A pH-Sensitive Spirocyclization Strategy for Constructing a Single Fluorescent Probe Simultaneous Two-Color Visualizing of Lipid Droplets and Lysosomes and Monitoring of Lipophagy.

Lipid droplets (LDs) and lysosomes are crucial for maintaining intracellular homeostasis. But single fluorescent probes (SFPs) capable of simultaneous and discriminative visualizing of two organelles above and their interaction in living cells are still challenging due to the lack of rational design strategies. To break this bottleneck, herein, we develop a reliable strategy based on a pH-sensitive intramolecular spirocyclization. As a proof of concept, an SFP CMHCH, which possesses a switchable hemicyanine/spiro-oxazine moiety induced by pH, has been designed and synthesized. In acidic environments, the ring-open form CMHCH exhibits red-shift emission and low logP value, whereas the ring-closed form CMHC displays blue-shift emission and high logP value in neutral or basic environments. Thus, the distinct different hydrophilicity/hydrophobicity and absorption/emission properties of these two forms enable targeting LDs and lysosomes simultaneously and discriminatingly. Very importantly, the dynamic process of lipophagy can be directly monitored with CMHCH. The success of CMHCH indicated that the spirocyclization strategy is efficient for constructing SFPs to LDs and lysosomes.

Permeability-Controllable Potentiometric Fluorescent Probes Enable Visually Discriminating Near-Zero and Normal Situations of Cell Membrane Potential.

The permeability-controllable potentiometric fluorescent probes that can visually discriminate near-zero and normal situations of cell membrane potential were reported for the first time. Different from traditional potentiometric probes that utilize fluorescence intensity to reflect membrane potential, CQ12 and CP12 have different localizations under the two situations of cell membrane potential. Thus, the two situations can be point-to-point indicated by two fluorescent images with an obvious difference, avoiding complex operations and calibration of conventional methods.

Colocalization Coefficients of a Target-Switchable Fluorescent Probe Can Serve As an Indicator of Mitochondrial Membrane Potential.

The mitochondrial membrane potential (MMP) definitely reflects mitochondrial function. Thus, it is very essential to found a physical parameter as MMP indicator. At present, available parameters are either fluorescent intensity of monochromatic probes such as rhodamine 123 or a ratio of fluorescent intensity at different wavelengths of dual-color dyes such as JC-1, but the inconvenience in practice as well as serious effect of loading concentrations on experimental results limited their application. To address this concern, herein,we found a reliable and easily obtainable colocalization coefficient (CLC) of a fluorescent probe as new MMP indicator and developed a target switchable fluorescent probe (Mito-Lyso) to attain the aim. Because of its intrinsic nature, Mito-Lyso exclusively stains mitochondria with normal MMP and a subsequent decreasing of MMP results in release of some Mito-Lyso. Importantly, the released Mito-Lyso can reversibly transfer between mitochondria and lysosomes. Thus, CLCs of Mito-Lyso and a commercial lysosomal probe (NIR-Lyso) can be MMP-dependent. CLCs gradually increased from 0.20 to 0.8 with the decreasing of MMP and then returned to 0.3 with the recovering of MMP, which better proves that the CLC is a valuable MMP indicator. Furthermore, both the design principle and action mechanism of Mito-Lyso has been explained in detail for the development of this type of probes.

Neutrophil Targeting Platform Reduces Neutrophil Extracellular Traps for Improved Traumatic Brain Injury and Stroke Theranostics.

Traumatic brain injuries (TBI) and stroke are major causes of morbidity and mortality in both developing and developed countries. The complex and heterogeneous pathophysiology of TBI and cerebral ischemia-reperfusion injury (CIRI), in addition to the blood-brain barrier (BBB) resistance, is a major barrier to the advancement of diagnostics and therapeutics. Clinical data showed that the severity of TBI and stroke is positively correlated with the number of neutrophils in peripheral blood and brain injury sites. Furthermore, neutrophil extracellular traps (NETs) released by neutrophils correlate with worse TBI and stroke outcomes by impairing revascularization and vascular remodeling. Therefore, targeting neutrophils to deliver NETs inhibitors to brain injury sites and reduce the formation of NETs can be an optimal strategy for TBI and stroke therapy. Herein, the study designs and synthesizes a reactive oxygen species (ROS)-responsive neutrophil-targeting delivery system loaded with peptidyl arginine deiminase 4 (PAD4) inhibitor, GSK484, to prevent the formation of NETs in brain injury sites, which significantly inhibited neuroinflammation and improved neurological deficits, and improved the survival rate of TBI and CIRI. This strategy may provide a groundwork for the development of targeted theranostics of TBI and stroke.

