Publications by authors named "Hwan Myung Kim"

93 Publications

An azo dye for photodynamic therapy that is activated selectively by two-photon excitation.

Chem Sci 2020 Nov 6;12(1):427-434. Epub 2020 Nov 6.

Department of Chemistry, Department of Energy Systems Research, Ajou University Suwon 443-749 South Korea

Two-photon photodynamic therapy (TP-PDT) is a promising approach for the treatment of cancer because of its better penetration depth and superior spatial selectivity. Here, we describe an azo group containing cyclized-cyanine derivatives ( and ) as a two-photon activated, type I based photosensitizer (PS). These small-molecule and heavy atom-free organic dyes showed marked reactive oxygen species (ROS)-generating ability under physiological conditions, as well as fast loading ability into the cells and negligible dark toxicity. Live cell analyses with one- and two-photon microscopy revealed that these dyes showed higher ROS generation ability upon two-photon excitation than upon one-photon excitation the type I process. The PSs have superior PDT properties compared to conventional Visudyne and under mild conditions. These characteristics allowed for precise PDT at the target region in mimic tumor spheroids, demonstrating that the developed TP PS could be useful in efficient PDT applications and in designing various PSs.
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http://dx.doi.org/10.1039/d0sc05686cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178981PMC
November 2020

Highly selective two-photon fluorescent off-on probes for imaging tyrosinase activity in living cells and tissues.

Chem Commun (Camb) 2021 Jul;57(56):6911-6914

Department of Chemistry and Nanoscience, Ewha Womans University, Seoul, 120-750, Korea.

A coumarin-based two-photon (TP) fluorescent off-on probe has been developed for detecting tyrosinase activity. High selectivity, sensitivity and biocompatibility enable the probes to successfully image tyrosinase activity in live cells and tissues using TP microscopy.
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http://dx.doi.org/10.1039/d1cc02374hDOI Listing
July 2021

A fluorescent ESIPT-based benzimidazole platform for the ratiometric two-photon imaging of ONOO and .

Chem Sci 2020 Jul 16;11(28):7329-7334. Epub 2020 Jun 16.

Department of Chemistry , University of Bath , BA2 7AY , UK . Email: ; Email:

In this work, we have developed an ESIPT-based benzimidazole platform ( and ) for the two-photon cell imaging of ONOO and a potential ONOO-activated theranostic scaffold (). Each benzimidazole platform, , were shown to rapidly detect ONOO at micromolar concentrations (LoD = 0.28 μM, 6.53 μM and 0.81 μM respectively). The potential theranostic was shown to release the parent fluorophore and drug indomethacin in the presence of ONOO but unfortunately did not perform well due to low solubility. Despite this, the parent scaffold demonstrated its effectiveness as a two-photon imaging tool for the ratiometric detection of endogenous ONOO in RAW264.7 macrophages and rat hippocampus tissue. These results demonstrate the utility of this ESIPT benzimidazole-based platform for theranostic development and bioimaging applications.
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http://dx.doi.org/10.1039/d0sc02347gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499849PMC
July 2020

Design and synthesis of efficient heavy-atom-free photosensitizers for photodynamic therapy of cancer.

Chem Commun (Camb) 2020 Sep;56(77):11489-11492

Institute of Research and Development, Duy Tan University, Da Nang 550000, Vietnam.

Novel thiocarbonyl derivatives (NIS and CRNS) with excellent ROS generation abilities are synthesized and studied as potential photosensitizers for one- and two-photon excited photodynamic therapy. In particular, NIS-Me and CRNS display outstanding phototoxicity toward HeLa cells under two-photon excitation (800 nm) with negligible dark toxicity.
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http://dx.doi.org/10.1039/d0cc04644bDOI Listing
September 2020

Screening of Drug-Induced Steatosis and Phospholipidosis Using Lipid Droplet-Selective Two-Photon Probes.

Anal Chem 2020 08 28;92(16):11223-11231. Epub 2020 Jul 28.

Department of Energy Systems Research and Department of Chemistry, Ajou University, Suwon 16499, Korea.

