Electrospun polycaprolactone nanofibrous membranes loaded with baicalin for antibacterial wound dressing

Due to the rise in bacterial resistance, the antibacterial extractions from Chinese herbs have been used more frequently for wound care. In this work, baicalin, an extraction from the Chinese herb Scutellaria baicalensis, was utilized as the antibacterial component in the poly(ε-caprolactone)/MXene (PCL/Ti3C2TX) hybrid nanofibrous membranes for wound dressing. The results revealed that the presence of Ti3C2TX aided in the diameter reduction of the electrospun nanofibers. The PCL hybrid membrane containing 3 wt% Ti3C2TX nanoflakes and 5 wt% baicalin exhibited the smallest mean diameter of 210 nm. Meanwhile, the antibacterial tests demonstrated that the PCL ternary hybrid nanofibers containing Ti3C2TX and baicalin exhibited adequate antibacterial activity against the Gram-positive bacterial S. aureus due to the good synergistic effects of Ti3C2TX naoflakes and baicalin. The addition of Ti3C2TX nanoflakes and baicalin could significantly improve the hydrophilicity of the membranes, resulting in the release of baicalin from the nanofibers. In addition, the cytotoxicity of the nanofibers on rat skeletal myoblast L6 cells confirmed their good compatibility with these PCL-based nanofibrous membrances. This work offers a feasible way to prepare antibacterial nanofibrous membranes using Chinese herb extraction for wound dressing applications.

www.nature.com/scientificreports/ had good antibacterial activity against E. coli and S. aureus 14 . In addition, the nanofiber of the hybrid scaffold exhibited good biocompatibility and cell adhesion to fibroblast L929 cells in vivo tests. Ekram et al. demonstrated that the presence of zinc chloride (ZnCl 2 ) reduced the diameter of the PCL/ZnCl 2 nanofibers while increasing the degradation rate and mechanical properties 15 . Moreover, the antibacterial composite nanofiber was found to greatly boost the proliferation of stem cells. Trcin et al. found that the PCL scaffolds containing TiO 2 nanoparticles could provide statistically significant antimicrobial activity against different types of bacteria 16 . Furthermore, the PCL/TiO 2 scaffolds with a maximum porosity of 93%, on the other hand, were found to be capable of supporting the adhesion and proliferation of limbal stem cells.
MXene is a new family of two-dimensional (2D) materials that integrates the transition metals M (Ti, Cr, V, Nb and Mo etc.) with huge amounts of X (carbides, nitrides, or carbonitrides) by removing the A-element in the MAX phase 17,18 . MXene (Ti 3 C 2 T X ) exhibits superior biocompatibility and antibacterial efficiency against both Gram-negative and Gram-positive bacteria than graphene oxide due to its ultrathin structure and unique physiochemical properties 19,20 . Awasthi et al. found that the addition of MXene into PCL nanofibers maintained good biocompatibility in vitro with ibroblasts (NIH-3T3) and preosteoblasts (MC3T3-E1) cell lines 21 . In addition, the presence of Ti 3 C 2 T X nanosheets contributed to reducing the diameter and improving the morphology of PCL/Ti 3 C 2 T X nanofibers. However, no data on the antibacterial activities of PCL/Ti 3 C 2 T X nanofibers have been reported to far.
Herbal extracts have been widely utilized to cure various diseases since ancient times. Plant-derived phytochemicals can serve as potential antibacides with fewer side effects. Baicalin, a flavonoid extracted from the Chinese herb Scutellaria baicalensis, has been regarded as a multitherapeutic agent in the field of biomedicine 22 . It shows various positive benefits on wounds, including anti-oxidative, anti-bacterial, and anti-inflammatory properties 23,24 . However, little research has been performed to date on the use of baicalin in electrospun fibers for wound dressings.
In this work, MXene (Ti 3 C 2 T X ) was first exfoliated to obtain Ti 3 C 2 T X nanoflakes. Then the resulting Ti 3 C 2 T X nanoflakes were incorporated into the PCL matrix with herbal extraction baicalin by electrospinning. The morphology, thermal stability, hydrophilicity, and mechanical properties of the electrospun nanofibers consisting of PCL/Ti 3 C 2 T X /baicalin ternary composites were investigated. The addition of Ti 3 C 2 T X and baicalin was expected to have synergistic effects on improving the wound dressing's antibacterial performance against gram-positive bacterial S. aureus. Furthermore, the biocompatibility in vitro of this wound dressing was also evaluated by using rat skeletal myoblast L6 cells.
