CN105012991B - Antibacterial anti hemorrhagic material with non-woven fibrous fabric structure and preparation method thereof - Google Patents

Abstract

An antibacterial-hemostatic material with a non-woven fabric structure and a preparation method thereof belong to the technical field of biomedical materials. The antibacterial-hemostatic material is characterized by dissolving aliphatic polyester in a solvent, first making an electrospun fiber membrane through an electrospinning process, and loading chitosan on the surface of the electrospun fiber membrane, and then further loading thrombin made. Taking advantage of the material characteristics of aliphatic polyester and chitosan, as well as the structural characteristics of the formed electrospun fiber membrane such as high porosity and high specific surface area, thrombin was stably loaded through the crosslinking of chitosan and glutaraldehyde , forming an antibacterial‑hemostatic material with a nonwoven fiber fabric structure. The material has good antibacterial and hemostatic effects, which are obviously superior to commercially available gelatin sponges, and has the advantages of good mechanical properties, easy storage, and easy removal after use, and the preparation method is simple to operate and the amount of raw materials is saved. The products can be used in surgical operations, trauma first aid and other fields.

Description

Antibacterial-hemostatic material with non-woven fabric structure and preparation method thereof

technical field

The invention relates to an electrospun fiber antibacterial-hemostatic material with a non-woven fabric structure and a preparation method thereof, belonging to the technical field of biomedical polymer materials.

Background technique

Hemostatic materials and antibacterial materials play a very important role in post-injury first aid or medical rescue. At present, the hemostatic materials commonly used in clinical practice include gelatin, oxidized cellulose, fibrin glue, chitosan, etc., and the forms are often sponges, gauze, etc. The hemostatic properties of these commonly used hemostatic materials, especially the hemostatic speed, cannot fully meet all emergency medical procedures. the needs of the situation. As a biological agent, thrombin has a good hemostatic effect, but it is commercially available as a freeze-dried powder, which has disadvantages such as complicated application methods and easy inactivation. In addition, there are a large number of research reports on various antibacterial materials. Among them, chitosan, a natural polymer material, not only has good biocompatibility and biodegradability, but also has good antibacterial function, and has a wide range of applications in the field of biomedicine. Applications. However, due to the high viscosity, high charge and strong intramolecular and intermolecular hydrogen bonding of chitosan in its acidic aqueous solution, it is difficult to spin successfully alone.

Electrospinning is a process that utilizes the jet action of a polymer solution or melt under the action of a strong electric field for spinning. The fiber products processed by electrospinning are called electrospun fibers. The biggest feature of electrospun fibers is that the diameter is very small, which is several orders of magnitude smaller than the diameter of fibers prepared by conventional methods, and its diameter generally ranges from 3nm to 1μm. Electrospun fiber non-woven fabrics have high porosity, large specific surface area, high fiber fineness and uniformity, and large aspect ratio. These excellent characteristics that cannot be obtained by traditional spinning methods endow electrospun fibers with broad application prospects. At present, electrospun fibers have been widely used in the fields of sensors, wound dressings, membrane separation, and tissue engineering.

From the material point of view, aliphatic polyester has good biodegradability and good biocompatibility, and has certain mechanical strength, flexibility and spinnability. If aliphatic polyester can be prepared into electrospinning If the advantages of aliphatic polyester and chitosan are combined by coating chitosan on the surface, firstly, the structural characteristics of the very high porosity of the electrospun fiber membrane can be used to make it Like commonly used hemostatic materials such as hemostatic gauze and gelatin sponge, it is easy to adhere to the complex wound surface through the porous physical structure; secondly, it can overcome the difficulty of chitosan which is not easy to spin, and use chitosan as an antibacterial substance. The product has antibacterial properties. The third is that chitosan can be used to stably and efficiently load the hemostatic drug thrombin to achieve rapid hemostasis, so that the product can fill the gap in rapid hemostasis materials.

Compared with the currently existing hemostatic materials containing thrombin or coagulation factors, the present invention has better hemostatic performance, and uses the non-woven fiber fabric structure as the carrier to make the product more convenient to use and easy to remove after hemostasis . In addition, using chitosan as a stabilizer increases the storage stability of thrombin, making the antibacterial-hemostatic product easier to store. And the preparation method has the advantages of simple operation and saving raw material consumption.

