Preparation Of Novel Graphene-based Composite Materials And Experimental Study On Using Them To Promote Skin Wound Repair

Background:Once the integrity of the skin is destroyed,cutaneous wounds will form and lose their protective mechanisms.On one hand,dehydration of wounds may not guarantee an ideal moist environment for wound healing,and excessive water loss can also cause breakdown of internal environment.On the other hand,cutaneous wounds can easily cause microbial invasion,serious infection,hinder wound healing process,and even threaten the life.How to protect and close the wound quickly,and control bacterial infections,are very important issues in the field of skin wound repair.Large area deep wounds and chronic wounds cannot heal quickly.Thus,it is necessary to use wound dressings as temporary skin substitutes to protect wounds and accelerate wound healing.However,clinical products still have some defects,such as sole function,wound soakage,high prices and so forth.Thus,manufacturing an ideal multifunctional wound dressing still remains a great challenge for us.Graphene is an allotrope of carbon consisting of a single layer of carbon atoms arranged in a hexagonal lattice.It has special two-dimensional structure,large specific surface area,outstanding mechanical stiffness,high photothermal conversion efficiency,excellent biocompatibility,strong antibacterial activity and other physical,chemical or biological properties.Polydimethylsiloxane?PDMS?/silicon rubber,as a commonly used polymer material,exhibits lots of extraordinary properties,including relatively low price,outstanding flexibility,high gas permeability and good biocompatibility.It can be used as an ideal wound dressing material.If graphene were incorporated into PDMS matrix,it might be worth finding out whether the resulting graphene-PDMS composite material could possess more comprehensive features in favor of potential applications as wound dressing.There is no relevant research report at present.This issue is of great significance to expand the application of graphene in skin wound repair.Therefore,in the first part of the study,we made our great efforts to fabricate the graphene-PDMS composite material,systematically explored the aforementioned questions via in vitro and in vivo experiments,and finally confirmed that it could be considered as a promising multifunctional wound dressing.Furthermore,the antibacterial effect of graphene is often subjected to many factors,such as concentration,size,number of layers,type of bacteria and experimental surroundings.We also found that the in vivo antibacterial activity of the graphene-PDMS composite wound dressing was not completely satisfactory,which limited its application in infected skin wounds.Infection is another important factor affecting skin wound repair.Clinical treatment for bacterial infections of skin wounds still relies heavily on the use of antibiotics,but often accompanied by the emergence and prevalence of multidrug-resistant strains.At present,due to the lack of new antibiotics,even no drug can be used against some pandrug-resistant strains.Thence,we're trying to continue using graphene as a research object to find novel therapeutic strategies for bacterial infections of skin wounds or subcutaneous tissues from the perspective of biomaterials and nanomedicine.Graphene is an excellent photothermal agent,and the antibacterial PTT based on graphene has become a research hotspot in recent years.However,graphene-based antibacterial PTT still has some defects.The greatest defect could be the difficulty of targeting pathogenic bacteria specially to avoid undesirable thermal damages of the surrounding healthy tissues.Therefore,in the second part of the study,we struggled to conjugate glycol chitosan?the targeted moiety?with carboxyl graphene?the substrate material?by the condensation reaction between amino groups and carboxyl groups,to prepare a novel targeted antimicrobial nanomaterial GCS-CG.When located in the acid environment,it became positively charged rapdily,due to the protonation of free amine groups on the GCS backbone,and then presented strong adherence to bacteria surface via electrostatic interactions,ensuring targeted heating and eradicating the bacteria,controlling MRSA infection in vivo and promoting wound healing.This part of the study focuses on a