Preparation Of Nano-silver-incorporated Wound Dressing Based On Dopamine Modification And Its Role In Wound Healing

BackgroundCutaneous wounds caused by burns,trauma,venous ulcers and diabetic ulcers will cause serious infection and loss of body fluids,bringing great pain and heavy medical burden to patients.Biological wound dressings could effectively protect the wounds from bacterial infection and keep a natural microenviroment for the regeneration of epidermis and dermis,which play an important role in wound management.Therefore,developing a novel wound dressing is of great significance for clinical wound care.Bacterial infection is the major hurdle for wound healing.Thus,an ideal wound dressing must possess excellent antibacterial activity.Nano silver(NS)is a nanoparticle with a size of 1-100 nm,which has robust bactericidal capacity against Gram-positive bacteria and Gram-negative bacteria.More noteworthy,nano silver could kill multi-drug-resistant bacteria,which makes it exhibit unique advantages over traditional antibiotics as the increasing emergence of multi-drug-resistant bacteria worldwide.Therefore,nano silver is an ideal antibacterial agent for the preparation of wound dressings.However,the traditional methods for nano silver synthesis have some disadvantages such as complicated operation procedure,high energy consumption and environmental pollution.In recent years,the surface modification technology of biomimetic dopamine,which is efficient,eco-friendly and simple,has attracted wide attention in the fields of materials science and biomedicine.Inspired by the phenomenon that marine mussels tightly adhere on the rocks,researchers discover that dopamine(DA)undergoes self-polymerization to form a polydopamine layer on the surface of materials under alkaline condition.The polydopamine layer has good reductive ability and super adhesivity,which could effectively in situ reduce the metal ions to metal elements and immobilize them on the materials.More importantly,many studies have demonstrated that eco-friendly polydopamine is biocompatible with human body,and beneficial to cell adhesion and proliferation.In the first part of our research,we aimed to prepare wound dressings composed of a natural eggshell membrane(ESM)containing different concentrations of nano silver using the biomimetic dopamine modification technology.We expected to select an optimal ESM/NS film with good biosafety through cytotoxicity evaluation,and detect its physical and chemical properties,antibacterial activity and effect on wound healing,so as to explore the feasibility of dopamine modification technology to prepare nano-silver-incorporated wound dressings.The scaffold materials used for the preparation of wound dressings are generally divided into natural materials and synthetic materials.In the first part of our research,as a natural material,ESM is limited in the practical application at present due to the difficulty of quantity production and formability,as well as the poor mechanical property.In contrast,synthetic materials generally possess good mechanical property and formability,and are easy for quantity production,which make them more likely to be used in practice.Therefore,in the second part of our research,we aimed to prepare wound dressings composed of an electrospinning polycaprolactone(PCL)nanofibrous film—a synthetic material scaffold,containing different concentrations of nano silver using the biomimetic dopamine modification technology.We expected to evaluate their mechanical properties,biosafety,antibacterial activity and effect on wound healing,and further explore the feasibility and clinical applicability of dopamine to modify synthetic materials to prepare nano-silver-incorporated wound dressings.ObjectiveIn this study,we aim to prepare wound dressings composed of a natural material/synthetic material containing different concentrations of nano silver using the biomimetic dopamine modification technology,and then investigate their mechanical properties,biosafety,antibacterial activity and effect on wound healing,so as to explore the feasibility of dopamine modification technology to prepare nano-silver-incorporated