Contents of the invention

The purpose of the present invention is to propose a kind of electrospun fiber antibacterial-hemostatic material with non-woven fiber fabric structure and preparation method thereof, hoping to utilize the material characteristics of aliphatic polyester and chitosan, and the formed electrospun fiber membrane With the structural characteristics of high porosity and high specific surface area, the cross-linking effect of chitosan and glutaraldehyde is used to stably load thrombin, improve the storage stability of thrombin, and make the material achieve the application purpose and effect of antibacterial-hemostatic materials.

Technical scheme of the present invention is as follows:

An antibacterial-hemostatic material with a non-woven fabric structure is characterized in that the antibacterial-hemostatic material is a porous non-woven structure and contains aliphatic polyester fibers, chitosan and thrombin; aliphatic polyester The fiber diameter is 100nm-1500nm, the porosity is 50%-90%, the static contact angle is 0°-130°, and the water absorption rate of the material is 2-20;

The material is prepared by the following method: first, the aliphatic polyester is made into an aliphatic polyester fiber film by electrospinning, and then fully immersed in a chitosan solution, so that the surface of the aliphatic polyester fiber is covered with nano Then the chitosan-loaded aliphatic polyester fiber membrane is completely immersed in the thrombin aqueous solution, and after drying, an antibacterial-hemostatic material with a non-woven fiber fabric structure is obtained.

A kind of preparation method of antibacterial-hemostatic material with non-woven fiber fabric structure provided by the present invention is characterized in that, the method comprises the steps:

1) dissolving the aliphatic polyester in a solvent to obtain a spinning solution, and then using an electrospinning method to make the spinning solution into an aliphatic polyester electrospun fiber membrane;

2) fully immersing the dried aliphatic polyester electrospun fiber membrane in a chitosan solution, taking it out and drying at room temperature to obtain an aliphatic polyester electrospun fiber membrane loaded with chitosan;

3) Completely immerse the chitosan-loaded aliphatic polyester electrospun fiber membrane in the glutaraldehyde aqueous solution, take it out and dry it at room temperature, then completely immerse it in the thrombin aqueous solution, take it out and dry it at room temperature , that is, an antibacterial-hemostatic material with a non-woven fabric structure is obtained.

In the method of the present invention, preferably: the molecular weight of the chitosan described in step 2) is 50,000～500,000, and the degree of deacetylation is more than or equal to 80%, and the chitosan solution is an acidic aqueous solution, and the mass percentage of the chitosan solution is The concentration is 0.5wt%-15wt%. The concentration of the thrombin aqueous solution is 0.5U/mL˜10U/mL. The mass percent concentration of the glutaraldehyde aqueous solution is 0.0001wt%-1wt%.

In the above technical solution of the present invention, it is characterized in that the aliphatic polyester refers to a polyester formed by polycondensation of monomers having hydroxyl and carboxyl groups at the same time, or aliphatic dibasic acid and aliphatic dibasic acid The polyalcohol is polycondensed into polyester, or the polyester or copolyester is formed from aliphatic lactone through ring-opening polymerization. The molecular weight of aliphatic polyester is 50,000-250,000. The polyester formed by polycondensation of monomers having both hydroxyl and carboxyl groups is polylactic acid formed by direct polycondensation of lactic acid; the polyester formed by polycondensation of aliphatic dibasic acid and aliphatic dibasic alcohol It is polybutylene succinate, polyhexamethylene sebacate, polyethylene succinate or polyhexamethylene succinate; a polymer formed by ring-opening polymerization of aliphatic lactones The ester is polylactic acid formed by ring-opening polymerization of lactide, and polycaprolactone formed by ring-opening polymerization of caprolactone; the copolyester is polylactide.

