Bilobalide A modified polycaprolactone nanofiber membrane as well as preparation method and application thereof

Abstract

The invention relates to a bilobalide A modified polycaprolactone nanofiber membrane as well as a preparation method and application thereof, and relates to the technical field of composite biological materials. The nanofiber membrane is prepared from the following components: polycaprolactone and bilobalide A. The invention further discloses a preparation method of the nanofiber membrane. The bilobalide A is modified on the polycaprolactone nanofiber membrane through an electrostatic spinning technology, and the obtained functional bilobalide A modified polycaprolactone nanofiber membrane increases the hydrophilicity of polycaprolactone, promotes cell adhesion and inhibits generation of macrophages and inflammatory factors; after being implanted in a body wrapping the tissue-engineered cartilage, the material can inhibit immune inflammatory response of the tissue-engineered cartilage, maintain the form of the engineered cartilage and promote cartilage regeneration.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of composite biomaterials, and particularly to a ginkgolide A modified polycaprolactone nanofiber membrane, a preparation method and an application thereof. BACKGROUND

[0002] Tissue engineered cartilage has been widely used in joint cartilage, trachea, intervertebral disc repair, and nasal and auricular cartilage reconstruction. In the process of constructing tissue engineered cartilage, morphological maintenance is a key factor. It not only maintains the structural integrity of the engineered cartilage and promotes functional recovery, but also regulates cell behavior, reduces inflammatory response, promotes tissue integration, and prevents cartilage degradation.

[0003] In the construction of morphologically stable tissue engineered cartilage, high molecular weight materials are usually required to provide structural support. Polycaprolactone (PCL) is a synthetic aliphatic polyester with the characteristics of low cost, easy availability, high biocompatibility and plasticity. In the construction of morphological cartilage tissue, PCL has been widely used as a supporting material. However, PCL has a slow degradation rate and a hydrophobic surface structure, which is not conducive to cell growth and integration with other materials and surrounding tissues. More importantly, long-term implantation of PCL inevitably causes an inflammatory response, leading to material collapse and poor morphological maintenance. Therefore, inhibiting the immune inflammatory response caused by PCL is crucial for maintaining the morphology of engineered cartilage.

[0004] Many studies have attempted to combine immunologically bioactive agents with biomaterials to reduce the overreaction of the host immune system. However, most of them are cytokines or compounds that cannot be clinically applied in the short term. Common clinical anti-inflammatory drugs often have side effects such as individual differences, gastrointestinal reactions, and bleeding. Ginkgolides are traditional Chinese medicine compounds extracted from ginkgo leaves, including ginkgolide A, B, C, J, M and bilobalide. Ginkgolides are natural platelet activating factor receptor specific antagonists, which have benefits for the treatment of asthma, endotoxin shock, organ transplant rejection, cardiovascular and cerebrovascular diseases, and various inflammatory diseases. Ginkgolide injection has been widely used in clinical practice. Ginkgolide A (GA) is one of the core components of ginkgolides. Currently, a few studies have confirmed that GA has the ability to inhibit inflammation and myocardial fibrosis. In view of this, the present application provides a ginkgolide A modified polycaprolactone nanofiber membrane, a preparation method and an application thereof. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a ginkgolide A (GA) modified polycaprolactone (PCL) nanofiber membrane, a preparation method and an application thereof. The purpose is to inhibit the immune inflammatory response of tissue engineered cartilage in vivo, maintain the morphology of engineered cartilage, and promote cartilage regeneration.

[0006] The technical scheme for solving the above technical problems is as follows:

[0007] In a first aspect, the ginkgo biloba extract A modified polycaprolactone (PCL-GA) nanofiber membrane has components including polycaprolactone and ginkgo biloba extract A, and has hydrophilicity, promotes cell adhesion, and inhibits macrophages and inflammatory factor production, wherein the molecular weight of the polycaprolactone is about 60000-80000.

[0008] On the basis of the above technical scheme, the present application can also be improved as follows.

[0009] Further, the mass ratio of the polycaprolactone and the ginkgo biloba extract A is 1:0.001-0.1.

[0010] Further, the thickness of the nanofiber membrane is 20um-100um.