Preparation of electrospun PCL composite nanofibrous membranes. The exfoliation of Ti 3 C 2 T X was performed by high energy ball milling using a Miqi YXQM-1L planetary micromill (Changsha, China). Ti 3 C 2 T X (4 g) and zirconium oxide milling balls (70 g) were placed in a 250 ml grinding bowl and performed at 600 rpm for 4 h. Then the exfoliated Ti 3 C 2 T X was removed from the grinding bowl with DI water, followed by sonication in DI water for 30 min to exfoliate completely. Finally, the resulting Ti 3 C 2 T X nanoflakes were centrifuged at 3500 rpm for 10 min, and the obtained suspension was freeze-dried overnight.
The electrospun nanofibers of the PCL/Ti 3 C 2 T X /baicalin ternary composite were prepared using a commercially available electrospinning machine (TL-Pro, Tongli Weina Co., Ltd., Shenzhen, China), as shown in Fig. 1. The formulations of PCL based nanofibers are shown in Table 1. The concentration of PCL was fixed at 100 mg/mL in a mixed chloroform/DMF (8:2, v/v) solution. Subsequently, the desired amount of Ti 3 C 2 T X and baicalin was added into the PCL solution with vigorous stirring. The prepared PCL solution was loaded into a 10 mL plastic syringe with a metal capillary needle (0.50 mm inner diameter, and 30 mm length). The applied electrospinning voltage was fixed at 15 kV and the flow rate was kept at 1 mL/h. The obtained nanofibers were subsequently placed in a vacuum oven at 50 ℃ for 6 h to remove the remaining solvent.
Scanning electron microscope (SEM) observation. The morphology of Ti 3 C 2 T X and the prepared nanofibers was observed by a scanning electron microscope (SEM, FEI Quata 250, USA). Prior to observation, the specimens were sputtered with a thin layer of gold to avoid charge accumulation. www.nature.com/scientificreports/ Thermogravimetric analysis (TGA). The thermal stability was evaluated by a thermogravimetric analyzer (TGA, Netzsch TGA-209F1). The specimens were heated from room temperature to 600 ℃ at a ramping rate of 10 ℃/min.

Water contact angels (WCA).
The wettability of the nanofibers was evaluated via the measurement of the water contact angle using a See System E instrument (Advex Instruments, Czech Republic).

Minimum inhibitory concentration (MIC) determination. The standard broth microdilution method
was applied to determine the MIC value of baicalin toward S. aureus as described in the previous reports 25,26 . Briefly, the S. aureus was inoculated and grown in a broth subculture inside a flask. The bacteria were then incubated at 37 °C for 24 h. Then, the bacterial concentration was adjusted to a density of 1.0 × 10 6 CFU/ml. The baicalin solution, with a concentration ranging from 1 to 1024 mg/ml, was added into the S. aureus solution to observe the bacteria's growth. The lowest concentration, at which no visible bacterial growth was observed in the plate, was considered as the MIC.
Antibacterial tests. The antibacterial properties of the PCL based nanofiebers against S. aureus were evaluated by a standard "SNV 195920-1992" evaluation model 26 . The evaluation standard of inhibition zone is summarized in Table S1. The Firstly, 100 µL of 10 8 CFU/mL bacteria suspension was spread on an LB agar plate, and then the nanofibrous membrane samples with a diameter of 1.0 cm were placed on the surface of the agar. The bacteria suspension with the PCL-based films were incubated at 37 °C, followed by the digital images of the PCL-based nanofibrous membranes on the agar plate with bacteria were recorded at 24 h, 72 h, and 120 h, respectively. All samples were tested in triplicate.

