Preparing A Photothermal-induced Targeted Graphene Nano-Antibacterial Materials For Treatment Of Skin Infection

Background:Bacterial infectious disease,in particular multi-drug resistance,has turned into one of the world wide serious problems.Multi-drug resistant bacterial infection of the wound will not only aggravate the deterioration of the wound,delay the healing,but also lead to invasive infections and even the death of sepsis.Such worry boosts the improvement of alternative efficient therapeutic approaches to treat drug-resistant bacteria infections.Photothermal therapy and photodynamic therapy are new methods to diagnose and treat diseases by using their photothermal and photodynamic effect,respectively.Photodynamic therapy is recognized as the best treatment strategy of invasive tumor or bacterial infection via the production of free radical.In general,light energy in photothermal therapy can undergo the transfer by photo-sensitizers?PSs?to produce reactive oxygen species?ROS?for ablating of tumor cell or bacteria.Nevertheless,ROS generation in photodynamic therapy depends on oxygen,significantly limiting the therapeutic effects in hypoxic bacteria infected wounds.Photothermal therapy materials with high photothermal conversion efficiency and converts light energy into heat energy when exposed to an external light source,killing tumors or bacteria.When used alone,the radiation intensity usually required is higher than tolerance of the skin,and the resulting local high temperature may cause serious damage to nearby healthy tissue.Carbon-based free radicals are also highly reactive and can be formed without oxygen.However,controlling these radicals'generation at the bacteria infection sites still remains a great challenge.As a class of two-dimensional nanomaterials,graphene has aroused wide interests in drug delivery fields due to its large specific surface area.Graphene derivatives have good biocompatibility.Thus,graphene nanosheets are well suited as the heat source and initiator carrier.Through dopamine self-polymerization in the alkaline case,polydopamine can adhere to the surface of a variety of materials.It is a very effective photosensitizer with remarkable light absorption performance.GCS,a water-soluble chitosan derivative featuring pH-sensitive charge reversa,has many excellent properties such as biofunctionality,compatibility,blood compatibility,safety and biodegradability.It can bind to bacteria that with negative charge.A new therapeutic strategy for the eradication of multidrug-resistant bacteria is proposed in this study according to alkane free radicals'light-induced generation.The characterization in the synthesis process,the antibacterial properties,antibacterial mechanism,ability of targeting bacteria in vitro and in vivo,biocompatibility and effects of alkanes on wound healing of multi-drug resistant bacteria were studied under normal oxygen condition and oxygen-free condition.Graphene nanocomposites provides a new oxygen independent strategies,and reveals the various free radicals in biomedical application potential,but also offers a detailed experimental basis for the future research and development of new functional graphene antimicrobial dressings or coatings based on free radical initiators.It also provides a new idea and method for the treatment of infected wound.Objective:The purpose of this study is to develop an initiator-losded and pH-sensitive heat-producible hybrid of bactericides based on the light-activated alkyl free-radical generation?AIBI-GCS-PDA@CG?,and the antibacterial effect,biological safety and the influence on infected wound healing of the prepared AIBI-GCS-PDA@CG will be investigated.The accomplishment of this study will provide a new therapeutic direction and strategy for the treatment of multiple drug-resistant