Click-free imaging of carbohydrate trafficking in live cells using an azido photothermal probe.

Real-time tracking of intracellular carbohydrates remains challenging. While click chemistry allows bio-orthogonal tagging with fluorescent probes, the reaction permanently alters the target molecule and only allows a single snapshot. Here, we demonstrate click-free mid-infrared photothermal (MIP) imaging of azide-tagged carbohydrates in live cells. Leveraging the micromolar detection sensitivity for 6-azido-trehalose (TreAz) and the 300-nm spatial resolution of MIP imaging, the trehalose recycling pathway in single mycobacteria, from cytoplasmic uptake to membrane localization, is directly visualized. A peak shift of azide in MIP spectrum further uncovers interactions between TreAz and intracellular protein. MIP mapping of unreacted azide after click reaction reveals click chemistry heterogeneity within a bacterium. Broader applications of azido photothermal probes to visualize the initial steps of the Leloir pathway in yeasts and the newly synthesized glycans in mammalian cells are demonstrated.

Synthesis of Glycopolymer Micelles for Antibiotic Delivery.

In this work, we designed biodegradable glycopolymers consisting of a carbohydrate conjugated to a biodegradable polymer, poly(lactic acid) (PLA), through a poly(ethylene glycol) (PEG) linker. The glycopolymers were synthesized by coupling alkyne end-functionalized PEG-PLA with azide-derivatized mannose, trehalose, or maltoheptaose via the click reaction. The coupling yield was in the range of 40-50% and was independent of the size of the carbohydrate. The resulting glycopolymers were able to form micelles with the hydrophobic PLA in the core and the carbohydrates on the surface, as confirmed by binding with the lectin Concanavalin A. The glycomicelles were ~30 nm in diameter with low size dispersity. The glycomicelles were able to encapsulate both non-polar (rifampicin) and polar (ciprofloxacin) antibiotics. Rifampicin-encapsulated micelles were much smaller (27-32 nm) compared to the ciprofloxacin-encapsulated micelles (~417 nm). Moreover, more rifampicin was loaded into the glycomicelles (66-80 µg/mg, 7-8%) than ciprofloxacin (1.2-2.5 µg/mg, 0.1-0.2%). Despite the low loading, the antibiotic-encapsulated glycomicelles were at least as active or 2-4 times more active than the free antibiotics. For glycopolymers without the PEG linker, the antibiotics encapsulated in micelles were 2-6 times worse than the free antibiotics.

Imaging, Identification and Inhibition of Microorganisms Using AIEgens.

Microorganisms, including bacteria, viruses and fungi, are ubiquitous in nature. Some are extremely beneficial to life on Earth, whereas some cause diseases and disrupt normal human physiology. Pathogenic microorganisms can also undergo mutations and develop resistance to antimicrobial agents, which complicates diagnostic and therapeutic regimens. This calls for continuing efforts to develop new strategies and tools that can provide fast, sensitive and accurate diagnosis, as well as effective treatment of ever-evolving infectious diseases. Aggregation-induced emission luminogens (AIEgens) have shown promise in imaging, identification and inhibition of various microbial species. Compared to conventional organic fluorophores, AIEgens can offer improved photostability, and have found utilities in imaging microorganisms. AIEgens have been shown to detect microbial viability and differentiate among different microbial strains. Theranostic AIEgens that integrate imaging and killing of microbes have also been developed. This review highlights examples in the literature where AIEgens have been employed as molecular probes in the imaging, discrimination and killing of bacteria, viruses and fungi.