Preparation and application of antibacterial and anti-inflammatory fibrous membrane skin dressing

Abstract

The invention belongs to the field of medical biological materials, and particularly relates to preparation and application of an antibacterial and anti-inflammatory fibrous membrane skin dressing. The invention provides a fibrous membrane skin dressing, the fibrous membrane skin dressing is fiber coated with tannic acid TA and polylysine PLys, the polylysine PLys is grafted to carboxyl of the tannic acid TA through EDC / NHS mediated amide condensation reaction, and the selected bioactive polylysine cooperates with the tannic acid to play a stronger antibacterial effect. Meanwhile, the fibrous membrane skin dressing disclosed by the invention is well applied to skin dressings for preventing bacterial infection of wounds, reducing inflammatory response of the wounds and promoting healing of the wounds.

Description

Technical Field

[0001] The invention belongs to the field of medical biomaterials, and particularly relates to the preparation and application of an antibacterial and anti-inflammatory fiber membrane skin dressing. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Wound healing often faces excessive inflammatory response leading to immune dysfunction, and is often accompanied by the risk of bacterial infection, which causes the wound to persist and turn into chronic wounds or pathological scars. With the aging population, the increasing number of diabetes and obesity, the management and treatment of skin wounds face huge challenges in clinical practice. How to reduce inflammation, prevent infection, and accelerate wound healing are issues that need to be considered in the current development of wound dressings, and are also key factors to be considered in the clinical treatment of chronic wounds. Traditional dressings such as oil gauze and cotton cloth have played a historic role in wound treatment. However, the single function leads to limited treatment effects, and continuous dressing changes increase the burden on patients and medical care. Therefore, in addition to protecting wounds and reducing external stimuli, wound dressings should also have good exudate absorption capacity, breathability and infection prevention capabilities, and comfort.

[0004] The fibers prepared by electrospinning have the structural characteristics of the extracellular matrix of natural skin tissue, and are considered to be an important type of wound dressing construction material that can regulate cell behavior, promote skin repair and functional regeneration. They have the characteristics of easy processing, controllable performance, and a wide range of material sources. In addition, the fiber material constructed by a single polymer has also been proven to be highly breathable and prevent bacteria from penetrating the membrane due to its ultrafine porous fiber structure size, which plays an important role in protecting the wound from external bacterial invasion and reducing wound infection. How to further improve the biocompatibility of fiber dressings, enhance their bactericidal ability after skin colonization, regulate wound inflammatory response, and promote skin tissue regeneration are currently problems to be solved in the construction of wound prevention skin dressings (especially chronic wounds). Based on this, the present invention uses a tannic acid (TA) coating to graft the antibacterial molecule cationic polymer polylysine onto nanofibers, providing a method for preparing a dressing with antibacterial, anti-inflammatory and skin healing properties for wound healing. The specific functional analysis is as follows:

[0005] In terms of antibacterial properties, the nanostructure size of the fiber membrane has a physical isolation effect, providing the first layer of protection for skin healing; TA is a natural plant polyphenol, with abundant -OH groups on the benzene ring that can form hydrogen bonds with body tissues, produce adhesion to achieve rapid hemostasis and close wounds, thereby reducing the risk of bacterial infection; TA is also a dopamine derivative, and its abundant pyrogallol groups can adsorb bacterial cell walls, physically kill bacteria and show a certain antibacterial effect, and TA can also bind to cell receptors to inhibit microbial proliferation; on the other hand, polyamino acids (PAAs) are an important class of biopolymers. Among all types of PAAs, poly(ε-lysine) (PLys) is a cationic linear polymer composed of 25 to 30 lysines covalently linked by amino and carboxyl groups, which has beneficial antibacterial effects, can inhibit microbial respiration, lead to anabolism, destroy dynamic membrane structures to produce cell autolysis, and bind to ribosomes to inhibit the synthesis of biomacromolecules such as microbial proteins and enzymes.