bacterial infections.Methods:1.Synthesis and characterization of AIBI-GCS-PDA@CG nano-antibacterial materials:1.1 Synthesis of AIBI-GCS-PDA@CG nano-antibacterial materials:AIBI-GCS-PDA@CG was synthesized by using carboxyl graphene as the substrate,coated with polydopamine and crosslinked with ph-sensitive glycol chitosan molecules through non-covalent bond loading of free radical initiator.1.2 Characterization of AIBI-GCS-PDA@CG nano-antibacterial materials:The hydrodynamic diameters and Zeta-potentials of AIBI-GCS-PDA@CG were measured by nanoparticle size analyser.The photothermal ability of AIBI-GCS-PDA@CG were characterized by an 808 nm NIR laser and an IR thermal camera.The surface size and morphology of the prepared samples were characterized under AFM and TEM.Using an IR Prestige-21 infrared spectrophotometer,the FTIR spectra were recorded.With the use of a UV-vis-NIR spectropho-tometer,the UV-vis-NIR optical properties of AIBI-GCS-PDA@CG in PBS were recorded.The contents of nanocomposites were acquired using TGA in a N₂ environment.The Raman spectra of graphene composites were recorded using a Raman spectrometer.2.Study on the targeted effect of AIBI-GCS-PDA@CG nano-antibacterial materials:2.1 In vitro AIBI-GCS-PDA@CG's Specific Targeting to Bacteria:By incubating them together in an acidic environment?pH 6.3?and a physiological environment?pH 7.4?,the interactions between synthetic nanomaterials and bacteria were assessed in vitro.The Zeta-potentials of the bacteria together with nanomaterials were detected by the nanoparticle size analyzer.Meanwhile,the morphology of synthetic nanomaterials with bacteria and also NIH 3T3 fibroblasts were detected under SEM.2.2 In vivo AIBI-GCS-PDA@CG's Specific Targeting to Bacteria:The stability of fluorescent dyes combined with nanomaterials was tested by a UV-vis-NIR spectropho-tometer.The NIRF and fluorescent imaging of bacterial infection in vivo were detected by with an IVIS Lumina II imaging system.The thermographic images of bacterial infection were taken with an IR thermal camera,and the photothermal ability and targeting of bacterial infection site of the nanomaterials was also evaluated.3.Study on the photothermal-induced targeted antibacterial activity of AIBI-GCS-PDA@CG nano-antibacterial materials:3.1 In vitro antibacterial effect of AIBI-GCS-PDA@CG nano-antibacterial materials:AIBI-GCS-PDA@CG's in vitro antibacterial efficiency was assessed with the standard plate count.3.2 In vivo antibacterial effect of AIBI-GCS-PDA@CG nano-antibacterial materials:The model of MRSA subcutaneous abscess in mice was established.The photothermal antimicrobial effect was observed after tail vein injection,and the colony counts of abscess tissues were quantitatively analyzed.The healing of abscess tissues was analyzed by H&E staining.4.Mechanism of antibacterial action of AIBI-GCS-PDA@CG nano-antibacterial materials:4.1 Detection of alkanes free radicals of AIBI-GCS-PDA@CG nano-antibacterial materials:The ESR signal of AIBI-GCS-PDA@CG was detected using an ESR spectrometer.The formation of alkanes free radicals after AIBI-GCS-PDA@CG combined with bacteria was detected by ESR.The mechanism of alkanes free radical treatment under the normal oxygen and hypoxia conditions was detected by ESR.4.2 Molecular biological effects of free radicals produced by AIBI-GCS-PDA@CG nano-antibacterial materials:Detection of reactive oxygen species?ROS?,glutathione?GSH?and DNA damage under normal and anaerobic conditions to investigate the molecular biological effects of alkanes free radicals on bacterial oxidative stress.5.Biocompatibility Evaluation of AIBI-GCS-PDA@CG nano-antibacterial materials:5.1 Effects of AIBI-GCS-PDA@CG nano-antibacterial materials on the proliferation of 3T3 fibroblasts:After co-culture with 3T3 fibroblasts for 24 