[0011] In a second aspect, a preparation method of the ginkgo biloba extract A modified polycaprolactone nanofiber membrane includes the following steps:

[0012] (1) dissolving polycaprolactone in a hexafluoroisopropanol solvent to obtain a polycaprolactone solution; dissolving ginkgo biloba extract A in the polycaprolactone solution to obtain a mixed solution;

[0013] (2) electrospinning the mixed solution to prepare a ginkgo biloba extract A modified polycaprolactone blended membrane;

[0014] (3) vacuum drying the ginkgo biloba extract A modified polycaprolactone blended membrane to obtain a nanofiber membrane.

[0015] Further, the use amount ratio of the polycaprolactone and the hexafluoroisopropanol solvent in step (1) is 1g:8-12mL; and the mass ratio of the polycaprolactone and the ginkgo biloba extract A is 1:0.001-0.1.

[0016] Further, the parameters of the electrospinning are as follows: the spinning distance is 10cm-20cm; the spinning voltage is 10kV-20kV; and the flow rate is 4.5-13.5mm / min.

[0017] Further, the parameters of the vacuum drying in step (3) are as follows: the vacuum degree is not greater than 0.08MPa, the room temperature, and the time is 12h-36h. Specifically, the vacuum degree can be 0.08MPa, the room temperature, and the time can be 24h.

[0018] In a third aspect, the ginkgo biloba extract A modified polycaprolactone nanofiber membrane is used to prepare a cell-loaded nanofiber membrane composite.

[0019] In a fourth aspect, a nanofiber membrane cell-laden composite comprises the ginkalide A-modified polycaprolactone nanofiber membrane.

[0020] Further, the nanofiber membrane is prepared by a method comprising: wrapping the ginkalide A-modified polycaprolactone nanofiber membrane around a tissue engineering cartilage.

[0021] The oriented PCL-GA nanofiber membrane designed and produced by electrospinning technology is a good carrier for sustained release of GA; the oriented PCL-GA nanofiber membrane exhibits a more ordered, uniform and aligned structure than the reticular structure of biomaterials, and a stronger ability to promote cell adhesion and growth; in addition, by adjusting the thickness of the PCL nanofiber, the degradation time of PCL can be adjusted to match the regeneration of cartilage; GA has a dual effect of anti-inflammation and inhibition of the production of cartilage matrix degradation enzymes; loading GA into the PCL electrospun membrane can change the hydrophobicity of PCL to hydrophilicity. After implantation in vivo, the PCL-GA nanofiber membrane prevents the infiltration of inflammatory cells, the presence of GA successfully reduces the immune inflammatory response of the implanted material, promotes the secretion of cartilage matrix, and maintains the morphology of cartilage.

[0022] The present application has the beneficial effects that: the present application modifies ginkalide A to PCL nanofiber membrane by electrospinning technology, the obtained functional PCL-GA nanofiber membrane not only increases the hydrophilicity of PCL, promotes cell adhesion, inhibits the production of macrophages and inflammatory factors, but also promotes the secretion of cartilage cell matrix, which can inhibit the immune inflammatory response of tissue engineering cartilage, maintain the morphology of engineering cartilage, and promote cartilage regeneration after wrapping tissue engineering cartilage and implanting in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 4 is a characteristic result graph of the GA-loaded PCL membrane of the present application; wherein (A) is a process diagram for preparing a composite scaffold; (B) is a SEM micrograph and distribution diagram of the nanofiber diameter of different groups; (C) is a WCA optical image of the nanometer structure membrane of the PCL, PCL-GA20 mg and PCL-GA100 mg groups; (D) is the GA release curve of the drug-loaded membrane soaked in PBS for up to 11 days; (E) is the FTIR of the GA, PCL, PCL-GA20 mg and PCL-GA100 mg groups, and the red arrow indicates the peak near 3600-3300 cm -1 (OH), 1750-1800 cm -1 (C=O pentavalent saturated γ-lactone); data are expressed as mean ± standard deviation; statistically significant difference compared with the PCL group is indicated by **** P<0.0001;

