Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump, leading to autophagy induction through the activation of the Ca2+/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.

The majority of near-infrared (NIR) fluorophores are organic molecules that show significant overlap between the excitation and emission spectra and therefore exhibit high fluorescence backgrounds during in vivo imaging. Recently, cyanine dyes with a large Stokes shift have shown great promise for NIR imaging but often undergo rapid photodegradation and nonspecific protein adsorption. Alternatively, fluorescence resonance energy transfer (FRET) is a promising technique to generate a larger gap between the excitation and emission maxima and thus can reduce the background signal. Here, we report the rational design of FRET-based polymeric nanoparticles for NIR and FRET imaging. The particles were assembled from diblock copolymers of poly(d,l-lactic-co-glycolic acid) and maleimide-activated poly(ethylene glycol), which were also encapsulated with both the donor (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine) and acceptor (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine) fluorophores. Because of their extreme hydrophobicity, thousands of fluorophores could be encapsulated inside a single particle without causing leakage. FRET resulted in a large Stokes shift (>100 nm) of the emission maxima, and the transfer efficiency could be fine-tuned by further adjusting the doping ratio of the donor and acceptor fluorophores. The optimized formulation was less than 100 nm in size, brighter than quantum dots, stable in biological media, and demonstrated similar biodistribution to most nanomaterials. Additional animal phantom studies demonstrated that the FRET imaging platform developed could have far-reaching applications in optical imaging.

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