Lipid droplets (LDs) are organelles that play a major role in regulating the storage of neutral lipids. Dysregulation of LDs is associated with metabolic disorders, such as fatty liver diseases, obesity, diabetes, and atherosclerosis. We have developed LD-selective small-molecule fluorescence probes (probes and ) that are available for both one- and two-photon microscopy, employing live or fixed cells. We found that probes and sensitively detect the increased LDs in response to oleic acid or endoplasmic reticulum stress, both in cells and tissues of the liver. The narrow absorption and emission bands of probes and allow multicolor imaging for the study of the role of LDs in pathophysiology and LD-associated signaling by the coapplication of the probes for different organelles or antibodies against specific proteins. In addition, we show here, for the first time, that two-photon microscopy imaging using our LD-selective probes with LysoTracker provides a novel method for screening drugs to potentially induce steatosis and/or phospholipidosis.
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http://dx.doi.org/10.1021/acs.analchem.0c01728DOI Listing
August 2020

Two-Photon and Multicolor Fluorogenic Bioorthogonal Probes Based on Tetrazine-Conjugated Naphthalene Fluorophores.

Bioconjug Chem 2020 05 23;31(5):1545-1550. Epub 2020 Apr 23.

Department of Chemistry, Seoul National University, Seoul 08826, Korea.

Herein, we report the use of two-photon fluorogenic probes using tetrazine-based bioorthogonal reactions with multicolor emissions that cover nearly all of the visible region. New fluorogenic probes were designed based on donor-acceptor-type naphthalene structures conjugated with a fluorescence-quenching tetrazine moiety for turn-on properties in one- and two-photon fluorescence. Our fluorescent probes showed a moderate to good turn-on ratio after bioorthogonal inverse electron demand Diels-Alder cycloaddition with cyclooctenol in one- and two-photon fluorescence. We successfully applied our probes to mitochondria- and lysosome-selective bioorthogonal imaging in live cells with one-/two-photon and one-photon microscopy, respectively.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00197DOI Listing
May 2020

Combining hydrophilic and hydrophobic environment sensitive dyes to detect a wide range of cellular polarity.

Chem Sci 2020 Jan 25;11(2):596-601. Epub 2019 Nov 25.

Department of Chemistry , Department of Energy Systems Research , Ajou University , Suwon 443-749 , Korea . Email:

Intracellular polarity is an important parameter of pathological and biological phenomena of cells; abnormal polarities are associated with diabetes, neurological diseases, and cancer. However, previously reported polarity probes have issues with quantitatively detecting intracellular polarities, can measure only a limited range of polarities, and can only detect specific intracellular regions. Here, we developed a novel two-dye system, , that contains a new "turn-on" polarity probe () based on a spiropyran intramolecular ring closing-opening system activated in polar protic solvents, and a benzothiadiazole containing dye (), which emits only in non-polar solvents with a large stoke shift. Individually, and selectively localized to lysosome and lipid droplets, respectively; however, combining these dyes, which have completely different characteristics, a piperazine linker resulted in the staining of various intracellular organelles. Therefore, as and have the same absorption but different emissions, combining them resulted in a ratiometric polarity probe that could quantitatively measure a wider polarity range inside the cell using a single excitation source. In addition, ratiometric imaging using our probe to quantitatively detect the distribution of polarity in different cell lines indicated that lysosomes were the most polar organelles in the cell.
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http://dx.doi.org/10.1039/c9sc04859fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069515PMC
January 2020

Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury.

J Neurosci 2020 02 23;40(9):1943-1955. Epub 2020 Jan 23.

Department of Neurosurgery, CHA University School of Medicine, CHA Bundang Medical Center, Seongnam-si, Gyeonggi-do, Republic of Korea, 13488,

Currently, the role of transient receptor potential vanilloid type 4 (TRPV4), a nonselective cation channel in the pathology of spinal cord injury (SCI), is not recognized. Herein, we report the expression and contribution of TRPV4 in the pathology of scarring and endothelial and secondary damage after SCI. TRPV4 expression increased during the inflammatory phase in female rats after SCI and was expressed primarily by cells at endothelial-microglial junctions. Two-photon microscopy of intracellular-free Ca levels revealed a biphasic increase at similar time points after SCI. Expression of TRPV4 at the injury epicenter, but not intracellular-free Ca, progressively increases with the severity of the injury. Activation of TRPV4 with specific agonist altered the organization of endothelial cells, affected tight junctions in the hCMEC/D3 BBB cell line , and increases the scarring in rat spinal cord as well as induced endothelial damage. By contrast, suppression of TRPV4 with a specific antagonist or in female KO mouse attenuated inflammatory cytokines and chemokines, prevented the degradation of tight junction proteins, and preserve blood-spinal cord barrier integrity, thereby attenuate the scarring after SCI. Likewise, secondary damage was reduced, and behavioral outcomes were improved in KO mice after SCI. These results suggest that increased TRPV4 expression disrupts endothelial cell organization during the early inflammatory phase of SCI, resulting in tissue damage, vascular destabilization, blood-spinal cord barrier breakdown, and scarring. Thus, TRPV4 inhibition/knockdown represents a promising therapeutic strategy to stabilize/protect endothelial cells, attenuate nociception and secondary damage, and reduce scarring after SCI. TRPV4, a calcium-permeable nonselective cation channel, is widely expressed in both excitable and nonexcitable cells. Spinal cord injury (SCI) majorly caused by trauma/accidents is associated with changes in osmolarity, mechanical injury, and shear stress. After SCI, TRPV4 was increased and were found to be linked with the severity of injury at the epicenter at the time points that were reported to be critical for repair/treatment. Activation of TRPV4 was damaging to endothelial cells that form the blood-spinal cord barrier and thus contributes to scarring (glial and fibrotic). Importantly, inhibition/knockdown of TRPV4 prevented these effects. Thus, the manipulation of TRPV4 signaling might lead to new therapeutic strategies or combinatorial therapies to protect endothelial cells and enhance repair after SCI.
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http://dx.doi.org/10.1523/JNEUROSCI.2035-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046444PMC
February 2020

