Research And Application Of Photodynamic Antibacterial Chemotherapy In The Treatment Of Burn Infection

Background and objective:Burns can lead to severe skin defects and susceptibility to bacterial infections.Burn-wound infections often cause systemic sepsis and severe shock that can be fatal.In accordance with the latest report from the World Health Organization,about 265,000 deaths occur due to burn wounds each year.Burn wound excision and skin grafting combined with antibiotics are common clinical treatments.Staphylococcus aureus(S.aureus)are early colonizers and accounts majority of burn wounds,moreover,they are responsible for many other skin and soft tissue infections and life-threatening infections.However,the overuse of antibiotics in the last decades has led to the emergence of multiple drug resistant(MDR)strains.The strains often form recalcitrant biofilms which can protect the resident bacteria from antimicrobial drugs and phagocytosis.There is a pressing clinical need to explore alternative strategies for controlling and eradicating infections caused by MDR bacterial strains and their biofilms.Administration of these PSs within a suitable pharmaceutical formulation is essential for their clinical efficacy.Topical drugs come in liquid,semisolid,and solid formulations,and they can be classified as emulsions,gels,liposomes,and transdermal patches according to the structural matrix.Some of these formulations have been used in PACT due to favorable properties such as penetration and deep contact with damaged tissues and a prolonged remain during treatment.Thus,topical treatment such as PACT is a less invasion and more attractive alternative for clinical eradication of microorganisms.Compared with conventional drug carriers,hydrogels-based drug delivery systems have several distinct advantages in PACT.First,a hydrogel matrix is usually composed of compounds with diverse polarities,making them suitable for encapsulating various molecules and nanoparticles,including PSs and other functional agents.Second,thanks to the low fluidity of the hydrogels,the PSs in the hydrogel matrix can stay in situ for a long time with low unwanted diffusion,which can reduce the side effects of PACT.Wound healing is a complicated biological process involving a series of dynamic events,including hemostasia,inflammation,cell proliferation and differentiation,neovascularization,granulation tissue formation,collagen synthesis,epithelialization,and wound contraction.Increasing evidence has established the hypothesis that basic fibroblast growth factor(bFGF)are key regulators of normal and abnormal angiogenesis,and tissue repair in animals and humans.Therefore,increasing the levels of bFGF potentially has clinical superiority in cutaneous wounds.In addition,bFGF stimulates collagen synthesis and alleviates the scarring observed in wound healing,which has clinical application value of rapid burn wound healing.However,since growth factor is easy to denatured in presence of water and under higher temperature,bioactivity of the growth factor cannot maintain for a long term when it is used in the wound site in aqueous solution.As a result,it can only remain at the wound sites for a very short time,and the half-life of the growth factor becomes very short.Poly(lactic-co-glycolic-acid)(PLGA)is a biodegradable material,which has been widely used in nanoparticles as carriers to extend the sustained release time of drugs and promote tissue regeneration and repair.Based on the above research objectives,we screened the concentration of in vivo and in vitro photodynamic of DVDMS and the photodynamic antibacterial of hydrogel encapsulated DVDMS and nanomaterials.The specific research contents covered the following five parts:Methods and Results:(1)Effect of DVDMS-PACT on S.aureus/MDR-S.aureus:The type of free radicals of photosensitizers after light treatment have been detected by ESR,the enrichment time of DVDMS in bacteria was detected by alkaline lysis method,selected the maximum enrichment of the drug for PACT treatment.After PACT treatment,counting colony-forming units(CFU)was applied to estimate the bactericidal effect.SYTO 9/PI was used to detect the changes in the permeability of bacterial membranes.Scanning electron microscopy and atomic force microscopy were used to observe the bacterial cell walls and damage to biofilms.H2-DCFH-DA was used to detect the changes in intracellular reactive oxygen species(ROS).The results showed that with the increase of incubation time,the relative fluorescence intensity of DVDMS in bacteria increased and reached a peak at 75 minutes.Compared with the control group,DVDMS alone has no obvious toxicity,and DVDMS-PACT has a significant killing effect on S.aureus/MDR-S.aureus,and showed a dose-dependent(including light and drug dose).DVDMS combined with 50 J/cm2 can kill 99.99%of bacteria.After DVDMS-PACT treatment,the cell membrane,cell wall and biofilm showed obvious damage.During the DVDMS-PACT,a large amount of reactive oxygen species was generated,and the ROS production of drug-resistant bacteria was lower than that of S.aureus when treated with the same dose.By sequencing the bacterial transcriptome,it could provide the basis for the molecular mechanism and target of PACT antibacterial,and which could better reveal the mechanism of action of PACT to overcome bacterial resistance.In this study,the RNA-seq of multidrug-resistant S.aureus was compared between the DVDMS-PACT and the untreated control group,and a total of 222 differentially expressed genes were obtained.The expression of 111 genes in MDR-S.aureus was up-regulated,and the expression of 111 genes was up-regulated.Among them,ribosome,amino acid and other pathways were down-regulated,indicating that DVDMS-PACT may inhibit the regeneration of bacteria through the formation of proteins and cell wall membranes,which provided a basis for antibiotic resistance targeting ribosome.In addition,the pentose phosphate pathway increased bacteria's tolerance to oxidative stress through the production of NADPH,and the expression of genes related to the pentose phosphate pathway was upregulated after DVDMS-PACT treatment,indicating that oxidative stress in MDR-S.aureus is increased after PACT treatment,which reduced its DVDMS-PACT kill rate.