a thickness of 0.02mm. The nanofiber non-woven film contains 0.1-1.5 mg of antibacterial drugs per square centimeter;

10. Put the fiber non-woven membrane prepared in step 9 into a vacuum desiccator for 8 hours, pack it in a double-layer plastic bag and seal it on a high-frequency sealing machine, and then sterilize it with γ-ray irradiation for use.

Referring to accompanying drawing 2, according to the antibacterial silk composite nanofiber material that the technical scheme provided in this embodiment makes, its fiber diameter is about 96nm, has the hole that runs through up and down between the nanofiber non-woven membrane, and average aperture is about 1.2 μ m, The porosity is about 80%.

After testing, the antibacterial zones of the nanofiber membrane to Staphylococcus aureus and Escherichia coli were 16mm and 13mm respectively (according to the test standard, the antibacterial zone is greater than 7mm to have antibacterial properties), while the inhibition zone of the pure silk nanofiber nonwoven membrane The bacterial circle is 0, which shows that the nanofiber material prepared according to the technical solution of this embodiment has excellent antibacterial properties. The test of drug release performance shows that the drug release concentration of the antibacterial silk composite nanofiber material has reached the minimum bactericidal concentration of the drug within 1 hour, and the drug is still released continuously after 48 hours, maintaining a long-lasting drug effect. The release rate can be controlled by adjusting the diameter of nanofibers, and the smaller the diameter, the faster the release due to the larger specific surface area, thereby achieving the effect of controlled drug release. And because the non-woven membrane has a large number of micropores penetrating up and down and has excellent water permeability and air permeability, it can be used as medical materials such as antibacterial wound medical dressings and oral ulcer patches.

Claims (3)

1. the method for preparing anti bacteria natural silk composite nano-fiber material, earlier with silk through come unstuck, dissolving, dialysis, purifying, drying, obtain pure fibroin albumen, it is characterized in that carrying out again the processing of following steps:

A. be dissolved in pure fibroin albumen, shitosan or chitin or biguanides acetate in the polar solvent respectively, make homogeneous solution after they fully being mixed, described polar solvent is one or more in glacial acetic acid, dimethyl formamide, anhydrous formic acid, the trifluoroethanol again;

B. in above-mentioned solution, add conductive agent, crosslinking agent, the preparation quality mark is 8～20% spinning solution, component in the solute and each component ratio by weight are: fibroin albumen 58～90%, shitosan or chitin 0～40%, biguanides acetate 0～5%, conductive agent 1～4%, crosslinking agent 1～4%, wherein, the content of shitosan, chitin, biguanides acetate is not 0% simultaneously;

Described conductive agent is one or more in borate, the acetate; Described crosslinking agent is one or more in tannic acid, pentaerythrite, the boric acid;

C. above-mentioned spinning solution is adopted electrostatic spinning process, obtaining average pore size on dash receiver is 0.5～5.0 μ m, and porosity is 60～90%, and average fibre diameter is the anti bacteria natural silk composite nano-fiber nonwoven film of 40～100nm.

2. the method for preparing anti bacteria natural silk composite nano-fiber material according to claim 1 is characterized in that described electrostatic spinning process condition is: voltage 15～35KV, spinning distance 7～20cm, spinning solution feed quantity 0.1～3.0ml/L.

3. the method for preparing anti bacteria natural silk composite nano-fiber material according to claim 1 is characterized in that: with the anti bacteria natural silk composite nano-fiber nonwoven film that makes again through gamma-ray irradiation.

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