[0006] In terms of anti-inflammatory, TA can activate the ERK1 / 2 pathway to downregulate inflammatory cytokines such as IL-1 and IL-6 to regulate the inflammatory microenvironment, and accelerate wound healing, re-epithelialization and hair follicle growth by upregulating the expression of growth factors such as basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β) and VEGF. It is reported that TA can also induce human macrophages to produce nitric oxide in a concentration-dependent manner, showing effective anti-inflammatory activity. In addition, PLys has rich amide groups, which can effectively neutralize the acidic products accumulated by cell metabolism in the wound, thereby reducing the inflammatory response. The above provides other advantages for promoting skin wound healing. The above provides other advantages for promoting skin wound healing.

[0007] Based on the above advantages, the present invention proposes to introduce PLys as a functional coating for fiber dressings by relying on the rich -COOH groups on the TA benzene ring, which not only protects the -OH on the TA benzene ring from being consumed, maintains efficient adhesion and bactericidal ability, but also combines the antibacterial and anti-inflammatory effects of the physical structure of the fiber membrane, TA and PLys, to achieve the antibacterial and anti-inflammatory effects of skin dressings. Therefore, the skin dressing preparation method of the present invention is an efficient and quick way to construct a skin dressing that prevents bacterial infection of wounds and promotes wound healing. Summary of the invention

[0008] The present invention relies on the rich -COOH groups on the TA benzene ring to introduce PLys as the functional coating of the fiber dressing, grafts the bioactive molecule polylysine on the basis of the tannic acid coating, and cooperates with tannic acid to exert stronger anti-inflammatory and antibacterial effects, thereby preparing a fiber membrane skin dressing with a tannic acid and polylysine coating.

[0009] In one aspect, the present invention provides a fiber membrane skin dressing, which is a fiber with surface functional modifications of TA and PLys, wherein the PLys is grafted onto the carboxyl group of TA through an amide condensation reaction mediated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS).

[0010] Furthermore, a method for preparing a fiber membrane skin dressing is provided, and the specific steps are as follows:

[0011] (1) Preparation of polylactic acid-caprolactone (PLCL) nanofiber membrane: PLCL was dissolved in hexafluoroisopropanol and stirred overnight to prepare a 5% spinning solution, and electrospinning was used to spin the nanofiber membrane the next day;

[0012] (2) Preparation of TA surface-coated nanofiber membrane (TA@PLCL): Prepare 8 mg / mL TA solution (solvent: Tris-HCl, pH 8.5, 10 mM), immerse the membrane in the solution and shake for 24 h, and wash with ultrapure water 2-3 times;

[0013] (3) Preparation of nanofiber membranes grafted with PLys on the surface (PLys@TA@PLCL): Prepare a 2 mg / mL PLys solution and add the corresponding EDC (57%) and NHS (3 mg / ml), soak the TA-modified fiber membrane in the solution and shake for 6 h, and wash it with ultrapure water 2-3 times to obtain functionalized fibers.

[0014] Furthermore, the concentrations of TA and PLys in the fiber membrane skin dressing are 1 mg / mL to 25 mg / mL and 1 mg / mL to 25 mg / mL, respectively.

[0015] Furthermore, the electrospinning process parameters in the preparation step (1) of the fiber membrane skin dressing are: spinning rate 20uL / min, applied voltage 6-7kV, using a flat plate to receive the jet monofilament, receiving distance 15cm, receiving device winding rate 10rpm, ambient temperature 0-1000℃, ambient relative humidity 0-100%.

[0016] Furthermore, the reaction in step (3) of preparing the fiber membrane skin dressing is as follows: the reaction time is 1-24 hours; the reaction temperature is 0-100°C.

[0017] Another aspect of the present invention provides an application of the fiber membrane skin dressing in the construction of a skin dressing for preventing bacterial infection of wounds.

[0018] Another aspect of the present invention provides a use of the fiber membrane skin dressing in a skin dressing for reducing inflammatory response of wounds.