hours,AIBI-GCS-PDA@CG's in vitro cytotoxicity was ascertained by a cell counting kit-8?CCK-8?assay.5.2 Study on blood compatibility of AIBI-GCS-PDA@CG nano-antibacterial materials:The blood compatibility of AIBI-GCS-PDA@CG was tested by hemolysis assay.5.3 In vivo safety testing of AIBI-GCS-PDA@CG nano-antibacterial materials:After AIBI-GCS-PDA@CG injected into the mice through tail vein for 1 and 30 days,the pathological changes of the main organs and blood systems of mice were observed to explore their acute and chronic toxic effects.Result:1.Development and characterization of AIBI-GCS-PDA@CG nano-antibacterial materials:1.1 The particle size of 37.38%AIBI-GCS-PDA@CG was 712nm after 2 hours ultrasound,39.12%AIBI-GCS-PDA@CG was 459nm after 6 hours ultrasound,32.18%AIBI-GCS-PDA@CG was 220nm after 10 hours ultrasound.1.2 AIBI-GCS-PDA@CG solutions increased rapidly in the case of the NIR light?0.5or 0.75 W/cm 2?,achieving concentration-dependent plateaus in 7 min?AIBI-GCS-PDA@CG exhibited both laser energy-and concentration-dependent temperature change.The AIBI-GCS-PDA@CG has a high PTC of 41.96%.1.3 After five cycles of irradiation at 0.5 W/cm 2,AIBI-GCS-PDA@CG suspensions'absorbance?808 nm?loss was below 5%,illustrating AIBI-GCS-PDA@CG had a prominent photothermal stability.After being cultured in 10%serum-containing PBS,the hydrodynamic diameter of AIBI-GCS-PDA@CG showed negotiable change with the incubation time increasing,indicating that the nanosheets in serum-containing medium were very stable for at least 7 days.1.4 TEM showed that the surface of AIBI-GCS-PDA@CG was smooth and transparent with wrinkles,and AFM showed the thickness of AIBI-GCS-PDA@CG was?2.1 nm,with a single layer with slightly rough surface.1.5 The nanoparticle size meter showed that the surface of AIBI-GCS-PDA@CG was positively charged at pH 5.0-7.0 and negatively charged at pH 7.4.1.6 FTIR-ATR showed that the characteristic absorption peaks of CG,PDA,GCS and AIBI all appeared in AIBI-GCS-PDA@CG.1.7 The UV-vis-NIR spectrometer suggested that the absorption peak of AIBI-GCS-PDA@CG increased significantly at 808nm.1.8 TGA indicated that AIBI loading in AIBI-GCS-PDA@CG was 5.84%.1.9 The Raman spectra indicated that the G peak of graphene in AIBI-GCS-PDA@CG was near 1580cm^-1,and the D peak was near 1350cm^-1.2.Study on the targeted antibacterial effect of AIBI-GCS-PDA@CG nano-antibacterial materials:2.1 In vitro,the solution of aibi-gcs-pda@cg mixed with bacteria?pH 7.4?was turbid without aggregation and precipitation.Under pH 6.3,the mixture rapidly accumulated and precipitated to the bottom of the tube within 30 min.2.2 The pH-responsive GCS-PDA@CG and AIBI-GCS-PDA@CG showed a net-positive surface charge under the aforementioned acidic environment?pH 6.3?,causing close electrostatic interaction with the MRSA membrane charged negatively,which converts their surface charge from negative to positive.2.3 SEM showed that bacteria?S.aureua,MRSA?were closely combined with GCS-PDA@CG and AIBI-GCS-PDA@CG under acidic conditions,but GCS-PDA@CG and AIBI-GCS-PDA@CG was not found under neutral conditions.AIBI-GCS-PDA@CG or GCS-PDA@CG nanosheets had a little negative charge and were not attached to the cultured cells charged negatively when exposed to 3T3 fibroblasts at 7.4.2.4 The Cy5 molecules were conjugated on the nanosheets through the covalent bond;therefore,Cy5 molecules could not dissociate with the complex at least 24 hours,which was also confirmed by the fluorescence spectroscopy characterizations.It is proved that f-AIBI-GCS-PDA@CG can effectively play an imaging role in vivo.2.5 Much fluorescence could be observed via Live animal imaging system at 2 h after AIBI-GCS-PDA@CG injection,and it gradually reached the maximum at 6 h after injection,indicating that NPs accumulated continuously at the