Novel fabrication and development of multifunctional Zn/Fu@Cs nanofibers material for wound care and operate room infection control

Treatment of operate room wounds infections is a clinically more challenging process. Therefore, several techniques and wound care materials have been taken to improve wound healing mechanisms. The present study is mainly focused on zinc oxide and fucoidan-loaded chitosan nanofiber scaffolds fabricated by the electrospinning method. The functional groups, morphology, and hydrothermal stability of fabricated Zn/Fu@Cs were observed and investigated by microscopic and some spectroscopic techniques. The wound healing potential of Zn/Fu@Cs nanofiber has been evaluated by various in vitro biological experiments. In addition, the prepared nanofiber showed suitable bacterial growth inhibition against P. aeruginosa, B. subtilis, S. aureus, and E. coli wound infecting bacteria and in vitro studies confirmed the excellent cell proliferation, and cytocompatibility. In vitro study exhibited significant cell proliferation and viability is observed in Zn/Fu@Cs nanofiber treated L929 cells within 3 days, which is comparable to the control it is higher. In wound scratch assay the wound healing efficiency has been monitored on the human skin fibroblast L929 cell line. The wound scratch experiment results revealed that the Zn/Fu@Cs nanofiber shows quick cell regeneration without bacterial infections. The biodegradation study concluded the biocompatibility of nanofibers under physiological condition. The overall results suggest that the Zn/Fu@Cs nanofiber is a potential material for wound care with enhanced antibacterial property against operate room pathogens.


Introduction
Wound healing without operate room infection is the biggest medical challenge in patients. which increasing risk in health and socio-economic status. Wounds are painful hurdles encompassed with the extent of chronic inflammation and bacterial infection [1,2]. These non-healable operate room wounds infections lead to repeated hospitalization and severe damage to an organ [3][4][5]. The factors such as excess secretion of degrading enzymes, bacterial infection, and poor vascularization mechanism of epithelial cells play important roles. Ideal therapeutic techniques and innovative materials are necessary to wound healing and facilitate the regeneration of affected tissues [6][7][8]. Nanoparticles, nanogels, bandages, and nanofibrous coatings are common options for prolonged wounds. Nanofiber mats composed of marine products and metal nanoparticles can be helpful for wound dressings because of their medicinal value and bioactivity [9][10][11][12][13]. Marine products were renowned as a promising resource for agrochemicals, cosmetics, and biomedical applications. Especially, seaweeds, such as green, red, and brown algae species, can produce various biomolecules and they have a broad spectrum in biomedicine [14][15][16]. For about more than 2000 years of human evolution, marine algae have great impact on traditional medicine. In addition, it was used traditionally to treat cancer, cardiovascular diseases, ulcer, asthma, and psoriasis. The therapeutic effects have been explained and approved by employing pharmacological experiments, such as antimicrobial, anticancer, anti-viral, and neuroprotective activities [17][18][19][20]. Some reports show the biological activity of marine sulfated polysaccharide fucoidan in anti-oxidant, immunoregulation, and anti-coagulant effects. In this context, the marine compound fucoidan has been used in a variety of biomedical applications because of cytocompatibility and bioactivity. Although fucoidan-based hydrogels are used in wound dressings, the combination of nanofiber with metal oxide promotes wound healing in a variety of ways [21][22][23][24]. Due to functional qualities such as water uptake capacity, maintaining a moist, adhesive property, nontoxicity, and superior mechanical strength, polymers are more easily applied for wound treatments. Chitosan is a natural polymer derived from marine species with considerable anticancer, antioxidant and antimicrobial capability [25][26][27][28]. It has been reported as a harmless agent for tissue regeneration, and slow delivery of bioactive substances. Chitosan is an effective wound healing material as it can increase fibroblast multiplication and macrophage migration. During wound healing, chitosan stimulates fibroblast proliferation, collagen formation, and hyaluronic accumulation in the wound area. Chitosan-based electrospinning nanofibrous nets possess huge potential for wound healing [29][30][31][32]. Zinc plays a prominent role regulatory phases of wound healing process and tissue re-epithelialization mechanism. In previous clinical trials zinc oxide enhance the cell replication and migration. The anti-inflammatory property of zinc leads the edges of wounds. Food and drug administration (FDA) has registered zinc oxide as a safe material for biomedical uses. We aim to discover a multifunctional wound healing agent for wound care without operate room infections. Doping of fucoidan with chitosan and zinc blended nanofibers to improve the mechanical and biological properties of composite nanofibers. It helps in the formation of uniform nanofibers and biodegradability [33][34][35][36][37][38]. In addition, the incorporation of fucoidan with chitosan/zinc nanofibers enhances the cell viability and proliferation of fibroblast cells. Electrospinning nanofibers made by chitosan scaffolds have excellent potential in skin tissue regeneration and wound healing applications [39][40][41][42]. Therefore, we aimed that the electrospinning nanofiber scaffolds of Zn/Fu@Cs will be a promising agent for wound care and may be used in the future for post cesarean wound care. In this study, we fabricated Zn/Fu@Cs nanofibers scaffold for wound healing process mediated antibacterial activity. The results of current research manifested that Zn/Fu@Cs nanofiber can improve the healing efficacy. And the high degree of wound closure was enhanced by fucoidan incorporation with Zn/Cs nanofibers scaffold could be an efficient antibiotic agent for wound care and operate room infection control.

