3.3. Assessment of antibacterial activity
The in vitro antibacterial activity of OHH NPs was investigated by bacteriostatic ring test using MRSA and K.Pneumoniae as model organisms. After 24 hours of culture, there was not any inhibitory area observed in the tested bacteria after treatment with PBS or HMPB NPs. By contrast, free ofloxacin and OHH NPs treatment groups showed obvious inhibition zone (Fig. 3A) owing to antibacterial activity of ofloxacin. Fig.3B and Fig.3C showed that the diameters of inhibition zones of ofloxacin towards MRSA and K.Pneumoniae were ~1.70 cm, ~3.77 cm, respectively and the diameters of inhibition zones of OHH NPs towards MRSA andK.Pneumoniae were ~1.48 cm, ~3.33 cm, respectively. It should be noted that the inhibition ring of free ofloxacin at an equivalent concentration with loading ofloxacin of OHH NPs was slightly larger than that of OHH NPs, which may be due to slower ofloxacin release of OHH NPs. Meanwhile, the inhibition ring of ofloxacin for K.Pneumoniae (Gram-negative bacteria) was much larger than that for MRSA(Gram-positive bacteria), which was attributed to ofloxacin with stronger antibacterial activity towards Gram-negative bacteria. Additionally, the chemo-photothermal synergistic bactericidal efficacy of OHH NPs was investigated using MRSA and K.Pneumoniaeby the spread plate technique. Specifically, MRSA andK.Pneumoniae cells treated with different agents were diluted 105 and 104 times, respectively. In Fig. 3D and 3F, the number of colonies after treatment with NIR or HMPB NPs hardly reduced compared with control group (PBS). However, the significant decrease of the number of bacterial colonies was observed for HMPB NPs+NIR, ofloxacin, ofloxacin+NIR and OHH NPs treated groups. The bacteria-killing efficacy of HMPB NPs+NIR, ofloxacin, ofloxacin+NIR and OHH NPs treated groups reached 28.38%, 53.90%, 49.35% and 27.76% for MRSA, respectively and it reached 51.63%, 75.61%, 73.52% and 72.56% for K.Pneumoniae , respectively. These data demonstrated that, in the tested dose range, both sole ofloxacin and photothermal treatment could not eliminate all the bacterial colonies. Surprisingly, the OHH NPs combined with NIR laser (OHH NPs+NIR) exhibited high antibacterial efficiency towards MRSA and K.Pneumoniae . Almost no bacterial colonies were observed in OHH NPs+NIR treated groups and the inactivation of bacteria was highly up to 99.99% (Fig. 3E and 3G). The ultrahigh antibacterial efficiency of OHH NPs+NIR could be attributed to the effective synergistic effect of ofloxacin and PTT. Compared with the loading rates of two different Prussian blue (Fig.S3), we can observe that the hollow mesoporous structure can effectively increase the loading of antibiotics. Due to the laser irradiation and the secretion of HAase in bacterial cells, ofloxacin is released from the nanoparticles about 80% for 24 hours.