COMPOSITE FIBER

Abstract

The present invention provides a composite fiber which comprises an alginate fiber, a polymer material, an antibacterial agent, and a plasmid encoding growth factor-gene. The present invention also provides a wound dressing, wherein the wound dressing comprises a composite fiber as described above. The composite fibers prepared according to the present invention are capable of releasing the antibacterial agent and the growth factor gene, not only to reduce microorganism growth, but also to secrete growth factors in a wound site through transfection, thereby promoting wound healing.

Claims

1. A composite fiber, wherein the composite fiber comprises an alginate fiber, a polymer material, an antibacterial agent and at least one plasmid encoding growth factor-gene.

2. The composite fiber of claim 1, wherein the antimicrobial agent is a metal ion, nanoparticle, or an oxide thereof, an antibiotic, graphene or carbon nanotubes or a combination thereof.

3. The composite fiber of claim 1, wherein the polymer material comprises polyester, polyamide, polycarbonate, polyurethane, or a combination thereof.

4. The composite fiber of claim 1, wherein the growth factor-gene is a gene encodes a platelet-derived growth factor, an epidermal growth factor, a keratinocyte growth factor, a fibroblast growth factor, a transforming growth factor-β1, a vascular endothelial growth factor, an insulin-like growth factor growth factor or a combination thereof.

5. The composite fiber of claim 1, wherein the weight ratio of the alginate fiber and the polymer material ranges from 1:9 to 9:1.

6. The composite fiber of claim 5, wherein the weight ratio of the alginate fiber and the polymer material is 8:2.

7. The composite fiber of claim 2, wherein the antibacterial agent is nano-silver.

8. The composite fiber of claim 1, wherein the alginate fiber crosslink by using a calcium salt.

9. The composite fiber of claim 8, wherein the calcium salt is calcium carbonate, calcium phosphate, calcium oxalate, calcium chloride, calcium sulfate or calcium nitrate.

10. The composite fiber of claim 1, wherein the plasmid encoding growth factor-gene is encapsulated by a non-viral vector.

11. The composite fiber of claim 10, wherein the non-viral vector comprises a liposome complex, a cationic polymer, a peptide or a chitosan polymer.

12. A method for producing a composite fiber, wherein the method step comprises: step (a) providing an alginate solution and a polymer material solution, wherein alginate and polyoxyethylene (PEO) or polyvinyl alcohol (PVA) are mixed to obtain a solution having a concentration of 1 to 10 wt % of alginate, preferably a alginate solution of 2 to 8 wt %; polymer material and polyoxyethylene (PEO) or polyvinyl alcohol (PVA) are mixed to obtain a polymer material solution; step (b) providing a nano-silver solution, wherein the nano-silver solution is formed through a redox reaction of a silver salt and a reducing agent; step (c) mixing the nano-silver solution with wherein the polymer material solution to obtain a silver-loaded polymer solution; and step (d) producing the composite fiber from the alginate solution and the silver-loaded polymer solution.

13. The method for producing a composite fiber of claim 12, which further comprises a step (e) of adsorbing positively charged complexes formed by combining a non-viral vector and a plasmid onto the composite fiber produced in step (d).

14. The method for producing a composite fiber of claim 12, wherein the concentration of the nano-silver solution ranges from 5 mM to 75 mM.

15. The method for producing a composite fiber of claim 14, wherein the concentration of the nano-silver solution is 30 mM.

16. The method for producing a composite fiber of claim 12, wherein the reducing agent comprises sodium borohydride, hydrazine hydrate, sodium citrate or dimethylformamide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is a schematic drawing showing the preparation of a composite fiber by a co-electrospinning technology.

[0045] FIG. 2 is an SEM image showing the appearance of the composite fibers.

[0046] FIG. 3 is a TEM image showing the nano-silver in a composite fiber.

[0047] FIG. 4 is a photograph showing the antibacterial effect of the composite fibers at different proportions against Staphylococcus epidermidis by using a disk diffusion method.

[0048] FIG. 5 is a photograph showing the antibacterial effect of the composite fibers at different proportions against Escherichia coli by using a paper ingot diffusion method.

[0049] FIG. 6 is a graph showing the analysis of the bactericidal rates of the composite fibers at different proportions.

[0050] FIG. 7 is a graph showing the analysis of the bactericidal rates of the composite fibers having different concentrations of nano-silver.

