Saran Long

Imaging γ-Glutamyltranspeptidase for tumor identification and resection guidance via enzyme-triggered fluorescent probe

Development of high selectivity, accurate targeted and noninvasive fluorescent probe for monitoring specific enzyme activity associated with the tumor is urgent needed for early diagnosis of cancer and clinical fluorescence interventional resection guidance treatment. Owing to the invasion of malignant tumor cells, tumor cells are mixed with normal cells in the actual tumor location, which make it quite difficult for clinician to diagnose early diagnosis of tumor as well as resection of tumor. To overcome aforementioned obstacle, herein, an ingenious enzyme-activated one and two-photon fluorescent probe TCF-GGT was constructed and synthesized including γ-GGT enzyme specific identification site and far-red fluorophore for imaging. Under simulative physiological condition, probe TCF-GGT demonstrated high selectivity, sensitivity (DL 0.014 mU/mL), rapid response (Te 14u202fmin) for the detection of γ-GGT enzyme. By virtue of its biocompatibility, probe was employed for the identification of ovarian cancer cells (A2780u202fcells) from normal cells (NIH-3T3 cells), particularly in mixed cultivation dish (simulate the actual environment of tumor) through 2D&3D fluorescence imaging with dual mark (Nucleic acid labeling used by Hoechst 33342 dye and γ-GGT enzyme labeling used by probe TCF-GGT) for the first time. Probe TCF-GGT could be visualize endogenous γ-GGT activity in HepG-2u202fcells and zebrafish on the two-photon confocal platform, which is conduce to estimate the inhibitor of γ-GGT enzyme inxa0vivo. Through NaBu (a potential anticancer drug) stimulation, the changes of γ-GGT activity were observed in living MCF-7u202fcells by using this probe. More importantly, the deep tissue penetration ability of far-red fluorescence allowed the two-photon fluorescent probe TCF-GGT to real-time track γ-GGT activity in tissue slices and tumor xenotransplantation model of mice by the tail vein injection, which showed that this enzyme-triggered fluorogenic probe would be a potential tool for preclinical applications of tumor resection.

Recognition of Exogenous and Endogenous Nitroxyl in Living Cells via a Two-photon Fluorescent Probe

Nitroxyl (HNO), one electron reduced and protonated form of nitric oxide (NO), plays vital in various biological functions and pharmacological activities, such as mediating β-agonist dobutamine, inhibiting the activity of enzyme and treating cardiovascular diseases. However, the accurate mechanism of HNO in living cells is not thoroughly understood due to lacking of effective methods. In this work, a novel two-photon fluorescent probe TP-HNO was designed and synthesized based on 6-hydroxyl-quinonline-2-benzothiazole derivatives through introducing 2-(diphenylphosphino)benzoate as the ideal HNO recognition unit, which demonstrated the merits of outstanding selectivity, excellent sensitivity (DL 0.19 μM) and rapid response (20 min). In addition, owing to the high cell permeability and low biotoxicity of probe TP-HNO, it was successfully used for the qualitative and bioimaging of exogenous and endogenous nitroxyl concentration fluctuations in living cells via a two-photon laser confocal fluorescence microscopy, respectively, which is of importance for revealing the biological of HNO in the further.

A ratiometric fluorescence probe for lysosomal polarity

Lysosomal polarity affects the interaction activities between enzymes and substrates at the cellular level. Abnormal lysosomal polarity closely linked with disorders and diseases is worthy of attention. The first fluorescence probe, which can image polarity ratiometrically and detect lysosomal polarity quantitatively, is reported herein. The probe termed NOH can emit dual-peaks both in solvents (λemu202f=u202f474, 552u202fnm) and in micro-environment. NOH exhibits the Boltzmann function response of the fluorescence intensity ratio to the polarity in a wide range and localizes at lysosomes specifically (Rru202f=u202f0.97). In the method of ratiometric fluorescence imaging with NOH, the variation of lysosomal polarity (Δf) can be directly discerned by the color changes. In virtue of ratiometric fluorescence imaging and the Boltzmann function relationship between the fluorescence intensity ratio and the polarity, lysosomal polarity in MCF-7u202fcells was calculated to be 0.224 and the polarity in the condition of lysosomal storage disorders (or cell death) could also be obtained. This probe will be a promising tool for studying lysosome-related physiological or pathological processes.

