Title：pH-Responsive AIE Photosensitizers for Enhanced Antibacterial Therapy

Journal: Angewandte Chemie International Edition

IF: 16.1

Original link：https://onlinelibrary.wiley.com/doi/10.1002/anie.202506505

Reporter：Yiwen Xie-23-master

Bacterial infections significantly alter the local microenvironment, with acidic byproducts from bacterial metabolism leading to a pronounced pH reduction. Leveraging this characteristic, we synthesized and identified DHTPA, a near-infrared (NIR) fluorescent and pH-responsive aggregation-induced emission (AIE) photosensitizer, for enhanced photodynamic therapy against bacterial infections. DHTPA aggregates exhibit a 2.1-fold increase in ROS generation under weakly acidic conditions (pH 5.5) compared to neutral conditions (pH 7.4), attributed to its pH-dependent electronic structure modulation. Upon NIR light irradiation, DHTPA aggregates precisely respond to the acidic microenvironment, significantly boosting ROS production for efficient bacterial eradication. In vitro studies demonstrated that DHTPA achieved over 99.9% bactericidal efficiency against both gram-negative and gram-positive bacteria. In a murine infected wound model, DHTPA treatment accelerated wound healing by 2.4 times, markedly reduced bacterial burden, and alleviated inflammatory responses, highlighting its therapeutic potential. By integrating NIR activation, pH responsiveness, and AIE properties, DHTPA presents a precise and efficient antibacterial therapeutic strategy, offering an innovative solution for the clinical management of bacterial infections.

Bacterial infections pose a severe global health challenge, particularly due to the increasing prevalence of antibiotic-resistant pathogens. Photodynamic therapy (PDT) has emerged as a promising non-invasive antibacterial strategy that utilizes photosensitizers (PSs) to generate cytotoxic reactive oxygen species (ROS) upon light activation, causing irreversible damage to bacterial membranes, proteins, and DNA. Unlike conventional antibiotics that target specific cellular pathways, PDT exerts a broad-spectrum antibacterial effect through oxidative stress, significantly reducing the risk of bacterial resistance development. However, despite its potential, the clinical application of PDT in bacterial infection treatment remains limited due to challenges such as insufficient ROS generation under complex biological conditions and poor photosensitizer performance in acidic and hypoxic environments.

Bacterial-infected tissues typically exhibit an acidic microenvironment (pH 4.5–6.5) due to bacterial metabolism and inflammation. Conventional PS often suffer from aggregation-induced fluorescence quenching and reduced ROS generation in such environments, limiting their therapeutic efficacy. To address these challenges, researchers have explored the development of pH-responsive and aggregation-induced emission (AIE) PSs, which can specifically activate in acidic conditions and enhance ROS generation through aggregation effects. AIE-based PS overcome the limitations of aggregation-caused quenching (ACQ) observed in traditional fluorophores, enabling enhanced fluorescence emission and photodynamic efficiency in biological systems. Although pH-responsive AIE PS have been investigated for tumor therapy, their application in antibacterial PDT remains largely unexplored.

By integrating NIR activation, pH responsiveness, and AIE effects, DHTPA represents a precise and efficient antibacterial PDT strategy, offering a novel solution for the clinical management of bacterial infections.

1. Material synthesis and characterisation

A series of donor–acceptor (D–A) organic small molecules (DHCZS, DHCZV, DTPA, and DHTPA) with NIR absorption were designed and synthesized using benz[c,d]indole as the core acceptor unit (A), coupled with triphenylamine and carbazole derivatives as strong donor units (D). A series of characterisations were performed.

The UV–Vis absorption spectra revealed a progressive red shift with structural variation, with DHTPA showing a maximum absorption and emission peak at ∼680 and 730 nm, respectively. The results of activated oxygen detection experiments show that these PS generate multiple ROS through both type I and type II photo-chemical pathways, enabling robust therapeutic efficacy under both normoxic and hypoxic conditions. Among them, DHTPA aggregates exhibited the most pronounced ROS generation capability. Finally, to explore the acid-responsive properties of the “star molecule” DHTPA, UV–Vis-NIR spectroscopy was employed to study its absorption and emission behavior in buffers of varying pH.

