Facile construction of electrospun zein nanofiber loaded with Rana chensinensis skin collagen for wound care after caesarean section surgery

Numerous types of biomedical applications have found success using electrospun nanofibers. However, electrospinning techniques overlook Zein fibers favoring monolithic and coaxial fibers. Uniaxial electrospinning was used to construct Zein-structured nanofibers for wound healing, with the phase separation of electrospun polycaprolactone (PCL)/polyvinyl alcohol (PVA) in solution, providing the basis for the investigation. Successful loading of silver nanoparticles (AgNPs) and Rana chensinensis skin collagen (RCSCs) into Zein nanofibers (NFs) improved their bioactivity and antibacterial activity as wound dressings. Zein nanofibers were investigated for their heat conductivity, wettability, and mechanical characteristics. The cytotoxic effects of fabricated nanofibers were examined by using L929 fibroblast cells. Ag@RCSCs-NFs also increased cell migration and proliferation, and the wound scratch model was significantly reduced in size using an in vitro scratch assay. The antibacterial studies showed that the Ag@RCSCs-NFs have a potent antibacterial effect against the tested microbial pathogens (Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus)). Finally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PCL/PVP, RCSCs, and Ag@RCSCs against two different bacteria were determined. These results suggest that wound care using Zein nanofibers loaded with RCSCs and AgNPs during cesarean section surgery has great promise.