Preferably, the solvent used to dissolve the aliphatic polyester described in step 1) is one or a mixture of hexafluoroisopropanol, tetrahydrofuran, N,N-dimethylformamide and trifluoroacetic acid; The mass percentage concentration when forming the spinning solution is 5wt%-30wt%. The voltage in the electrospinning method is 10KV-30KV, the spinning distance is 10cm-20cm, the injection speed is 0.3mL/h-2mL/h, and the receiving roll speed is 100rpm-500rpm.

The present invention has the following advantages and outstanding effects: the antibacterial-hemostatic material with a non-woven fiber fabric structure proposed by the present invention can fully combine the performance of raw materials and the structural characteristics of the electrospun fiber membrane to achieve the purpose of antibacterial-hemostatic . The antibacterial and hemostatic material with non-woven fiber fabric structure obtained by the present invention uses chitosan and glutaraldehyde to carry out stable loading on thrombin to improve the storage stability of thrombin, and uses thrombin to improve the hemostatic performance, which is obviously better than commercially available gelatin sponge. The product uses non-woven fabric as the carrier, which is more convenient to use and store and easy to remove after hemostasis. Simultaneously, the preparation method adopted in the present invention has the advantages of simple operation, convenient batch production and saving raw material consumption.

Description of drawings

Fig. 1 is the scanning electron micrograph of the polybutylene succinate (PBS) nonwoven fabric structure that the present invention prepares according to embodiment 1.

Fig. 2 is the scanning electron micrograph of the polybutylene succinate (PBS) non-woven fabric structure prepared according to Example 1 after dipping chitosan in the present invention.

3 is a scanning electron micrograph of the polybutylene succinate (PBS) non-woven fabric structure prepared according to Example 1 after dipping chitosan and thrombin in the present invention.

detailed description

A kind of antibacterial-hemostatic material with non-woven fabric structure provided by the invention is a porous non-woven structure, containing aliphatic polyester fiber, chitosan and thrombin; the diameter of aliphatic polyester fiber is 100nm ～1500nm, porosity 50%～90%, static contact angle 0°～130°, material water absorption rate 2～20; Spinning fiber membrane, and after chitosan is loaded on the surface of the electrospun fiber membrane, further loading thrombin is formed; the specific preparation method is as follows:

First, the aliphatic polyester is dissolved in a solvent to prepare a spinning solution, and then the spinning solution is made into an aliphatic polyester electrospun fiber membrane by electrospinning; the types of the aliphatic polyester solvent include but Not limited to hexafluoroisopropanol, tetrahydrofuran, N,N-dimethylformamide or trifluoroacetic acid. Electrospinning conditions are not limited, but the recommended conditions are: the mass percentage concentration when forming the spinning solution is 5wt%-30wt%, the voltage is 10KV-30KV, the spinning distance is 10cm-20cm, and the injection speed is 0.3mL/h- 2Ml/h, the speed of receiving roll is 100rpm-500rpm.

The above dried aliphatic polyester electrospun fiber membrane is fully immersed in the chitosan solution, taken out and dried at room temperature to obtain an electrospun fiber membrane loaded with chitosan; the degree of deacetylation of the chitosan is ≥ 80% and molecular weight 50000-500000, the chitosan solution is generally an acidic aqueous solution, but the type of acid used is not limited, and the mass percentage concentration when forming the chitosan solution for dipping is 0.5wt%-15wt%.

The chitosan-loaded aliphatic polyester electrospun fiber membrane is first fully impregnated with an appropriate concentration of glutaraldehyde aqueous solution and dried at room temperature, and then fully impregnated with an appropriate concentration of thrombin aqueous solution and dried at room temperature. Just obtain the antibacterial-hemostatic material with non-woven fiber fabric structure; The mass percent concentration of described glutaraldehyde aqueous solution is 0.0001wt%-1wt%; The concentration of described thrombin aqueous solution is 0.5U/mL-10U /mL.

