CN105316933A - Preparation method of antibacterial electrospun fibrous membrane - Google Patents

Abstract

The invention provides a method for preparing an antibacterial electrospun fiber membrane, which belongs to the technical field of biomedical materials. The antibacterial electrospun fiber membrane of this method is characterized by taking the electrospun fiber membrane of biodegradable synthetic polymer as the matrix, adding soluble protein to it, and using the peptide bond of the soluble protein on the surface of the composite fiber and the synthesized metal nanoparticles The hydrogen bonds between the groups on the surface make the antibacterial metal nanoparticles firmly loaded on the surface of the electrospun fiber, and the good biocompatibility of the soluble protein can be used to expand its application in the field of biomedical materials. The antibacterial electrospun fiber membrane prepared by the method can be well used not only in the field of biomedicine, but also in other fields.

Description

A kind of preparation method of antibacterial electrospun fiber membrane

technical field

The invention relates to a method for preparing an antibacterial electrospun fiber membrane, which belongs to the technical field of biomedical materials.

Background technique

Antibacterial materials play an important role in food, electrical appliances, medical equipment and other fields. At present, most of the antibacterial materials used in the market are organic antibacterial agents, but organic antibacterial agents generally have poor antibacterial effects, and only have a strong anti-mold effect, and there are very few organic antibacterial agents that have excellent inhibitory effects on bacteria and mold. Moreover, the heat resistance and stability of organic antibacterial agents are poor, and their decomposition products and volatiles may be harmful to the human body. Therefore, inorganic antibacterial agents with strong inhibitory effect on bacteria stand out. Compared with organic antibacterial agents, inorganic antibacterial materials have the characteristics of persistence, persistence, broad spectrum, resistance to drug resistance, good heat resistance, and high safety. However, when the inorganic antibacterial agent is used alone, it has the disadvantages of poor stability and difficult recycling.

As a new polymer processing method, electrospinning plays an important role in the preparation of new polymer composites. Due to the advantages of high specific area, uniform diameter, and feasibility for a variety of polymers, electrospun fibers account for a non-negligible proportion in the preparation of composite materials containing metal nanoparticles. Electrospun fibers can provide a stable environment for metal nanoparticles (various chemical bonds or non-chemical bonds can be formed between metal nanoparticles and discharge fibers), so that metal nanoparticles can be stably dispersed on electrospun fibers.

There are different combinations of electrospinning and metal nanoparticles, which can be roughly divided into three types. The first one is to mix the metal salt in the spinning solution, and reduce the metal salt after spinning (using bulk reduction or additional reducing agent reduction) ; The second is spinning after the reduction of metal salts into nanoparticles in the spinning solution. Both of the above two methods introduce metal nanoparticles, but most of the silver nanoparticles are located inside the electrospun fiber matrix, which cannot fully and efficiently play the role of silver nanoparticles. The third method: adsorption or spraying, this method can overcome the shortcomings of the first two methods, and introduce silver nanoparticles to the surface of the electrospun fiber, although the method of sputtering and spraying can introduce silver nanoparticles on the surface of the electrospun fiber membrane Particles, but must rely on special equipment and strict operating conditions, the cost is high and difficult to operate; and adsorption is to adsorb the pre-synthesized silver nanoparticles on the surface of the electrospun fiber through some kind of chemical bond or non-chemical bond. Dong et al. used sodium citrate as a stabilizer and synthesized silver nanoparticles with carboxyl groups on the surface. Using the hydrogen bond between the carboxyl groups on the surface of silver nanoparticles and the amide bond on the surface of PA ₆ electrospun fiber membrane, the silver nanoparticles were loaded on The surface of the electrospun fiber membrane. However, the polyamide used in the above experiments is a non-biodegradable polymer, which limits the application of the antibacterial electrospun fiber membrane prepared with this material in the field of biomedicine.

Contents of the invention

The purpose of the present invention is to propose a method for preparing an antibacterial electrospun fiber membrane, so that the prepared antibacterial electrospun fiber membrane can be well used not only in the field of biomedicine, but also in other fields.