[0024] Figure 2Representative histological images of acute phase immune inflammatory response after 1 week of in vivo implantation of the cell-scaffold construct of the present application; Wherein, (A) Representative appearance image of the cell-scaffold construct implanted for 1 week; (B) Representative image of hematoxylin-eosin (H&E) staining of the cell-scaffold construct at 1 week post-surgery; Scale bar: 800 pm and 200 pm; (C) Quantitative analysis of the depth of inflammatory cell infiltration in HE staining of the implanted cell-scaffold construct; (D-E) Representative images and quantitative analysis of IHC staining of CD86 and CD206 around the membrane at 1 week post-surgery; Scale bar: 200 pm and 50 pm; Data are presented as mean ± standard deviation; Statistically significant differences compared with the GelMA group are represented by ** P < 0.01, *** P < 0.001, **** P < 0.0001; ## P < 0.01, ### P < 0.001, #### P < 0.0001 compared with the GelMA+PCL group;

[0025] Figure 3 Cartilage regeneration results of the cell-scaffold construct of the present application in vivo for 16 weeks; Wherein, (A) Macroscopic view of the engineered cartilage-like tissue after in vivo culture for 16 weeks; (B) Cross-section of the engineered cartilage-like tissue was stained with H&E; (C) Quantitative evaluation of the volume of regenerated cartilage; (D) Histological staining of H&E, Safranin-O, Alcian blue, Collagen II and Collagen I of the engineered cartilage-like tissue cultured in vivo for 16 weeks; (E) Biochemical quantitative evaluation of the total weight, GAG content, DNA content and GAG / DNA of the regenerated cartilage after in vivo culture for 16 weeks (n = 5); Data are presented as mean ± standard deviation; Statistically significant differences compared with the GelMA group are represented by * P < 0.05, ** P < 0.01; # P < 0.05, ## P < 0.01 compared with the GelMA+PCL group. DETAILED DESCRIPTION

[0026] The principles and features of the present application are described below, and the examples are only used to explain the present application and are not intended to limit the scope of the present application. If the specific technology or condition is not specified in the examples, it is performed according to the technology or condition described in the literature in the art or according to the product instruction. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be commercially available through a regular channel.

[0027] 1. Experimental methods.

[0028] 1.1 Preparation and property analysis of GA-loaded PCL membrane

[0029] (1) Preparation of GA-loaded PCL membrane

[0030] To prepare GA-loaded PCL nanofibers, 1 gram of PCL (polycaprolactone, molecular weight of about 80,000 Dalton, Sigma-Aldrich, USA) was dissolved in 10 milliliters of hexafluoroisopropanol mixed solution. The mixture was then stirred at room temperature overnight. Next, GA (200 milligrams and 1 gram, purity > 98%, Aladdin, China) was directly added to the mixed solution and stirred for 24 hours until uniform. After that, the mixture was loaded into a 5 milliliter syringe. The syringe was loaded into an electrospinning pump with a flow rate set at 9 millimeters per minute, and a high-voltage power supply of 12 kilovolts was set between the needle and the nanofiber collector for electrospinning. The ginkgolide A modified polycaprolactone blended film was vacuum dried to obtain a nanofiber film. PCL films loaded with 0, 20, 100 milligrams of GA were referred to as PCL, PCL-GA20 milligrams, and PCL-GA100 milligrams, respectively.

[0031] (2) Drug release analysis

[0032] Seven and a half milligrams of nanofiber film were cut from different PCL-GA groups and placed in a 10 milliliter centrifuge tube. Then, after adding 1.5 milliliters of PBS (pH = 7.4, 0.01M) to the tube, it was continuously shaken and cultured at 37°C. At different time points (1, 2, 3, 4, 5, 6, 7, and 11 days), the supernatant was extracted for drug concentration analysis using high-performance liquid chromatography (HPLC). The formula for calculating the cumulative release amount of GA is as follows.

[0033] Cumulative release rate (%) = [V1C n + V2∑C(n-1)] / w x 100;

[0034] In the formula, C n is the concentration of GA in the supernatant at the predetermined time point. V1 is the total volume of PBS (1.5 milliliters), and V2 is the volume of supernatant extracted each time (1 milliliter). w is the amount of GA initially loaded in the electrospun film.