The aliphatic polyester mentioned in this preparation method refers to the polycondensation of monomers having both hydroxyl and carboxyl groups, or polycondensation of aliphatic dibasic acids and aliphatic dibasic alcohols, or polycondensation of fatty Polyesters or copolyesters formed by ring-opening polymerization of family lactones, with a molecular weight of 50,000-250,000, among which polyesters formed by polycondensation of monomers with both hydroxyl and carboxyl groups include but are not limited to direct polycondensation of lactic acid polylactic acid (L-PLA, D-PLA); polyesters formed by polycondensation of aliphatic dibasic acids and aliphatic dibasic alcohols, including but not limited to polybutylene succinate (PBS), poly Hexamethylene sebacate (PHS), polyethylene succinate (PES), polyhexylene succinate (PHS); polyesters formed by ring-opening polymerization of aliphatic lactones include But not limited to polylactic acid (PLA) formed by ring-opening polymerization of lactide, polycaprolactone (PCL) formed by ring-opening polymerization of caprolactone; or copolyesters including but not limited to polylactide ( PLGA).

The following examples will further illustrate the present invention:

Example 1: Preparation of electrospun fiber membrane: Add 9 grams of hexafluoroisopropanol to a beaker, add 1 g of polybutylene succinate (PBS) (molecular weight Mw=65000), stir at room temperature to make it Dissolving to obtain a spinning solution with a mass percentage concentration of 10 wt%. The prepared spinning solution is subjected to electrospinning to obtain an electrospun fiber membrane, and the prepared electrospun fiber membrane is desolventized in a vacuum oven for 24 hours.

Preparation of chitosan solution: dissolving glacial acetic acid in deionized water to prepare a concentration of 5 wt% acetic acid solution, weighing 1.5g of chitosan powder (deacetylation degree>90%, molecular weight 200000) was dissolved in 100g of the above-mentioned acetic acid solution A chitosan solution with a mass fraction of 1.5wt% was obtained.

A certain area of the electrospun fiber membrane was cut and dipped into the chitosan solution, soaked for 30 minutes, taken out and dried at room temperature to obtain the chitosan-loaded electrospun fiber membrane.

A glutaraldehyde solution with a mass fraction of 0.01% was prepared, and the chitosan-loaded electrospun fiber membrane prepared above was immersed in the glutaraldehyde solution for 30 minutes, taken out, and then dried at room temperature. The thrombin freeze-dried powder was prepared with deionized water to prepare a thrombin solution with an enzyme activity concentration of 2 U/mL, and the above-mentioned electrospun fiber membrane was soaked in the thrombin solution for 30 minutes, and then dried at room temperature after taking it out to obtain a Antibacterial-hemostatic material of woven fiber fabric structure.

Hemostatic performance: The hemostatic performance of the electrospun fibrous membrane was studied using a rabbit liver injury bleeding model. Compared with the market product gelatin sponge, the hemostatic time of the electrospun fibrous membrane was shorter than that of the gelatin sponge.

Antibacterial performance: The prepared electrospun fiber membrane has antibacterial ability, and the 2-h contact sterilization rate for E.coli (DH5α) is determined to reach 80% according to the following characterization method.

Antibacterial assay method:

LB medium: Add 1g NaCl, 1g tryptone, and 0.5g yeast extract to 100mL deionized water, stir and dissolve, then divide into 50mL Erlenmeyer flasks, 5mL per bottle, in high-pressure steam Sterilize in a sterilizer at 120°C for 15 minutes.

LB-agar medium (LB plate): add 1g NaCl, 1g tryptone, 0.5g yeast extract (yeast extract) to 100mL deionized water, add 2g agar (agar) after stirring and dissolving, Sterilize in a sterilizer at 120°C for 15 minutes. 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 1cm×1cm, sterilize them in 75% ethanol for 30min, and dry them in a clean bench. The sterilized and dried sample was placed on the LB plate to ensure that it was completely attached to the surface of the culture medium. Inoculate 10 μL of pre-cultured bacteria solution on each sample, incubate in a 37°C incubator for 2 hours, remove the sample, put it into a test tube that has been added with 1 mL of deionized water, and keep shaking for 5 minutes to completely remove the bacteria on the sample. Eluted to obtain a 100-fold diluted bacterial suspension. After the bacterial suspension 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 on an LB plate, 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:

Example 2: Change the molecular weight of polybutylene succinate in Example 1 to Mw=100000, the preparation method is the same as Example 1, and the hemostatic time of the electrospun fiber membrane is shorter than that of gelatin sponge. The 2h contact sterilization rate of E.coli(DH5α) reached 83%.