(2)DVDMS-PACT for the treatment of burn infection:The infection model of deep second degree burns in mice was established by controlling different pressures and temperatures to study the effects of DVDMS-PACT on wound healing,bacterial colonization and skin factor levels in the infected area of burns to evaluated the efficacy and biological safety of DVDMS-PACT in the treatment of burn infection.Compared with the model group,the growth factors VEGF,bFGF,HYP and TGF-?1 levels was increased in the skin tissue of mice,the content of skin colonization bacteria and inflammatory factors TNF-?,IL-6,MDA were reduced and quickly promoted wound healing after CSDP hydrogel-PACT treatment.And DVDMS-PACT had no significant effect on the body weight of the mice or the histological features of the major organs,indicating good biosafety.(3)Synthesis and characterization of CSDP hydrogels(co-loaded with DVDMS and PLGA-bFGF nanoparticles):First,the PLGA nanoparticles loaded with bFGF were constructed by double emulsification method,and the basic properties of the nanoparticles were characterized by scanning electron microscopy,dynamic laser particle size analyzer,Zeta potentiometer.Second,CS hydrogel was formed by EDC/NHS catalysis.In the process of hydrogel formation,DVDMS and PLGA-bFGF nanoparticles were added to synthesize CSDP hydrogels.The basic properties of the hydrogels were characterized using infrared spectroscopy,rheometer,universal stretcher,and the drug stability and response release potential were detected under different pH conditions.The results showed that synthesized PLGA-bFGF nanoparticles sustained-release system with uniform and stable particle size.PLGA-bFGF nanoparticles could effectively promote the proliferation and migration of NIH-3T3 cells,increase the content of collagen and growth factors in skin tissues,and effectively promote skin wound healing.CSDP hydrogel still retained DVDMS spectral characteristics after being encapsulated with DVDMS.It has obvious pH response and could release DVDMS responsively in the wound.In addition,the hydrogel has inherent antibacterial properties,hemostatic properties,oxygen permeability,and mechanics.The properties,rheological properties and adhesion showed that the hydrogel has better wound adaptability.The fluorescence imaging results showed that CSDP hydrogels with the same concentration had higher fluorescence intensity and stability than free DVDMS in vivo and in vitro.The 1O2 yield of CSDP hydrogels after combined light treatment was 1.9 folds than that of free DVDMS-PACT.In addition,the hydrogel system has no obvious toxic and side effects on red blood cell,fibroblast cell,and skin of mice.(4)Killing effect of CSDP hydrogel on S.aureus/MDR-S.aureus:Using S.aureus/MDR-S.aureus as a model,the CFU was used to analyze the killing efficiency of CSDP hydrogel-PACT to S.aureus/MDR-S.aureus;SYTO 9/PI staining was used to detect bacterial cell membrane permeability after different treatments;Scanning electron microscopy and laser confocal microscopy were used to observe changes in the bacterial biofilm structure.Crystal violet staining and CFU were used to quantitatively analyze the bacterial biofilm;H2-DCFH-DA staining combined with flow cytometry to detect CSDP hydrogel-PACT treatment.The study found that:CSDP hydrogel-PACT can effectively kill S.aureus/MDR-S.aureus,the killing effect was better than Free DVDMS-PACT and clinical antibiotics at the same conditions;SYTO 9/PI staining observed bacteria membrane permeability change,and a large amount of ROS is produced in the process;Scanning electron microscope and laser confocal microscope have observed the destruction of bacterial biofilm structure,crystal violet quantitative and CFU experiments confirmed that CSDP hydrogel-PACT could effectively destroy the structure of bacterial biofilm and reduce the number of live bacteria in the biofilm.(5)Treatment of burn infection with CSDP hydrogel:The treatment of burn infection by CSDP hydrogel-PACT was judged from skin wound healing rate,skin colonization bacteria,pro-inflammatory factors,anti-inflammatory factors,and slice staining in skin tissue.The results showed that CSDP hydrogel-PACT could better promote skin healing after burn infection and reduce the content of skin colonization bacteria compared with free DVDMS-PACT and CSD hydrogel-PACT treatment.Skin sections and ELISA results showed that CSDP hydrogel could promote the contraction of wound area,increase the content of collagen and hair follicles in the skin,reduce the levels of skin inflammatory factors,and be beneficial to wound healing.And there were no obvious changes in the weight and organs of the mice during the treatment,indicating good biosafety.Conclusion:(1)DVDMS-PACT could effectively kill S.aureus/MDR-S.aureus,causing damage to the cell membrane,cell wall and biofilm of bacteria and accompanied by the production of a large number of ROS.RNA-seq results showed that 222 differentially expressed genes were detected after DVDMS-PACT treatment.The differentially expressed genes of MDR-S.aureus mainly focused on metabolic process,molecular function and cell components,etc.The mechanism of action of DVDMS-PACT damaged bacteria was related to the expression changes of these genes.(2)DVDMS-PACT could reduce the number of colonizing bacteria and the content of inflammatory factors on skin wounds,increase the level of growth factors in skin tissues,and promote the healing of skin at the infected site of burns.(3)The synthesized CSDP hydrogel has good mechanical properties,adhesion,hemostatic properties,biocompatibility and the stability of the loaded drugs,which can be used for the environmental responsive drug release on the wound surface of burn infection.(4)CSDP hydrogel-PACT treatment had a significant killing effect on S.aureus/MDR-S.aureus,and the effect was superior to the Free DVDMS-PACT group.(5)CSDP hydrogel-PACT could promote the rapid healing of skin after burn infection,reduce the content of skin colonization bacteria and inflammatory factors,and improve the level of growth factors in skin tissue.The treatment effect of CSDP hydrogel-PACT was better than that of Free DVDMS-PACT and CSD hydrogel-PACT.