[0035] (3) Scaffold surface morphology

[0036] The microstructure of PCL-GA fibers was observed by scanning electron microscopy (Philips XL-30, Netherlands). Simply put, the PCL-GA film of each group was dried in a vacuum overnight. After gold plating, the film surface was observed using a scanning electron microscope at an acceleration voltage of 5 kilovolts. Images were captured at 1000 times and 20000 times magnification. Then, the fiber diameter was measured using Nano Measurer software.

[0037] (4) Scaffold hydrophilicity evaluation

[0038] The water contact angle (WCA) of the nanofiber membranes was measured by a video contact angle instrument (HARKE-SPCA, Beijing, China). Specifically, a nanostmctured membrane was fabricated on a glass slide to create a plane, and then a water droplet (3 μL) was placed vertically on the membrane before capturing the WCA.

[0039] (5) Composition analysis

[0040] The composition analysis of the membranes was performed using a Fourier transform infrared spectrometer (FTIR) (6800-50 / NEXUS, Nicolet, USA) in the attenuated total reflection mode. The samples of PCL and PCL-GA membranes were prepared. In the spectral region of 4000-500 cm, the transmittance readings were measured with a resolution of 0.09 cm -1 .

[0041] 1.2 Preparation and in vivo evaluation of the cell-laden composite with GA-PCL nanofiber membranes

[0042] (1) Preparation of the cell-laden composite with GA-PCL nanofiber membranes

[0043] The pre-gel solution was prepared 12 hours before cell embedding. Briefly, the lyophilized GelMA (methacrylated gelatin) was completely dissolved in the culture medium to achieve a final GelMA concentration of 10%. LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) powder, a photoinitiator, was completely dissolved in the solution to achieve a final LAP concentration of 0.25%. PEO (polyethylene glycol, average molecular weight = 300,000) powder, a pore-forming agent, was dissolved in the solution to achieve a final PEO concentration of 1%. Based on the phase separation void formation strategy, the aqueous two-phase emulsion of GelMA and PEO polymers was prepared together, followed by a photo-crosslinking and leaching procedure. All the hydrogel formulations were mixed thoroughly and continuously stirred, sterile filtered with a syringe filter (0.45 μm pore size), and stored in an incubator in the dark. The expanded rabbit chondrocytes were mixed with the GelMA microporous hydrogel at a concentration of 2 x 10 7 / ml and poured into a cylindrical mold with a diameter of 1 centimeter and a thickness of 2 millimeters, and then the PCL-GA membranes carrying cells were covered on the upper and lower surfaces of the hydrogel. Finally, the photo-crosslinking hydrogel formulation was induced by exposure to blue light (wavelength: 405 nanometers; light source: LED (Uvata Precision Optoelectronics Co., Ltd.); intensity: 20 mW / cm 2 ; distance: 10 centimeters; exposure time: 60 seconds) to obtain the composite cell scaffold.

[0044] (2) In vivo implantation of the composite cell scaffold

[0045] To test the chondrogenic and immunobiological activities of the composite cell scaffold in vivo, 2-month-old female rabbits were chosen to establish an in vivo implantation model. Briefly, after the rabbits were anesthetized, a 1.5 cm incision was made on the back. The scaffolds cultured in the medium for 24 hours were removed of PEO, and then subcutaneously transplanted into the rabbits to regenerate cartilage tissue. The rabbits were sacrificed at each time point (1 and 16 weeks).

[0046] (3) Histological and immunohistochemical analysis

[0047] The samples were collected and subjected to histological and immunohistochemical analysis. Briefly, the samples were fixed with 4% formaldehyde, embedded in paraffin, and sectioned. Then, the sections stained with H&E, Safranin O, Alcian blue, and type II collagen (type II collagen, 1:100, Abeam, UK) immunohistochemistry were applied to evaluate the histological structure and cartilage-specific ECM deposition in the regenerated tissue. Sections stained with picrosirius red, CD86, CD206, and type I collagen (CD86 / CD206 / type I collagen, 1:100, Abeam, UK) immunohistochemistry were used to evaluate the inflammatory response and fibrous capsule formation.

[0048] (4) Biochemical analysis

[0049] The samples were collected and the surrounding fibers were thoroughly removed, the remaining cartilage weight was accurately weighed, and then the cartilage was dropped into a 1 ml container containing PBS buffer. The liquid level rise was measured to determine the volume of the regenerated cartilage, and then the sample was chopped to determine the DNA content and glycosaminoglycan (GAG) content. Briefly, the Quant-iT TM DNA content was determined using dsDNA assay and GAG content was determined using dimethylmethylene blue assay.