Azulene-Derived Fluorescent Probe for Bioimaging: Detection of Reactive Oxygen and Nitrogen Species by Two-Photon Microscopy.

J Am Chem Soc 2019 12 27;141(49):19389-19396. Epub 2019 Nov 27.

Department of Chemistry , University of Bath , Bath BA2 7AY , United Kingdom.

Two-photon fluorescence microscopy has become an indispensable technique for cellular imaging. Whereas most two-photon fluorescent probes rely on well-known fluorophores, here we report a new fluorophore for bioimaging, namely azulene. A chemodosimeter, comprising a boronate ester receptor motif conjugated to an appropriately substituted azulene, is shown to be an effective two-photon fluorescent probe for reactive oxygen species, showing good cell penetration, high selectivity for peroxynitrite, no cytotoxicity, and excellent photostability.
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http://dx.doi.org/10.1021/jacs.9b09813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909233PMC
December 2019

Discrimination between Human Colorectal Neoplasms with a Dual-Recognitive Two-Photon Probe.

Anal Chem 2019 11 4;91(22):14705-14711. Epub 2019 Nov 4.

Department of Energy Systems Research and Department of Chemistry , Ajou University , Suwon 16499 , Korea.

Colorectal cancer is a major cause of cancer-related deaths worldwide. Histologic diagnosis using biopsy samples of colorectal neoplasms is the most important step in determining the treatment methods, but these methods have limitations in accuracy and effectiveness. Herein, we report a dual-recognition two-photon probe and its application in the discrimination between human colorectal neoplasms. The probe is composed of two monosaccharides, d-glucosamine and β-d-galactopyranoside, in a fluorophore for the monitoring of both glucose uptake and β-gal hydrolysis. In vitro/cell imaging studies revealed the excellent selectivity and sensitivity of the probe for glucose transporter-mediated glucose uptake and β-gal activity. Cancer-specific uptake was monitored by increased fluorescence intensity, and additional screening of cancer cells was achieved by changes in emission ratio owing to the higher activity of β-gal. Using human colon tissues and two-photon microscopy, we found that the plot of intensity versus ratio can accurately discriminate between colorectal neoplasms in the order of cancer progression (normal, adenoma, and carcinoma).
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http://dx.doi.org/10.1021/acs.analchem.9b03951DOI Listing
November 2019

Two-Photon Fluorescence Probe for Selective Monitoring of Superoxide in Live Cells and Tissues.

Anal Chem 2019 11 1;91(22):14691-14696. Epub 2019 Nov 1.

Department of Chemistry and Nano Science , Ewha Womans University , Seoul 120-750 , Korea.

The abnormal location or generation of superoxide radical anion (O) are implicated in many diseases, including cancers; thus, development of an efficient method to detect O is of great importance. Inspired by the fluorophore-governed selective manner to O and peroxynitrite (ONOO) of previously reported phosphinate-based fluorescence probes, in this contribution, a phosphinothioate-containing probe, , was designed. The probe exhibited easy accessibility through a one-step sequence and good photostability and biocompatibility. Interestingly, showed high specificity and sensitivity to O over other reactive oxygen species/nitrogen species including ONOO. Furthermore, with the assistance of two-photon microscopy, was successfully applied for imaging endogenous O in live cells and tissues.
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http://dx.doi.org/10.1021/acs.analchem.9b03937DOI Listing
November 2019

Ratiometric Detection of γ-Glutamyltransferase in Human Colon Cancer Tissues Using a Two-Photon Probe.