Technical scheme of the present invention is as follows:

A method for preparing an antibacterial electrospun fiber membrane, characterized in that said method comprises the steps of:

1) Dissolving biodegradable synthetic polymers and soluble proteins in a solvent to make a spinning solution, and then using an electrospinning device to make the spinning solution into an electrospun fiber membrane, and drying the electrospun fiber membrane, wherein, the electrospun fiber membrane is The mass fraction of soluble protein in the spun fiber membrane ranges from 7% to 50%; the biodegradable synthetic polymer is 93% to 50%;

2) Use chemical methods to prepare metal salts with antibacterial effects into metal nanoparticle sols, use peptide bonds on protein molecules and groups on the surface of metal nanoparticles to form hydrogen bonds, and make metal nanoparticles loaded on the electrospun fiber membrane After washing and drying, the antibacterial electrospun fiber membrane is obtained.

The biodegradable synthetic polymer refers to aliphatic polyester, polyether or polyvinyl alcohol, or a copolymer of aliphatic polyester and polyamide, or a copolymer of aliphatic polyester and aromatic polyester.

Preferably, the metal nanoparticles are silver, copper, zinc or titanium nanoparticles, or titanium oxide nanoparticles.

Preferably, the method of loading metal nanoparticles on the electrospun fiber membrane adopts a method of filtration, soaking or suction filtration.

The present invention has the following advantages and outstanding effects: the preparation method of an antibacterial electrospun fiber membrane proposed by the present invention. Since the peptide bonds on the protein molecules and the groups on the surface of the metal nanoparticles form hydrogen bonds, the stable dispersion of the metal nanoparticles on the surface of the electrospun fiber membrane can be ensured. At the same time, the electrospun fiber membrane is composed of biodegradable synthetic polymers and soluble proteins. Among them, soluble proteins, as natural polymers, have many advantages that synthetic and semi-synthetic polymers do not have, such as: good biocompatibility, etc. Therefore, , the prepared antibacterial electrospun fiber membrane can be well used not only in the field of biomedicine, but also in other fields.

Description of drawings

Fig. 1 is the transmission electron microscope of the antibacterial electrospun fiber membrane obtained according to the preparation method of Example 1 of the present invention, and the sample membrane is: polycaprolactone/soluble egg shell membrane protein-silver nanoparticles.

detailed description

A kind of preparation method of antibacterial electrospun fiber membrane provided by the invention, the method comprises the steps:

1) Dissolving biodegradable synthetic polymers and soluble proteins in an organic solvent to make a spinning solution, then using an electrospinning device to make the spinning solution into an electrospun fiber membrane, and drying the electrospun fiber membrane. Among them, the mass fraction of soluble protein in the electrospun fiber membrane ranges from 7% to 50%, and the biodegradable synthetic polymer is 93% to 50%. The biodegradable synthetic polymer refers to aliphatic polyester, polyether or polyethylene Alcohol, or aliphatic polyester and polyamide copolymer, or aliphatic polyester and aromatic polyester copolymer.

2) Using a chemical method to prepare the metal salt with antibacterial effect into nanoparticles of silver, copper, zinc, titanium or titanium oxide. Using the method of filtration, immersion or suction filtration, and using the peptide bond on the protein molecule and the group on the surface of the metal nanoparticle to form a hydrogen bond, the metal nanoparticle is loaded on the electrospun fiber membrane, and the antibacterial property is obtained. Electrospun fiber membrane.

Example 1: Preparation of electrospun fiber membrane: add hexafluoroisopropanol, polycaprolactone (PCL) and soluble egg shell membrane protein (SEP) in the sample bottle, the mass ratio of the three is: hexafluoroisopropanol Propanol:PCL:SEP=133:9:1, so the mass fraction of soluble egg shell membrane protein is: 10%. Stir at room temperature until dissolved. The prepared spinning solution was subjected to electrospinning to obtain an electrospun fiber membrane, and the prepared electrospun fiber membrane was desolventized in a vacuum oven at 40° C. for 24 hours.