[0051] FIG. 8 is a graph showing the analysis of the survival rates of NIH 3T3 cells cultured in the composite fibers at different proportions.

[0052] FIG. 9 is a graph showing the analysis of the survival rates of NIH 3T3 cells cultured in the composite fibers having different concentrations of nano-silver after 1 and 5 days.

[0053] FIG. 10 is a fluorescence photograph showing in situ transfection of cells in the composite fibers at a ratio of A8P2.

[0054] FIG. 11 is a fluorescence photograph showing in situ transfection of cells in the composite fibers at a ratio of A2P8.

[0055] FIG. 12 is a graph comparing the coagulation rates of the composite fibers.

[0056] FIG. 13 is a graph comparing the appearances of wounds treated with different composite fibers on days 7 and 11.

[0057] FIG. 14 is a graph comparing the wound healing rates treated with different composite fibers on day 7 and day 11.

[0058] FIG. 15 showing H&E staining images of sections of wound tissues treated with different composite fibers on day 7 and day 11.

[0059] FIG. 16 is a schematic diagram showing the multifunctions of the composite fiber.

EXAMPLES

[0060] Production of Composite Fibers

[0061] Preparation of Alginate/Polyethylene Oxide (PEO) Spinning Solution

[0062] A 5 g of spinning solution was prepared by mixing 3.33 g of alginate stock solution, 1.0 g of PEO stock solution and 0.525 g of co-solvent (dimethyl sulfoxide)/surfactant (Triton X-100), and adding 0.145 g of water, so that the final concentration of alginate was 4 wt %, PEO was 2 wt %, dimethyl sulfoxide was 10%, and Triton X-100 was 0.5%, and the solution was heated and stirred (at 50° C., 60 rpm) for 2 days, the bubbles were removed by centrifugation.

[0063] Preparation of PCL/PEO Solution

[0064] 4 g of solution was prepared from 1.8 g of polycaprolactone (PCL) stock solution and 1.8 g of PEO stock solution, and then 0.4 g of dimethylformamide (DMF) was added to obtain a solution, of which the final concentration of PCL was 4.5 wt %, PEO was 3.6 wt %, then the solution was heated and stirred (40° C., 60 rpm) for one day.

[0065] Preparation of 30 mM of Ag PCL/PEO Spinning Solution

[0066] 25.48 mg of silver nitrate was added to 0.5 ml of dimethylformamide (DMF) and stirred at 60 rpm for 5 min at room temperature, then 0.4 ml of silver-containing DMF solution was added dropwise to 3.6 g of PCL/PEO solution, the solution was finally stirred and heated at 40° C., 60 rpm for one day to complete the preparation.

[0067] The composite fiber of alginate spinning solution and Ag PCL/PEO spinning solution was synthesized by co-electrospinning (FIG. 1).

[0068] In the present invention, nano-silver was introduced into PCL, and then co-electrospun with alginate to form the composite fibers (FIG. 2). It was confirmed that the composite fibers were indeed a nano-network structure and the PCL had nano-silver (FIG. 3).

[0069] In order to increase the effect of wound healing, the platelet-derived growth factor B (PDGF B) was added to the composite fibers in the present invention, wherein PDGF B was a chemoattractant of neutrophils and capable of inducing the proliferation and differentiation of fibroblasts, which in turn promoted wound repair.

[0070] In the manufacturing method, plasmid DNA encoding PDGF B-gene was encapsulated by cationic polymer to form positively charged complex, which was adsorbed onto the negatively-charged alginate fiber in the composite fiber.

[0071] Antibacterial Experiments

[0072] In the present invention, nano-silver was introduced into the PCL fibers, and the composite fibers produced from alginate/PCL at a weight ratio of 8:2 (A8P2), 6:4 (A6P4), 4:6 (A4P6), and 2:8 (A2P8) were subjected to antibacterial experiments. It was found that the growth of Staphylococcus epidermidis (FIG. 4) and Escherichia coli (FIG. 5) were inhibited, and the effect increased with an increase in the proportion of PCL, and the inhibitory effect was effectively achieved even with only 20% of PCL (A8P2), whereas pure alginate (pure A) fiber showed no antibacterial effect.