Anal Chem 2019 07 2;91(14):9246-9250. Epub 2019 Jul 2.

Department of Energy Systems Research and Department of Chemistry , Ajou University , Suwon 16499 , Korea.

γ-Glutamyltransferase (GGT) plays a role in cleaving the γ-glutamyl bond of glutathione. The GGT is known to be overexpressed in some tumors and has been recognized as a potential biomarker for malignant tumors. Colon cancer is one of the most common cancers worldwide; however, there is no quantitative method for detecting cancer cells in human colon tissues. In this study, we report a ratiometric two-photon probe for GGT that can be applied in human colon tissues. The probe (Probe 2) showed high fluorescence efficiency, marked fluorescence changes, excellent kinetics, and selectivity for the GGT in live colon cells. Additionally, we obtained ratiometric two-photon microscopy images of GGT activity in human colon tissue. We used this method to compare normal and cancer tissues based on their ratio values; the ratio value was higher in cancer tissue than in normal tissue. This study provides a method for quantitative analysis of GGT, particularly in human colon cancer, which will be useful for studying GGT-related diseases and diagnosing colon cancer.
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http://dx.doi.org/10.1021/acs.analchem.9b02137DOI Listing
July 2019

Unusual fluorescence of o-phenylazonaphthol derivatives with aggregation-induced emission and their use in two-photon cell imaging.

Chem Commun (Camb) 2019 Jun;55(47):6747-6750

Organic and Optoelectronic Materials Laboratory, Department of Advanced Organic Materials and Textile System Engineering, Chungnam National University, Daejeon 34134, Korea.

o-Phenylazonaphthol (o-PAN) derivatives including 6-bromo-1-((4-bromophenyl)diazenyl)naphthalen-2-ol (AN-Br-OH) and 1-phenylazo-2-naphthol (AN-OH, known as Sudan I (Color Index 12055)) were synthesized to investigate their fluorogenic behaviors, in which their aggregated-induced emission (AIE) is reported. The o-PANs showed a two-photon absorption. The protection of hydroxyl groups in o-PANs was used for fluorescence imaging of esterase-expressed HepG2 cells, which is potentially suitable for sensing and two-photon cell imaging applications.
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http://dx.doi.org/10.1039/c9cc03106eDOI Listing
June 2019

A two-photon fluorescent probe for colorimetric and ratiometric monitoring of mercury in live cells and tissues.

Chem Commun (Camb) 2019 Feb;55(12):1766-1769

Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 120-750, Korea.

Owing to the extreme toxicity of mercury, methods for its selective and sensitive sensing in solutions, and in live cells and tissues are in great demand. In this study, we developed a naphthalimide-based diphenylphosphinothioyl group-containing fluorescent and colorimetric probe that selectively detects mercury (Hg2+). Upon addition of mercury (Hg2+) to a solution of the probe, both a colorimetric change from colorless to yellow and a fluorescence change from blue to green (under a 365 nm hand-held UV lamp) occur, both of which can be observed using the "naked-eye". Furthermore, the probe possesses the capability of sensing intracellular mercury in both live cells and tissues using dual-emission channels and two-photon microscopy.
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http://dx.doi.org/10.1039/c8cc08608gDOI Listing
February 2019

High-depth fluorescence imaging using a two-photon FRET system for mitochondrial pH in live cells and tissues.

Chem Commun (Camb) 2018 Dec 15;54(96):13531-13534. Epub 2018 Nov 15.

Department of Chemistry, Sookmyung Women's University, Seoul, 04310, Korea.

We developed a fluorescent pH probe (1) capable of two-photon excitation and far-visible-emission based on FRET, composed of naphthalimide-piperazine-rhodamine. It exhibited a pH-dependent reversible and fast ratiometric fluorescence change in the rhodamine emission. Probe 1 was applied to image the pH perturbations of mitochondria in living cells and tissues.
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http://dx.doi.org/10.1039/c8cc07934jDOI Listing
December 2018

Endoplasmic Reticulum-Targeted Ratiometric N-Heterocyclic Carbene Borane Probe for Two-Photon Microscopic Imaging of Hypochlorous Acid.

Anal Chem 2018 11 23;90(21):12937-12943. Epub 2018 Oct 23.

Department of Chemistry and Nano Science , Ewha Womans University , Seoul 03760 , Korea.