Introduction
The skin is the first of the body's functional biobarriers, protecting the internal organs from outside contaminants and regulating the body's water balance [1]. Daily living might present opportunities for even minor injuries to the skin. Skin injuries in humans may be less detrimental to health if they heal more quickly. Therefore, scientists are looking at ways to speed up the recovery process after skin wounds [2][3][4]. Today, the risks of different diseases brought on by a delay in skin wound healing are further reduced thanks to targeted drugs created based on the investigation of wound healing processes [5]. Internal medicine performed in reaction to inflammation induced by a wound, for example, has ancillary therapeutic roles in addition to its primary function of reducing inflammation [6][7][8]. Most wound care drugs are used externally, and this category of medicine includes liquid drugs, ointments, and dressings [9][10][11]. In addition to reducing swelling and pain, these medicines can function as a barrier against outside pollutants, safeguard wounds, and hasten their closure [12]. The topic of wound dressings has recently become a center of attention in wound healing research [13]. To be effective, a wound dressing must combine several properties, including physical protection, bacterial blockage, humidity maintenance, gas exchange without obstruction, moderate flexibility, and good biocompatibility [14].
Electrospinning is a cutting-edge area of study in biomedicine and the materials sciences [15]. More specifically, the high-voltage electrostatic field may be used to manufacture fibers with a wide range of mechanical characteristics, from microscale to nanoscale, and single-layer to multilayer structures can be gained by varying the design of the process and the choice of equipment [16][17][18]. The use of this material in wound dressings has garnered a lot of interest. Nanofibers made by electrospinning are widely utilized and investigated because of their high porosity, low weight, and huge surface area [19]. Electrospun nanofiber membranes provide outstanding specific surface areas, nanoscale porosity architectures, and mechanical strength [20][21][22]. To promote skin wound healing, this approach may be modified to use a variety of drug loading strategies, including embedding, adsorption, and mosaic, all of which can be acquired through the optimization of spinning equipment or post-processing [23].
Silver has been one of the most effective antibacterial drugs for many years. It has been employed with various natural and artificial polymers in composite form or as nanoparticles. In 1974, researchers began exploring silver sulfadiazine's potential as a drug for treating burn wounds [24]. Results showed that sulfadiazine alone was not very efficient against germs but was helpful when combined with silver. Researchers in 1982 looked at whether adding cerium salts rendered silver sulfadiazine ineffective. We infer that the inactivation of silver sulfadiazine was caused by adding cerium salt, bringing the inhibitory zone closer to the infected region [25]. The significance of silver nanoparticles as a potent antibacterial agent was briefly discussed. Electrospinning has led to a rise in silver utilization even though its use has decreased overall due to the availability of new antibacterial agents. Beneficial utilization of metallic nanoparticles is demonstrated in electrospinning [26]. In 2010, a nylon and silver composite membrane was created and described. The tensile strength of composite membranes was higher than that of nylon membranes. The antibacterial activity of nonwoven polyvinyl alcohol (PVA), chitosan (CS), and silver nitrate (AgNO 3 ) blends was synthesized and described [27]. It was shown that the blends exhibited more antibacterial activity than the individual components. Onestep electrospinning of nylon was used to include silver nanoparticles (AgNPs), which were then evaluated for antibacterial activity in addition to their morphological and surface features [28]. Besides that, AgNPs have also been employed in a wide variety of applications when combined with a wide range of natural and synthetic polymers, such as poly methyl methacrylate (PMMA), polyvinylpyrrolidone (PVP), Zein, polyacrylonitrile (PAN), chitosan, and others [29,30].
Natural polymer zein is derived from corn. It is a polymer that is both biodegradable and biocompatible. Because of its biocompatible and biodegradable qualities, Zein has been widely used in the biomedical industry [31][32][33]. The parameters that influence the structure and morphology of electrospun zein nanofibers were studied, and an ultrafine membrane of Zein was created via electrospinning. Nanofibers made of zein prolamine were also formed and analyzed for their thermal and morphological characteristics [34]. The antimicrobial properties of silver and zein composites were studied after they were prepared in two distinct pH ranges [35]. Incorporating Zein with other antibacterial drugs, such as cyclodextrins, eudragit, ketoprofen (KET), and ferulic acid, has proven beneficial in biomedical applications. In addition to its usage in controlled drug administration and tissue engineering, Zein has been incorporated with various natural and synthetic polymers [36][37][38][39].
The excellent nutritional value of Rana chensinensis, a prehistoric species found in northeastern China, has led to its widespread usage in the food and medicine industries. However, R. chensinensis wastes skin in significant amounts and becomes an issue [40][41][42]. R. chensinensis has several bioactive compounds in its skin that have considerable commercial potential. Several valuable substances, including polysaccharides and antibacterial peptides, have been isolated from the skin of abandoned Rana chensinensis [43]. Prior work involved the isolation and purification of collagen from the skin of the Rana chensinensis [44,45]. Complementary performance and synergy may be brought between collagen and Poly(L-lactide) (PLLA), a synthetic material that increases mechanical qualities and strong biodegradability and bioabsorbability. Collagen is commonly utilized in wound care because they speed up healing and enhance wound healing. For this work, we developed a multifunctional dressing for wound healing by combining Zein nanofibers generated by electrospun polycaprolactone (PCL)/polyvinyl alcohol (PVA) with RCSCs and AgNPs. We expected that the Zein structure nanofibers would increase wound healing in a full-thickness skin defect mouse model by releasing drugs continuously, promoting the L929 cells proliferation and adherence, and inhibiting bacterial development. . DMEM high glucose medium, phosphate-buffered saline (PBS, pH=7.4), 0.05% trypsin-EDTA, and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, NY, USA). All materials were used as received without any further purification. Deionized water (Millipore, 18 MΩ) was used to prepare the buffer and physiological media.
The morphological examination was performed using SEM apparatus Quanta 250 (FEI Company Inc., Thermo Fisher Scientific) at 500×magnification and 2.80 kV voltage. A light microscope Imager.M1 with a digital camera AxioCam MRc5 (Carl Zeiss, Oberkochen, Germany) was used for the diameter determination of a light microscope. UV-vis spectra were measured using a Shimadzu UV-3600 spectrophotometer to measure 200 nm and 800 nm absorbance wavelengths. Specific surface area and corresponding pore-size distribution of nanoparticles were measured by a Micromeritics Tristar 3000 system (Micromeritics, Norcross, GA, USA). TGA was carried out for each coating with a heating rate of 10°C min −1 up to 700°C using a SETARAM Labys SDT Q600 Simultaneous Thermal Analyzer device under the nitrogen atmosphere. Fourier transforms infrared (FTIR) spectroscopy (Nicolet iS10, Thermo Fisher Scientific). A total of 64 scans were taken and averaged from 4000 to 600 cm−1 with a resolution of 4 cm−1 for each spectrum. Cell viability at 570 nm by a microplate reader Synergy HTX Multi-Mode Microplate Reader, BioTek).

