Fig. 3. In vitro antibacterial activity test. (A)Photographs of antibacterial results of PBS(a),HMPB(b), Ofloxacin(c) and OHH NPs(d), against MRSA and K.pneumoniae . (B–C) Quantitative measurement of the inhibition zones. (D,F)Photographs of the agar plates of MRSA and Klebsiella Pneumoniae treated with PBS,HMPB,Ofloxacin and OHH NPs with/without 808 nm light irradiation (1 W/cm2, 5 min). (E, G) Treatment with PBS was set up as control. Effect of OHH NPs on bacterial counts of MRSA and K.pneumoniae . The all concentration above of HMPB is 100μg/mL and ofloxacin is 10 μg/mL.
Next, the viability of K.pneumoniae was further investigated by a fluorescence staining assay based on the integrity of bacterial membrane with HMPB,Ofloxacin and OHH at the same antibiotic concentration (10μg/mL)and the same HMPB concentration (100 μg/mL).As shown as Fig. 4A, almost no visible red fluorescence was observed after treatment with PBS, NIR and HMPB, indicating nearly all of the bacteria survived in these groups. In comparison, the bacteria treated with HMPB NPs+NIR, ofloxacin, ofloxacin+NIR and OHH NPs+NIR emitted visible red fluorescence, which demonstrated a part of bacteria were killed. However, the bacreria treated by OHH NPs+NIR presented the strongest red fluorescence(Fig. 4B), suggesting most of the bacreria were inactive38. Additionally, SEM images of Fig. 4C further verified that sole PBS and HMPB NPs had no antibacterial ability, which was confirmed by the intact and smooth bacterial membranes, while the bacterial membranes after treatment with OHH NPs+NIR were obviously shrinking. Collectively, all of the results provided confirmative evidence of OHH NPs having highest bacteria-killing ability.