2.5.2 Antibacterial ability assay
The ROS generation capability of THB was initially tested employing
commercially available dichlorodihydrofluorescein diacetate (DCFH) and
9,10-anthracenediylbis(methylene)-dimalonic acid (ABDA) (Figure 4D, 4E).
The fluorescence of DCFH rapidly increased with white light irradiation
time at the presence of low concentration THB, demonstrating the good
total ROS generation efficiency of THB. The absorbance of ABDA also
decreased as the increasing generation of singlet oxygen, suggesting the
potential photodynamic antibacterial performance of THB. S.
aureus or E. coli was incubated with THB NPs, THB@ANVs or
THB@ANVs hydrogels under the same conditions to investigate the
photodynamic killing effect on Gram-negative and Gram-positive bacteria
by traditional plate method. All groups exhibit high photodynamic
antibacterial ability againstS. aureus under white
light irradiation with a killing
rate of nearly 100 % (Figure 4F, S14). Besides, we were surprised to
find that the antibacterial ability of THB against Gram-negative
bacteria was notably enhanced after encapsulated into ANVs.
Both Gram-positive and Gram-negative bacteria are negatively charged,
which facilitates the positively charged THB to stain, but the thick
outer membranes of Gram-positive bacteria might obstruct the binding
process. To figure out the reason for the increased antibacterial
efficacy change of THB ANVs on Gram-positive bacteria and Gram-negative
bacteria, the surface charges of the bacteria were measured by Zeta
potentials (ζ ), aiming to investigate the interactions between
the bacteria and THB (Figure 4G). Apparent positive potential shift of
both S. aureus and E. coli was observed after being incubated with
THB@ANVs, while the ζ potentials of E. coli incubated with THB
NPs remain almost constant compared with that of E. coli alone.
The above results indicate that the ANVs encapsulation process of
photosensitizers may enhanced their antibacterial activity by increasing
the binding ability of THB with Gram-negative bacteria. The aggregation
induced luminescence properties of THB was also used for bacteria
imaging. It was visualized in Figure 4H that both the fluorescent signal
of S. aureus and E. coli showing excellent image contrast
to the background after incubation with THB@ANVs. This proves the
bacterial targeting ability of THB@ANVs on both kind of bacteria. The
higher labeling efficiency of S. aureus than E. coli can
also be observed, which is consistent with the better killing effect onS. aureus . As shown in Figure 4I, SEM was further applied to gain
insights into the morphology changes of S. aureus and E.
coli upon treatment with THB@ANVs, followed by white light irradiation.
After THB@ANVs-based PDT treatment, the bacteria underwent shape change
of cell walls shrinkage and fusion, providing direct evidence of the
bacteria killing effects.
2.6 In vivo wound healing assessment of deep second-degreescald wound