Samples PCL (wt%) Ti 3 C 2 T X (wt%) Baicalin (wt%)
PCL-0 100 -- www.nature.com/scientificreports/ Cytocompatibility evaluation. The cytotoxicity of the nanofibers on rat skeletal myoblast L6 cells was evaluated using the CCK-8 method with a leaching pattern. The sterilized nanofibers extract solutions (10 mg/ mL) were prepared by immersing the dried nanofiber in medium for 12 h at 37 ℃ with ultrasonic extraction. The L6 cells were seeded in a 96-well plate at a density of 5000 cells/well, and pre-cultured for 24 h before replacing the culture medium with the fresh medium and extract solutions to make the final sample concentration of 0.2 mg/mL, 1 mg/mL, and 5 mg/mL. Each sample to be tested (PCL-0, PCL-1, PCL-2, PCL-3, and PCL-4), blank control (culture medium) and positive control (culture medium and cell) were incubated for 48 h and repeated four times. The results were recorded as the absorbance at 450 nm through an ultraviolet spectrophotometer by the following formula: Statistical analysis. All experiments were carried out in triplicate. The data was analyzed by the SPSS software (IBM Analytics, USA). Significance of all the statistical tests was predetermined at P < 0.05. Results were expressed as mean ± standard deviation (SD).

Results and discussion
Characterization of MXene nanoflakes. In Fig. 2A, the pristine MXene (Ti 3 C 2 T X ) shows a typical organ-like structure with gaps of tens of nanometers wide. The exfoliated Ti 3 C 2 T X nanoflakes in Fig. 2B show that the layers are clearly separated from each other. In addition, some small nanoflakes in the range of tens of nanometers are observed due to the grinding. The EDX elemental mapping images in Fig. 2C confirm the presence of Ti, C, and O, which is consistent with the chemical structure of Ti 3 C 2 T X

20
. The appearance of F on the surface of Ti 3 C 2 T X nanoflakes is due to the remaining F element after etching with LiF/HCl 17 . In the AFM image in Fig. 2D, it is observed that the exfoliated Ti 3 C 2 T X nanoflake has a thickness of 1.5 nm, which is similar to the reported results 27,28 . The XRD patterns of pristine Ti 3 C 2 T X and exfoliated Ti 3 C 2 T X nanoflakes are shown in Fig. 2E. It is noted that the characteristic peak at 6.9° in pristine MXene indicates the interlayer spacing of www.nature.com/scientificreports/ 1.28 nm. In addition, the prominent peaks at 9.4°, 19.1°, 34.0°, 38.7°, 41.7° and 44.9°, which correspond to the diffraction of (002), (004), (101), (008), (104), and (105) planes of Ti 3 C 2 T X , respectively 29 . As for the exfoliated Ti 3 C 2 T X nanoflakes, the characteristic peak shifts from 6.9° to 5.4° due to the exfoliation. In addition, this peak becomes board and weak, which is ascribed to the ball milling and exfoliation reducing the size of the nanoflakes and enlarging the interlayer spacing.
Characterization of PCL nanofibrous membranes. The SEM images of PCL nanofibrous membranes are presented in Fig. 3. It is noted that the nanofibers of PCL-0 with a mean diameter of 269 nm (Fig. 3B) have a smoother surface and non-beaded structures. With the addition of 3 wt% Ti 3 C 2 T X nanoflakes, the mean diameter of PCL-1 decreases from 269 to 217 nm as compared with that of PLA-0. In Fig. 3F,H,J, the mean diameters of PCL-2, PCL-3 and PCL-4 are 254 nm, 223 nm, and 210 nm, respectively. It is believed that the presence of conductive Ti 3 C 2 T X nanoflakes can increase the charge density of the electrospinning solution due to the polar groups on their surface, thus enhancing the electrostatic force of the applied electric field and reducing the diameter of the nanofibers 30,31 . On the other hand, the introduction of baicalin has little effect on the diameter of the nanofibers. However, the surface of the PCL nanofibers containing baicalin is relatively smoother than that of PCL-1. It is speculated that baiclin is a type of small molecule with many hydroxyl groups that can reduce the viscosity of the electrospinning solution and react with the polar groups of Ti 3 C 2 T X nanoflakes to form hydrogen bonds, resulting in the homogeneous dispersion of Ti 3 C 2 T X nanoflakes in the PCL matrix. The typical tensile stress versus strain curves for PCL-based nanofibrous membranes are shown in Fig. S1A, and the corresponding tensile properties are summarized in Fig. S1B. It is noted that the pure PCL membrane shows high ductility (elongation at break of 305%), which is consistent with other published works 32,33 . For PCL-1, the addition of Ti 3 C 2 T X nanoflakes results in strong reinforcing effects, increasing the tensile modulus significantly. Although the abundant polar groups on the surface of Ti 3 C 2 T X nanoflakes can react with the ester bonds of PCL, the inhibition effects of the rigid nanosheets will be more profound, lowering the plastic flow ability of PCL molecular chains 34 . On the other hand, the PCL-2 shows a clear decrease in both elasticity and strength. In Figure S1B, the elongation at break and tensile strength of PCL-2 are reduced to 178% and 2.58 MPa, respectively. This is because baicalin is a small molecule that has a plasticizing effect on PCL. As for PCL-3, the tensile strength of the samples improved obviously, with a little sacrifice in elongation at break as compared with those of PCL-2. It is speculated that the presence of Ti 3 C 2 T X nanoflakes can react with baicalin to form hydrogen bonds to some extent. With the further increase in the baicalin content, the tensile properties of PCL-4 deteriorate progressively, indicating the baicalin exceeds the reaction sites of Ti 3 C 2 T X nanoflakes.