Electrospinning synthesis of Zn/Fu@Cs nanofibers
One gram of chitosan powder was dissolved in 10 ml of acidic acid, and the chitosan solution was agitated with a magnetic stirrer to produce a homogenous chitosan solution [39]. Then after, 0.1M of zinc nitrate hexahydrate was steadily added to the chitosan solution while being agitated magnetically. This mixture was held at room temperature with continuous stirring for 2 h. A brownish-white colloid was obtained. Furthermore, 20 mg of fucoidan was dropped into the Zn/Cs colloid at ambient temperature. Finally, the polymeric mixture colloid was poured into a 15 ml syringe. The resulting polymer reaction mixture was then used to create nanofiber by vertical type electrospinning techniques at room temperature. The nanofiber collector was covered with aluminium foil at a distance of 15 cm, and a voltage of 15 kV was provided during the electrospinning process.

Characterization techniques
The crystalline structure of synthesized Zn/Fu@Cs nanofiber was analysed by Powder X-Pert PRO X-ray diffractometer at 40 kV using Cu-Kα radiation (λ=0.15418 nm) with scan rate 0.01 s step, and scan range 10-90 degrees. The surface morphology of the nanofiber scaffold was examined, and images were taken by using FE-SEM (Carl Zeiss) microscopy with sputtering time 10 min and voltage 30 kV. Functional groups of Zn/ Fu@Cs nanofibers identified Jasco (Model-FT/IR-4100), FTIR spectroscopy with wavenumber range of 400-4500 cm −1 . The thermal stability of nanofiber was analysed by thermogravimetric analysis (TGA) (Pyris 1, Perkin Elmer, USA) under a nitrogen atmosphere. The samples were excited 50°C-800°C at a heating rate of 10°C min −1 . The water contact angles of Zn/Fu@Cs nanofiber were measured using a contact angle measurement instrument (SmartDrop, Femtobiomed, Korea).

Encapsulation efficiency of Zn/Fu@Cs nanofibers
The encapsulation efficiency of nanofibers was measured according to previous method. Initially, 10 mg of fucoidan was dissolved in ethanol and the characteristic absorbance of fucoidan was identified UV-vis spectroscopy at the range of 200-800 nm (T60 UV-Visible spectrophotometer, USA). The maximum absorbance of fucoidan was observed at 312 nm. About 10 mg of Zn/Fu@Cs nanofiber was dissolved in with 5 ml ethanol under slow magnetic agitation at 120 rpm speed for 15 min. Then, the nanofiber suspension was filtered by using syringe filter (0.45 μm pore size) and the absorbance of solution was measured at 312 nm single wavelength. The bare Zn/Cs nanofibers without fucoidan considered as a reference. Total percentage of fucoidan loading were quantified from the standard calibration curve of fucoidan. The encapsulation efficiency (EE) was performed by following equation: where Ce and Ct are the concentration of loaded fucoidan and the initial concentration of fucoidan in the nanofibers respectively.