Ligustrazine Nanoparticle Hitchhiking on Neutrophils for Enhanced Therapy of Cerebral Ischemia-Reperfusion Injury.

Ischemic stroke is a refractory disease that endangers human health and safety owing to cerebral ischemia. Brain ischemia induces a series of inflammatory reactions. Neutrophils migrate from the circulatory system to the site of cerebral ischemia and accumulate in large numbers at the site of inflammation across the blood-brain barrier. Therefore, hitchhiking on neutrophils to deliver drugs to ischemic brain sites could be an optimal strategy. Since the surface of neutrophils has a formyl peptide receptor (FPR), this work modifies a nanoplatform surface by the peptide cinnamyl-F-(D)L-F-(D)L-F (CFLFLF), which can specifically bind to the FPR receptor. After intravenous injection, the fabricated nanoparticles effectively adhered to the surface of neutrophils in peripheral blood mediated by FPR, thereby hitchhiking with neutrophils to achieve higher accumulation at the inflammatory site of cerebral ischemia. In addition, the nanoparticle shell is composed of a polymer with reactive oxygen species (ROS)-responsive bond breaking and is encased in ligustrazine, a natural product with neuroprotective properties. In conclusion, the strategy of hitching the delivered drugs to neutrophils in this study could improve drug enrichment in the brain, thereby providing a general delivery platform for ischemic stroke or other inflammation-related diseases.

A general strategy to increase emission shift of two-photon ratiometric pH probes using a reversible intramolecular reaction of spiro-oxazolidine.

Fluorescent pH probes have been served as powerful tools in biological and pathological studies in recent years due to the important roles of pH values in various physiological processes. Although plenty of pH probes have been delivered, development of two-photon ratiometric pH probes with large emission shift for detecting the variation of intracellular pH values is still a greatly challenging task. To address this concern, in this work, we have discovered a general strategy designing pH probes by means of a pH-dependent reversible intramolecular reaction of spiro-oxazolidine which can efficiently change their conjugation length and the electronic effect concurrently. To display the generality of the strategy, we have synthesized six pH probes, and all these probes exhibit short emission in basic conditions and dramatically red-shifted emission in acid environments. The emission shift of the six probes is more than 150 nm and even up to 210 nm, much larger than shift of all commercial and reported pH probes. The chemical sensing mechanism of intramolecular ring opening/closing reaction of spiro-oxazolidine has been confirmed with 1H NMR spectra and density functional theory (DFT) calculations. Finally, we have used one of six with one- and two-photon properties to successfully image lysosomal pH changes under confocal and two-photon microscopes in a ratiometric manner. We believed that this spiro-oxazolidine strategy can serve as a general and powerful platform for the design of ideal pH probes.

Reaction-free and MMP-independent fluorescent probes for long-term mitochondria visualization and tracking.

Visualizing and tracking mitochondrial dynamic changes is crucially important in the fields of physiology, pathology and pharmacology. Traditional electrostatic-attraction based mitochondrial probes fail to visualize and track the changes due to their leakage from mitochondria when mitochondrial membrane potential (MMP) decreases. Reaction-based MitoTracker probes can realize visualization and tracking of mitochondria changes independent of MMP changes. However, such probes impair mitochondrial proteins and exhibit high cytotoxicity. Therefore, it still remains challenging to explore reaction-free and highly biocompatible probes for visualizing and tracking mitochondrial dynamics independent of MMP fluctuations. Herein we synthesized two reaction-free fluorescent mitochondrial probes ECPI-12 and IVPI-12 bearing a long C12-alkyl chain. These cationic probes can firmly immobilize in the mitochondrial inner membrane by strong hydrophobic interaction between the C12-alkyl chain and lipid bilayer, resulting in high specificity and long-term mitochondrial staining regardless of MMP changes. They also exhibit large two-photon absorption cross-sections and show deep penetration in live tissues in two-photon microscopy. Furthermore, they display excellent biocompatibility and realize in situ and real-time mitophagy tracking in live cells. These excellent properties could make ECPI-12 and IVPI-12 the first selective tools for long-term visualization and tracking of mitochondrial dynamics.