The FT-IR spectra of PCL-based nanofibrous membranes are presented in Fig. 4. It is clearly observed that the two characteristic peaks located at 2943 cm −1 and 2863 cm −1 of PCL-0 correspond to the stretching bands of CH 2 groups. The absorption peak at 1737 cm −1 is ascribed to the stretching of the C=O groups, while the peaks at 1294 cm −1 and 1184 cm −1 belong to the asymmetric and symmetric stretching of the C-O-C groups, respectively. In addition, the characteristic peak at 1243 cm −1 denotes the CH 3 vibrations, and 1045 cm −1 belongs to C-O stretching and C-H bending 35 . With the incorporation of Ti 3 C 2 T X and baicalin, no obvious change is observed in the FT-IR spectra. The ternary composites show a weak and broad peak between 3600 and 3300 cm −1 , which is ascribed to the overlapping of the O-H polar groups on the surface of Ti 3 C 2 T X and the O-H stretching vibration of baicalin 36 . Furthermore, there is a new characteristic peak at 1609 cm −1 that corresponds to the stretching vibration of C=C of phenyl groups from baicalin 37 .
The thermal stability of PCL-based nanofibrous membranes was measured by TGA, as shown in Fig. 5. The corresponding data, including the initial weight loss temperature (T 10 , the temperature at 10% weight loss), the peak weight loss temperature (T p , the temperature at maximum weight loss rate), and char residues at 600 ℃ are listed in Table 2. It can be observed that PLA-0 shows a distinct weight loss stage from 300 to 420 ℃, which was associated with the pyrolysis of the chemical bond cleavage of PCL chains 38 . The thermal decomposition curve of PCL-1 shifts to a higher temperature, indicating the presence of Ti 3 C 2 T X can increase the thermal stability of PCL nanofibers. According to Table 2, the T 10 and T p of PCL-1 increases from 361.8 to 365.8 ℃ and from 403.2 to 415.7 ℃, respectively, as compared with that of PLA-0. This is due to the Ti 3 C 2 T X nanoflakes serving as a thermal barrier to protect the underlying PCL matrix. In the case of the PCL/Ti 3 C 2 T X /baicalin ternary nanofibers, the thermal decomposition curves can be roughly divided into two major stages. The initial weight loss stage occurs in the range of 200-370 ℃ due to the thermal decomposition of baicalin. The second stage occurs at around 370-420 ℃, which is related to the pyrolysis of PCL chains. In addition, the T 10 and T p of PCL-4 show a decreasing tendency as compared with those of PLA-0, which is ascribed to the low thermal stability of baicalin. Figure 6 depicts the XRD patterns of PCL-based nanofibrous membranes. As shown in Fig. 6, the PCL-0 has three significant diffraction peaks at 2θ = 21.3°, 22.0° and 23.7°, which correspond to the (110), (111), and (200) planes, respectively 39 . In addition, there is also a relatively weak and broad peak at 11.8° in the PCL-0 pattern. The above results indicate that the pristine PCL is in a semi-crystalline state. In the XRD pattern of PCL-1, one additional peak appears at 2θ = 6.4°, which is attributed to the (002) plane of Ti 3 C 2 T X 20 . After the introduction of baicalin, the diffraction angle of PCL at 2θ = 11.8° becomes weaker and shifts to a lower angle, indicating that the physical interaction between PCL and baicilin is altered.
