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