[0019] Another aspect of the present invention provides a use of the fiber membrane skin dressing in a skin dressing for promoting wound healing.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention not only imparts antibacterial and anti-inflammatory effects to the fiber membrane by introducing the active ingredient TA, but also provides a bridge for PLys grafting. The introduction of PLys not only effectively enhances the antibacterial ability of the dressing, but also significantly improves the anti-inflammatory and skin regeneration-inducing functions of the fiber after coating modification. The composite wound dressing prepared by combining TA, PLys and fiber membrane provided by the present invention has a simple process and high efficiency, and can effectively solve the defects of the skin dressing's antibacterial, anti-inflammatory, and skin regeneration-inducing functions, and is expected to promote the research and development of skin dressings for preventing bacterial infection of wounds and their transformation into clinical applications;

[0022] (2) The present invention uses PLys grafting and TA (PLys@TA) coating methods, which are convenient, fast, economical, and can effectively enhance the antibacterial and anti-inflammatory effects of the dressing. Moreover, with the addition of PLys, the dressing can have the functional activity of promoting skin healing.

[0023] (3) The PLys@TA coating method of the present invention can enhance the application efficacy of biomaterials in the field of skin dressings, such as enhancing the biological activity of biologically inert synthetic polymers in preventing bacterial infection and repairing chronic wounds;

[0024] (4) The method for preparing functionalized fibers that can be used for skin dressing construction of the present invention can effectively improve the potential application prospects of fibers with biologically inert synthetic polymer structures in the field of skin wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 : Scanning electron microscopy image of PLCL fibers;

[0027] Figure 2 : Scanning electron microscopy image of pure TA modified PLCL fiber;

[0028] Figure 3 : Scanning electron microscopy image of PLys@TA modified PLCL fibers;

[0029] Figure 4 : X-ray spectra XPS of PLCL, TA-modified PLCL, and PLys@TA-modified PLCL fibers;

[0030] Figure 5 : Staphylococcus aureus (S. aureus) was cultured on PLCL, TA-modified PLCL, and PLys@TA-modified PLCL fibers for 2 hours to isolate bacteria;

[0031] Figure 6 : Antibacterial effect of Staphylococcus aureus (S. aureus) after being cultured on PLCL, TA-modified PLCL, and PLys@TA-modified PLCL fibers for 12 hours;

[0032] Figure 7 : General image of PLCL, TA-modified PLCL and PLys@TA-modified PLCL fibers promoting wound healing in diabetic mice;

[0033] Figure 8 : Histological staining analysis of PLCL, TA-modified PLCL and PLys@TA-modified PLCL fibers promoting wound healing in diabetic mice;

[0034] Fig. 9 : Immunofluorescence analysis of the in vivo anti-inflammatory effects of PLCL, TA-modified PLCL, and PLys@TA-modified PLCL fibers. DETAILED DESCRIPTION

[0035] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with embodiment, but the content of the present invention is not limited to the following embodiment. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0036] Example 1. Preparation of PLCL fibers

[0037] 1.1 Preparation of polylactic acid-caprolactone (PLCL) fiber

[0038] At room temperature, 0.5 g of PLCL (copolymer ratio of lactic acid and caprolactone is 50:50, viscosity is 2.9 dl / g) was dissolved in 10 mL of hexafluoroisopropanol, and stirred for 12 hours to obtain a spinning solution with a mass fraction of 5%; electrospinning was carried out at room temperature and humidity of 40-60%, with an applied voltage of 6-7 kV, a receiving distance of 15 cm, a receiving speed of 10 rpm, and an injection rate of 20 uL / min. The scanning electron microscope image of the prepared pure PLCL fiber is shown in Figure 2. Figure 1 As shown, the fiber diameter is 1.67 ± 0.17 μm.