Preparation of Zein nanofibers
Uniaxial electrospinning was used to fabricate zein nanofibers based on a previously published process [45]. Briefly, 0.50 g of PVP and 0.50 g of PCL were immersed in a mixture of 8 g of chloroform and 2 g of dimethylformamide (DMF) and agitated at 30°C to achieve a homogenous solution (a). To make solution b, we first dissolved 1 g of PVP in 10 g of DMF and stirred the mixture for 4 h. To achieve solution c, 0.4 g RCSCs were added to solution a. In the dark, 0.2 g of AgNO 3 was added to solution b, yielding solution d. The AgNO 3 @RCSCs solution was prepared by thoroughly combining solutions c and d.
At ambient conditions, Zein nanofibers were electrospun. A schematic of the experiment's fundamental procedure is depicted in figure 1. After 20 h of stratification, the AgNO 3 @RCSCs solution was injected into a 10-mL plastic syringe. The working distance between the nozzle and grounding collector was 10 cm, and the supplementary voltage was 13-15 kV. Using the conditions, AgNO 3 @RCSCs Zein nanofibers were produced. Solution a was used to make PCL/PVP Zein nanofibers, whereas solution c was used to make RCSCs Zein nanofibers. For the in vitro analysis, all nanofibers were dehydrated for 2 days at RT under a vacuum.

Release of RCSCs
The following protocol investigated the RCSCs release from Ag@RCSCs-NFs in PBS solution at 37°C. Absorbance at 593 nm following the addition of Coomassie brilliant blue reagent was used to generate a standard curve, which was then used to calibrate the concentration gradient used in the RCSCs standard. The 5 mg nanofiber membrane was submerged in 3 ml PBS solution (pH 7.4) at 37°C. After a predetermined period, releasing solution (1 ml) was withdrawn and restored with fresh PBS. UV-Visible Spectrophotometry at 593 nm was used to determine the relative concentrations of the obtained solution with a normal curvature.

Cell proliferation assay
L929 fibroblast cells were maintained in a DMEM medium supplemented with 1% penicillin-streptomycin and 10% FBS and an atmosphere of 5% CO 2 and 95% air at 37°C. All cells were cultured on 75 cm 2 culture flasks, and when used for imaging, cells were sub-cultured on 35 mm glass-bottom culture dishes for 1-2 days to reach ∼80% confluence. For evaluating the cell proliferation of PCL/PVP, RCSCs, and Ag@RCSCs on L929 cells, cells were seeded into a 96-well plate (3×10 3 per well) and cultured for 1, 3, and 7 days. CCK 8-contained culture medium (10 μl CCK 8) was added to a 96-well plate and maintained for 3 h. Next, the absorbance of each group was detected [46][47][48].
Further, the cell proliferation was examined by acridine orange/ethidium bromide (AO/EB) staining. The different days' culture media was eliminated from the 96-well plates, and the AO/EB staining was added to the same well plates for 15 min in dark conditions. Finally, the stained cells were examined under the fluorescence microscope.

