The Study Of Antimicrobial Electrospun Nanofiber And Its Mechanism In Promoting Wound Healing

Wound healing occupies a remarkable place in clinical and remains a challenging problem. In this study, a mixture of poly（vinyl alcohol）（PVA） and chitosan oligosaccharides（COS） was electrospun with silver nanoparticles（AgNPs） to produce fibrous mats for use in wound healing. The AgNPs were reduced by COS prior to electrospinning or Ag+ was reduced via ultraviolet irradiation in nanofibers. The optimal formulation was screened using spinnability of solution, conductivity, dosage as index. The optimal formulation was as follows:volume ratio of 11% PVA to 3% COS was 2:1, drug-loading amount was 3%, ultraviolet irradiation for 8 hours.The morphologies of the PVA/COS/AgNO₃ and PVA/COS-AgNP nanofibers were analyzed by scanning electron microscopy. The nanofibers had a smooth surface with an average diameter of 130–192 nm. Formation of the Ag NPs was investigated by field emission transmission electron microscopy, ultraviolet-visible spectroscopy, fourier transform infrared spectroscopy, and X-ray diffraction. The diameters of the AgNPs were in the range of 15–22 nm, and the AgNPs was bonded to nanofibers by means of Van der Waals force. According to the results of permeability and swelling ratio, the optimal crosslinking condition was as follows:glutaraldehyde vapor steam for 48 hours. In order to study the stability of the nanofibers, we used spinning appearance, the permeability and the drug content as indicators and conducted accelerated test. The results showed that the nanofibers had good stability after three months. The Ag in vitro release of the nanofibers was determined using an atomic absorption spectrophotometer. The results showed that the release of Ag from both samples was rapid during the first 8 hours, the maximum release of Ag from the PVA/COS-AgNPs nanofiber exceeded the quantity released from the PVA/COS/AgNO₃ nanofiber. Combining with analysis results, we discovered that the drug release process was dissolution, diffusion and corrosion.In this study, we have conducted safety evaluation of the nanofibers. The MTT results showed that the PVA/COS-AgNP nanofiber has excellent in vitro biocompatibility and has marked benefits over the PVA/COS/AgNO₃ nanofiber at the concentrations of 0.8–1.0 mg/mL. The results of the skin irritation test for single and multiple applications showed that both PVA/COS/AgNO₃ and PVA/COS-AgNP nanofiber caused no irritation to normal skin after single and multiple applications.The pharmacology in vitro and vivo are the important indices about pharmaceutic preparation assessment. Our study used S.aureus and E.coli to detect the bacteriostatic ability of the nanofibers in vitro. The results showed that the two kinds of nanofibers had good antibacterial effect, however, the PVA/COS-AgNP nanofiber had better antibacterial effect than the PVA/COS/Ag NO₃ nanofiber. Meanwhile, the back wounds of Sprague Dawley rats were given PVA/COS/AgNO₃ nanofiber, gauze, PVA/COS-AgNP nanofiber and a commercially available woundplast, and pharmacodynamics in vivo was investigated. The results showed that the PVA/COS-AgNP nanofiber group had no phenomenon of festering at the early stage of healing process, while the other groups had a different infection. These results indicated that the PVA/COS-AgNP nanofiber accelerated the early stage of wound healing and received better effect than the PVA/COS/AgNO₃ nanofiber.To clarify the mechanism underlying the accelerated healing effect of the PVA/COS-AgNPs nanofiber, in this study, we used a Sprague Dawley rat skin wound model and a potent inhibitor of the TGFβ1 receptor, SB431542, which can efficiently inhibit the activation of the TGFβ1/Smad signaling pathway. Hematoxylin–eosin, Masson’s trichrome, immunofluorescent staining, quantitative real-time polymerase chain reaction, and western blot analyses were used to examine the influence of the signaling molecules involved in the TGFβ1/Smad signaling pathway with the usage of the PVA/COS-AgNPs nanofiber. The results showed that the PVA/COS-AgNPs nanofiber exerted a potent effect on wound closure and remodeling, the nanofiber plus SB431542 group presented festering wounds during the early stage, and wound remodeling was the slowest throughout the entire healing period. PVA/COS-AgNPs nanofiber treatment resulted in significantly increased TGFβ1, TGFβRI, TGFβRII, collagen I, collagen III, and fibronectin mRNA expression, meanwhile, the protein levels of TGFβ1, collagen I, collagen III, phospho-Smad2 and phospho-Smad3 also increased during the early stage of wound healing. On the contrary, the inhibition of the TGFβ1/Smad signaling pathway down-regulated the above gene and protein levels. In conclusion, our results demonstrated that the promoting healing effect of PVA/COS-AgNPs nanofiber involved the activation of the TGFβ1/Smad signaling pathway.