wound dressings.On this basis,we expected to select the optimal dressings with excellent biosafety to provide new ideas and strategies for clinical wound treatment.Part One Preparation of nano-silver-incorporated eggshell membrane based on dopamine modification and its effect on wound healingMethods1.Preparation and optimization of nano-silver-incorporated eggshell membrane1.1 ESMs were treated with an aqueous solution of deionized water(pH=7.0),hydrochloric acid(pH=3.0)and sodium hydroxide(pH=11.0)for 72 hours,and then observed under scanning electron microscopy(SEM).1.2 The hydrochloric acid treated ESM was modified with dopamine.Then the polydopamine formed by self-polymerization of dopamine was able to in situ reduce different concentrations of silver ions into nano silver and anchor them on the surface of ESM,preparing dressings of ESM containing different concentrations of nano silver(ESM/NS).1.3 The fibroblasts isolated from GFP transgenic neonatal mice were seeded on different ESM/NS films.Next,the cytotoxicity of ESM/NS films was evaluated using an MTT test,fluorescence microscope observation and SEM observation.Finally,the optimal ESM/NS film without obvious cytotoxicity was selected for following studies.2.The characterizations of ESM/NS filmThe characterizations of ESM/NS film were analyzed using SEM,Scanning transmission electron microscopy(STEM),Energy-dispersive X-ray spectroscopy(EDS),Fourier transform infrared spectroscopy(FTIR)and Inductively coupled plasma-atomic emission spectrometry(ICP-AES).3.The antibacterial activity of ESM/NS filmThe antibacterial activity of ESM/NS film against S.aureus,E.coli and multi-drug-resistant A.baumannii was detected using a disc diffusion method and a bacterial suspension assay.4.The effect of ESM/NS film on wound healing4.1 The effect of ESM/NS film on healing of uninfected woundUsing a murine full-thickness skin defect wound model,the effects of ESM/NS film on wound healing rate,re-epithelialization,granulation tissue formation,and expression of PCNA and IL-1?were evaluated in vivo.4.2 The effect of ESM/NS film on healing of infected woundUsing a murine A.baumannii-infected wound model,the in vivo antibacterial activity of ESM/NS film and the effects of ESM/NS film on healing rate of infected wound,re-epithelialization and granulation tissue formation were evaluated.5.In vivo biosafety of ESM/NS filmThe heart,liver,spleen,lung,kidney of the mice treated with ESM/NS film for 7 days and 28 days were analyzed histopathologically.Results1.The porosities of ESM treated with deionized water,sodium hydroxide and hydrochloric acid were 42.1%,45.0%and 46.1%,respectively,and then the hydrochloric acid treated ESM was selected for preparation of ESM/NS.The cytotoxicity of different ESM/NS films was nano-silver-dose dependent,and the ESM/NS film containing low dose(30?M)nano silver was selected as the optimal one for following studies as it had excellent cytocompatibility.2.The results of SEM,STEM and FTIR showed that ESM/NS film was characterized with a three-dimensional fibrous network structure,and spherical nano silver was successfully incorporated onto its surface.The result of ICP-AES revealed that the loading amount of silver on the ESM/NS film was 3.66?g/cm~2,the concentration of silver ions released from the ESM/NS film at day 1 was 0.35?g/mL,and the constant release could last to 7 days(0.50?g/mL).3.The results of antibacterial activity test indicated that ESM/NS film possessed excellent bactericidal capacity against S.aureus,E.coli and multi-drug-resistant A.baumannii.4.The in vivo animal test indicated that ESM/NS film could promote keratinocyte proliferation and control inflammation response via enhancing the expression of PCNA and decreasing the expression of IL-1?in uninfected cutaneous wounds,respectively.Finally,it promoted wound healing through accelerating re-epithelialization and granulation tissue formation.At day 7 post-surgery,the wound healing rates of control group,Vaseline gauze group,ESM group,ESM/DA group and ESM/NS group were 78.3%,81.6%,81.9%,82.7%and 88.6%.On the other hand,ESM/NS film could effectively eliminate the