Specific discrimination and efficient elimination of gram-positive bacteria by an aggregation-induced emission-active ruthenium (II) photosensitizer

Highlights

• A multifunctional ruthenium (II) polypyridine complex with aggregation-induced emission (AIE) characteristic was synthesized.

• The selective discrimination and imaging of gram-positive bacteria was with Ru2.

• Ru2 under light irradiation had the best antibacterial activity in vitro and in vivo.

• Ru2 altered Gram-positive bacterial gene expression and successfully applied to S. aureus-infected wound treatment.

Abstract

The infections caused by Gram-positive bacteria (G⁺) have seriously endangered public heath due to their high morbidity and mortality. Therefore, it is urgent to develop a multifunctional system for selective recognition, imaging and efficient eradication of G⁺. Aggregation-induced emission materials have shown great promise for microbial detection and antimicrobial therapy. In this paper, a multifunctional ruthenium (II) polypyridine complex Ru2 with aggregation-induced emission (AIE) characteristic, was developed and used for selective discrimination and efficient extermination of G⁺ from other bacteria with unique selectivity. The selective G⁺ recognition benefited from the interaction between lipoteichoic acids (LTA) and Ru2. Accumulation of Ru2 on the G⁺ membrane turned on its AIE luminescence and allowed specific G⁺ staining. Meanwhile, Ru2 under light irradiation also possessed robust antibacterial activity for G⁺ in vitro and in vivo antibacterial experiments. To the best of our knowledge, Ru2 is the first Ru-based AIEgen photosensitizer for simultaneous dual applications of G⁺ detection and treatment, and inspires the development of promising antibacterial agents in the future.

Introduction

Bacterial infections caused by Gram-positive bacteria (G⁺) remain a leading cause of morbidity and mortality in humans [1,2]. G⁺ causes a variety of infections that include skin and soft tissue infections [3], surgical site infections [4], bloodstream infections [5] and pneumonia infections [6], which are closely related to endocarditis, pneumonia, arthritis, osteomyelitis, and sepsis [7]. Notably, some G⁺ existing in tumors may inhibit the curative efficacy and promote chemoresistance of chemotherapy drugs [8,9]. Therefore, it is of great significance to develop new antibacterial agents, especially those that can accurately identify and kill G⁺ [10,11].

The main methods for G⁺ identification include the Gram staining [12], polymerase chain reaction (PCR) [13], nucleic acid-related methods [14], immunological techniques [15], and surface enhanced Raman spectroscopy (SERS) [16], which have generally long running time, low accuracy, complicated operation and instrumental dependence [10]. Compared to these aforementioned methods, fluorescent detection techniques are an alternative approach due to their high sensitivity, fast response, non-invasiveness, and direct observation, and have been rapidly developed and applied for bacteria detection and identification [17]. Additionally, the effective killing of G⁺ based on accurate identification was indeed the ultimate goal to combat G⁺ for humans. Antibiotics are the most widely accepted bactericide at present. However, the occurrence of multidrug-resistant strains associated with the abuse of antibiotics narrows the clinical option of traditional antibiotics [18,19]. Antimicrobial photodynamic therapy (APDT) has been considered one of the most promising methods to overcome bacterial resistance [[20], [21], [22]]. Through irradiation, the activated photosensitizers (PSs) can transform oxygen molecules to reactive oxygen species (ROS), mainly singlet oxygen (¹O₂), which can disrupt bacterial cell membranes and irreversibly damage biomolecules, such as nucleic acids, lipids, and proteins, eventually leading to bacterial cell death [23]. Besides, PSs have been utilized in fluorescence image-guided antibacterial studies [22]. However, most of the PSs are hydrophobic and tend to aggregate when interacting with bacteria, leading to fluorescence quenching and low efficiency of ROS generation, which attenuated the imaging performance and antibacterial activity [24,25]. Aggregation-induced emission luminogens (AIEgens) possessed enhanced fluorescence intensity and high ¹O₂ generation efficiency in the aggregated state through the intramolecular rotation/motion restriction, which could be a potential solution against bacteria [26]. Up to now, some elegant AIEgens have been developed for bacterial detection or ablation [[27], [28], [29]]. Among the multifarious AIEgens, heavy metal-based AIEgens gained wide interest due to their rich luminescent properties, and straightforward synthesis routes, high photo-induced ROS yields, which made them promising alternatives to organic AIEgens for the bacteria detection, imaging and therapy [29]. In this field, a few rare examples of AIE-derived transition metal complexes were those of Ir(III) [30], Pt(II) [31], Zn(II) [32] and Au(I) [33]. Such complexes have been used as probes for the detection and eradication of bacteria. However, the metal ions employed in these complexes were not abundant and were often absent from the selective discrimination ability of bacteria. It was, therefore, essential to develop other transition metal complexes that can discriminate and have pharmacologic activity for bacteria.

The cell wall of G⁺ contains multiple thick peptidoglycans and teichoic acid layers (LTA), while the cell wall of G⁻ is composed of 2–3 layers of peptidoglycan surrounded by a second outer membrane containing lipopolysaccharide (LPS) and lipoprotein. As a result, the specific outer wall structures of bacteria play a crucial role in determining the different binding interactions of metal-based AIEgens with bacteria and enable us to discriminate them by AIEgens [29].

Herein, a series of multifunctional ruthenium-based AIE probes Ru1-3 were designed and synthesized; their ability of imaging and efficient eradication of G⁺ were also investigated (Fig. 1). Ru2 could rapidly distinguish G⁺ through specific binding to the LTA in the G⁺ cell wall without an extra targeting group. More importantly, Ru2 could selectively discriminate G⁺ even in the presence of G⁻. Besides, the effective growth inhibition of G⁺ upon Ru2 treatment under light irradiation was validated in vitro and in vivo. To the best of our knowledge, this was the first Ru(II) complex for specific discrimination, imaging, and sterilization of G⁺. This study offered useful guidance for designing the next-generation antibacterial agents.