[0073] In the present invention, the composite fibers produced from alginate/PCL at a weight ratio of 8:2 (A8P2), 6:4 (A6P4), 4:6 (A4P6), and 2:8 (A2P8) were subjected to tests for bactericidal rate of Staphylococcus epidermidis and Escherichia coli, and comparisons to pure alginate were also conducted. Even with only 20% of PCL (A8P2), a bactericidal rate of 83% of Staphylococcus epidermidis and 71% of Escherichia coli were able to be achieved after 12 hours (FIG. 6).

[0074] In the present invention, the composite fibers having a concentration of 0 mM, 10 mM, 30 mM, and 50 mM of nano-sliver were subjected to evaluate their bactericidal rate against Escherichia coli and Staphylococcus epidermidis. After 11.5 hours, the Escherichia coli bactericidal rates of the composite fibers having 30 mM and 50 mM of nanosilver were 83% and 95%, respectively, and the Staphylococcus epidermidis bactericidal rate were 71% and 73%, respectively (FIG. 7), both of which were over 70%.

[0075] Cell Survival Rate Test (MTT Assay)

[0076] Since the release of nano-silver from composite fibers might cause cytotoxicity, the cell survival rate test was performed using the MTT assay. It was found that the higher the proportion of PCL, the more significant the toxicity of nano-silver, however, A6P4 and A8P2 were able to maintain more than 60% of cell survival rates (FIG. 8).

[0077] NIH 3T3 cells were cultured on the composite fibers for 1 and 5 days, and the cell survival rate was analyzed by MTT. Compared to the control group on day 5 (FIG. 9), it was found that the cytotoxicity of composite fibers containing 50 mM of nano-silver was very significant, but no significant difference was found for composite fibers containing 10 mM and 30 mM of nano-silver.

[0078] Although composite fibers containing 50 mM of nano-silver had the best antibacterial effect, the cytotoxicity of this concentration was too high to be suitable for wound dressing.

[0079] On the other hand, plasmid DNA containing genes of green fluorescent protein and PDGF B was encapsulated by positively charged non-viral vector to form a positively charged complex, and adsorbed onto the composite fibers for in situ transfection.

[0080] Since alginate was negatively charged, it was able to promote the adsorption of positively charged complexes. The results showed that the higher the proportion of alginate, the better the transfection effect (FIGS. 10 and 11).

[0081] The results confirmed that the present invention was able to regulate the composition ratio of the fiber, thereby controlling the composite fiber to have both antibacterial and gene delivery capabilities, avoiding the side effect of cytotoxicity caused by the antibacterial nano-silver. The ratio of A8P2 was the one that had the best overall performance in this embodiment.

[0082] Blood Coagulation Test

[0083] Since slow coagulation might hinder wound healing and increase the risk of infection, blood coagulation function of the composite fiber was tested. 100 μl of human whole blood containing anticoagulants was first added to the composite fibers, placed at room temperature for 5, 10, and 20 minutes, and then the blood coagulation rate was measured spectrometrically (FIG. 12).

[0084] Because the crosslinked composite fiber was able to release calcium ions, the coagulation rate was significantly higher than that of gauze and uncrosslinked composite fibers.

[0085] Wound Healing Test

[0086] Two 5 mm-diameter wounds were created on the back of C57BL/6 mice, the wound dressings were placed on the wounds, the wound sizes were recorded on day 7 and day 11, respectively. Based on the wound appearance (FIG. 13) and wound healing rate (FIG. 14), it was found that the wound healing of the control group (wounds without dressing coverage), even after 11 days, was less than 60%. In contrast, the PDGF gene-loaded composite fibers caused wound healing of 77% and 95% on day 7 and day 11, respectively, and the wound was almost invisible after 11 days. After being stained by H & E staining (FIG. 15)), it was found that the epidermis had formed in the PDGF gene-loaded composite fiber group on day 7, suggesting that PDGF B gene was able to deliver to the wound site, so that the transfected cells secreted PDGF B to promote wound healing.

[0087] In sum, the present invention, after the above-described tests, showed that the composite fiber had high mechanical strength, and the functions of hemostasis acceleration, wound exudate absorption, bacteria inhibition and promotion of wound tissue regeneration (FIG. 16), and the composition ratio of the components was adjustable to make the composite fiber to perform even better.

[0088] Although the present invention has been disclosed using the above-mentioned preferred embodiments, it is not intended to limit the present invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the appended claims.