Heat-Enhanced Bacterial Penetration Of Photosensitizers And Antibacterial Application

Bacterial infection is still a clinical challenge even though some advanced antibacterial materials and techniques have been proposed.With the development of nanomedicine technology,polymeric hydrogel materials have been widely used in the field of microbial diagnosis and prevention due to their irreplaceable and unique merits.The development of new optical diagnosis and treatment techniques have also greatly promoted the biomedical applications of hydrogels.In this thesis,the minimal temperature at which the photosensitizer can effectively penetrate the biofilm of Gram-positive bacteria and Gram-negative bacteria was determined by thermal regulation.Besides,the combination of mild-temperature photothermal therapy and photodynamic therapy(PDT)of bacteria and bacterial biofilms was realized.In addition,we prepared a supramolecular bio-hydrogel loaded with photosensitizer and photothermal reagent,and demonstrated that it could reazlie the combined photothermal and photodynamic therapy(PTDT).Specifically,this thesis mainly includes the following two sections:(1)Hyperthermia-enhanced antibacterial effect based on rose bengal(RB).We explored the minimal temperature at which RB could effectively penetrate the biofilm of Gram-positive and Gram-negative bacteria,and realized the hyperthermia-promoted photodynamic therapy of bacteria Gram-negative bacteria,such as E.coli,have an outer membrane,which cannot be penetrated by nanodrugs at room temperature.Hyperthermia effectively enhanced the penetration of RB into E.coli’s wall/membrane.After E.coli and RB were incubated at 53℃for 15 min,the permeability of the bacterial cell wall/membrane increased,and a large amount of RB entered the Gram-negative bacteria that were difficult to be eliminated.After 15 min illumination with a 660 nm laser(0.2W/cm~2),massive ROS were generated to disrupt the bacterial structure and finally achieve a 99%inactivation efficiency.In addition,we also compared the penetration amount of rhodamine B,fluorescein isothiocyanate(FITC),and SYTO9 toward the heat-treated E.coli,and found that RB,which is more water-soluble,has a penetration advantage compared with other hydrophobic dyes.Interestingly,co-incubation of SYTO9 and E.coli at 53℃could achieve stable fluorescence staining for more than 72 h,which could lead to long-term fluorescence labeling of E.coli.(2)Photothermal and photodynamic antibacterial therapy based on a supramolecular hydrogel loaded with RB and cypate(a cyanine dye and a commonly used photothermal agent).We first purified Nap FFY(Nap-Phe-Phe-Tyr-OH)by solid-phase polypeptide synthesis(SPPS)and high performance liquid chromatography(HPLC).Then we prepared the injectable hydrogel by mixing RB and cypate.Dense nanofiber network structure was found by transmission electron microscopy.In this work,upon 808 nm(1 W/cm~2,15 min)light irradiation,the hydrogel was heated,which increased the permeability of E.coli cell wall/membrane and the amount of RB entering E.coli.On the other hand,heat itself killed part of E.coli.With the help of further PDT,this hydrogel system could kill 98%of the bacteria.Moreover,the synergistic action mechanisms of the RB/cypate@Nap FFY with bacteria and biofilms were explored in detail from membrane disruption and ROS generation.It is expected that the supramolecular injectable hydrogel loaded with RB and cypate can release singlet oxygen in situ in infected sites to achieve local sterilization,and will not lead to bacterial resistance,which is of great significance for developing highly effective antibacterial materials.