breakthrough in the bacterial targeting of photothermal agents,and further expands the application of graphene in the field of skin wound repair.Objectives:In this study,two kinds of novel graphene-based composite materials?graphene-PDMS and graphene-chitosan?were prepared,providing fresh ideas and experimental basis for the application of graphene in the field of skin wound repair,as well as new strategies for the clinical treatment of skin wound diseases.Part?Fabrication of a novel wound dressing based on graphene-PDMS and investigation of its facilitating effect on skin wound healingMethods:1.Preparation of the porous rGO-PDMS composite membrane using a“three-step”method.2.SEM observation of the rGOAMs and rGO-PDMS mixture.3.Determination of the residual amounts of organic solvents in the porous rGO-PDMS composite membrane by a headspace gaschromatography.4.TEM and AFM observations of the free rGO sheets;TEM observation of the rGO sheets in the porous rGO-PDMS composite membrane.5.Structural observations of the porous rGO-PDMS composite membrane by SEM and digital microscope.6.Mechanical property measurements of the porous rGO-PDMS composite membrane.7.Determination of water vapor transmission rates?WVTR?of the porous rGO-PDMS composite membrane.8.Assessment of cytotoxicity of the porous rGO-PDMS composite membrane using CCK-8 assays.9.SEM observation of interactions between rGO and bacteria;investigation of reactive oxygen species?ROS?production in bacterial cells.10.Evaluation of in vitro antibacterial activity of the porous rGO-PDMS composite membrane using standard plate method.11.A mouse full thickness skin defect wound model was used to study the effects of the porous rGO-PDMS composite membrane on the wound healing;the wound size,length of the newly formed epithelium tongue and granulation tissue thickness were determined based on the photographs of macroscopic appearance of wounds and hematoxylin and eosin?HE?sections using the Image Pro Plus 6.0?IPP6.0?software,respectively;the wound healing rate and wound closure time were also calculated.Results:1.The rGOAMs exhibited a unique well-defined“center-diverging microchannel”dandelion-like microstructure.Under negative pressure,PDMS solution was easily sucked into the microspheres.The rGO sheets could be well dispersed in PDMS matrix through microsphere fragmentation after sonication.2.The rGO?0,0.1,0.2 and 0.5wt%?-PDMS composite membranes were successfully fabricated.3.The residual contents of THF and hexane in the rGO-PDMS composite membrane were?2 ppm and?0.6 ppm,respectively.4.The rGO?0,0.1,0.2 and 0.5 wt%?-PDMS composite membranes exhibited a porous structure.The pore sizes of the top surfaces of the membranes made from rGO?0,0.1,0.2and 0.5 wt%?-PDMS are 18.64±2.67?m,17.52±2.75?m,15.57±1.87?m and14.94±2.35?m,respectively;the pore sizes of main pores of the bottom surfaces of the rGO?0,0.1,0.2 and 0.5 wt%?-PDMS composite membranes are 77.95±9.83?m,72.97±8.92?m,68.37±6.33?m and 60.67±9.09?m,respec-tively.The pore sizes of the top surfaces are significantly smaller than those of the bottom surfaces.5.Addition of the rGO sheets resulted in significant increase in tensile strength and Young's modulus.6.WVTRs of the rGO?0,0.1,0.2 and 0.5 wt%?-PDMS composite membranes are 119±2 g/m²/24h,122±1 g/m²/24h,124±2 g/m²/24h and 137±2 g/m²/24h,respectively.7.There were no significant differences in cell viability among the three groups?rGO?0,0.1 and 0.2 wt%?-PDMS?on days 1,3,5 and 7 post-seeding;however,on day 5 and 7post-seeding,the fibroblast viability in the rGO?0.5 wt%?-PDMS group was reduced to88.54±0.04%and 83.43±0.06%of the rGO?0 wt%?-PDMS group,respectively.8.Most of E.coli cells lost their cellular integrity after exposure to rGO.Besides,some E.coli cells were embedded in the rGO sheets.The levels of ROS in rGO treated cells were 2.44-fold higher,compared to the level of ROS in control cells.9.In vitro,the rGO-PDMS composite membranes performed much better than the pure PDMS membrane in reducing bacterial number,and it was found that bacterial viability decreased with increasing rGO content in the membranes.10.On the 7th day post surgery,87.37±2.75%and 87.63±3.72%wound size reduction was found in the rGO?0.2 wt%?-PDMS and rGO?0.5 wt%?-PDMS groups,respectively?87.37±2.75%vs 87.63±3.72%,P=1.000?,which was significantly higher those of the rest groups.Meanwhile,the wound closure time in the rGO?0.2 wt%?