Synthesis of silver nanoparticles: Dissolve AgNO ₃ in 45mL deionized water, add Na ₃ C ₆ H ₅ O ₇ 2H ₂ O to the solution under stirring, the mass ratio of the three is AgNO ₃ : deionized water: Na ₃ C ₆ H ₅ O ₇ ·2H ₂ O=1:5294:1.73; weigh NaBH ₄ and dissolve it in 5mL deionized water, the mass ratio of the two is: NaBH ₄ : deionized water=1:526; , quickly pour the _NaBH4 solution into the _AgNO3 solution. After continuing to stir rapidly at room temperature for a period of time, the reaction is ended, and the final product is a dark brown dispersion liquid, which is silver nanoparticle sol. Before using the silver nanoparticle sol, adjust its pH to 5, and the silver nanoparticle sol is ready-to-use.

Because the stabilizer used for synthesizing silver nanoparticles is sodium citrate, there are carboxyl groups on the surface of the prepared silver nanoparticles, and the carboxyl groups can form hydrogen bonds with the peptide bonds on the protein (also with the esters on the polycaprolactone). groups form hydrogen bonds, but in small numbers). So the electrospun fiber membrane can adsorb silver nanoparticles.

Cut a certain area of the electrospun fiber membrane, under the condition of suction filtration, saturately adsorb (10ml) silver nanoparticles, and then wash with distilled water. Take it out and dry it at room temperature to obtain an electrospun fiber membrane loaded with silver nanoparticles.

Antibacterial performance: The prepared electrospun fiber membrane was tested for its antibacterial ability, and the contact sterilization rate for E.coli (DH5α) was determined to reach 98% according to the following characterization method.

Antibacterial assay method:

LB medium: add NaCl, tryptone (tryptone), and yeast extract (yeastextract) to 50mL deionized water, the mass ratio of the four is: deionized water:NaCl:tryptone:yeastextract=200:2:2:1 , Stir to dissolve and put into 25mL Erlenmeyer flasks, 5mL per bottle, and sterilize in a high-pressure steam sterilizer at 121°C for 15min.

LB-agar medium (LB plate): add NaCl, tryptone (tryptone), and yeast extract (yeastextract) to 50mL deionized water, stir and dissolve, then add agar (agar), the mass ratio of the five is: deionized Water:NaCl:tryptone:yeastextract:agar＝200:2:2:1:4, sterilized in a high-pressure steam sterilizer at 121°C for 15min. The sterilized culture medium is divided into 9 cm culture dishes sterilized by high pressure steam before being cooled and solidified, and about 20 mL of culture medium is added to each culture dish, and it is advisable to completely cover the culture dish, and it is cooled and solidified in an ultra-clean bench.

Gram-negative Escherichia coli (E.coli, DH5α) was used to test the antibacterial ability.

Bacterial pre-cultivation: Inoculate the bacteria into LB medium, incubate for 18 hours at 37° C. in a shaker at a speed of 230 rpm, and set aside. The bacterial concentration is about 109cfu/mL.

The antibacterial performance determination experiment was determined by the contact sterilization method. Cut the sample to be tested and the reference sample into a size of 1.5cm×1.5cm, sterilize them in 75% ethanol for 30 minutes, and dry them in a clean bench for a period of time. The sterilized and dried samples were placed on the LB plate to ensure that it was completely attached to the surface of the culture medium. Each sample was inoculated with 10 μL of pre-cultured bacterial solution, and after incubating in a 37°C incubator for 2 hours, the sample was removed and placed into a PE tube pre-added with 1 mL of deionized water, and continuously vibrated for 5 minutes to remove the bacteria on the sample. The bacteria were completely eluted to obtain a 100-fold diluted bacterial solution. After the bacterial solution was serially diluted to 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , and 10 ⁷ times, 100 μL of each was taken and spread evenly on LB plates, and the bacterial concentration on each sample was measured by plate counting method. The bacterial concentration on the test sample is recorded as A ₁ (cfu/mL), and the corresponding bacterial concentration on the reference sample is recorded as A ₀ (cfu/mL), then the bactericidal rate P is calculated by the following formula:

$\mathrm{<span\; class="notranslate">\; P</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}\mathrm{<span\; class="notranslate">\; \%</span>}$

Example 2: The polycaprolactone in Example 1 was replaced with polybutylene succinate, the preparation method was the same as in Example 1, and the 2-h contact sterilization rate for E.coli (DH5α) reached 97%.