abscess site and decreased gradually at 24 h after injection.Ex vivo fluorescence images of 24 h post injection further demonstrated enrichment of AIBI-GCS-PDA@CG in abscess and some metabolic organs like the liver owing to the reticuloendothelial system.2.6 Thermal imaging figure further showed a good abscess-targeting efficiency in vivo,and indicated that the AIBI-GCS-PDA@CG nanosheets possessed remarkable photothermal conversion capacity in vivo.3.Study on the photothermal-induced targeted antibacterial activity of AIBI-GCS-PDA@CG nano-antibacterial materials:3.1 Standard plate counting showed that AIBI-GCS-PDA@CG had antibacterial activity under photothermal condition.3.2 In vivo animal experiments showed that the anti-bacterial rate of AIBI-GCS-PDA@CG was 99%.H&E staining indicated that the abscess wound healed basically.The effect of AIBI-GCS-PDA@CG in treating subcutaneous abscess caused by multi-drug resistant bacterial infection was very significant.4.Mechanism of antibacterial action of AIBI-GCS-PDA@CG nano-antibacterial materials:4.1 The ESR spectra showed that AIBI-GCS-PDA@CG could generate alkanes free radicals under 808nm NIR.4.2 ESR showed that the spectrum intensity of alkanes free radicals decreased after AIBI-GCS-PDA@CG combined with bacteria,which proved that alkanes free radicals played a role.4.3 Under the normal oxygen and hypoxia conditions ESR showed that the generation of R·is oxygen independent and can easily be transferred to RO·by oxygen under air environment because of its high reactivity.4.4 Under normal oxygen conditions RO·produced by AIBI-GCS-PDA@CG could directly lead to the increase of bacterial ROS level,and under hypoxia conditions R·could lead to oxidative stress reaction of bacteria through cumulative effect,which both eventually leaded to bacterial death.4.5 The generated alkyl radicals could effectively cleave the DNA strands under both normoxic and hypoxic conditions,indicating the high DNA-damaging activity of AIBI-GCS-PDA@CG in bacteria.5.Biocompatibility Evaluation of AIBI-GCS-PDA@CG nano-antibacterial materials:5.1 CCK-8 assay showed that AIBI-GCS-PDA@CG had negligible toxicity to 3T3fibroblasts when the concentration was lower than 1.0 mg/mL.5.2 When AIBI-GCS-PDA@CG concentration was 0.1mg/ml-1.0mg/mL,the hemolysis rate was less than 5%,showed no damage to red blood cells and good blood compatibility.5.3 AIBI-GCS-PDA@CG at concentrations of 0.20 mg/mL and 0.50 mg/mL was injected into the tail vein of mice for 1 day and 30 days,and no significant pathological changes were observed in the main organs,indicating that the mice had good biocompatibility in vivo.After 30 days,the blood routine,liver function and kidney function of the mice were examined,and there was no obvious chronic toxicity,which further proved the good biological safety of AIBI-GCS-PDA@CG.Conclusion:A novel therapeutic strategy based on light-induced free radicals is proposed to therapy the infection of drug-resistant bacteria.Combining the photothermal effect of our bactericidal depot with the thermal decomposability of AIBI,AIBI-GCS-PDA@CG could produce free radicals in the case of NIR light.The produced free radicals show equivalent therapeutic efficacy under hypoxia and normoxia yet with various mechanisms.On exposure to normoxia,the generated alkyl radicals?R·?are able to undergo conversion into alkoxyl radicals?RO·?by oxygen,directly causing bacteria damage.Under hypoxia,the produced alkyl radicals?R·?also show DNA-damaging activity.This is a cause of bacterial death.Besides,the AIBI-GCS-PDA@CG has a significant therapeutic effect on drug-resistant bacteria in vivo.The results show that this method provides the ability and possibility for the application of phototherapy under more complex conditions and is of great value for subsequent research.