TWI714373B - A composite fiber - Google Patents

Abstract

The present invention provides a composite fiber, which contains alginic acid fiber, a polymer material, an antibacterial agent, and a plastid containing growth factor genes. The present invention also provides a wound dressing, wherein the wound dressing comprises the aforementioned composite fiber. The composite fiber prepared by the present invention can release antibacterial agents and genes containing growth factors, in addition to reducing the colonization of microorganisms, and can secrete growth factors at the wound site by transfection, thereby promoting wound healing.

Description

A composite fiber

The present invention discloses a composite fiber made of alginic acid and polymer materials prepared by a composite electrospinning technology, which is characterized in that the composite fiber is mixed with an antibacterial agent and a plastid containing growth factor genes. The prepared fiber can not only reduce the growth of microorganisms, but also can secrete growth factors at the wound site through gene transfection, thereby promoting wound healing.

Traditional dressings are made of cotton or synthetic fibers, such as gauze, cotton sheets, etc. Its advantages are that it can quickly absorb wound exudate and the processing process is simple, but due to its high permeability, the wound is too dry and microorganisms are easy to pass through. Infection, and more importantly, will stick to the wound when removed, causing great pain when changing the dressing.

The common form of synthetic dressing is polyurethane (PU) as a material film. It is a waterproof dressing that can keep the wound moist and prevent bacteria from passing through. However, this dressing cannot absorb wound exudate and will cause cell tissue when torn off. Damaged.

These dressings can only provide passive protection and cannot promote wound regeneration, so they cannot be applied to chronic wounds such as those commonly seen in diabetic patients.

In addition, dressings with silver ions or nanosilver added are also used in medical devices to reduce the risk of infection. However, excessively high concentrations of silver are proven to be cytotoxic, which may lead to failure to achieve healing when applied to some chronic wounds. purpose.

At present, most of the products on the market are mainly passive or protective, and some emphasize antibacterial effects. However, none of them has the effect of promoting wound tissue regeneration.

In order to combine the advantages of wound dressings on the market mentioned above and overcome their shortcomings, it is necessary to develop a new type of multifunctional wound dressing.

Nanofiber scaffolds can mimic the structure of extracellular matrix. In addition, they are believed to increase cell attachment, migration, differentiation and proliferation. In addition, because nanofiber scaffolds have high surface area and porosity, they can make wounds It has better air permeability and prevents liquid accumulation in the wound site, so it has the ability to promote and increase wound healing. The nanofiber yarn prepared by electrospinning technology is regarded as the simplest and most cost-effective method.

The composite fiber of the present invention is produced by methods including electrospinning or electrospray.

The composition of electrospinning used in the field of tissue engineering can be divided into two categories: natural polymer and synthetic polymer, and each has its own advantages and disadvantages.

Natural polymers are obtained from plants and animals and have good biocompatibility and biodegradability, but their poor mechanical properties limit the development of natural polymers; synthetic polymers are synthesized artificially and have good mechanical properties, but degrade It is easy to cause cell death.

Electrospinning has the characteristics of high surface area to volume ratio, small pore size and high porosity, so it has great potential in many applications, especially wound dressings.

The slow healing of chronic wounds may cause many risks and inconveniences in life. Therefore, the present invention intends to develop a multifunctional wound dressing to promote tissue regeneration.

The composite fiber composition of the present invention includes alginate fibers, polymer materials, antibacterial agents, and plastids containing growth factor genes.

In one embodiment, the antibacterial agent is a metal ion, nanoparticle or its oxide, antibiotic, graphene, carbon nanotube, or a combination thereof.

In one embodiment, the antibacterial agent is silver ion, titanium dioxide, zinc oxide nanoparticle, copper oxide nanoparticle, ferroferric oxide nanoparticle, nanosilver or a combination thereof.

In one embodiment, the polymer material is biodegradable.

In one embodiment, the polymer material is polyester, polyamide, polycarbonate, polyurethane, or a combination thereof.

In one embodiment, the polyester is polylactic acid (Polylactide, PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), or polyglycolic acid (PLGA). Ester (polycaprolactone, PCL).