The naphthoimidazolium borane 4 is shown to be a selective probe for HOCl over other reactive oxygen species. Unlike other boronate-reactive oxygen species (ROS) fluorogenic probes that are oxidized by HOCl through a nucleophilic borono-Dakin oxidation mechanism, probe 4 is distinguished by its electrophilic oxidation mechanism involving B-H bond cleavage. Two-photon microscopy experiments in living cells and tissues with the probe 4 demonstrate the monitoring of endogenous HOCl generation and changes in HOCl concentrations generated in the endoplasmic reticulum during oxidative stress situations.
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http://dx.doi.org/10.1021/acs.analchem.8b03565DOI Listing
November 2018

Near-IR Fluorescent Tracer for Glucose-Uptake Monitoring in Live Cells.

Bioconjug Chem 2018 10 12;29(10):3394-3401. Epub 2018 Sep 12.

Center for Systems Biology, Massachusetts General Hospital , Harvard Medical School , Boston , Massachusetts 02114 , United States.

Fluorescent tracers for glucose-uptake monitoring could be used as chemical tools for diagnosis and for discovery of novel therapeutic agents via the development of phenotypic screening systems. Here we present a new near-infrared fluorescent glucose tracer, Glc-SiR-COH, for monitoring the cellular glucose uptake. By conjugating glucosamine with two different silicon rhodamine fluorochromes, we found that the net charge of fluorochromes has considerable effects on cellular uptake of the probe. Competition assay with d/l-glucose as well as Western blot analysis implied GLUT-dependent uptake mechanism of this probe. Finally, Glc-SiR-COH not only differentiates cancer cells from normal cells, but also allows monitoring anticancer effects in live cells.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00558DOI Listing
October 2018

New Six-Membered pH-Insensitive Rhodamine Spirocycle in Selective Sensing of Cu through C-C Bond Cleavage and Its Application in Cell Imaging.

ACS Omega 2017 Nov 20;2(11):8167-8176. Epub 2017 Nov 20.

Department of Chemistry, University of Kalyani, Kalyani 741235, India.

A new rhodamine-based chemosensor with a six-membered spirocyclic ring has been synthesized, which exhibits excellent pH stability and shows selective "turn-on" fluorescent detection of Cu ions over a series of other metal ions including Cu ions. The expansion of spirocycle improves the stability and selectivity of the chemosensors in sensing of metal ions. Till today only few rhodamine structures - with thiourea-, hydrazine amide-, or pyrrole-decorated six-membered spirocyclic rings are known that exhibit metal-ion sensing via C-N bond cleavage of the spiro ring. In this context, rhodamine compound that responds to the metal ion through C-C bond cleavage of the six-membered spiro ring is completely unknown. The present example is a first-time report that demonstrates selective sensing of Cu ions through C-C bond cleavage over the conventional existing systems in the literature. The chemosensor is cell permeable and can detect Cu in live cells using confocal microscopy in the biologically relevant pH range with high photostability.
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http://dx.doi.org/10.1021/acsomega.7b01324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045328PMC
November 2017

Carboxylesterase-2-Selective Two-Photon Ratiometric Probe Reveals Decreased Carboxylesterase-2 Activity in Breast Cancer Cells.

Anal Chem 2018 08 27;90(15):9465-9471. Epub 2018 Jul 27.

Department of Energy Systems Research , Ajou University , Suwon 443-749 , Korea.

Human carboxylesterase-2 (CE2) is a carboxylesterase that catalyzes the hydrolysis of endogenous and exogenous substrates. Abnormal CE2 levels are associated with various cancers, and CE2 is a key mediator of anticancer prodrugs, including irinotecan. Here, we developed a two-photon ratiometric probe for detecting CE2 activity using succinate ester as a recognition site for CE2. The probe showed high selectivity to CE2, a clear emission color change, high photostability, and bright two-photon microscopy (TPM) imaging capability, allowing the quantitative detection of CE2 activity in live cells. Using TPM ratio analysis, we show for the first time that CE2 activity was much lower in breast cancer cells than in normal cells. In CE2 overexpression studies, cancer cells had a markedly enhanced sensitivity to the cytotoxic effect of irinotecan, corresponding well with the TPM ratio of the probe. These results may provide useful information for quantitatively measuring CE2 activity in situ and predicting the responsiveness to anticancer drugs.
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http://dx.doi.org/10.1021/acs.analchem.8b02101DOI Listing
August 2018

Ratiometric Two-Photon Fluorescent Probe for Detecting and Imaging Hypochlorite.

Anal Chem 2018 08 24;90(15):9510-9514. Epub 2018 Jul 24.