Wound scratch assay
A wound scratch assay was used in vitro to assess the ability of PCL/PVP, RCSCs, and Ag@RCSCs to heal wounds [49][50][51]. Briefly, L929 fibroblast cells were seeded with DMEM containing 10% FBS and 1% pen/strep in a 6-well plate at the cell density of 10 6 cells per ml and kept in a CO 2 incubator at 37°C. When the monolayer of cells covered the wells, a scratch was made with 200 μl pipette tips, followed by washing cells with FBS-free DMEM. Next, the wells added the sterilized solution of the required concentration of PCL/PVP, RCSCs, and Ag@RCSCs in DMEM (without FBS). A control group was also run in which cells were cultured in DMEM (without FBS) and left untreated. After being cultured for 0, 6, 12, 24, and 48 h at 37°C with 5% CO 2 , the images of cells were taken with a fluorescence microscope.

Antibacterial activity
The PCL/PVP, RCSCs, and Ag@RCSCs generated from aqueous solutions were tested against Gram-negative bacteria, Escherichia coli (ATCC: 25922) and Gram-positive bacteria, Staphylococcus aureus (ATCC: 29213) using the agar diffusion method. The bacteria (E. coli and S. aureus) were grown overnight, and of which 150 μl (∼10 7 CFU ml −1 of E. coli and ∼10 9 CFU ml −1 of S. aureus) were poured on the Luria-Bertani (LB) agar plates. Afterward, the circular nanofiber samples (diameter=6 mm) were placed on the agar plates and incubated at 37°C for 24 h. Then, the diameters of the inhibition zone were measured. All experiments were performed in triplicates [52][53][54].
Minimum inhibitory concentration (MIC) tests were carried out using 96-well plates. Each well contained 100 μl Mueller-Hinton broth. The stock solutions of PCL/PVP-NFs, RCSCs-NFs, and Ag@RCSCs-NFs were diluted, and 100 μl of each diluted solution was transferred into the first well of each row. Then, dilutions were performed to obtain final concentrations of 3.9-2000 μg ml −1 for pure curcumin and nanocomposites containing curcumin. PCL/PVP-NFs, RCSCs-NFs, and Ag@RCSCs-NFs were used at final concentrations of 13.76-7000 μg ml −1 .
The plates were then incubated at 37°C for 48 h after adding 100 μl of the bacterial suspensions to each well. MIC, which the sample's clarity may determine, is the lowest concentration of an antibacterial agent that prevents the development of bacteria visible under the microscope. Blood agar plates were used to culture wells without growth, and the MBC was then calculated by overnight incubation at 37°C. MBC is the lowest test substance concentration that decreases bacterial growth by 99.9%.

