Highly efficient phototheranostics of macrophage-engulfed Gram-positive bacteria using a NIR luminogen with aggregation-induced emission characteristics
Introduction
Immune response system serves as the front line to attack invading bacterial pathogens, in which macrophages take the major roles to recognize, engulf and digest bacteria through a type of cell endocytic process, namely phagocytosis [[1], [2], [3], [4], [5], [6], [7], [8]]. Although phagocytosis is a promising immediate response to bacterial infections, its bacterial encounter and killing rates are relatively low in comparison to the bacterial reproduction rate. Moreover, macrophage-engulfed bacteria could survive and reproduce against the digestion process in phagolysosomes, which largely hinder the effectiveness of macrophages, probably even lead to severe infectious diseases such as tuberculosis [[9], [10], [11], [12], [13], [14]]. Given the circumstances, developing multifunctional theranostic agent for simultaneous detection and ablation of macrophage-engulfed bacteria remains a supremely important task [11,15]. Unlike manipulating extracellular bacteria, the shielding effect of host macrophages has considerably hampered the detection and ablation of macrophage-engulfed bacteria [16,17], making this task particularly challenging with very limited success achieved even though enthusiastic efforts have been devoted by scientists.
Fluorescent bioimaging is a promising approach in effective bacterial detection and behavioral monitoring due to the excellent sensitivity, selectivity and non-invasiveness [[18], [19], [20], [21]]. However, the previously developed fluorescent probes (such as bacteria BioParticles indicators and pHrodo dye series) [[22], [23], [24]] for detecting macrophage-engulfed bacteria often suffer from their respective and collective drawbacks including short emission wavelengths, complicated experimental protocols, multiple washing procedures, long incubation time, lack of bactericidal function, as well as the tendency to exhibit aggregation-caused quenching (ACQ) phenomenon [25]. In particular, the ACQ tendency has been a long-term unresolved issue, in which the fluorophores experience strong emission quenching in high concentrations or in aggregate formation, preventing their practical employment as effective bacterial targeting probes. Recently, a novel class of fluorophores namely aggregation-induced emission (AIE) luminogens which perform oppositely to ACQ fluorophores have captivated much interest as more ideal fluorescent probes [26,27]. AIE luminogens (AIEgens) are weakly emissive in solution state, but become strongly emissive upon aggregation or binding to certain biomolecules due to the restriction of intramolecular motions (RIMs), which give great potential applications in biological system [28,29]. Furthermore, well-tailored AIEgens exhibit efficient photosensitizing ability by generating reactive oxygen species (ROS) in aggregation state, which are suitable to participate in photodynamic bacterial killing [[30], [31], [32], [33], [34], [35]]. Therefore, AIEgens become ideal candidates as efficient phototheranostic agents for bacteria.
Indeed, the recent emergence of several AIE-active fluorescent probes based on metabolic labeling technique or conjugated peptide has partially addressed those problems in handling macrophage-engulfed bacteria, by virtue of their high signal-to-noise ratio, high photostability, and photosensitizing capability [36,37]. Nevertheless, the employment of metabolic labeling technique takes considerable time for the AIE probe to target engulfed bacteria or phagosomes, resulting in the incapability of real-time phagocytosis study and ineffective anti-bacterial treatment. In addition, those AIE probes are incapable of discriminating different types of intracellular bacteria, and their bacterial ablation efficiencies are yet to be improved. Evidently, developing a versatile AIE bioprobes for highly efficient theranostics of macrophage-engulfed bacteria is still vitally desirable.
In this contribution, we demonstrated the application of an AIEgen, namely TTVP with excellent water solubility, near-infrared (NIR) emission and high ROS generating efficiency as a simple and effective probe for simultaneous detection and photodynamic ablation of macrophage-engulfed Gram-positive bacteria (Fig. 1). TTVP was capable of tracing both engulfing process and initiation of digestion process of Gram-positive bacteria by macrophages with high signal-to-noise ratio. Meanwhile, TTVP exhibited excellent performance as photosensitizer to eliminate cell-entrapped bacteria through photodynamic therapy pathway without attacking host macrophages. Compared to previously reported AIEgens, this work demonstrates the use of TTVP as a novel and versatile AIE intracellular Gram-positive bacterial bioprobe, which can perform efficient phagocytosis tracking and facilitate highly effective theranostics to cell-engulfed bacteria. This work thus gives potential application to boost the intracellular bacterial killing efficiency of macrophages in immune system.
Section snippets
Materials
Ultra-pure water was supplied by Milli-Q Plus System (Millipore Corporation, United States). Dulbecco's modified essential medium (DMEM), fetal bovine serum (FBS), penicillin, calcein AM, ethidium homodimer-1 and streptomycin were purchased from Thermo Fisher Scientific. Phosphate buffered saline (PBS) was purchased from Sigma-Aldrich. Lysogeny broth (LB) medium, LB agar, Hoechst 33258, Lysotracker Green, MitoTracker Green FM, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Detection of macrophage-engulfed Gram-positive bacteria
Bacteria-macrophages co-culture system was established by using Raw264.7 cells as macrophage model and Gram-positive S. aureus as bacteria model. Raw264.7 cells were first incubated with S. aureus for 30 min, which allows enough time for Raw264.7 cells to initiate phagocytosis and engulf extracellular live S. aureus. After that, 1 μM of TTVP with maximum emission at 704 nm was added to the S. aureus-macrophages culture for different incubation time. The nucleus of Raw264.7 cells and the invaded
Conclusions
In summary, we demonstrated the use of a NIR AIEgen, TTVP, as a multi-functional probe for macrophage-engulfed bacteria discrimination, bacterial phagocytosis tracking and intracellular bacterial elimination. This work provides a versatile and facile strategy to trace phagocytosis process of Gram-positive bacteria by macrophages through ultrafast and selective staining of TTVP to Gram-positive bacteria over macrophages with wash-free and low staining concentration protocol. Besides, the superb
CRediT authorship contribution statement
Michelle M.S. Lee: Methodology, Software, Formal analysis, Data curation, Resources, Investigation, Writing - original draft. Dingyuan Yan: Methodology. Joe H.C. Chau: Formal analysis, Investigation. Hojeong Park: Formal analysis, Investigation. Charlie C.H. Ma: Investigation. Ryan T.K. Kwok: Data curation. Jacky W.Y. Lam: Project administration. Dong Wang: Conceptualization, Funding acquisition, Writing - review & editing. Ben Zhong Tang: Conceptualization, Supervision, Funding acquisition,
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was partially supported by the National Science Foundation of China (21788102), the Research Grant Council of Hong Kong (16305518 and C6009-17G), the Innovation and Technology Commission (ITC-CNERC14SC01 and ITCPD/17-9), and National Key Research and Development Program of China (2018YFE0190200). the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province (2020B1515020011), and the Science and Technology Foundation of Shenzhen City (JCYJ20190808153415062).
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2022, Dyes and PigmentsCitation Excerpt :Regarding that AIE-based PSs show strong fluorescence and high ROS production capability in the aggregated state, two main strategies, i.e., the chemical and physical methods, have been developed for preparation AIE PS nanoparticles, which endows the PSs with a good water dispersibility and biocompatibility. In the chemical method, the AIE core is covalently linked with the hydrophilic group, including neutral polymers, charged chains and biomolecules, to form amphiphilic molecular, which can automatically self-assemble nano-aggregation in aqueous solution [35–39]. While the physical one is encapsulation the AIE core with some amphiphilic polymer matrixes, with more widely used matrixes being pluronic F-127, polyvinyl alcohol and DSPE-PEG 2000 [40–42].