Department of Chemistry and Nano Science , Ewha Womans University , Seoul 120-750 , Korea.

The ratiometric fluorescent probe B6S, which contains pyrene as a fluorophore and imidazoline-2-thione as a reactive site, was developed for detection of hypochlorite (OCl). B6S displays a high specificity toward OCl in contrast to other reactive oxygen species and reactive nitrogen species. The probe has a low detection limit and operates under biological conditions. Moreover, the low cytotoxicity of B6S enables it to be utilized effectively for OCl imaging in living cells and tissues by using two-photon microscopy. The findings indicate that B6S has the capability of serving as a probe to explore the biological functions of OCl in living systems.
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http://dx.doi.org/10.1021/acs.analchem.8b02195DOI Listing
August 2018

A Two-Photon Fluorescent Probe for Imaging Endogenous ONOO near NMDA Receptors in Neuronal Cells and Hippocampal Tissues.

Anal Chem 2018 08 13;90(15):9347-9352. Epub 2018 Jul 13.

Department of Chemistry and Nano Science , Ewha Womans University , Seoul 120-750 , Korea.

In this study, we developed a two-photon fluorescent probe for detection of peroxynitrite (ONOO) near the N-methyl-d-aspartate (NMDA) receptor. This naphthalimide-based probe contains a boronic acid reactive group and an ifenprodil-like tail, which serves as an NMDA receptor targeting unit. The probe displays high sensitivity and selectivity, along with a fast response time in aqueous solution. More importantly, the probe can be employed along with two-photon fluorescence microscopy to detect endogenous ONOO near NMDA receptors in neuronal cells as well as in hippocampal tissues. The results suggest that the probe has the potential of serving as a useful imaging tool for studying ONOO related diseases in the nervous system.
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http://dx.doi.org/10.1021/acs.analchem.8b01960DOI Listing
August 2018

Carboxylate-Containing Two-Photon Probe for the Simultaneous Detection of Extra- and Intracellular pH Values in Colon Cancer Tissue.

Anal Chem 2018 07 11;90(13):8058-8064. Epub 2018 Jun 11.

Department of Gastroenterology , Ajou University School of Medicine , Suwon 443-721 , Korea.

Acidified extracellular pH (pHe) is directly related to various disorders such as tumor invasion and the resistance to drugs. In this study, we developed two-photon-excitable emission ratiometric probes (XBH1-3) for the in situ measurement of pHe. These probes, based on benzimidazole and polar solubilizing groups, exhibited a strong two-photon-induced fluorescence and sensitive blue-to-green emission color changes with p K values of 5.1-5.7. XBH1, containing a carboxylic acid, stained the extracellular region in neutral media; it entered the cell under acidic media, thereby allowing a precise measurement of the extra- and intra-cellular pH values in the acidified tissue. XBH2, containing the sulfonate peripheral unit, facilitated the monitoring of the pHe value only. Ratiometric two-photon microscopy imaging revealed that XBH1 can directly monitor the pH values both inside and outside the cells in colon cancer tissue; there is also the morphological aspect. This could be useful for cancer analyses and drug development.
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http://dx.doi.org/10.1021/acs.analchem.8b01114DOI Listing
July 2018

Naphthalene-based fluorescent probes for glutathione and their applications in living cells and patients with sepsis.

Theranostics 2018 3;8(5):1411-1420. Epub 2018 Feb 3.

Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea.

Among the biothiols-related diseases, sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection and can result in severe oxidative stress and damage to multiple organs. In this study, we aimed to develop a fluorescence chemosensor that can both detect GSH and further predict sepsis. In this study, two new naphthalene dialdehyde compounds containing different functional groups were synthesized, and the sensing abilities of these compounds towards biothiols and its applications for prediction of sepsis were investigated. Our study revealed that the newly developed probe 6-methoxynaphthalene-2, 3-dicarbaldehyde (MNDA) has two-photon is capable of detecting GSH in live cells with two-photon microscopy (TPM) under the excitation at a wavelength of 900 nm. Furthermore, two GSH detection probes naphthalene-2,3-dicarboxaldehyde (NDA) and 6-fluoronaphthalene-2,3-dicarbaldehyde (FNDA) not only can detect GSH in living cells, but also showed clinical significance for the diagnosis and prediction of mortality in patients with sepsis. These results open up a promising direction for further medical diagnostic techniques.
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http://dx.doi.org/10.7150/thno.22252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835946PMC
January 2019

Pyrrolidine dithiocarbamate reverses Bcl-xL-mediated apoptotic resistance to doxorubicin by inducing paraptosis.