Embodiment 3: The polycaprolactone in embodiment 1 is replaced by polylactic acid, the preparation method is the same as that of embodiment 1, and the 2h contact sterilization rate for E.coli (DH5α) reaches 98%.

Embodiment 4: change saturated adsorption (10ml) silver nanoparticle into unsaturated adsorption (5ml) silver nanoparticle in embodiment 1, preparation method is the same as embodiment 1, reaches 92% for the 2h contact sterilization rate of E.coli (DH5α) %.

Example 5: Change the mass concentration fraction of soluble protein in the electrospun fiber membrane from 10% to 7% in Example 1, the preparation method is the same as in Example 1, and the 2h contact sterilization rate for E.coli (DH5α) reaches 90% .

Example 6: The mass fraction of the soluble protein in the electrospun fiber membrane in Example 1 was changed from 10% to 50%, the preparation method was the same as in Example 1, and the 2-h contact sterilization rate for E.coli (DH5α) reached 99%.

Example 7: The soluble egg shell membrane protein in Example 1 was replaced with soluble collagen, the preparation method was the same as in Example 1, and the 2-h contact sterilization rate for E.coli (DH5α) reached 99%.

Example 8: E.coli (DH5α) in Example 1 was replaced with B.Subtilis (168), the preparation method was the same as in Example 1, and the 2-h contact sterilization rate for E.coli (DH5α) reached 90%.

Example 9: The pH of the silver nanoparticles in Example 1 was adjusted to 3, the preparation method was the same as in Example 1, and the 2-h contact sterilization rate for E.coli (DH5α) reached 99%.

Example 10: The method of absorbing silver nanoparticles in Example 1 was replaced by filtration, the preparation method was the same as in Example 1, and the 2-h contact sterilization rate for E.coli (DH5α) reached 99%.

Example 11: The mass concentration of the soluble eggshell membrane protein in Example 1 was adjusted to 30%, the preparation method was the same as that in Example 1, and the 2-h contact sterilization rate for E.coli (DH5α) reached 99%.

Example 12: The mass concentration of the soluble eggshell membrane protein in Example 1 was adjusted to 50%, the preparation method was the same as that in Example 1, and the 2-h contact sterilization rate for E.coli (DH5α) reached 99%.

Example 13: Silver nanoparticles in Example 1 were replaced with copper nanoparticles, the preparation method was the same as in Example 1, and the 2-h contact sterilization rate for E.coli (DH5α) reached 82%.

Comparative Example 1: If the silver nanoparticles are directly used for sterilization, the silver nanoparticles will not be recovered effectively and will be mixed in the bacterial sample, which is expensive and potentially dangerous.

Claims (4)

1. a preparation method of antibacterial electrospun fiber membrane, is characterized in that described method comprises the steps: 1) Dissolving biodegradable synthetic polymers and soluble proteins in a solvent to make a spinning solution, and then using an electrospinning device to make the spinning solution into an electrospun fiber membrane, and drying the electrospun fiber membrane, wherein, the electrospun fiber membrane is The mass fraction of soluble protein in the spun fiber membrane is 7%-50%; the mass fraction of biodegradable synthetic polymer is 93%-50%; 2) Use chemical methods to prepare metal salts with antibacterial effects into metal nanoparticle sols, use peptide bonds on protein molecules and groups on the surface of metal nanoparticles to form hydrogen bonds, and make metal nanoparticles loaded on the electrospun fiber membrane After washing and drying, the antibacterial electrospun fiber membrane is obtained. 2. by the preparation method of a kind of antibacterial electrospun fiber membrane described in claim 1, it is characterized in that: biodegradable synthetic polymer refers to aliphatic polyester, polyether or polyvinyl alcohol, or aliphatic polyvinyl alcohol Copolymers of esters and polyamides, or copolymers of aliphatic polyesters and aromatic polyesters. 3. according to the preparation method of a kind of antibacterial electrospun fiber membrane described in claim 1 or 2, it is characterized in that: described metal nanoparticle is the nanoparticle of silver, copper, zinc or titanium, or titanium oxide nanometer particle. 4. According to the preparation method of a kind of antibacterial electrospun fiber membrane described in claim 1 or 2, it is characterized in that: the method for metal nanoparticles loaded on the electrospun fiber membrane adopts the method of filtering, soaking or suction filtration.