Photosensitizer Nanofibers For Treatment Of Wound Infection And Enhancement Of Wound Healing

Skin wounds are very common in social life.Serious wounds can lead to skin barrier defect.If no timely and reasonable medical intervention is carried out,wound infection is likely to occur,which not only affects wound healing,but also may cause infection or even sepsis in the body.The widespread use of antibiotics promotes the emergence of drug-resistant bacteria,which brings more challenges to the treatment of wound infection.Clinical wound infection is mostly treated by oral or injection an-tibiotics and a small amount of topical antibiotics,but it does not promote wound healing.Wound healing often uses wound dressing,but the general dressing does not have antibacterial effect.Based on the advantages of photodynamic antibacterial ac-tion and nanofiber dressing,photosensitizer nanofiber was prepared,which can obtain the effective antibacterial effect of wound surface.What’s more,the high specific surface area and porosity of the nanofiber with similar structure to the cellular matrix can also be used to obtain the excellent healing promoting function and to realize the combination of the treatment of wound infection and the function of promoting healing.1.Research on nanofibersUsing electrospinning process,acetic acid solution of chitosan and polyvinyl alcohol（PVA）were as the spinning material with spinnability,fiber morphology,and diameter as the main indicators.The formulation and preparation technologies were optimized.The optimized electrospinning solution consisted of 10%（v/v）acetic acid solution,2%（w/v）chitosan and 7%（w/v）PVA.The best preparation parameters were the voltage of 15 kV,the receiving distance of 15 cm and the pushing rate of 0.4 ml/h.The nanofibers observed by scanning electron microscope showed a three-dimensional cross-network with smooth surface.The average diameter was 252 nm±38 nm,and the water absorption ratio of the nanofibers was up to 220%.Fourier transform infrared spectrum（FTIR）and X-ray diffraction（XRD）showed that the intermolecular hydrogen bonding happened between chitosan and PVA in electrospinning process,which was favorable for the formation of nanofibers.2.Antibacterial effect of electrospinning nanofibersThe methicillin-resistant Staphylococcus aureus（MRSA）and meropenem-resistant Pseudomonas aeruginosa（MRPA）were cultured in vitro.5-aminolevulinic acid（5-ALA）and indocyanine green（ICG）solutions were prepared.The bacteria and photosensitizers solution were mixed and incubated in the dark,irradiated with 630 nm and 785 nm lasers and counted.Illumination alone has no antibacterial effect.Compared with the negative control group,40 mM 5-ALA solution in the absence of light（dark toxicity）reduced the number of MRSA and MPSA colony survival by 1.51 log₁₀-units and 1.34 log₁₀-units.40 mM 5-ALA at the light of 144 J/cm²,The number of colonies of MRSA decreased by about 2.6 log₁₀-units.Under the light irradiation of 192 J/cm²,the number of colonies of MRPA decreased by about 2.03 log₁₀-units.Compared with the negative control group,25.6μM ICG at the light of 120 J/cm²,The number of colonies of MRSA decreased by about 4.39 log₁₀-units.194μM ICG under the light irradiation of 192 J/cm²,the number of colonies of MRPA decreased by about 4.28 log₁₀-units.ICG had no dark toxicity to MRSA and MRPA.This may be due to that 5-ALA should be converted into protoporphyrin by a series of enzymes,especially in mitochondria.But bacteria do not have mitochondria and related enzymes.ICG exhibits excellent PACT,especially gram-positive bacteria better than gram-negative bacteria.3.Characteristics of photosensitizer nanofibersICG nanofibers were prepared by the optimized electrospinning technology and prescription.Scanning electron microscopy showed nanoscale fibers of smooth surface.The FTIR showed no interaction of ICG with chitosan and PVA.The visible spectrophotometric method was established to determine the concentration of ICG in the release solution at 781 nm.The drug release results showed that ICG nanofibers is a good slow-release fertilizer.Simulation may reveal the mechanisms of drug release.The highest r values of ICG nanofibers as the Ritger-Peppas equation.The results showed that Fick’s diffusion dominated ICG released from ICG nanofibers.ICG nanofibers have high effective antibacterial function.4.Pharmacodynamics and safety of photosensitizer nanofibersA 2×2 cm² wound of total cortical resection was made on both sides of the rat’s back,and 100μL MRSA broth was added to each wound.After the wound was closed for 48 hours,a yellowish pus was formed on the wound.A wound infection rat model was established successfully.The wound were covered by ICG nanofibres,and were added with physiological saline in advance.Under the light dose of 240 J/cm²,the 785nm laser was used to irradiate the wound on the third,fifth,and seventh days respectively.Nanofibers were replaced before irradiating on the fifth day.The experiment ended on the 15th day.Compared with fusidic acid cream,blank nanofibers,non-illuminated ICG nanofibers,and ICG solutions,ICG nanofibers with light irridation had the higher wound healing ratio at first and decreased the expression of inflammatory factors including F4/80,TNF-αand IL-6 obviously.This indicated the infection was controlled effectively.On the 7th and 15th days,the number of colony decreased and the platelet-endothelial cell adhesion molecule（CD31）increased,which indicated promoted angiogenesis.The newborn epidermis and dermis structure were complete and arranged in order.Collagen fiber had clear morphology and arranged closely,which was advantageous for enhanced wound healing.There were no obvious abnormalities in the main organs of the rats in the administration group,demonstrating that the safety of ICG nanofibers was good.In this study,a novel photosensitizer nanofiber was prepared,which provided a new method for antibacteria and wound healing.