Carcinogenesis 2018 03;39(3):458-470

Department of Biochemistry, Ajou University School of Medicine, Suwon, Korea.

Elevated Bcl-xL expression in cancer cells contributes to doxorubicin (DOX) resistance, leading to failure in chemotherapy. In addition, the clinical use of high-dose doxorubicin (DOX) in cancer therapy has been limited by issues with cardiotoxicity and hepatotoxicity. Here, we show that co-treatment with pyrrolidine dithiocarbamate (PDTC) attenuates DOX-induced apoptosis in Chang-L liver cells and human hepatocytes, but overcomes DOX resistance in Bcl-xL-overexpressing Chang-L cells and several hepatocellular carcinoma (HCC) cell lines with high Bcl-xL expression. Additionally, combined treatment with DOX and PDTC markedly retarded tumor growth in a Huh-7 HCC cell xenograft tumor model, compared to either mono-treatment. These results suggest that DOX/PDTC co-treatment may provide a safe and effective therapeutic strategy against malignant hepatoma cells with Bcl-xL-mediated apoptotic defects. We also found that induction of paraptosis, a cell death mode that is accompanied by dilation of the endoplasmic reticulum and mitochondria, is involved in this anti-cancer effect of DOX/PDTC. The intracellular glutathione levels were reduced in Bcl-xL-overexpressing Chang-L cells treated with DOX/PDTC, and DOX/PDTC-induced paraptosis was effectively blocked by pretreatment with thiol-antioxidants, but not by non-thiol antioxidants. Collectively, our results suggest that disruption of thiol homeostasis may critically contribute to DOX/PDTC-induced paraptosis in Bcl-xL-overexpressing cells.
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http://dx.doi.org/10.1093/carcin/bgy003DOI Listing
March 2018

A Two-Photon Ratiometric Fluorescent Probe for Imaging of Hydrogen Peroxide Levels in Rat Organ Tissues.

ChemistryOpen 2018 01 22;7(1):53-56. Epub 2017 Nov 22.

Department of Energy System Research and Department of Chemistry Ajou University Suwon Gyeonggi-do 443-749 Republic of Korea.

Hydrogen peroxide (HO) is important in the regulation of a variety of biological processes and is involved in various diseases. Quantitative measurement of HO levels at the subcellular level is important for understanding its positive and negative effects on biological processes. Herein, a two-photon ratiometric fluorescent probe (SHP-Cyto) with a boronate-based carbamate leaving group as the HO reactive trigger and 6-(benzo[]thiazol-2'-yl)-2-(,-dimethylamino) naphthalene (BTDAN) as the fluorophore was synthesized and examined for its ability to detect cytosolic HO in situ. This probe, based on the specific reaction between boronate and HO, displayed a fluorescent color change (455 to 528 nm) in response to HO in the presence of diverse reactive oxygen species in a physiological medium. In addition, ratiometric two-photon microscopy (TPM) images with SHP-Cyto revealed that HO levels gradually increased from brain to kidney, skin, heart, lung, and then liver tissues. SHP-Cyto was successfully applied to the imaging of endogenously produced cytosolic HO levels in live cells and various rat organs by using TPM.
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http://dx.doi.org/10.1002/open.201700155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754560PMC
January 2018

N-Heterocyclic Carbene Boranes as Reactive Oxygen Species-Responsive Materials: Application to the Two-Photon Imaging of Hypochlorous Acid in Living Cells and Tissues.

Angew Chem Int Ed Engl 2018 02 9;57(6):1567-1571. Epub 2018 Jan 9.

Department of Chemistry and Nano Science (BK 21 Plus), Ewha Womans University, Seoul, 03760, Korea.

N-Heterocyclic carbene (NHC) boranes undergo oxidative hydrolysis to give imidazolium salts with excellent kinetic selectivity for HOCl over other reactive oxygen species (ROS), including peroxides and peroxynitrite. Selectivity for HOCl results from the electrophilic oxidation mechanism of NHC boranes, which stands in contrast to the nucleophilic oxidation mechanism of arylboronic acids with ROS. The change in polarity that accompanies the conversion of NHC boranes to imidazolium salts can control the formation of emissive excimers, forming the basis for the design of the first fluorescence probe for ROS based on the oxidation of B-H bonds. Two-photon microscope (TPM) ratiometric imaging of HOCl in living cells and tissues is demonstrated.
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http://dx.doi.org/10.1002/anie.201711188DOI Listing
February 2018

A ratiometric two-photon probe for Ca in live tissues and its application to spinal cord injury model.