Evaluation of the antibacterial activity of Zn/Fu@Cs nanofibers
The bactericidal potential of prepared Zn/Fu@Cs nanofibers was investigated against wound infecting bacteria Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis. The efficiency of bacterial inhibition was quantified by a Well diffusion assay [40]. Bacterial strains were grown overnight culture in nutrient agar. A total of 25 ml of muller Hinton agar was poured into bacteriological Petri dishes. After the solidification 5 X, 5mm holes were made in the bottom of the plat. The overnight cultured bacterial strains were swabbed on the surface of the agar medium by sterile swabbing puts. The different concentrations of Zn/Fu@Cs nanofibers were loaded on the wells. Finally, the nanofiber-treated Petri dishes were incubated at 37°C for 12 h. After the incubation, the zone of bacterial inhibition was measured by a zone scale.

Assessment of cell viability by using MTT assay
The cytotoxicity and cell viability of Zn/Fu@Cs nanofibers were quantified by MTT assay [41]. The human fibroblast L929 cells were cultured in a DME medium under the CO 2 flow at 37°C. After 48 h of culture, the media was discarded from the wells using PBS solution. Then 100 μg ml −1 concentration of nanofiber was loaded into 12-well plates each well contain 1ml of media and L929 cells. The treated cells are allowed for 24 h of incubation for cell proliferation. Then, the treated cells were stained using MTT (0.5 mg ml −1 ) and incubated for four hours. The excess MTT stain was removed from the treated cells by using a DMSO solution. The total percentage of cell viability was quantified at different time intervals (24, 48, and 72 h) after cell seeding. Finally, the percentage of cell viability rate was measured by using a microplate reader at 570 nm under a triplicates experiment.

= -Ć T%
Absorbance of control Absorbance of treated Absorbance of control 100

Assessment of apoptotic morphological changes in nanofibers treated cells
The AO/EtBr dual staining approach [42] was used to assess the cytotoxic effects and morphological changes of Zn/Fu@Cs nanofibers treated fibroblast cells. L929 cells were sown and cultured for 48 h at 37 degrees Celsius under CO 2 flow. After incubation, AO/EtBr dual stains (20 g ml −1 ) were applied for 10 min to well-containing nanofiber-treated cells. Centrifugation was used to remove excess dye from treated cells. Fluorescence microscopy revealed cell nuclear damage and morphological alterations. ROS generation and nuclear deformity were detected using a fluorescent microscope, and photos were captured. Image J software was used to calculate the fluorescence intensity of treated and untreated cells. The percentage of cell migration rate was calculated by the fluorescence intensity of translated cells and cellular DNA count.

In vitro wound scratch assay
On L929 human fibroblast cells, the wound healing efficacy of Zn/Fu@Cs nanofibers was investigated [42]. The Fibroblast cells cell line was seeded in a 12-well plate with 2 ml of DME media and cultured at 37°C for 24 h. Following the incubation time, wells were scratched with 200l micro tips and a 100 g ml −1 dose of Zn/Fu@Cs nanofibers was added. Without the addition of nanofiber, this is termed a negative control. Under an inverted phase contrast microscope, cell multiplication and migration were detected. The amount of cell regeneration was calculated at various time intervals of 24, 48, and 72 h. All tests were carried out in triplicate. T232 Scratch software and Image J have been used to measure the migration rate of treated cells.

= -Ẃ ound closure%
A0 At At 100 Where, A0=0 h is the average area of the wound after scratching (time zero), and At=Δh is the average area of the wound measured at different hours after the scratch.

