Design strategies for organelle-selective fluorescent probes: where to start?

Abstract

Monitoring physiological changes within cells is crucial for understanding their biological aspects and pathological activities. Fluorescent probes serve as powerful tools for this purpose, offering advantageous characteristics over genetically encoded probes. While numerous organelle-selective probes have been developed in the past decades, several challenges persist. This review explores the strategies and key factors contributing to the successful rationale design of these probes. We systematically discuss the typical mode of cellular uptake generally adopted by fluorescent probes and provide a detailed examination of the key factors to consider in design rationale from two perspectives: the properties of the target organelle and the physicochemical properties of the probe itself. Additionally, recent examples of organelle-targeted probes are presented, along with a discussion of the current challenges faced by fluorescent probes in the field.

Fig. 1. (A) Schematic representation of a typical fluorescent probe design for targeting organelles. (B) Illustrative depiction of fluorescent probe targeting and visualizing specific intracellular organelles. Some organelles, along with their structures and roles, are depicted.

Fig. 2. Transport mechanisms of probes into cell: passive or equilibrative diffusion, facilitated diffusion, and active transport.

Fig. 3. (A) Chemical structure of PKMO, a cyclooctatetraene-conjugated Cy3.5 with Abs/Em of 591/608 nm. (B) 2D-STED nanoscopic recording of mitochondrial cristae of a COS-7 cell labeled with PKMO (scale bar, 10 μm). Inset (i) and (ii) are magnified image.

Fig. 4. The recognition mechanism of probe RS1 for lysosomal pH changes involves a strategy, indicated by the orange circle, where the ring remains closed in a neutral/basic environment but opens under acidic conditions.

Fig. 5. (A) Reversible Michael addition reaction between MitoRT and GSH. TPP is used as mitochondria-directing moiety. (B) Proposed mechanism of reaction between probe ER-F and cysteine (Cys).

Fig. 6. (A) Membrane diffusion vs. molecular volume. Small molecules conforming to the Ro5 rule (green dots) approximately follow an exponential trend, while larger molecules beyond the Ro5 rule (bRo5, blue dots) experience steep decline in permeability. To ensure the generality of these trends, a variety of unrelated synthetic and natural products were subjected to the same analysis (orange dots). Reproduced with permission from ref. , Copyright 2017 American Chemical Society. (B) Structure of nucleus-targeting probe Hoechst and its derivatives. Typical nucleus-targeting probe features hydrophilic cations, hydrophobic chains, and planar aromatic structure, to bind to minor groove of dsDNA and/or intercalate between the base pairs of DNA helix.

Fig. 7. (A) QSAR model: mapping probe physicochemical properties onto preferred probe's intracellular localization. (B) 3D scatter plot of QSAR analysis for three properties: lipophilicy (S log P), molecular charge (Q_VSA_FNEG) and solubility (log S). Blue and red dots represent probes that are cell permeable. Reproduced with permission from ref. , copyright 2016 Nature Publishing.

Fig. 8. Fluorescent probes targeted toward lysosome and mitochondria. (A) Association of mitochondrial and lysosomal dysfunction and the onset of numerous diseases. (B) The typical structure of turn-on or ratiometric fluorescent probes, as a general example of organelle probes, consists of fluorophore (F), recognition unit (R), organelle targeting moiety (O), and target molecule (T). (C) Lysosome-targeted probes (LP). (D) Mitochondria-targeted probes (MP).