[0039] 1.2 Preparation of TA-modified PLCL fibers

[0040] 80 mg of TA was dissolved in 10 mL of Tris buffer with a concentration of 10 mM and a pH of 8.5, and the TA solution was prepared by stirring thoroughly. 1 cm × 1 cm PLCL fiber dressing was immersed in the TA solution and oscillated at a vibration rate of 100 times / min at room temperature for 24 hours to obtain TA-modified fiber. The scanning electron microscopy image of the TA-modified fiber is shown in the figure. Figure 2 As shown, the presence of granular aggregates on the fiber surface was confirmed, and the increase in fiber diameter (1.89±0.13 μm) indicated that TA was successfully coated on the fiber surface.

[0041] 1.3 Preparation of PLys@TA modified PLCL fibers

[0042] Dissolve 20 mg of PLys in 10 mL of PBS and stir thoroughly to dissolve to prepare a PLys solution. After adding 570 uL of EDC and 0.3 g of NHS, immerse the 1 cm × 1 cm TA-modified fiber dressing in the PLys solution and oscillate at room temperature for 6 hours at a vibration rate of 100 times / min to obtain PLys@TA-modified fibers. The scanning electron microscopy image of the PLys@TA-modified fibers is shown in Figure 2. Figure 3 As shown, it shows that the granular aggregates on the fiber surface begin to increase, and the further increase in fiber diameter (1.98±0.11μm) indicates that the coating effect of PLys@TA is enhanced. Example 2 Characterization of X-ray spectra of different PLCL fibers by XPS

[0043] The prepared PLCL fibers were characterized by XPS, such as Figure 4 The elemental composition of the PLCL group shown is mainly CC, CO, and C=O structures. The TA@PLCL group has a stronger CO structure, indicating that TA adheres to PLCL through strong hydrogen bonding. Compared with the first two groups, the PLys@TA modified PLCL fiber group has a stronger N1s signal (CN, -N=, -NH-, -NH2), which shows that PLys is successfully grafted to the surface of TA@PLCL.

[0044] Example 3: Characterization of antibacterial properties of different PLCL fibers in vitro

[0045] 3.1 Characterization of antibacterial properties of PLCL fibers in vitro

[0046] Staphylococcus aureus was grown on the fibers with different modifications, and the bacterial proliferation of the fiber dressings was observed after 2 hours. Figure 5 The results show that PLCL has a certain effect of isolating bacteria. Staphylococcus aureus was inoculated on different modified PLCL fibers and co-cultured for 12 hours to observe the antibacterial effect of the fiber dressing scaffold. Figure 6 As shown, compared with the blank group, the antibacterial effect of PLCL alone was not ideal.

[0047] 3.2 Characterization of in vitro antibacterial properties of TA-modified PLCL fibers

[0048] After the stent was inoculated with Staphylococcus aureus for 2 hours, the isolated bacteria on the stent were observed. Figure 5 As shown in the figure, it shows that compared with pure PLCL, the bacterial isolation effect of TA-modified PLCL is significantly enhanced. Similarly, the antibacterial effect of fiber dressings modified by TA is also significantly enhanced, such as Figure 6 shown.

[0049] 3.3 Characterization of in vitro antibacterial properties of PLys@TA modified PLCL fibers

[0050] The isolated bacteria of the stent were observed 2 hours after the stent was inoculated with Staphylococcus aureus. Figure 5 As shown in the figure, it shows that compared with PLCL and TA@PLCL, PLys@TA@PLCL has a better effect of isolating bacteria. The antibacterial effect of the stent after the stent was inoculated with Staphylococcus aureus for 12 hours is shown in the figure. Figure 6 As shown, PLys@TA@PLCL has better antibacterial effect compared with PLCL and TA@PLCL.