-PDMS and rGO?0.5 wt%?-PDMS groups was 8.46±0.30 days and 8.62±0.30 days,respectively?8.46±0.30 days vs 8.62±0.30 days,P=1.000?,which was significantly shorter than those of the rest groups.11.The length of the newly formed epithelium tongue was similar in the rGO?0.2wt%?-PDMS and rGO?0.5 wt%?-PDMS groups?2325.31±208.08?m vs 2362.64±111.22?m,P=1.000?,which was significantly longer compare to those of the rest groups at day 7post-surgery.There was no distinction between the granulation tissue thickness in the rGO?0.2 wt%?-PDMS and rGO?0.5 wt%?-PDMS groups?1932.06±136.32?m vs 1825.31±225.37?m,P=1.000?,which was significantly larger than those of the rest groups at day 7post-surgery.Part?Development of a novel targeted antimicrobial material based on graphene-chitosan with charge conversion and study on its effect of treating skin infectionMethods:1.Synthesis of carboxyl graphene-glycol chitosan?GCS-CG?:GCS was grafted onto CG by the condensation reaction of amine groups on GCS and carboxyl groups on CG.2.Fourier transform infrared?FTIR?spectra of GCS-CG were recorded.3.The morphology,size and thickness of GCS-CG were measured by TEM,dynamic light scattering?DLS?technique and AFM,respectively.4.The pH-dependent changes of zeta potential of GCS-CG were measured.5.The UV–Vis-NIR spectra of GCS-CG were recorded.6.The photothermal ability of GCS-CG was evaluated.7.SEM and laser confocal microscopy observations of interactions between GCS-CG and bacteria.8.The zeta potentials of the bacteria before and after being incubated with GCS-CG were measured.9.SEM observation of interactions between GCS-CG and 3T3 cells.10.The cytotoxicity of GCS-CG was evaluated using CCK-8 assays.11.The in vivo biosafety of GCS-CG was evaluated using adult BALB/c mice:Different concentrations of GCS-CG were injected into mice through the tail vein.After being treated for 7 or 21 days,the heart,liver,spleen,lung and kidney of the mice were subjected to histological examination.The blood samples were also analyzed according to the standard procedures of serum biochemistry.12.The in vitro antibacterial activity of GCS-CG was investigated using standard plate method.13.A mouse subcutaneous abscess model was developed.To examine the in vivo bacteria-targeting ability of GCS-CG,GCS-CG was injected into the abscess on one flank of the test mouse.As a control,the same amount of GCS-CG was injected subcutaneously into the normal skin on another flank of the same mouse.Each side of the back of each test mouse was separately irradiated with the NIR laser at the predetermined time intervals,and the temperatures of the two flanks were compared.14.For the in vivo anti-MRSA study,the abscess was injected with GCS-CG and irradiated using the NIR laser.At the indicated time points of treatment,the tissues of infected site of the testing mice were collected for following histology examination and bacteria colony counting.Results:1.The appearance of C=O—N(1633 cm^-1)and C—N—H(1532 cm^-1)and the decreased intensity of carboxylic groups C=O—O(1726 cm^-1)in the spectrum of GCS-CG suggests the successfully synthesis of GCS-CG.2.GCS-CG was a sheet-like structure with a thickness of¹.7 nm.The size of²50nm corresponds to the maximum number of sheets with this size.3.The GCS-CG converted from a slight net-negative charge?-3 mV?at pH 7.4 to a net positive one?9 mV at pH 6.3?as pH was reduced.4.GCS,CG and GCS-CG exhibit optical absorption from the UV to the NIR region with 0.008,0.252 and 0.441 absorbance at 808 nm,respectively.5.Upon NIR laser?808 nm,0.5 W/cm²?irradiation,the temperature of GCS-CG increased rapidly,reaching concentration-dependent plateaus within 3 min.When being irradiated at 0.75 W/cm² for 10 min,GCS-CG with the concentration of 0.1 and 0.2 mg/mL increased up to 56.2?and 66?from 29?,respectively.GCS-CG possesses a high photothermal conversion efficiency?PTC?of 39.6%.6.Under the acidic environment?pH 6.3?,GCS-CG exhibited a strong positive surface charge that resulted in strong electrostatic interactions with the negatively-charged bacteria?S.aureus,E.coli and MRSA?,thus the bacteria were encapsulated by GCS-CG,converting their surface charge from negative to positive.In the normal physiological environment?pH7.4?,almost no GCS-CG was observed on the surface of bacteria.Upon