Self-Assembly of Silole-Based Aggregation-Induced Emission Compounds with Green Fluorescent Protein under Physiological Conditions for Traceable and Versatile Drug Delivery.

Biomacromolecules are viewed as promising drugs due to their specific functions in biological processes, biocompatibility, and pharmacological efficacy. Injective administration, chosen to avoid intestinal barriers, may in turn lead to immediate decay in the circulation system, unreliable targeting performance, or the induction of immune responses. For some biomacromolecules, chemically modified proteins have been developed for practical use. Various cargo or carrier systems are under development but have been delayed by technical difficulties. We present self-assembled nanocapsules with diameters ranging from 100 to 500 nm that can be deployed in physiological buffers to enclose various substances present in the buffers at the same time. Our amphiphilic nanocapsule, consisting of silole-core dendrimer products as the hydrophobic part and green fluorescent protein (GFP) derivatives as the hydrophilic part, connects and assembles spontaneously when mixed in solutions while engulfing dissolved or dispersed compounds together in a dose-dependent manner and shows unique optical characteristics because the dendrimer products exhibit aggregation-induced emission. Furthermore, the emission of the dendrimer causes considerable fluorescence resonance energy transfer (FRET) to GFP derivatives upon association. We could easily monitor assemblies by FRET states and particle sizes and have confirmed a stable presence in the buffer for at least a month. Further tracking of nanocapsules by fluorescence confirmed efficient uptake into some cancer cells. Nanocapsules based on GFP variants with or without a cell-surface-specific tag demonstrated that the tag improved the potential for specific targeted delivery. There were also indications that the nanocapsules became unstable after cellular uptake in the intracellular environment. We report here the simple preparation of traceable, stable, and biocompatible self-assembled nanocapsules as the basis for a versatile drug delivery system.

Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.