Characterizations of Zein-NFs
Uniaxial electrospinning methods effectively synthesized the PCL/PVP, RCSCs, and Ag@RCSCs-NFs. The NFs morphology was linear in the SEM images, with no beads or spindles (figure 2). Image-J analysis revealed that the size distributions of PCL/PVP-NFs, RCSCs, and Ag@RCSCs-NFs were 1012±115 nm, 1320±173 nm, and 912±98 nm, respectively. PCL/PVP, RCSCs, and Ag@RCSCs-NFs porosity is 22.54±3.24%, 17.48±5.21%, and 15.47±3.01%, respectively. The morphology of Ag@RCSCs-NFs was drastically altered upon the incorporation of AgNPs. Comparatively to PCL/PVP-NFs and RCSCs-NFs, the size distribution seemed more skewed, the porosity appeared somewhat lower, and the surface roughness appeared slightly greater. Adding Ag + causes a shift in crystallinity, which alters the conductivity and viscosity, providing a possible explanation for this result. Figure 2 demonstrates that Ag@RCSCs-NFs take on a crimped and grooved appearance after being immersed in water and dried, which may be attributable to the Zein nanofibers' unique surface chemistry. While PCL is very hydrophobic, PVP is highly hydrophilic. Nanofibers' specific surface area increased because the PVP component was progressively destroyed upon submerging in the water while the PCL remained unaltered, giving the fibers a hollow U-shaped structure. The surface shape of Ag@RCSCs-NFs, which is rough and has a high specific surface area, may facilitate adhesions with tissues through physicochemical and mechanical methods, making them useful for biological applications like wound healing. Consequently, the pace at which a wound heals may be tracked based on the physicochemical characteristics of the NFs scaffold, which can be tailored by adjusting the concentration of additive AgNPs. The Zein nanofibers in this study were electrospun in a uniaxial direction following phase separation in the solution. This differs from the side-by-side orientation employed in earlier investigations [42]. Since the two working fluids in side-by-side electrospinning linear contact and pumped out of side-by-side spinneret syringes, they can combine into Zein nanofibers may be hindered by their tendency to separate during the early stages of Taylor cone formation. To get around this problem, we presented a novel approach to fabricating Zein nanofibers. Uniaxial electrospinning following phase separation in solution is a novel method that allows the two working fluids to flow over a vast arched region, maximizing contact and preventing the fluids from being separated. In addition, the outside surface of the spinneret components can be arranged in a uniaxial configuration had a whole circular surface charged, which helped develop the Zein structure. This is because PCL is less dense than PVP due to gravity. The top PCL layer progressively covered the smaller PVP layer during electrospinning, producing a homogeneous Zein nanofiber. The study will intrigue finding the optimal concentration and volume ratios for PCL and PVP to create nanofibers with a Zein structure.
The connection and interaction between nanofiber membrane components were verified by FTIR spectroscopy. Figure 3(A) shows FTIR spectral analysis of the nanocomposites made from PVP, PCL, and RCSCs. PCL's spectra featured peaks attributable to C-O and C-C stretching at 1169 cm −1 and 1731 cm −1 and peaks attributable to asymmetric and symmetric -CH 2 stretching frequency (2859 cm −1 and 2949 cm −1 ). Several distinctive FTIR vibrational bands were displayed in PVP, confirming the presence of organic groups (figure 3(A)): at 1292 cm −1 (stretching of C-N), 1429 cm −1 , 1459 cm −1 (bending of -CH 2 ), 1661 cm −1 (stretching of C=O), and 2949-2878 cm −1 (stretching frequency of -CH or -CH 2 ). PVP's C=O stretching vibration (1657 cm −1 ) and PCL's C-C stretching vibration (1734 cm −1 ) explain the corresponding peaks in the FTIR spectrum of PCL/PVP-NFs ( figure 3(A)). Typical RCSCs peaks were observed at 3284, 1652, 1536, and 1397 cm −1 ( figure 3(A)) assigned to the amide bonds and -NH stretching vibrations. Spectral analysis of RCSCs-NFs and Ag@RCSCs-NFs showed the presence of all the typical bands of RCSCs, albeit with modest shifts, as shown in figure 3(A). The interaction between C=O and Ag+is evident in shifting the PVP absorption peak from 1657 to 1663 cm −1 assigned to Ag@RCSCs-NFs.
PCL/PVP-NFs, RCSCs-NFs, and Ag@RCSCs-NFs are depicted in figure 3(B), along with their respective TGA curves. The initial phase of weight loss (10.22%) for the PCL/PVP-NFs occurred between 40°C and 110°C. Weight loss of 2.04% and 4.07% occurred between 40 and 80°C for RCSCs and Ag@RCSCs-NFs. The solvent (DMF and DCM) on the nanofiber film surfaces and insides were responsible for these losses. The second phase of weight loss for the PCL/PVP-NFs, at 280°C-460°C, was attributed to the breakdown of PCL and PVP due to the breaking of −CH 2 , C-N, and C=O bonds. At temperatures between 460 and 580°C, the carbides of PVP and PCL underwent further oxidative breakdown, resulting in a 9.88% weight loss. At temperatures between 50 and 190°C, the initial stage of weight loss (6.84%) for RCSCs. In another phase, which took place between 190 and 479°C, RCSCs organic moieties were carbonized. The carbide underwent additional oxidation and decomposition in the third stage, between 470°C and 580°C. RCSCs and Ag@RCSCs-NFs had weight loss curves comparable to those of PCL/PVP-NFs. As a result of the presence of RCSCs and AgNPs, a second stage was observed between 280°C (weight loss of 72.04%) and 440°C (weight loss of 65.11%) for RCSCs and Ag@RCSCs-NFs. The third stage was detected between 440°C and 580°C. This data demonstrated that the structured NFs could be reliably retained in these temperatures. When heated over 440°C, organic nanofiber films decomposed and carbonized, contributing to a rapid mass loss. The outcomes showed that all three composite nanofiber materials (PCL/PVP, RCSCs, and Ag@RCSCs ) performed well thermally.
DSC was also used to assess the nanofibers' thermal efficiency. The differential scanning calorimetry (DSC) curves from 20°C to 700°C for PCL/PVP-NFs, RCSCs-NFs, and Ag@RCSCs-NFs are shown in figure 3(C). The glass transition temperature (Tg) of pure RCSCs was determined using DSC to be 190.4°C ( figure 3(C)). This might be attributed to the evaporations of balance moisture inside the RCSCs. 62.35°C (Tg) and 564.87°C melting temperature (Tm) were calculated from the DSC curve of PCL/PVP-NFs ( figure 3(C)). Additionally, the Tm of RCSCs-NFs decreased to 545.74°C without any discernible difference in Tg. Two distinct peaks could be shown in the Tm of the Ag@RCSCs-NFs, at 395.41°C and 535.49°C, respectively ( figure 3(C)). The PCL/PVP Tm was located at the higher temperature peak, whereas the other peak was associated with another phase resulting from a mechanical change following encapsulation. The electrospun nanofibers of RCSCs and Ag@RCSCs were combined with PCL/PVP, such their melting peaks lie between these molecules, demonstrating compatibility.
Nanofibers' mechanical qualities, such as their strength and flexibility, are crucial to their widespread use. Tensile stress, shown in figure 3(D) as a function of strain (%), was used to compare the PCL/PVP, RCSCs, and Ag@RCSCs-NFs. Scaffolds' physical properties may impact cell behavior and tissue regeneration. There was no variation in the highest tensile strength between the PCL/PVP, RCSCs, and Ag@RCSCs-NFs (4, 3.79, and 3.82 MPa, respectively). In addition, the ultimate tensile strain of PCL/PVP-NFs was 29%, whereas that of RCSCs-NFs was 148%, and that of Ag@RCSCs-NFs was 58%. Improving the maximum tensile strain of fiber may benefit from incorporating RCSCs, whereas comprising AgNPs may have unintended consequences. Human skin has ultimate tensile stress and strain of between 1 and 32 MPa and 35 and 115%, respectively. Therefore, the composite Ag@RCSCs-NFs developed in this work might be used for wound dressings due to their favorable mechanical characteristics.
A dressing's wettability determines how well it absorbs exudate and how well it keeps a wound moist as it heals. The wettability of PCL/PVP, RCSCs, and Ag@RCSCs-NFs was characterized in this work using the contact angle. The composite membrane of PCL/PVP-NFs absorbed the water droplet in less than 10 sec due to the vinyl and polyamide groups of PVP, which shows excellent hydrophilicity of the membrane. The wettability of RCSCs-NFs was lower than that of PCL/PVP-NFs, possibly because of the larger nanofiber diameter and reduced porosity that resulted from the incorporation of RCSCs. Equated to PCL/PVP-NFs, Ag@RCSCs-NFs had a reduced wettability. Additionally, the Ag@RCSCs-NFs maintained strong hydrophilicity, measured by a contact angle of less than 90°, promoting cell attachment. While the Ag@RCSCs-NFs could fully absorb water thanks to the hydrophilicity of PVP, the fibers mats remained insoluble.