[0051] Example 4: Investigation of different PLCL fibers promoting wound healing in diabetic mice

[0052] 4.1 Investigation of the performance of PLCL fibers in promoting wound healing in diabetic mice

[0053] A full-thickness skin defect with a diameter of 8 mm was prepared on the back of diabetic mice. The scaffold was applied to the skin wound of diabetic mice to observe and analyze the gross and histological effects of its promotion of wound healing. Figure 7 and Figure 8 and Fig. 9 As shown, compared with the blank group, PLCL fibers can promote wound healing and skin regeneration. 4.2 Investigation of the performance of TA-modified PLCL fibers in promoting wound healing in diabetic mice

[0054] The scaffold promoted wound healing in diabetic mice with full-thickness skin defects. Figure 7 , Figure 8 and Fig. 9 As shown, compared with the PLCL group, TA@PLCL fibers can provide anti-inflammatory and promote collagen regeneration in vivo, and can better promote skin regeneration and wound healing.

[0055] 4.3 Investigation of the wound healing performance of PLys@TA modified PLCL fibers in diabetic mice

[0056] The scaffold promoted wound healing in diabetic mice with full-thickness skin defects. Figure 7 , Figure 8 and Fig. 9 As shown, PLys@TA@PLCL fibers can provide anti-inflammatory, antibacterial and collagen regeneration effects, and can well promote the regeneration of skin and its appendages and wound healing.

[0057] The preparation method mainly improves the antibacterial, anti-inflammatory and skin regeneration-promoting properties of the fibers by introducing TA and PLys on the surface of the nanofibers. The present invention relates to a preparation method of functionalized fibers, which not only provides anti-inflammatory and antibacterial functions on the basis of simplified operation steps, but also further endows the fiber scaffold with the functions of promoting skin regeneration and wound healing, and has more application prospects in the fields of skin dressings and wound healing.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fiber membrane skin dressing, characterized in that: The fiber membrane skin dressing is a fiber coated with tannic acid and polylysine, wherein the polylysine is grafted onto the carboxyl group of the tannic acid through an amide condensation reaction mediated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide.

2. The method for preparing the fiber membrane skin dressing according to claim 1, characterized in that: The specific preparation steps are as follows: (1) Preparation of polylactic acid-caprolactone random fibers: polylactic acid-caprolactone was dissolved in hexafluoroisopropanol and stirred overnight to prepare a 5% spinning solution, and electrospinning was performed the next day; (2) Preparation of tannic acid-coated disordered fibers: Prepare 8 mg / mL tannic acid solution, the solvent is Tris-HCl, pH 8.5, 10 mM, soak the membrane in the solution and shake for 24 h, and wash with ultrapure water 2-3 times; (3) Preparation of polylysine-coated disordered fibers: Prepare a 2 mg / mL polylysine solution and add the corresponding 57% 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 3 mg / ml N-hydroxysuccinimide, soak the membrane in the solution and shake for 6 hours, and wash it with ultrapure water 2-3 times to obtain functionalized fibers.

3. The fiber membrane skin dressing according to claim 1, characterized in that: The concentrations of tannic acid and polylysine in the fiber membrane skin dressing are 1 mg / mL to 25 mg / mL and 1 mg / mL to 25 mg / mL respectively.

4. The method for preparing the fiber membrane skin dressing according to claim 2, characterized in that: The electrospinning process parameters in the preparation step (1) of the fiber membrane skin dressing are: spinning rate 20uL / min, applied voltage 6-7kV, using a flat plate to receive the jet monofilament, receiving distance 15cm, receiving device winding rate 10rpm, ambient temperature 0-1000℃, ambient relative humidity 0-100%.

5. The method for preparing the fiber membrane skin dressing according to claim 2, characterized in that: The reaction in step (3) of preparing the fiber membrane skin dressing is as follows: the reaction time is 6 hours and the reaction temperature is 0-100°C.

6. Use of the fiber membrane skin dressing as claimed in claim 1 in the construction of skin dressing for preventing bacterial infection of wounds.

7. Use of the fiber membrane skin dressing as claimed in claim 1 in skin dressing for reducing inflammatory response of wounds.

8. Use of the fiber membrane skin dressing as claimed in claim 1 in a skin dressing for promoting wound healing.

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