Biomaterials 2017 Oct 6;141:251-259. Epub 2017 Jul 6.

Department of Energy System Research and Department of Chemistry, Ajou University, Suwon, Gyeonggi-do, 443-749, Republic of Korea. Electronic address:

Ratiometric imaging with a small-molecule probe is important for the in-situ quantitative analysis of chemical events. We developed a ratiometric two-photon fluorescent probe (SCa1-I) derived from dual dyes with different Stokes shifts. This probe has two identical windows: a Ca-sensing window and an internal reference window, with eliminated FRET interference. SCa1-I shows a marked change in the ratio upon response with Ca, significant two-photon brightness, considerable selectivity for Ca, and cell loading ability with low cytotoxicity. The ratiometric two-photon microscopy images revealed that this probe could directly and quantitatively estimate Ca in live neurons and various tissues including rat spinal cord tissue. The studies of spinal cord injury model revealed that the Ca level was significantly affected by elapsed time after injury. These results will provide useful applications for in-situ [Ca] imaging and for the development of effective ratiometric probes.
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http://dx.doi.org/10.1016/j.biomaterials.2017.07.006DOI Listing
October 2017

Highly Selective and Sensitive Two-Photon Fluorescence Probe for Endogenous Peroxynitrite Detection and Its Applications in Living Cells and Tissues.

Anal Chem 2017 08 24;89(16):8496-8500. Epub 2017 Jul 24.

Department of Chemistry and Nano Science, Ewha Womans University , Seoul 120-750, Korea.

A new two-photon fluorescence probe for endogenous peroxynitrite (ONOO) detection was designed and synthesized. The probe exhibits good selectivity and sensitivity for ONOO in phosphate-buffered saline solution with a low detection limit (3.5 × 10 M). Furthermore, the probe displays good performance in detecting endogenous ONOO, not only in RAW 264.7 cells but also in rat hippocampal tissue, with a high two-photon cross-section value (δ ≈ 100 GM) at a deep depth of 120 μm.
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http://dx.doi.org/10.1021/acs.analchem.7b02059DOI Listing
August 2017

Visualization of vesicular transport from the endoplasmic reticulum to lysosome using an amidine derived two-photon probe.

Chem Commun (Camb) 2017 Jun;53(45):6097-6100

Department of Energy Systems Research and Department of Chemistry, Ajou University, Suwon 443-749, Korea.

We report an amidine-based small molecule two-photon fluorescent probe (ELP1) for monitoring vesicle transport from the ER to lysosome in live cells. Two-photon microscopy imaging studies indicated that this probe initially localized in the ER and subsequently transferred to lysosomal compartments through vesicular transport. These results may provide an effective tool for studying trafficking-related biology and pathology in real-time.
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http://dx.doi.org/10.1039/c7cc01518fDOI Listing
June 2017

α-Syntrophin stabilizes catalase to reduce endogenous reactive oxygen species levels during myoblast differentiation.

FEBS J 2017 07 4;284(13):2052-2065. Epub 2017 Jun 4.

Department of Biological Science, College of Natural Sciences, Ajou University, Suwon, Korea.

α-Syntrophin is a component of the dystrophin-glycoprotein complex that interacts with various intracellular signaling proteins in muscle cells. The α-syntrophin knock-down C2 cell line (SNKD), established by infecting lentivirus particles with α-syntrophin shRNA, is characterized by a defect in terminal differentiation and increase in cell death. Since myoblast differentiation is accompanied by intensive mitochondrial biogenesis, the generation of intracellular reactive oxygen species (ROS) is also increased during myogenesis. Two-photon microscopy imaging showed that excessive intracellular ROS accumulated during the differentiation of SNKD cells as compared with control cells. The formation of 4-hydroxynonenal adduct, a byproduct of lipid peroxidation during oxidative stress, significantly increased in differentiated SNKD myotubes and was dramatically reduced by epigallocatechin-3-gallate, a well-known ROS scavenger. Among antioxidant enzymes, catalase was significantly decreased during differentiation of SNKD cells without changes at the mRNA level. Of interest was the finding that the degradation of catalase was rescued by MG132, a proteasome inhibitor, in the SNKD cells. This study demonstrates a novel function of α-syntrophin. This protein plays an important role in the regulation of oxidative stress from endogenously generated ROS during myoblast differentiation by modulating the protein stability of catalase.
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http://dx.doi.org/10.1111/febs.14103DOI Listing
July 2017