The water contact angles (WCA) of the membrane surfaces were measured to evaluate the hydrophilicity of the PCL-based nanofibrous membranes, as shown in Fig. 7. It is observed that PCL-0 has a hydrophobic surface with a WCA of 127.1° ± 1.3°, which is consistent with previous reports 40,41 . With the addition of Ti 3 C 2 T X nanoflakes, the WCA of PCL-1 shows a slight decrease (115.3° ± 5.2°) due to the abundance of −OH groups on the surface of Ti 3 C 2 T X . When baicilin was introduced into the PCL matrix, the surfaces of the PCL nanofibrous membrances shifted to hydrophobicity. The contact angles for the PCL-2, PCL-3, and PCL-4 are 82.4° ± 5.4°,   Antibacterial activities of PCL nanofibrous membranes. Baicalin is the main antibacterial constituent of PCL-based membranes, which has broad-spectrum antibacterial activity, especially against Gram-positive bacteria like S. aureus 42 . In this work, the MIC of baicalin against S. aureus was found to be effective at 32 mg/ ml, which is similar to the reported literatures 43,44 . All PCL-based nanofibrous membranes were selected for a standard antibacterial test by the preincubation plate pouring method. In Fig. 8, the bacterial colonies appear clustered around the PCL-0 and PCL-1 nanofibrous membranes after 72 h in the detailed pictures, and more remarkable clustered colonies are observed after 120 h. This is due to PCL having no antibacterial ability, which is consistent with previous report 45 . On the other hand, PCL-2, PCL-3, and PCL-4 show obvious bacteriostatic activity at 120 h incubation time (The medium dried completely after 120 h, and further inhibited bacterial growth). It can be ascribed to the presence of baicalin can interrupt the formation of α-heptamer, hindering the cell lysis activity of α-Hemolysin of S. aureus 46 . In addition, it is observed that the surrounding areas of PCL-3 and PCL-4 exhibit a light yellow color at 24 h, indicating the addition of Ti 3 C 2 T X nanoflakes contributed to the spread of baicalin from the nanofibers. That is because baicalin has poor solubility in aqueous solution 47 .   assays so as to evaluate the biocompatibility of the as-prepared PCL-based nanofiber membrances. It is usually to measure the cell density after the cells have been exposed to the nanofibers' leaching liquor for 48 h to confirm the cytocompatibility of the material. In Fig. 9, it is noted that all groups show no significant cytotoxicity at different concentrations. The cell viability of all test groups modestly declines as the concentration increases. The group with the lowest survival rate remains close to 100% at the highest concentration (5 mg/mL), which suggests that the PCL/Ti 3 C 2 T X /baicalin ternary nanofibrous membranes can be utilized as safe wound dressings.

Conclusions
In this paper, the electrospun membrances based on PCL/Ti 3 C 2 T X /baicalin ternary composites were prepared for wound dressing applications. The SEM observation showed that the presence of Ti 3 C 2 T X nanoflakes could decrease in the diameter of the nanofibers due to the increase in the charge density of the electrospinning solution. The PCL nanofiber membrances containing 3 wt% Ti 3 C 2 T X flakes and 5 wt% baicalin had the smallest mean diameter of 210 nm. The thermal stability of the composite nanofibers was improved due to the barrier effect of Ti 3 C 2 T X nanoflakes. The addition of Ti 3 C 2 T X and baicalin could enhance the hydrophilicity, contributing to the release of baicalin from the nanofibrous membranes. Furthermore, the addition of baicalin could endow the PCL/Ti 3 C 2 T X /baicalin ternary nanofibrous membranes with good antibacterial properties. The cytocompatibility test confirmed that all of the PCL-based nanofibrous membranes had good compatibility. The antibacterial PCL/Ti 3 C 2 T X /baicalin ternary nanofibrous membranes have great potential for wound addressing applications.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.