Bacterial infections represent a leading cause of global human mortality. Conventional antibacterial strategies predominantly rely on antibiotics, yet their excessive use has precipitated an escalating crisis of drug resistance [1], [2]. Globally, approximately 4.95 million deaths are associated with antibiotic-resistant bacterial infections in 2019, with 1.27 million deaths directly attributable to antimicrobial resistance [3]. The sluggish development of novel antibiotics in recent decades has exacerbated this crisis [4]. Therefore, developing non-antibiotic antimicrobial agents and alternative therapeutic strategies have become imperative.

Advances in optical technology and development of novel photosensitizers (PSs) have established antibacterial photodynamic therapy (aPDT) as a highly promising strategy to fight against drug resistant pathogens [5]. This approach capitalizes on the interaction of light, PSs and oxygen to generate reactive oxygen species (ROS) that can destroy critical bacterial components including lipids, proteins, DNA and so on [6], [7], [8]. This multi-targeted mechanism of action enables aPDT to eliminate antibiotic resistant bacteria while inducing minimal resistance development [9], [10]. Currently, diverse classes of PSs have been developed for aPDT applications [11], [12]. Conventional PSs encompass compounds such as porphyrins [13], [14], [15], [16], [17], [18], cyanine dyes [19], [20], [21], methylene blue [22], [23], [24], [25], [26], boron-dipyrromethene (BODIPY) derivatives [27], [28], [29], [30], [31], [32] and transition metal complexes [33], [34], [35], [36], [37], [38], [39], [40]. On the other hand, aggregation-induced emission (AIE) agents as the new generation of PSs have recently demonstrated remarkable aPDT efficacy towards various pathogens, including drug-resistant strains [41], [42], [43], [44], [45], [46], [47], [48], [49].

ROS plays a pivotal role in aPDT, yet its dual nature as a double-edged sword is evident, as it can also inflict damage on normal tissues through analogous mechanisms. Therefore, selective bacterial eradication with little damage to normal tissues is a precondition for clinical applications. However, conventional PSs with persistent “always-on” photo-activity struggle to meet this requirement, as photo-toxicity to healthy tissues (e.g., skin) becomes inevitable due to non-specific PSs accumulation and unintended irradiation exposure [50]. To address this issue, development of switchable “OFF-ON” PSs has emerged as a promising solution [51]. The photo-activity of these PSs is quenched at the initial state but can be selectively switched on by intrinsic pathogen-specific or infection-related triggers. These include bacterial enzymes such as β-lactamase [52], [53], hyaluronidase [19], [54] and nitroreductase [55], [56], alongside pathophysiological microenvironmental cues like the acidic pH in biofilm-infected and inflammatory tissues [57], [58] and overexpressed hydrogen sulfide (H2S) and HClO at the infection sites [59], [60], [61].

The aforementioned strategy is able to effectively mitigate PSs-mediated side-effects prior to activation. Nevertheless, the activated PSs can be released from bacteria through metabolism or bacteria lysis induced by aPDT. As a result, these activated PSs remain susceptible to be internalized by healthy tissues, which can still induce phototoxic effects when exposed to undesired light irradiation [62]. Ideally, the development of “OFF-ON-OFF” PSs, which are exclusively active within pathogens and remain inert both prior to entering and subsequent to exiting bacteria, holds immense potential for minimizing possible side-effects. “OFF-ON-OFF” PSs designed for selective PDT treatment of tumors have been reported by Prof. Liu's group [63], [64], [65]. To the best of our knowledge, no documented investigations have been published regarding the application of such “OFF-ON-OFF” PSs in aPDT.

In this work, Ru-AzoCF3, a bacterial azoreductase-responsive “OFF-ON-OFF” photosensitizer (Scheme 1), was studied as a proof-of-concept. The azo group generally serves as a highly effective quencher for excited states, as its rapid E-Z photo-isomerization process dissipates the energy of the excited state, consequently inhibiting the luminescence and ROS generation of Ru-AzoCF3 [63]. Notably, azoreductase is widely distributed among bacterial species with high levels [66], whereas mammalian cells generally demonstrate basal expression under normoxic conditions [67]. The azo group in Ru-AzoCF3 can undergo specific reduction into hydrazine group forming Ru-HZCF3 within pathogens, thereby restoring its luminescence and ROS generation. Interestingly, upon exiting pathogens via metabolic processes or bacterial lysis induced by aPDT, the active Ru-HZCF3 undergoes re-oxidation to inert Ru-AzoCF3, which thus limits the ROS production exclusively within pathogens through this reversible “OFF-ON-OFF” activation cycle. In this proof-of-concept study, Ru-AzoCF3 demonstrates potent aPDT efficacy against Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) both in vitro and in vivo, while maintaining excellent biocompatibility. The photophysical properties, “OFF-ON-OFF” reversible transformation in solutions and within bacteria were studied in detail.