Embodiment 3: change polybutylene succinate into polycaprolactone in embodiment 1, use amount is 0.6g, preparation method is the same as embodiment 1, but change the thrombin solution concentration into 5U/mL, electric The hemostasis time of the spun fiber membrane is shorter than that of the gelatin sponge. The 2h contact sterilization rate of E.coli(DH5α) reached 72%.

Example 4: The polybutylene succinate in Example 1 was changed to polylactic acid, the usage amount was 0.6 g, the preparation method was the same as in Example 1, and the hemostasis time of the electrospun fiber membrane was shorter than that of gelatin sponge. The 2h contact sterilization rate of E.coli(DH5α) reached 68%.

Embodiment 5: Change the polybutylene succinate in embodiment 1 into polyhexamethylene sebacate of the same amount, and the solvent is changed into trifluoroacetic acid, and the preparation method is the same as in embodiment 1, the electrospun fiber membrane Hemostasis time is shorter than gelatin sponge. The 2h contact sterilization rate of E.coli(DH5α) reached 69%.

Embodiment 6: change the polybutylene succinate in embodiment 1 into the same amount of polylactide, chitosan (deacetylation degree is 80%, molecular weight is 300000), preparation concentration is 2wt% The other preparation methods are the same as in Example 1, and the hemostatic time of the electrospun fiber membrane is shorter than that of the gelatin sponge. The 2h contact sterilization rate of E.coli(DH5α) reached 90%.

Example 7: The fixed amount of thrombin in Example 5 was changed to 5 U/mL, the preparation method was the same as in Example 5, and the hemostatic time of the electrospun fiber membrane was shorter than that of the gelatin sponge. The 2h contact sterilization rate of E.coli(DH5α) reached 92%.

Example 8: The preparation method is the same as in Example 4, but the concentration of the glutaraldehyde solution is changed to 0.1%. Other preparation methods are the same as in Example 4, and the hemostatic time of the electrospun fiber membrane is shorter than that of the gelatin sponge. The 2h contact sterilization rate of E.coli(DH5α) reached 72%.

Example 9: Change the mass concentration of the solute in Example 1 to 15%, the preparation method is the same as in Example 1, and the hemostatic time of the electrospun fiber membrane is shorter than that of the gelatin sponge. The 2h contact sterilization rate of E.coli(DH5α) reached 82%.

Example 10: Change the 5wt% acetic acid solution in Example 6 to 1wt% hydrochloric acid aqueous solution, and other preparation methods are the same as in Example 6, and the hemostatic time of the electrospun fiber membrane is shorter than that of gelatin sponge. The 2h contact sterilization rate of E.coli(DH5α) reached 90%.

Comparative Example 1: Directly use medical gauze to stop the bleeding of the rabbit liver surface injury bleeding model, and the hemostatic time is much longer than that of the obtained electrospun fiber sample. It is ineffective for 2h contact sterilization of E.coli(DH5α).

Comparative Example 2: The gelatin sponge was used to stop the bleeding of the rabbit liver surface injury bleeding model, and the hemostatic time was longer than that of the obtained electrospun fiber sample. It is ineffective for 2h contact sterilization of E.coli(DH5α).

The present invention may be embodied in other specific forms without departing from the spirit or main characteristics of the invention. Therefore, no matter where

From a point of view, the above-mentioned embodiments of the present invention can only be considered as illustrations of the present invention rather than limiting the present invention.