2. Theoretical Calculations

To further elucidate the exceptional PDT performance of DHTPA, density functional theory (DFT) and time-dependent DFT (TD-DFT) were employed to systematically analyze the electronic structures of DHCZS, DHCZV, DTPA, and DHTPA, focusing on frontier molecular orbital distributions and their impact on ROS generation.

In summary, theoretical calculations unveiled DHTPA’s superior electronic structure and excited-state dynamics, further validating its potential as an efficient ROS generator. Through optimized electronic configurations and excitation energy transfer, DHTPA significantly enhances photodynamic efficacy, offering robust theoretical support for its application in PDT.

3. In Vitro Antimicrobial Experiments and Potential Mechanism Investigation

Given the remarkable ROS generating capacity of DHTPA aggregates, this study further investigated its antibacterial performance and underlying mechanisms under varying pH conditions. S. aureus and E. coli were selected as representative gram-positive and gram-negative bacteria, respectively.

Under both acidic (pH = 5.5) and neutral (pH = 7.4) conditions, the DHTPA + L group exhibited a significant reduction in CFUs of bacterial biofilms compared to the other groups, with a more pronounced antibacterial effect observed under acidic conditions. Scanning electron microscopy (SEM) was employed to observe morphological changes in bacterial biofilms. In the DHTPA + L group, bacterial cells exhibited the most pronounced membrane damage under acidic conditions (pH = 5.5), with numerous cells showing severe structural disruption or complete lysis. In contrast, the control and DHTPA groups displayed relatively minor membrane damage, with changes significantly less apparent than those observed in the DHTPA + L group. To further investigate the effects of DHTPA aggregates on biofilms, this study utilized fluorescent dyes combined with confocal laser scanning microscopy (CLSM) for 3D reconstruction analysis.

Conclusively, DHTPA aggregates demonstrated exceptional antibacterial performance under 660 nm laser irradiation and acidic pH conditions, particularly in the localized acidic microenvironment caused by bacterial infection metabolism. This underscores its potential as an effective therapeutic agent for biofilm-related infections.

4. Wound Healing Experiment in Mice

To further investigate the efficacy of DHTPA aggregates in combating wound infections, an in vivo study was conducted using a S. aureus-infected skin wound healing mouse model.

Compared with the control group, the DHTPA group exhibited notable wound size reduction and moderate healing.

To further assess the role of DHTPA aggregates in repairing infected wounds, histological evaluations of wound tissues were performed using hematoxylin and eosin (H&E) staining and Masson's trichrome staining.

The DHTPA + L group showed a marked reduction in inflammatory cell infiltration, signifying alleviated inflammation. The decrease in inflammation can be attributed to the robust ROS generation by DHTPA aggregates under 660 nm laser irradiation, which enhances its antibacterial potency and accelerates wound repair.

In summary, we have meticulously designed and identified a NIR photosensitizer, DHTPA, which integrates acid responsiveness and AIE properties. This photosensitizer significantly enhances ROS generation under acidic conditions and achieves highly efficient PDT upon NIR light activation in bacteria-infected tissues. DHTPA aggregates overcome the limitations of conventional PDT in complex biological environments and demonstrate remarkable potential in treating wound infections. It effectively eliminates bacteria, reduces the expression of virulence factors, alleviates tissue inflammation, promotes collagen deposition, and stimulates angiogenesis, thereby accelerating the healing of infected wounds. Against the backdrop of escalating antibiotic resistance, this dual-responsive AIE photosensitizer offers an innovative alternative strategy for antimicrobial therapy and provides robust support for future clinical translation.