Statistical analysis
GraphPad Prism was used in the statistical analysis. The mean standard deviation is used to calculate mean values. Statistical analysis was performed using one-way ANOVA software, at p < 0.05, considered the differences were significant. Diameter of the nanofibers were measured by using Image J software about 30-60 random points for each sample.

Results and discussions
3.1. XRD and FTIR analysis Through X-ray powder diffraction and FTIR spectroscopy method, crystalline structure and characteristic chemical bonding of the Zn/Fu@Cs nanofiber were investigated. Figure 1

Thermogravimetric and UV-vis spectroscopy analysis
Thermal stability of fabricated Zn/Fu@Cs nanofibers was examined by using TGA analysis. The thermograms and their first-order derivative curves are shown in figure 2(B). It found that the pure chitosan and fucoidan started the decomposition at 190°C and completely decomposed at 430°C. The bare zinc oxide nanoparticles showed a high level of thermal stability until 450°C with 12 % weight loss in total mass. In this context, Zn/ Fu@Cs nanofibers is stable until 200°C, gradual decomposition starts at 210°C. The presence of zinc oxide provides thermal stability to chitosan fibers. The decomposition of fucoidan and chitosan biopolymer leads to weight loss events in Zn/Fu@Cs nanofibers. The zinc oxide nanoparticles enhanced the stability of nanofiber under thermal conditions. The first stage was a weight loss of approximately 10 % between 180°C-210°C a continuous weight loss appeared until 750°C. These results concluded that the fabricated nanofiber is highly suitable for diabetic wound healing applications. The surface plasmon resonance of fabricated nanofiber was examined by UV-vis spectroscopy ( figure 2(A)). The results in UV-vis spectra encapsulation of fucoidan and chitosan showed a sharp absorbance at 312 and 322 nm. The pure ZnO nanoparticles show the characteristic peak at 372 nm. On the contrary, Zn/Fu@Cs nanofibers shows fucoidan and zinc-related peaks in 320 nm and 378 nm ranges. This absorbance peak shifting indicates the encapsulation of fucoidan within the Zn/Cs nanofiber at simultaneous fabrication. The encapsulation of fucoidan molecules suppressed the absorbance intensity of chitosan molecules and ZnO nanoparticles. The simultaneous encapsulation of fucoidan with nanofibers enhanced the drug loading capacity. The entrapment efficiency of nanofibers was calculated to be 95%. The percentage of drug entrapment calculated from comparison of standard calibration curve of fucoidan. The TGA and drug loading studies revealed that, the Zn/Fu@Cs nanofibers are more stable and efficient material for wound healing application.

Surface morphology and size assessments
In biomedicine, nanofibers have many advantages, such as the transport of anti-inflammatory, antibacterial, and anticancer medications. Strategies of tissue transplanting and wound repair incorporated with nanofibers. Compare to existing fibrous materials, nanofibers allow rapid response for accurate delivery of remedial medicines during the wound healing process. Hydrophilic and hydrophobic compounds encapsulated into nanofibers are employed in healthcare [45]. The targeted molecules are entrapped inside nanofiber frameworks by electrospinning. Nanofibers could be enhanced by trapping a variety of medicinal agents. The architectural characteristics of a fabricated Zn/Fu@Cs nanofibers utilized in this experiment presented evidence that target biomolecules were encapsulated within the nanofiber matrices. SEM images of fucoidan encapsulated Zn/Cs nanofiber and bare zinc/chitosan nanofibers are shown in figure 3. The pure Zn/Cs nanofibers show smooth and uniform fibrous matrices. There are no other particulate matters and droplets appeared on the nanofiber surface. The average diameter of Zn/Cs is 72±5 nm ranges ( figure 4(A)). In addition, fucoidan encapsulated ZnFu@Cs nanofibers are distributed almost uniformly in nanofiber formation. However, some aggregates and irregular shapes appeared in the intersections. In order, the encapsulation of dual molecules zinc and fucoidan enlarged the nanofiber size and leads to morphological disabilities (figures 3(C) and (D)). The average diameter of Zn/Fu@Cs is 86±3 nm. Fucoidan encapsulation increased the size of Zn/Fu@Cs nanofibers ( figure 4(B)).