Release of RCSCs
The RCSCs absorbance at 595 nm was monitored over time to examine the in vitro release profile of the RCSCs from Ag@RCSCs-NFs in PBS, and the cumulative release rate was computed. Figure 4 displays the dual-phase release profile of RCSCs-loaded composite nanofibers, consisting of an early burst release followed by a subsequent, delayed nonlinear release. After 35 min, the cumulative release ratio had nearly surpassed 100%, at 95.04%. For the incubation of an initial 5 min, surface drug release from the fiber occurred at 34.97%. Drugloaded fibers scaffolds frequently exhibit burst release, which may be traced to the drug's first escape from the surface of the fiber before the polymer's erosion mediates its release. Solubility of the drug, as well as transport properties and composite shape, had a role in the release rate.
Our system's initial explosion was caused by a concentration of RCSCs molecules on the surfaces of the fibers. The remainder of the drug molecules were sequestered at specific locations inside the nanofiber scaffold. Degradation features of the polymers were linked to the process of this another stage, which involves the breaking down scaffold and molecular links. Hydrophilic PVP dissolves slowly over time, allowing RCSCs to leak out of the scaffolds. The drug's water solubility and the nanofiber scaffolds' permeability to water molecules are other essential factors to consider. A-concentrations slope formed among the outside and inside of the NFs scaffold, limiting the delivery of the drugs. The nanofiber-based scaffold technology in this investigation suggested a gradual diffusion release mechanism for the RCSCs.