A.baumannii bacteria in vivo,keep a non-septic microenviroment for tissue regeneration,accelerate re-epithelialization and granulation tissue formation,and finally promote the healing of infected wounds and shorten the healing time.5.The result of in vivo biosafety evaluation showed that no pathological injuries such as inflammatory lesion and structural damage were found in the organs of mice treated with ESM/NS film,indicating that ESM/NS had good biosafety in vivo.Part Two Preparation of nano-silver-incorporated polycaprolactone nanofibrous film based on dopamine modification and its effect on infected wound healingMethods1.Preparation and characterizations of nano-silver-incorporated polycaprolactone(PCL)nanofibrous film1.1 The polycaprolactone nanofibrous film was modified with dopamine.Then the polydopamine formed by self-polymerization of dopamine was able to in situ reduce different concentrations of silver ions into nano silver and anchor them on the surface of PCL,preparing dressings of PCL containing different concentrations of nano silver(marked as PCL/NS0.5,PCL/NS1.0 and PCL/NS2.0,respectively).1.2 The characterizations of PCL/NS nanofibrous films were detected using SEM,EDS,FTIR,ICP-AES,water contact angle measurement and mechanical property test.2.The antibacterial activity of PCL/NS filmsThe antibacterial activity of PCL/NS films against S.aureus,E.coli and multi-drug-resistant A.baumannii was detected using a bacterial suspension assay.The bactericidal capacity was evaluated by absorbance measurement and live/dead bacteria staining method.The effect of PCL/NS films on the formation of bacterial biofilm was observed by SEM.3.The biosafety of PCL/NS films in vitro and in vivo3.1 Fibroblasts isolated from GFP transgenic neonatal mice were seeded on different PCL/NS films.Next,the cytotoxicity of PCL/NS films was evaluated using a CCK-8 test and fluorescence microscope observation.3.2 The leach liquors extracted from PCL,PCL/DA,PCL/NS0.5,PCL/NS1.0 and PCL/NS2.0 films,respectively,were used to culture HaCaT cells and NIH3T3 cells.Then the cytotoxicity was evaluated using a CCK-8 test and a flow cytometry apoptosis assay.3.3 The heart,liver,spleen,lung,kidney of the mice treated with PCL/NS1.0 film for 7days and 28 days were analyzed histopathologically.4.The silver ions released from PCL/NS1.0 filmThe PCL/NS1.0 film was immersed in PBS solution and the content of silver ions in PBS was detected by ICP-AES after 1,3,5 and 7 days.5.The effect of PCL/NS1.0 film on wound healingUsing a murine S.aureus-E.coli-infected wound model,the in vivo antibacterial activity of PCL/NS1.0 film and the effects of PCL/NS1.0 film on healing rate of infected wound,re-epithelialization,granulation tissue formation,and expression of PCNA and CD31 were evaluated.Results1.(1)The results of SEM,STEM and FTIR showed that PCL/NS0.5,PCL/NS1.0 and PCL/NS2.0 films were characterized with a three-dimensional nanofibrous network structure,and spherical nano silver was successfully incorporated onto its surface.The diameters of nano silver on PCL/NS0.5,PCL/NS1.0 and PCL/NS2.0 films were 73.3±33.6nm,74.3±30.4 nm and 72.2±26.3 nm,respectively.(2)The result of ICP-AES revealed that the loading amounts of silver on the PCL/NS0.5,PCL/NS1.0 and PCL/NS2.0 films were 1.89?g/cm~2,4.83?g/cm~2 and8.31?g/cm~2.(3)The result obtained by ImageJ software showed that the porosities of PCL, PCL/DA,PCL/NS0.5,PCL/NS1.0 and PCL/NS2.0 films were 54.1%,53.5%,53.4%,53.2%and 52.6%.(4)The water contact angles of PCL,PCL/DA,PCL/NS0.5,PCL/NS1.0 andPCL/NS2.0 films were 107.9°,27.7°,25.7°,27.5°and 29.2°,respectively,indicating the surface hydrophilicity of films was improved after dopamine modification.