Antibacterial activity of Zn/Fu@Cs nanofibers
Control of bacterial infection in wounds is the most challenging process. It is leading to many combined difficulties in wound healing. Therefore, we need an efficient antibacterial and wound care material for post-surgical wound. The antibacterial potential of Zn/Fu@Cs nanofibers was evaluated against different species of human infectious pathogens. The zone of bacterial inhibition against four microorganisms in the well diffusion assay was shown in figure 5 at different concentrations. The good diffusion assay results revealed that the nanofiber-treated pathogens show more than a 14 mm diameter zone of inhibition at 100 μg ml −1 concentration. The highest level of bacterial inhibition 16 mm diameter appeared in E. coli, S. aureus, and P. aeruginosa treated groups ( figure 6(A)). The synergistic effects of zinc and fucoidan molecules generated ROS affect the bacterial membrane, and protein and finally lead to bacterial cell death [46,47]. Another mechanism is chitosan a positively charged biopolymer, the amino groups of chitosan can form electrostatic interactions with the electronegative microbial cell wall and induce permeability of Zn/Fu@Cs  nanofiber molecules and metabolic disturbances. The detected antibacterial experiment results confirmed that fucoidan encapsulated Zn/Cs nanofibers have excellent antibacterial efficiency against human wound infecting bacteria, due to the synergistic effect of fucoidan and zinc oxide could efficiently support the wound healing process.   6(B)). The higher rate of biodegradability of fucoidan encapsulated Zn/Cs nanofibers is due to the release of zinc ions and fucoidan molecules from nanofibers [48]. The final results revealed that Zn/Cs and Zn/Fu@Cs nanofiber samples were biodegradables. Due to the lysozyme enzymatic process and the hydrophilic properties of biopolymers.

Cell Proliferation efficacy of Zn/Fu@Cs nanofibers
Fibroblast migration in wound sites is the most important mechanism of healing and cell regeneration. Migration of fibroblast depends on the interaction of specific molecules in the matrix. The accumulation of fibroblast and collagen matrix promotes the morphological changes in the wound site [45,49]. In this cell proliferation study, the cell proliferation experiment was obtained by using Zn/Fu@CS nanofiber at 100 μg ml −1 concentration in different time intervals (24,48, and 72 h) alone with Zn/Cs nanofiber considered as a control. The cell growth rate of Zn/Fu@Cs nanofibers is higher than Zn/Cs nanofibers ( figure 7(A)). The release of fucoidan leads to the enzymatic actions in the fibroblast matrix. Chitosan-based nanofiber matrix provides a suitable substrate for osteoblast and fibroblast proliferation for tissue culture. The cell proliferation percentage of Zn/Fu@Cs treated cells shows a significant increase in 24 h incubation. Compare this to the first day's threefold increase on the third day after seeding.