In one embodiment, the gene line of the growth factor has codes including platelet-derived growth factor, epidermal growth factor, keratinocyte growth factor, and fibroblasts. Fibroblast growth factor (Fibroblast growth factor), transforming growth factor β 1 (Transforming growth factor- β 1), vascular endothelial growth factor (Vascular endothelial growth factor), insulin-like growth factor (Insulin-like growth factor) or a combination thereof gene.

In one embodiment, the hydrophilic alginate fiber has high absorbency, can absorb wound exudate and provide a humid environment, and the polymer material can increase mechanical strength and promote cell adhesion.

The invention introduces the antibacterial agent into the polymer material so that it can continuously inhibit the growth of microorganisms.

In one embodiment, the qualitative system of the gene containing the growth factor of the present invention Coated in a non-viral vector.

In another embodiment, the non-viral carrier system liposome complex, cationic polymer, peptide or chitosan polymer.

In another embodiment, the complex formed by the non-viral vector and the plastid is a positively charged composite particle.

In one embodiment, the non-viral carrier system of the present invention is adsorbed on the alginic acid fiber, wherein the non-viral carrier includes the plastid containing the growth factor gene.

In another embodiment, the positively charged composite particles of the present invention are adsorbed on the alginate fibers through electrostatic force.

In one embodiment, the weight ratio of the alginic acid fiber to the polymer material of the present invention may range from 1:9 to 9:1.

In one embodiment, the weight ratio of the alginate fiber to the polymer material is 8:2.

In one embodiment, the present invention uses calcium salt to cross-link the alginate fibers with each other.

In one embodiment, the calcium salt is calcium carbonate, calcium phosphate, calcium oxalate, calcium chloride, calcium sulfate or calcium nitrate.

The alginic acid used in the present invention is easily soluble in water. In application, it will be cross-linked with calcium ions to produce an egg box structure. This problem can be solved, and then it can be released through electrospinning technology. Calcium ions promote blood clotting.

The invention can be applied to biomedical dressings. First, the composite fiber has high surface area and porous characteristics, which can make the wound have high air permeability. The composition contains antibacterial agents and plastids containing growth factor genes, and they are released together Calcium ions can promote blood clotting while reaching the wound Multifunctional repair, antibacterial and coagulation.

The present invention also provides a method for preparing composite fibers, wherein the method steps include:

Step (a) is to provide an alginic acid solution and a polymer material solution, and mix an alginic acid with a polyoxyethylene (PEO) or a polyvinyl alcohol (PVA) to prepare the alginic acid with a concentration of 1 to 10 wt % Of the alginic acid solution, preferably 2~8% by weight of the alginic acid solution; the polymer material is mixed with the polyoxyethylene (PEO) or the polyvinyl alcohol (PVA) to prepare the high Molecular material solution; step (b) is to provide a nanosilver solution, and form the nanosilver solution from the redox reaction of silver salt and reducing agent; step (c) is to mix the nanosilver solution and the polymer material solution, A silver-carrying polymer solution is obtained; step (d) is to prepare the composite fiber from the alginic acid solution and the silver-carrying polymer solution.

In one embodiment, the step (d) includes electrospinning technology or electrospray technology.

In one embodiment, the step (d) further includes syringes respectively set in two automatic sampling devices, with a sampling speed of 0.1 to 5.0 milliliters/hour (mL/h), a voltage of 12 to 24 kV and a voltage of 10 to With a collection distance of 25 cm, the spinning solution is sprayed out by electrostatic spinning technology.

In one embodiment, the method for preparing composite fibers may further include step (e) of adsorbing a positively charged composite particle formed by a non-viral carrier and a plastid to the composite fiber prepared in step (d) .

In one embodiment, the silver salt refers to a general term for ionic compounds formed by halogen or acid radicals and metallic silver, and is silver acetate, silver nitrite, silver nitrate, silver chloride or silver sulfate.

In one embodiment, the reducing agent is sodium borohydride, hydrazine hydrate, sodium citrate, or dimethylformamide.