Cell proliferation
The biocompatibility of the different samples was evaluated using a cell proliferation examination. The proliferation of PCL/PVP, RCSCs, and Ag@RCSCs-NFs was measured using the cell proliferation test CCK-8. Figure 5(A) shows that after the first 24 h, L929 cells could grow appropriately on all the evaluated nanofiber scaffolds, with cell viabilities of 94.68%, 95.12%, and 94.02% for the PCL/PVP, RCSCs, and Ag@RCSCs-NFs, respectively. After 3 days, the PCL/PVP-NF showed great cell vitality (96.95%) compared to the untreated (control) group. In contrast, the RCSCs and Ag@RCSCs-NFs showed slight declines in cell viability compared to the control (83.02% and 78.86%, respectively). Cell viability might be impacted by releasing RCSCs and AgNPs into the culture medium. After 7 days, the cell viability of the PCL/PVP-NFs was 91.94%, that of the RCSCs was 82.57%, and that of the Ag@RCSCs-NFs was 76.95%. Calcein-AM/PI staining of fluorescence microscopy images of L929 fibroblasts supported the results of the CCK-8 experiment, showing normal cell proliferation within 7 days ( figure 5(B)). AgNPs were crucial in developing effective antibacterial materials with their unique combination of physical and chemical characteristics. Furthermore, AgNPs' high oxidative activity led to the silver ions release, which had many harmful impacts on cell viability. Since AgNPs' cytotoxicity is dosedependent, particle concentration played a significant role in determining their effect on cells. To concurrently enhance bactericidal activity and cell survival.

Wound scratch assay
The easiest way to demonstrate the ability of drugs, molecules, or nanoparticles to heal a wound is through a scratch assay. As a result, an in vitro scratch test was used to establish the ability of PCL/PVP, RCSCs, and  Ag@RCSCs to repair wounds. The findings demonstrated that, compared to the control, the proliferation and migration of cells in the artificial wound region were much more significant when treated with PCL/PVP, RCSCs, and Ag@RCSCs ( figure 6). While the samples aided fibroblast migration, Ag@RCSCs restored cellular density more quickly than the rest of the samples. The percentage of scratch shrinking for the samples treated with PCL/PVP, RCSCs, and Ag@RCSCs was much more significant than for the untreated control, as shown in figure 6. A prior work that used collagen/chitosan scaffolds loaded with silver nanoparticles found comparable outcomes. It was shown that by utilizing an in vitro scratch assay, the silver nanoparticle-laden collagen/chitosan scaffolds promoted fibroblast migration. Combined, our findings lay the groundwork for further exploiting Ag@RCSCs promising antibacterial efficacy in wound healing applications.