Synthesis of an ultrasensitive BODIPY-derived fluorescent probe for detecting HOCl in live cells

Hypochlorous acid (HOCl) is a critical member of the reactive oxygen species (ROS) produced by immune cells to fight infections. On the other hand, HOCl in homeostasis causes oxidative damage to biomolecules and is linked to many diseases, including inflammatory, neurodegenerative, and cardiovascular diseases. Herein, we detail a procedure for the preparation of a boron-dipyrromethene (BODIPY)-derived fluorescent probe for HOCl (BClO) and its application as an imaging reagent in living cells. BClO is synthesized in one pot through a four-step procedure that is nearly the same as that for conventional BODIPY dye preparation, except for the ratio of starting materials. BClO has an extremely rapid response (saturated within seconds) and is ultrasensitive to HOCl. The detection limit of BClO reaches the subnanomolar range, which is the highest HOCl sensitivity to date. Taking advantage of the ultrasensitive character of BClO, we have previously demonstrated its ability to detect endogenous HOCl generated by macrophages and shown that it can also be used to discriminate cancer cell lines (which show high HOCl production) from non-cancer cell lines (which show low HOCl production). The protocol requires ~2 d for probe synthesis and up to ~18 h for fluorescence imaging and flow cytometry assays.This protocol describes the one-pot chemical synthesis and applications of BClO, an ultrasensitive fluorescent probe for detecting hypochlorous acid (HOCl) in live cells.

Near-Infrared Light-Initiated Molecular Superoxide Radical Generator: Rejuvenating Photodynamic Therapy against Hypoxic Tumors

Hypoxia, a quite universal feature in most solid tumors, has been considered as the Achilles heel of traditional photodynamic therapy (PDT) and substantially impairs the overall therapeutic efficacy. Herein, we develop a near-infrared (NIR) light-triggered molecular superoxide radical (O2-•) generator (ENBS-B) to surmount this intractable issue, also reveal its detailed O2-• action mechanism underlying the antihypoxia effects, and confirm its application for in vivo targeted hypoxic solid tumor ablation. Photomediated radical generation mechanism study shows that, even under severe hypoxic environment (2% O2), ENBS-B can generate considerable O2-• through type I photoreactions, and partial O2-• is transformed to high toxic OH· through SOD-mediated cascade reactions. These radicals synergistically damage the intracellular lysosomes, which subsequently trigger cancer cell apoptosis, presenting a robust hypoxic PDT potency. In vitro coculture model shows that, benefiting from biotin ligand, ENBS-B achieves 87-fold higher cellular uptake in cancer cells than normal cells, offering opportunities for personalized medicine. Following intravenous administration, ENBS-B is able to specifically target to neoplastic tissues and completely suppresses the tumor growth at a low light-dose irradiation. As such, we postulated this work will extend the options of excellent agents for clinical cancer therapy.

Imaging of Formaldehyde in Live Cells and Daphnia magna via Aza-Cope Reaction Utilizing Fluorescence Probe With Large Stokes Shifts

Formaldehyde (FA), a highly reactive carbonyl species, plays significant role in physiological and pathological functions. However, elevated FA will lead to cognitive impairments, memory loss and various neurodegenerative diseases due to its potent DNA and protein cross-linking mechanisms. In this work, a fluorescence probe, BD-CHO, based on benz-2-oxa-1, 3- diazole (BD) skeleton, was designed and synthesized for detection of FA via Aza-Cope reaction with high selectivity and large Stokes shifts (about 118 nm). BD-CHO was successfully applied to monitor the changes FA level in living cells, and kidney tissues of mice. Importantly it was the first time that BD-CHO was used for visualizing exogenous FA changes in Daphnia magna through fluorescence microscopy, demonstrating its potential application for studies of biological processes associated with FA.