In one embodiment, the nano-silver solution uses high-energy mechanical ball milling, evaporation condensation, photochemical reduction, liquid chemical reduction, electrochemical reduction, liquid chemical redox, microemulsion, or chemical precipitation. Prepared.

In one embodiment, the concentration of the nanosilver solution is 5 mM to 75 mM.

In one embodiment, the concentration of the nanosilver solution is 30 mM.

Figure 1 is a schematic diagram of composite fiber prepared by co-electrospinning.

Figure 2 is an SEM image of the appearance of the composite fiber.

Figure 3 is a TEM image of nanosilver in the composite fiber.

Figure 4 is a photograph of the antibacterial effect of different ratios of composite fibers on Staphylococcus epidermidis observed by the paper ingot diffusion method.

Figure 5 is a photograph of the antibacterial effect of different proportions of composite fibers on E. coli observed by the paper ingot diffusion method.

Figure 6 is an analysis diagram of the antibacterial rate of composite fibers.

Figure 7 is an analysis diagram of the antibacterial rate of composite fibers with different nanosilver concentrations.

Figure 8 is an analysis of cell survival rate of NIH 3T3 grown on composite fibers.

Figure 9 is an analysis of the survival rate of composite fibers grown on NIH 3T3 in different concentrations of nanosilver for 1, 5 days.

Figure 10 is a fluorescence photo of in situ transfection with composite fiber ratio of A8P2.

Figure 11 is a fluorescence photo of in situ transfection with composite fiber ratio of A2P8.

Figure 12 is a comparison diagram of coagulation tests of composite fibers.

Figure 13 is a comparison diagram of wound appearance of different composite fibers on 7 and 11 days.

Figure 14 shows the comparison of wound healing rates of different composite fibers on the 7th and 11th days.

Figure 15 shows H&E staining images of tissue sections of different composite fibers on the 7th and 11th days.

Figure 16 is a schematic diagram of the function of the composite fiber.

Preparation of composite fiber

Alginic acid/polyethylene oxide ((polyethylene oxide, PEO) spinning solution preparation

Prepared into 5g spinning solution, add 3.33g of alginic acid stock solution, 1.0g of PEO stock solution and 0.525g of cosolvent (dimethyl sulfide)/surfactant (Triton X-100), and supplement 0.145g of water was mixed to make the final concentration of alginic acid 4wt%, PEO 2wt%, dimethyl sulfite 10% and Triton X-100 0.5%, and this solution was heated and stirred (50℃, 60rpm ) After two days, remove air bubbles by centrifugation.

PCL/PEO spinning solution preparation

Prepare 4g spinning solution, weigh 1.8g each of PCL and PEO stock solution, and add 0.4g Dimethylformamide (DMF) to make the final concentration of PCL 4.5wt% and PEO 3.6wt%, this solution was heated and stirred (40°C, 60rpm) for one day.

Configure 30mM Ag PCL/PEO spinning solution

Weigh 25.48mg silver nitrate into the sample bottle, add 0.5ml DMF and stir at 60rpm for 5min at room temperature, add 0.4ml silver-containing DMF solution dropwise to 3.6g PCL/PEO solution, and finally stir at 40℃, 60rpm Heat for one day to complete the preparation.

The composite fiber of alginic acid spinning solution and Ag PCL/PEO spinning solution was synthesized by co-electrospinning (Figure 1).

The present invention introduces nanosilver into polycaprolactone and then mixes it with alginic acid to form The composite fiber is formed (Figure 2), and it is confirmed that the composite fiber is indeed a nano-network structure and that the polycaprolactone has silver nanoparticles (Figure 3).

In order to increase the efficacy of wound healing, the present invention adds platelet-derived growth factor B (PDGF B) to the composite fiber, wherein PDGF B is a chemical attractant for neutrophils and can induce fibroblasts Proliferation and differentiation, and then promote wound repair. In the preparation method, the positively charged composite particles formed by the cationic polymer and the plastid with PDGF B are adsorbed to the negatively charged alginic acid fibers in the composite fiber through electrostatic force.