(5)The result of mechanical property test showed that the tensile strength of PCL/NS0.5,PCL/NS1.0 and PCL/NS2.0 films were 0.52 MPa,0.58 MPa and 0.66MPa,respectively;Young's modulus were 0.32 MPa,0.37MPa and 0.39 MPa,respectively;elongation at break were 100.0%,104.4%and 105.6%,respectively.2.The results of absorbance measurement and live/dead bacteria staining method showed that PCL/NS1.0 and PCL/NS2.0 films had excellent antibacterial activity against S.aureus,E.coli and multi-drug-resistant A.baumannii.The result of SEM observation indicated that PCL/NS1.0 and PCL/NS2.0 films could effectively prevent formation of biofilms.3.PCL/NS0.5 and PCL/NS1.0 films had no obvious cytotoxicity towards fibroblasts inoculated to the surface,but PCL/NS2.0 film had a significant inhibitory effect on the growth of fibroblasts on its surface.The leach liquors extracted from PCL/NS0.5 and PCL/NS1.0 films had no obvious cytotoxicity towards HaCaT cells and NIH3T3 cells,but the leach liquor extracted from PCL/NS2.0 film induced significant apoptosis and necrosis,leading to cell death.Therefore,the PCL/NS2.0 film had obvious cytotoxicity,while PCL/NS0.5 and PCL/NS1.0 films showed good cytocompatibility.The result of in vivo biosafety evaluation showed that no pathological injuries such as inflammatory lesion and structural damage were found in the organs of mice treated with PCL/NS1.0 film,indicating that PCL/NS1.0 film had good biosafety in vivo.4.The result of ICP-AES revealed the concentrations of silver ions released from the PCL/NS1.0 film at day 1 and day 7 were about 0.30?g/mL and 0.57?g/mL,respectively.5.The in vivo animal test indicated that PCL/NS1.0 film could effectively eliminate the bacteria,keeping a non-septic microenviroment for tissue regeneration,and promote keratinocyte proliferation and agiogenesis via enhancing the expression of PCNA and CD31in infected cutaneous wounds.Finally,it promoted healing of infected wounds and shorten the healing time through accelerating re-epithelialization and granulation tissue formation.Conclusion1.In this study,we prepared two kinds of novel dressings of nano-silver-incorporated ESM and nano-silver-incorporated PCL using biomimetic dopamine modification for the first time.The preparation principle is that dopamine undergoes self-polymerization to form a polydopamine layer on the surface of ESM scaffold and PCL scaffold,which has good adhesive capacity and chemical reducibility,and can in situ reduce silver ions into nano silver and immobilize them on the surface of the material scaffold.This preparation method is simple,efficient and eco-friendly.Moreover,it is easy to be used widely due to the low energy consumption and cost.2.In this study,biomimetic dopamine modification technology was applied to not only effectively generate nano silver,but also significantly improve the hydrophilicity and mechanical properties of PCL,making it more conducive to wound repair.3.In this study,the biosafety of dressings incorporated with different concentrations of nano silver was evaluated using systematic experiments both in vitro and in vivo.Based on these data,the optimal nano-silver-incorporated ESM and nano-silver-incorporated PCL films with good biosafety were successfully selected.4.In this study,the optimal ESM/NS and PCL/NS1.0 films showed robust antibacterial activity against S.aureus,E.coli and multi-drug-resistant A.baumannii,and the PCL/NS1.0 film could effectively prevent formation of biofilms.Particularly,the excellent antibacterial activity of ESM/NS and PCL/NS1.0 films against multi-drug-resistant A.baumannii indicated that compared with traditional antibiotics,ESM/NS and PCL/NS1.0films had unique advantages.5.In this study,employing a murine full-thickness skin wound model,we demonstrated that optimal ESM/NS and PCL/NS1.0 films could effectively prevent bacterial infection in vivo and control inflammation response,keeping a non-septic microenviroment that suitable for wound tissue regeneration to promote infected/uninfected wound epithelial cell proliferation and vascularization.Finally,they promoted wound healing through accelerating re-epithelialization and granulation tissue formation.In summary,we demonstrated the feasibility of dopamine modification technology to prepare nano-silver-incorporated wound dressings.These nano-silver-incorporated wound dressings showed excellent antibacterial activity both in vitro and in vivo,and could effectively promote wound healing.These achievments provide new ideas and strategies for the preparation of wound dressings and are expected to play an important role in clinical wound treatment.