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.