Antibacterial experiment

The present invention introduces the antibacterial effect of nanosilver into the composite fiber, and the weight ratio of alginic acid/polycaprolactone is 8: 2 (A8P2), 6: 4 (A6P4), 4: 6 (A4P6) and 2: 8 ( A2P8) ratio of composite fiber made of antibacterial experiments, found that Staphylococcus epidermidis (picture 4) and E. coli (picture 5) can inhibit the growth, the effect increases with the proportion of polycaprolactone, only 20% Polycaprolactone (A8P2) can still successfully achieve antibacterial effect, while pure alginic acid (pure A) fiber has no antibacterial effect.

In the present invention, the weight ratio of alginic acid/polycaprolactone is 8:2 (A8P2), 6:4 (A6P4), 4:6 (A4P6) and 2:8 (A2P8) composite fibers to test E. coli and epidermal grapes respectively. Compared with pure alginic acid, after 12 hours, even only 20% of polycaprolactone (A8P2) can inhibit E. coli and Staphylococcus epidermidis at 83% and 71% respectively. % Inhibition rate (Figure 6).

In the present invention, composite fibers with nanosilver concentrations of 0 mM, 10 mM, 30 mM and 50 mM were tested for the inhibition rates of Escherichia coli and Staphylococcus epidermidis. After 11.5 hours, the inhibition rates of 30 mM and 50 mM composite fibers to E. coli were respectively Up to 83% and 95%, Staphylococcus epidermidis The bacteriostatic rates of the bacteriostasis were 71% and 73% (Figure 7), both of which exceeded 70% of the bacteriostatic effect.

Cell viability test (MTT assay)

The addition of nanosilver may cause cytotoxicity. Therefore, the cell viability test with MTT showed that the higher the proportion of polycaprolactone, the more significant the toxicity of nanosilver, but A6P4 and A8P2 still retain more than 60% biocompatibility (Figure 8).

After using NIH 3T3 cells to culture on the composite fiber for 1 day and 5 days, the cell survival rate was analyzed by MTT, and it was found from the results on day 5 (Figure 9) that the 50mM composite fiber was very toxic to cells. However, there is no significant difference between 10mM and 30mM composite fibers.

Although 50 mM composite fiber has the best antibacterial effect, this concentration is too toxic to cells, so it is not suitable as a wound dressing.

On the other hand, the gene containing green fluorescent protein and platelet-derived growth factor is coated with a positively charged non-viral vector to form positively charged composite particles, which are adsorbed on composite fibers for in situ transfection .

Because alginic acid is negatively charged, it can promote the adsorption of positively charged composite particles. The results show that the higher the ratio of alginic acid, the better the transfection effect (Figure 10, Figure 11).

The results prove that the present invention can control the fiber composition ratio, thereby controlling the composite fiber to have antibacterial and gene delivery capabilities at the same time, and avoid the side effects of cytotoxicity caused by the antibacterial agent nanosilver. The ratio of A8P2 is the best in this example. Overall performance.

Coagulation test

Slow coagulation may hinder wound healing and increase the risk of bacterial infection. Therefore, to test the coagulation function of the composite fiber, first add 100μl of human whole blood containing anticoagulant to the composite fiber and place it at room temperature for 5, 10 , 20 minutes later, the coagulation rate was tested by absorbance spectrum (Figure 12).

Since the cross-linked composite fiber can release calcium ions, its coagulation rate is significantly higher than that of ordinary gauze and uncross-linked fiber.

Wound healing test

Make 2 wounds with a diameter of 5mm on the back of C57BL/6 mice, place the dressing on the wound, and record the size of the wound on 7 and 11 days. The appearance of the wound (Figure 13) and wound healing rate (Figure 14) can be It can be seen that in the control group (which does not provide composite fibers at all), even by the 11th day, the wound has not healed to 60%. In contrast, the composite fiber with PDGF B gene was 77% and 95% respectively on 7 and 11 days. In addition, the wound is almost invisible after 11 days from the appearance of the wound. After staining with the H&E staining method (Figure 15), it is found that the epidermis has been formed in this group at 7 days, confirming that the composite fiber with PDGF B gene can indeed be In situ gene transfection is performed on the wound site so that cells in the wound site secrete PDGF B to promote wound healing.