Assessment of biocompatibility of Zn/Fu@Cs nanofibers
The MTT cell viability assay is a method for understanding the cytotoxicity of nanomaterials. The live and dead cell stains AO/EtBr was used for preliminary assessment of the biocompatibility of nanofibers. This MTT test results revealed that Zn/Fu@Cs nanofiber treated L929 cells show a mild toxic effect at 100 μg ml 3.8. In vitro wound healing potential of Zn/Fu@Cs nanofibers Cell migration and multiplication is the most prominent mechanism for tissue regeneration in wound healing. The in vitro cell scratch assay is a reliable mimic form of cell migration rate evaluation. When the cell monolayer is damaged, cell-to-cell interaction and cell signals will be lost. Stimulation of cytokines in the wound site increases cell proliferation and migration [29,35,40]. The cell proliferation and migration potential of Zn/ Fu@Cs nanofibers-mediated L929 cells were quantified from 24, 48, and 72 h post treatment. Interestingly, our Zn/Fu@Cs nanofibers efficiently increase cell migration and cell proliferation at 100 μg ml −1 concentration. The inverted phase contrast microscopic images of scratched cells figure 9 shows the high level of cell proliferation and cell moving in 42 h of incubation. The release of zinc and fucoidan promotes the mitogenic effect among scratched cells and leads to the wound healing process. Cell migration rate was analyzed by using ImageJ software. The viable cell migration has been gradually increased in Zn/Fu@Cs nanofibers treated group at 24 h. Compare to the control group nanofiber-treated cells showed a three-fold higher amount of cell  proliferation and movements. Granulation of tissues is the main process for wound healing. In this case, Zn/ Fu@Cs nanofibers influences the granulation in wound sites compared to the control group. The final in vitro scratch assay results profound that fabricated nanofiber matrix acts as a promising bioactive material for wound care applications.

Swelling index of Zn/Fu@Cs nanofibers
The swelling index plays a vital role in drug release and entrapment of the nanofibers. Figure 10(B) shows the swelling degree of fucoidan-loaded Zn/Fu@Cs and bare Zn/Cs nanofiber patches at different periods. The release of the drug depends on the swelling degree of nanofibers. The swelling index results revealed that the bare Zn/Cs are higher than fucoidan-loaded Zn/Fu@Cs nanofibers at an initial time of 4 h. This higher degree of swelling corresponds to the large surface area and pore size of Zn/Cs nanofibers. In this case, fucoidan-loaded Zn/Fu@Cs nanofiber shows decreased level of swelling index, due to the interaction of fucoidan molecules restricting the entry of water to swelling. However, after 4 h the swelling of Zn/Cs and Zn/Fu@CS both nanofibers decreased swelling degree in all stages. The drug-loaded polymeric nanofiber shows a slow erosion rate, which could be attributed to a net decrease in water flux in the presence of a hydrophobic drug in the drugloaded composite nanofiber. This decreased level of swelling is due to the degradation of polymer and water molecules by dissolution. Finally, Zn/Fu@Cs nanofibers have a high degree of swelling due to the slow dissolution of the polymer molecules over time due to erosion. Further, the hydrophilicity of Zn/Fu@Cs nanofibers was significantly creased after fucoidan encapsulation. The average contact angle of bare Zn/Cs nanofiber was 98±6.5°, in this case the contact angle of the Zn/Fu@Cs nanofibers was reduced to 47±5.3°( figure 10(A)). A low contact angle of fabricated Zn/Fu@Cs nanofiber implicit increased hydrophilicity of the nanofiber surface, which promotes the fibroblast cell adhesion and viability [50]. This study confirmed that fabricated nanofiber more stable and controlled drug-releasing material and it is more suitable for wound healing applications.

Conclusions
The multifunctional biocompatible nanofibers are fabricated by a simple elect spinning method. It showed great effects and different phases in the wound healing process. Therefore, Zn/Fu@Cs nanofibers enhanced the antibacterial effect against various human pathogens. At the same time, nanofibers suppress other adverse effects in wound sites and can speed up the healing process. Mainly, Zn/Fu@Cs nanofibers support cell proliferation and promote wound cell regeneration. The porous and biocompatible nature of nanofibers benefits new cell formation and granulation of tissue. The anti-inflammatory property of zinc enhances collagen deposition and fibroblast regeneration mechanisms. The antibacterial efficacy and biodegradation potential demonstrate very motivating results in diabetic wound healing. Experiments exhibited the highest performance in wound healing within three-day time intervals. From the present study, we concluded that the Zn/Fu@Cs nanofibers can serve as efficient biomaterials for wound care. The biological potential of zinc and fucoidan leads to the quick wound healing process. The fabricated nanofibers are the most suitable and multifunctional biomaterial for wound healing and operate room infection control applications.

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