Construction And Antibiofilm Application Of Antibacterial Carbon Dots And Its Composite Systems

Harmful bacteria invade organisms to form biofilm,which provides a protective barrier for the growth of internal bacteria,leading to bacterial infectious diseases.At present,the most effective method in clinic is using antibiotics,but the abuse of antibiotics leads to bacterial resistance,while the development of new antibiotics is very slow.In order to deal with the problem of the formation of bacterial biofilm and drug resistance,antibacterial nanomaterials with multiple antibacterial modes come into being.However,their biological toxicity problems need to be solved.Therefore,nanomaterials with antibacterial ability and biocompatibility,which can effectively antibiofilm without bacterial resistance,have become an important scientific problem in the field of antibacterial research.Carbon dots(CDs)have attracted the attentions of the scholars due to their low biological toxicity,easily modified surface and multi-function.Based on these,we have designed and constructed a series of antibacterial CDs and antibacterial composite systems based on CDs.Main work of this paper includes the following issues:Firstly,considering the resistance of bacteria and the impedance of biofilm to antibiotics,CDs are prepared from gentamicin sulfate by one-step calcination method inspired by the idea of nanocrystallization.If the calcination temperatures are lower than 210?,CDs will have good antibacterial activity even better than the precursors.When the calcination temperatures exceed 210?,the antibacterial activity of CDs will decrease rapidly until it disappears.Studies have shown that the antibacterial activity of some CDs comes from the retention of gentamicin groups on their surfaces,but they do not lead to bacterial resistance like gentamicin.It is the reason that the surface potentials of the CDs are positive and the CDs can produce a lot of reactive oxygen species(ROS)in bacteria.Due to the multiple antimicrobial modes,CDs will not cause bacterial resistance and are easier to penetrate biofilm compared with gentamicin.In addition,CD₁₈₀ has low cytotoxicity to HEK 293T,HUVEC,L929 and 3T3 cells.The survival rates are above 91%at 2000?g/m L.Considering the important effect of surface chemistry of CDs on their antibiofilm properties,polyamine-functionalized CDs(CD₆₀₀,CD_1w and CD_2.5w)are prepared by hydrothermal method using citric acid and polyethyleneimine(PEI)with molecular weights of600,10000 and 25000 as precursors.With the increase of molecular weight of polyethyleneimine,the antibacterial and antibiofilm activities of the CDs increase.The results show that although the surface groups and their existing forms of the three CDs are the same,the contents of these groups are different,especially the amino groups,which lead to different antibacterial properties.Different from the CDs obtained from the gentamicin,CDs modified by polyamine do not produce a large amount of ROS.Their antibiofilm activity is related to the surface chemistry,mainly the cation surface promotes the penetration of biofilm.At the same time,the surface properties also reduce the biocompatibility of CDs.Contrary to antimicrobial activity,the cytotoxicity of CD₆₀₀,CD_1w and CD_2.5w increases in turn.Considering antibacterial activity and biocompatibility simultaneously,CD_Lys and CD_Argare prepared by pyrolysis from L-arginine and L-lysine inspired by antimicrobial peptides.They have excellent antibacterial and antibiofilm properties.And the bacteria will not easy to develop drug resistance to these CDs.CD_Lys and CD_Arg can destroy the bacterial cell membrane by electrostatic interaction and ROS oxidation,resulting in the leakage of components,such as?–galactosidase.And these CDs can also inhibit the activity of TTC dehydrogenase in mitochondria.It is worth noting that CD_Lys and CD_Arg not only have no hemolytic activity and cytotoxicity,but also can promote the proliferation of 3T3,BMSCs and HUVECs,which show extraordinary cell selectivity.The reason is that CD_Lys and CD_Arg can effectively inhibit the activities of SOD and CAT in E.coli and S.aureus which leads to the lack of strong antioxidant capacity of bacteria to remove excessive ROS.However,they have no inhibitory effect on the activities of SOD and CAT in mammalian cells.Thus,these CDs achieving high cell selectivity.Animal studies have demonstrated that CD_Lys and CD_Arg can reduce inflammation,accelerate wound healing and promote tissue repair in the back wound of mice.On the basis of above reaserch,considering the slightly acidic environment of biofilm,we modify the surface of CD_Lys with the acid responsive and negatively charged triblock polymer(polyethylene glycol-polyethyleneimine-2,3-dimethylmaleic anhydride,PPD)by electrostatic self-assembly to prepare p H-responsive antibiofilm nanosystem(PPD@CD_Lys).In the neutral condition,PEG forms a hydrated layer on the surface of the nanosystem,which plays a"stealth"role.In the weak acid condition,the responsive component hydrolyzes and the charged property of PPD reverses,which leads to the disassembly of the nanosystem.Firstly,the outer hydrophilic polymer of the PPD@CD_Lys can rapidly diffuse in the biofilm to enhance the penetration of the nanosystem.At the same time,it will be dissociating into two antibacterial parts,m PEG-PEI and CD_Lys,in response to the acid environment.And then,through the effects of electrostatic contaction and ROS oxidation,the synergistic antibiofilm effect can be achieved.The mature S.aurues biofilm could be completely destroyed within 60 min by 250?g/m L PPD@CD_Lys.Under physiological conditions,the nanosystem shows good cytocompatibility to L929 cells.Even when the concentration of PPD@CD_Lys is as high as 2000?g/m L,the hemolytic activity is still at zero level.In order to expand the application of CDs,gentamicin-based CD_AG is prepared by two step calcination method to construct nanocomposite antibacterial Schiff-base hydrogel.On the one hand,CD_AG has high yield and multiple antibacterial mechanisms,which does not cause bacterial resistance and has excellent antibiofilm activity.On the other hand,the CD_AG-hydrogel has good viscoelasticity,injectable and self-healing properties.And it can release the CD_AG“on demand”according to the acid response function of Schiff base bond as the change of external p H.The composite hydrogel has a high inhibition rate of 93.6%for S.aureus biofilms and exhibit excellent biocompatibility.Compared with commercial antibacterial hydrogels,the epithelium and connective tissue of the wound after CD_AG-hydrogel treatment show better integrity and regularity,which form more fibroblasts,blood vessels,hair follicles and thicker granulation tissue.To sum up,this thesis focuses on the construction of new CDs-based antibacterial nanomaterials and their application of antibiofilm.Combined with the characteristics of CDs,such as low biological toxicity,easily modified and functionalized surface,these nanomaterials can achieve the purpose of antibacterial ability and biocompatibility,which will effectively resist biofilm without causing bacterial resistance.