[0050] 2. Statistical analysis

[0051] All quantitative data were collected from at least three repeated tests, and the values were expressed as mean ± standard deviation. After confirming the normal distribution of the data, one-way ANOVA was performed using GraphPad Prism 9 software to determine the statistical significance between groups, and statistically significant differences were indicated by * or # P<0.05, ** or ## P<0.01, *** or ### P<0.001, **** or #### P<0.0001.

[0052] 3. Experimental results.

[0053] 3.1 Characteristic analysis of GA-loaded PCL membrane.

[0054] The construction process of GA-loaded PCL membrane and microporous GelMA hydrogel is shown in the figureFigure 1 A). Scanning electron microscopy shows the morphological details of the nanostructured scaffolds with and without GA. The nanofibers are aligned and porous for directional chondrocyte adhesion and GA release ( Figure 1 B). With the addition of GA, the surface of PCL nanofibers became rougher, and the diameter of the nanofibers became thicker with increasing GA concentration. WCA was measured to evaluate the hydrophilicity of the nanostructured membranes. PCL exhibited its hydrophobic properties, while the addition of GA significantly increased its hydrophilicity, especially in the PCL-GA 20 mg group ( Figure 1 C). Cumulative release of GA showed that approximately 50% of GA was released within the first 7 days. A burst release was observed on the first day, which was stable over the next 11 days, indicating that GA was successfully released from the nanostructured membrane ( Figure 1 D) FTIR spectra showed that both GA powder and GA-containing film had 3600-3300 cm -1 (OH) and 1750~1800cm -1 (C=O pentavalent saturated γ-lactone) peak. These regions are the main characteristic peaks of GA ( Figure 1 E) PEO was dissolved in culture medium and then mixed with GelMA hydrogel.

[0055] 3.2 Acute immune response within 1 week after implantation.

[0056] In order to study the immune inflammatory infiltration of each group of scaffolds in the acute stage, the present invention collected samples 1 week after surgery and carefully preserved the fibrous tissue attached to the host tissue and scaffolds ( Figure 2 A). Although all groups showed cell aggregation around the implanted biomaterials, the depth of inflammatory cell infiltration (the thickness between the muscle layer and GelMA) around the GA-loaded scaffolds was lower ( Figure 2 B, 3C). In addition, CD86-positive M1 macrophages were distributed around all scaffolds, and a small number of CD206-positive M2 macrophages were present. However, the GelMA+PCL-GA group inhibited the infiltration of M1 macrophages and promoted the number of M2 macrophages ( Figure 2 D, 3E). This suggests that GA exerts its immunomodulatory effect by inhibiting M1 macrophages and converting scaffold-recruited M1 macrophages into an M2 phenotype, thereby alleviating the inflammatory response.

[0057] 3.3 Cartilage regeneration of cell-scaffold constructs after 16 weeks in vivo.

[0058] The cartilage regeneration effect was evaluated 16 weeks after the cell-scaffold constructs were implanted in vivo. A rough observation showed that the GelMA group had the greatest degree of deformation, with only a small amount of cartilage regeneration, while the cell-scaffold constructs wrapped in the other three groups maintained good morphology (Figure 3 A, Figure 3 B) Volume quantification results show that the residual volume percentage of the GelMA group is only 1.87 ± 0.57%, while the residual volume percentage of the group with PCL film is significantly higher than that of the GelMA group, reaching 7.79 ± 1.20%, 10.59 ± 0.61% and 24.48 ± 2.59% respectively Figure 3 C) HE, Alcian Blue, Safranine O staining all show that the ECM staining of the GelMA+PCL-GA 100 mg group is stronger than the other three groups. Immunohistochemical staining of type II collagen also gets similar results Figure 3 D) Biochemical quantification further clarifies the cartilage regeneration of each group, which is consistent with the results of appearance and histological examination. After completely removing the fibrous envelope, the weight of the regenerated cartilage in the four groups was measured as 12.00 ± 4.04 mg, 58.00 ± 4.16 mg, 61.33 ± 3.52 mg and 106.7 ± 13.33 mg respectively. The glycosaminoglycan (GAG) content of each group was 18.87 ± 3.24 ug, 60.29 ± 10.71 ug, 107.6 ± 7.45 ug and 130.2 ± 15.56 ug respectively. According to the DNA content standardization GAG content (GAG / DNA), the GAG content of the GelMA+PCL-GA group is higher than that of the other two groups, and the difference of the GelMA+PCL-GA 20 mg group is significant Figure 3 E) After 16 weeks of implantation, a large amount of collagen formation around the regenerated cartilage is observed, and immunohistochemistry shows that the expression level of type I collagen in the GelMA+PCL group is the highest. After loading GA, the expression level of type I collagen is significantly reduced Figure 3 F).