exposure to 3T3fibroblasts at pH 7.4,GCS-CG had a slightly negative charge and so did not adhere to the negatively charged cultured cells.7.The survival rates of bacteria with NIR or CG alone treatment had no significant difference from that of control without any treatment.Moreover,the bacteria that treated with GCS-CG exhibited a little decrease in the viability?ca.6–11%?.Compared with the others,GCS-CG with NIR irradiation showed the highest antibacterial efficiency with nearly 100%killing effect.However,CG with NIR irradiation showed only¹3–15%bacteria killing effects dependent on the type of bacteria.Compared with the untreated control,the viability of MRSA treated with different sizes of GCS-CG?small size range:190–342 nm;middle size range:396–615 nm;large size range:712–1106 nm?showed no significant reduction.Upon NIR irradiation,the antibacterial efficiencies of the three types of the GCS-CG were similar and all rose up to 100%.8.The infected abscess was obviously formed in the subcutaneous tissue on the back of the mouse.Immediately following the implantation of GCS-CG and NIR irradiation?0.75 W/cm²,10 min?,the temperatures of both the normal and the abscess rapidly increased to ca.56?.When NIR irradiation was applied at later times?2h and 4 h?,the temperature of normal skin significantly fell,while the temperature of the infected skin still kept at⁵6?.9.The CFU count in the group of GCS-CG+NIR was largely reduced to 5%of that in the control.After 10 days of treatment,the subcutaneous abscess infected wounds with GCS-CG+NIR treatment was mostly healed.Besides,a considerably decreased infiltration of inflammatory cells was observed in the skin with GCS-CG combined with NIR irradiation.10.GCS-CG had no significant impact on the viability of fibroblasts,regardless of the incubation time and GCS-CG concentration in vitro.No obvious damages of the major organs including heart,liver,spleen,lung and kidney were observed at 7 and 21 days after GCS-CG injection with concentrations from 0.5 to 1 mg/mL.Meanwhile,21 days post treatment,complete blood panel parameters,kidney function makers,liver function markers and myocardial injury markers were also measured and found to be normal compared to the control groups.Conclusions:Based on these results,the following conclusions can be drawn:1.The porous rGO-PDMS composite membrane was successfully fabricated.The rGO-PDMS composite membrane had an ordered porous structure.Small pores on the top surface could prevent wounds from excessive water loss.Meanwhile,large pores on the bottom surface were beneficial for cell adhesion and proliferation.The addition of rGO sheets into PDMS could improve the mechanical strength,thereby adding to the application of the rGO-PDMS composite membrane for wound dressing under high stresses.The rGO-PDMS composite membrane exhibited a suitable WVTR to supply an ideally moist environment for wound healing and avoid substantial accumulation of wound exudates.The rGO-PDMS composite membrane was biocompatible in vitro.The rGO-PDMS composite membrane showed significant inhibition of the growth of the S.aureus and E.coli bacteria in vitro.The rGO?0.2 and 0.5 wt%?-PDMS composite membrane remarkably accelerated wound healing via enhancement of the re-epithelialization and granulation tissue formation,and could be considered as a promising multifunctional wound dressing.2.GCS-CG with a special two-dementional structure was successfully synthesized.Following conjugated with GCS,the absorbance of CG obviously increases in the NIR region,enabling GCS-CG to possess a high PTC.GCS-CG shows fast pH-responsive surface charge transition from negative to positive,which presents strong adherence to negatively charged bacteria surface in abscess?the acidic environment?.Thus,GCS-CG based PTT can heat the pathogenic bacteria directly and significantly accelerate wound healing.However,exposure in normal physiological conditions,GCS-CG presents poor affinity for the neighboring host cells,therefore,avoiding the undesirable increases in the temperature of ambient tissue,which commonly damages the surrounding healthy tissues.GCS-CG was biocompatible both in vitro and in vivo.GCS-CG described here,has great potential in the treatment of bacterial infections and even multidrug-resistant bacterial infections in skin wounds or subcutaneous tissues.