Antibacterial activity
The antibacterial activity of PCL/PVP, RCSCs, and Ag@RCSCs-NFs was examined using S. aureus and E. coli. Several studies have examined Ag@RCSCs-NFs' ability to defend against various pathogenic microorganisms [55]. Gram-positive and Gram-negative pathogenic microorganisms of importance to public health were both significantly inhibited by the OPE-AgNPs ( figure 7). Ag@RCSCs-NFs demonstrated antibacterial action against E. coli and S. aureus, as shown in figure 8. Antibacterial activity of the Ag@RCSCs-NFs on the microorganisms E. coli and S. aureus has been shown in figure 8. It shows significant growth inhibition of bacterial culture by Ag@RCSCs-NFs concerning the control. The antibacterial activity in the case of E. coli indicates that 1 mg mL −1 concentration of Ag@RCSCs-NFs inhibits bacterial growth after 9 h. In the concentration of 4 mg mL −1 , growth inhibition starts after 3 h concerning the control culture. In the case of S. aureus, all the concentrations of Ag@RCSCs-NFs retard the growth at the same time interval. But in the case of S. aureus, there is a significant decrease in growth after 4 h in the highest concentration of Ag@RCSCs-NFs (4 mg mL −1 ). Accordingly, S. aureus shows growth inhibition approximately after 12 h in all the attention in the control experiment.
The antibacterial activity of PCL/PVP-NFs, RCSCs-NFs, and Ag@RCSCs-NFs against different bacteria was analyzed. Ag@RCSCs-NFs showed a higher antibacterial effect than PCL/PVP-NFs and RCSCs-NFs. Briefly, in the cases of E. coli, MIC values for Ag@RCSCs-NFs were 4 and 14 times lower compared to PCL/PVP-NFs, and RCSCs-NFs, respectively. In addition, in S. aureus, MIC values were 2 and 14 times more down when Ag@RCSCs-NFs was used to compare PCL/PVP-NFs, and RCSCs-NFs, respectively. MIC values of Ag@RCSCs-NFs against all bacteria were lower than those of PCL/PVP-NFs, and RCSCs-NFs (table 1).
Ag@RCSCs-NFs also showed the highest antibacterial effect against Staphylococcus aureus with a MIC value of 15.62 μg ml −1 . MBC values of this Ag@RCSCs-NFs against all bacteria were stable at 500 μg ml −1 . That was higher than the MBC values of PCL/PVP-NFs, and RCSCs-NFs against E. coli. According to the results, the preparation of Ag@RCSCs-NFs improved the antibacterial efficacy of PCL/PVP-NFs and RCSCs-NFs on the tested bacteria. The necessity for innovative approaches (such as discovering new antibacterial medications or utilizing novel drug delivery technologies to improve therapeutic efficiency) to combat these resistances and cure infectious diseases has expanded dramatically over the past several years.

Conclusion
Nanofibers with a zein structure were developed using uniaxial electrospinning for use in wound healing. After phase separation, the solution formed a robust interface in the uniaxial electrostatic spinneret, which allowed it to endure jet instability and electrical repulsion during electrospinning. Excellent mechanical properties, wettability, effective biocompatibility, and antibacterial properties to encourage wound healing were observed after loading with RCSCs and AgNPs in the Zein NFs. The unique Zein-structured NFs generated by uniaxial electrospinning is a potential nanofiber for wound care applications during surgery for patients. Ag@RCSCs-NFs also increased cell migration and proliferation, and the wound scratch model was significantly reduced in size using an in vitro scratch assay. Finally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PCL/PVP, RCSCs, and Ag@RCSCs against two different bacteria were determined. These results suggest that wound care using Zein nanofibers loaded with RCSCs and AgNPs during cesarean section surgery has great promise.

Data availability statement
No new data were created or analysed in this study.

Author contributions
The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript.
Disclosure statement