[0059] In summary, the ginkgo biloba A is modified to the PCL nanofiber membrane by the electrospinning technology in the application, the functional PCL-GA nanofiber membrane obtained not only increases the hydrophilicity of PCL, promotes cell adhesion, inhibits macrophages and inflammatory factor production, but also promotes cartilage cell matrix secretion; the PCL-GA nanofiber membrane can inhibit the immune inflammatory reaction of tissue engineering cartilage, maintain the morphology of the engineering cartilage and promote cartilage regeneration after wrapping the tissue engineering cartilage and implanting in vivo.

[0060] Although the embodiments of the application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the application.

Claims

1. Ginkgolide A modified polycaprolactone nanofiber membrane, characterized in that: The components of the nanofiber membrane include polycaprolactone and ginkgolide A. The nanofiber membrane is hydrophilic, promotes cell adhesion and inhibits the production of macrophages and inflammatory factors.

2. The polycaprolactone nanofiber membrane modified with ginkgolide A according to claim 1, characterized in that: The mass ratio of the polycaprolactone to the ginkgolide A is 1:0.001-0.

1.

3. The polycaprolactone nanofiber membrane modified with ginkgolide A according to claim 1, characterized in that: The thickness of the nanofiber membrane is 20um to 100um.

4. A method for preparing a polycaprolactone nanofiber membrane modified with ginkgolide A according to any one of claims 1 to 3, characterized in that: The steps include: (1) dissolving polycaprolactone in hexafluoroisopropanol solvent to obtain a polycaprolactone solution; dissolving ginkgolide A in the polycaprolactone solution to obtain a mixed solution; (2) electrospinning the mixed solution to prepare a polycaprolactone blended membrane modified with ginkgolide A; (3) The polycaprolactone blended membrane modified with ginkgolide A is vacuum dried to obtain a nanofiber membrane.

5. The method for preparing the polycaprolactone nanofiber membrane modified with ginkgolide A according to claim 4, characterized in that: In step (1), the dosage ratio of polycaprolactone to hexafluoroisopropanol solvent is 1 g: 8-12 mL; the mass ratio of the polycaprolactone to the ginkgolide A is 1: 0.001-0.

1.

6. The method for preparing the polycaprolactone nanofiber membrane modified with ginkgolide A according to claim 4, characterized in that: The electrostatic spinning parameters are: spinning distance of 10 cm to 20 cm; spinning voltage of 10 kV to 20 kV; and flow rate of 4.5 to 13.5 mm / min.

7. The method for preparing the polycaprolactone nanofiber membrane modified with ginkgolide A according to claim 4, characterized in that: The parameters of vacuum drying in step (3) are: vacuum degree not greater than 0.08 MPa, room temperature, and time of 12 h to 36 h.

8. Application of polycaprolactone nanofiber membrane modified with ginkgolide A, characterized in that: The polycaprolactone nanofiber membrane modified with ginkgolide A according to any one of claims 1 to 3 is used to prepare a nanofiber membrane-loaded cell complex.

9. A cell-supported nanofiber membrane complex, characterized in that: The cell-carrying complex of the nanofiber membrane comprises the polycaprolactone nanofiber membrane modified with ginkgolide A according to any one of claims 1 to 3.

10. The cell-supported nanofiber membrane complex according to claim 9, characterized in that: The nanofiber membrane is prepared by the following method: the tissue engineering cartilage is wrapped with the polycaprolactone nanofiber membrane modified with ginkgolide A by a wrapping method.

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