In summary, the composite fiber of the present invention, after the above-mentioned experimental tests, represents that the composite fiber has the functions of high mechanical strength, tissue hemostasis, absorbing wound exudate, antibacterial and promoting regeneration of injured tissue (Figure 16), and Adjusting the difference in composition ratios allows the composite fiber to perform better.

Although the present invention has been disclosed using the above-mentioned preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with the art without departing from the spirit and scope of the present invention may make various changes and modifications relative to the above-mentioned embodiments. The technical scope of the invention is protected. Therefore, the scope of protection of the invention shall be subject to the scope of the attached patent application.

Claims (15)

A composite fiber, wherein the composite fiber comprises an alginate fiber, a polymer material, an antibacterial agent and at least one growth factor gene plastid. The composite fiber described in item 1 of the scope of patent application, wherein the antibacterial agent is a metal ion, nanoparticle or its oxide, antibiotic, graphene, carbon nanotube, or a combination thereof. The composite fiber according to the first item of the scope of patent application, wherein the polymer material is polyester, polyamide, polycarbonate, polyurethane, or a combination thereof. The composite fiber described in item 1 of the scope of patent application, wherein the gene line of the growth factor has codes including platelet-derived growth factor (Platelet-derived growth factor), epidermal growth factor (Epidermal growth factor), keratinocyte growth factor (Keratinocyte growth factor), Fibroblast growth factor, Transforming growth factor β1 (Transforming growth factor-β1), Vascular endothelial growth factor, Insulin-like growth factor ) Or a combination of genes. In the composite fiber described in item 1 of the scope of patent application, the weight ratio of the alginate fiber and the polymer material ranges from 1:9 to 9:1. The composite fiber described in item 5 of the scope of patent application, wherein the weight ratio of the alginic acid fiber and the polymer material is 8:2. The composite fiber described in item 2 of the scope of patent application, wherein the antibacterial agent is a nano silver. The composite fiber described in item 1 of the scope of patent application, wherein the alginic acid fiber is cross-linked with each other by a calcium salt. The composite fiber described in item 8 of the scope of patent application, wherein the calcium salt is calcium carbonate, calcium phosphate, calcium oxalate, calcium chloride, calcium sulfate or calcium nitrate. The composite fiber described in item 1 of the scope of patent application, wherein the qualitative system containing the gene of the growth factor is coated with a non-viral vector. The composite fiber described in item 10 of the scope of patent application, wherein the non-viral carrier system liposome complex, cationic polymer, peptide or chitosan polymer. A method for preparing composite fibers, wherein the method steps include: step (a) is to provide an alginic acid solution and a polymer material solution, taking an alginic acid and a polyoxyethylene (PEO) or a polyvinyl alcohol (PVA) ) Mixing to prepare a solution with the alginic acid concentration of 1 to 10 wt%; taking the polymer material and mixing the polyoxyethylene (PEO) or the polyvinyl alcohol (PVA) to prepare the polymer material solution; step (b) To provide a nanosilver solution, form the nanosilver solution from a silver salt and a reducing agent through an oxidation-reduction reaction; step (c) is to mix the nanosilver solution and the polymer material solution to obtain a Silver-carrying polymer solution; step (d) is to make the alginic acid solution and the silver-carrying polymer solution into the composite fiber; and step (e) is to combine a non-viral carrier and a plastid to form a positive charge The composite particles are adsorbed on the composite fiber produced in step (d). A method for preparing composite fibers as described in item 12 of the scope of patent application, wherein the concentration of the nanosilver solution ranges from 5 mM to 75 mM. The method for preparing composite fibers as described in item 14 of the scope of patent application, wherein the concentration of the nanosilver is 30 mM. The method for preparing composite fibers as described in item 12 of the scope of the patent application, wherein the reducing agent is sodium borohydride, hydrazine hydrate, sodium citrate or dimethylformamide.

Images