3.4. Antibacterial mechanism exploration
In addition to the aforementioned results, the antibacterial mechanism was further explored by employing DCFH-DA probe to monitor ROS generation with different treatments on K.pneumoniae34, 39. As shown in Fig. 5A and Fig. 5B, the bacteria treated by OHH NPs +NIR exhibited the strongest green fluorescence signal, indicating the highest level of ROS produced, which was likely responsible for the bactericidal efficiency. As the major energy storage molecule, ATP plays a critical role in the various processes of physiology and pathology. Usually, ATP will decrease under conditions of necrosis or apoptosis 34, 40-42. We thus wish to explore if the decrease of the ATP level occurs after exposure to OHH NPs+NIR. Fig. 5C showed HMPB NPs treatment hardly caused the decline of ATP level in K.pneumoniae comparing with the control group. By comparison, a weak decrease of ATP level was caused after incubation with ofloxacin or OHH NPs. In contrast, ATP level dramatically declined about 92.3% after the treatment of OHH NPs+NIR, suggesting the best antibacterial efficacy of this strategy. Furthermore, protein leakage from K.pneumonia was also investigated, which was taken as a representative indicator of cell membrane damage 43. According to Fig. 5D, protein leakage from K.pneumonia of HMPB NPs-treated group was 10.07 μg/mL, which was similar with the control group. In comparison, protein leakage of bacteria after exposure to ofloxacin and OHH NPs increased to 24.97 μg/mL and 22.06 μg/mL, respectively. Notably, protein leakage in the OHH NPs+NIR-treated group was up to 42.26 μg/mL, which was about 5-fold that of the control group. These results indicated that the combination of ofloxacin and PTT could severely destruct bacterial cell membranes.