Claims (10)

1. an antibacterial-hemostatic material with non-woven fiber fabric structure is characterized in that, this antibacterial-hemostatic material is a porous non-woven structure, containing aliphatic polyester fiber, chitosan and thrombin; aliphatic The diameter of polyester fiber is 100nm-1500nm, the porosity is 50%-90%, the static contact angle is 0°-130°, and the water absorption rate of the material is 2-20; The material is prepared by the following method: first, the aliphatic polyester is made into an aliphatic polyester fiber film by electrospinning, and then fully immersed in chitosan solution, so that the surface of the aliphatic polyester fiber is covered with nano Then the chitosan-loaded aliphatic polyester fiber membrane is completely immersed in the thrombin aqueous solution, and after drying, an antibacterial-hemostatic material with a non-woven fiber fabric structure is obtained. 2. a preparation method of the antibacterial-hemostatic material with non-woven fiber fabric structure as claimed in claim 1, is characterized in that, the method comprises the steps: 1) dissolving the aliphatic polyester in a solvent to obtain a spinning solution, and then using an electrospinning method to make the spinning solution into an aliphatic polyester electrospun fiber membrane; 2) fully immersing the dried aliphatic polyester electrospun fiber membrane in a chitosan solution, taking it out and drying at room temperature to obtain an aliphatic polyester electrospun fiber membrane loaded with chitosan; 3) Completely immerse the chitosan-loaded aliphatic polyester electrospun fiber membrane in the glutaraldehyde aqueous solution, take it out and dry it at room temperature, then completely immerse it in the thrombin aqueous solution, take it out and dry it at room temperature , that is, an antibacterial-hemostatic material with a non-woven fabric structure is obtained. 3. according to the preparation method of a kind of antibacterial-hemostatic material with non-woven fabric structure according to claim 2, it is characterized in that, the molecular weight 50000～500000 of chitosan described in step 2), deacetylation degree ≥80%, the chitosan solution is an acidic aqueous solution, and the mass percent concentration of the chitosan solution is 0.5wt%-15wt%. 4. A method for preparing an antibacterial-hemostatic material having a non-woven fabric structure according to claim 2, wherein the concentration of the aqueous solution of thrombin is 0.5U/mL˜10U/mL. 5. according to the preparation method of a kind of antibacterial-hemostatic material with non-woven fiber fabric structure according to claim 2, it is characterized in that, the mass percent concentration of described glutaraldehyde aqueous solution is 0.0001wt%～1wt% . 6. according to the preparation method of a kind of antibacterial-hemostatic material with non-woven fabric structure according to claim 2,3,4 or 5, it is characterized in that, described aliphatic polyester refers to having simultaneously Polyesters formed by polycondensation of hydroxyl and carboxyl monomers, or polyesters formed by polycondensation of aliphatic dibasic acids and aliphatic diols, or polyesters formed by ring-opening polymerization of aliphatic lactones or Copolyester, the molecular weight of aliphatic polyester is 50,000-250,000. 7. according to the preparation method of a kind of antibacterial-hemostatic material with non-woven fiber fabric structure according to claim 6, it is characterized in that, the polyester that the monomer with hydroxyl and carboxyl group forms through polycondensation is through Polylactic acid formed by direct polycondensation of lactic acid; the polyester formed by polycondensation of aliphatic dibasic acid and aliphatic dibasic alcohol is polybutylene succinate, polyhexamethylene sebacate, Polyethylene succinate or polyhexamethylene succinate; polyester formed by ring-opening polymerization of aliphatic lactone is polylactic acid formed by ring-opening polymerization of lactide; Polycaprolactone formed by ring polymerization; copolyester is polyethylene lactide. 8. according to claim 2,3,4 or 5 described a kind of preparation method with the antibacterial-hemostatic material of non-woven fiber fabric structure, it is characterized in that, step 1) is used for dissolving the solvent of aliphatic polyester as One or a mixture of hexafluoroisopropanol, tetrahydrofuran, N,N-dimethylformamide and trifluoroacetic acid. 9. according to the preparation method of a kind of antibacterial-hemostatic material with non-woven fabric structure according to claim 2,3,4 or 5, it is characterized in that, the mass percentage when forming spinning solution in step 1) The concentration is 5wt%-30wt%. 10. According to claim 2, 3, 4 or 5, a method for preparing an antibacterial-hemostatic material with non-woven fabric structure, it is characterized in that, the voltage in the electrospinning method is 10KV～30KV, and the spinning The wire distance is 10cm-20cm, the injection speed is 0.3mL/h-2mL/h, and the speed of receiving roll is 100rpm-500rpm.