Sankarprasad Bhuniya

Hepatocyte-targeting single galactose-appended naphthalimide: A tool for intracellular thiol imaging in vivo

We present the design, synthesis, spectroscopic properties, and biological evaluation of a single galactose-appended naphthalimide (1). Probe 1 is a multifunctional molecule that incorporates a thiol-specific cleavable disulfide bond, a masked phthalamide fluorophore, and a single galactose moiety as a hepatocyte-targeting unit. It constitutes a new type of targetable ligand for hepatic thiol imaging in living cells and animals. Confocal microscopic imaging experiments reveal that 1, but not the galactose-free control system 2, is preferentially taken up by HepG2 cells through galactose-targeted, ASGP-R-mediated endocytosis. Probe 1 displays a fluorescence emission feature at 540 nm that is induced by exposure to free endogenous thiols, most notably GSH. The liver-specificity of 1 was confirmed in vivo via use of a rat model. The potential utility of this probe in indicating pathogenic states and as a possible screening tool for agents that can manipulate oxidative stress was demonstrated in experiments wherein palmitate was used to induce lipotoxicity in HepG2 cells.

A highly selective colorimetric and ratiometric two-photon fluorescent probe for fluoride ion detection.

A naphthalimide-based highly selective colorimetric and ratiometric fluorescent probe for the fluoride ion displayed both one- and two-photon ratiometric changes. Upon reaction with the F(-) (TBA(+) and Na(+) salts) anion in CH(3)CN as well as in aqueous buffer solution, probe 1 shows dramatic color changes from colorless to jade-green and remarkable ratiometric fluorescence enhancements signals. These properties are mechanistically ascribed to a fluoride-triggered Si-O bond cleavage that resulted in a green fluorescent 4-amino-1,8-naphthalimide.

Direct Fluorescence Monitoring of the Delivery and Cellular Uptake of a Cancer-Targeted RGD Peptide-Appended Naphthalimide Theragnostic Prodrug

Presented here is a multicomponent synthetic strategy that allows for the direct, fluorescence-based monitoring of the targeted cellular uptake and release of a conjugated therapeutic agent. Specifically, we report here the design, synthesis, spectroscopic characterization, and preliminary in vitro biological evaluation of a RGD peptide-appended naphthalimide pro-CPT (compound 1). Compound 1 is a multifunctional molecule composed of a disulfide bond as a cleavable linker, a naphthalimide moiety as a fluorescent reporter, an RGD cyclic peptide as a cancer-targeting unit, and camptothecin (CPT) as a model active agent. Upon reaction with free thiols in aqueous media at pH 7.4, disulfide cleavage occurs. This leads to release of the free CPT active agent, as well as the production of a red-shifted fluorescence emission (λ(max) = 535 nm). Confocal microscopic experiments reveal that 1 is preferentially taken up by U87 cells over C6 cells. On the basis of competition experiments involving okadaic acid, an inhibitor of endocytosis, it is concluded that uptake takes place via RGD-dependent endocytosis mechanisms. In U87 cells, the active CPT payload is released within the endoplasmic reticulum, as inferred from fluorescence-based colocalization studies using a known endoplasmic reticulum-selective dye. The present drug delivery system (DDS) could represent a new approach to so-called theragnostic agent development, wherein both a therapeutic effect and drug uptake-related imaging information are produced and can be readily monitored at the subcellular level. In due course, the strategy embodied in conjugate 1 could allow for more precise monitoring of dosage levels, as well as an improved understanding of cellular uptake and release mechanisms.

Gemcitabine–coumarin–biotin conjugates: a target specific theranostic anticancer prodrug

We present here, the design, synthesis, spectroscopic characterization, and in vitro biological assessment of a gemcitabine-coumarin-biotin conjugate (5). Probe 5 is a multifunctional molecule composed of a thiol-specific cleavable disulfide bond, a coumarin moiety as a fluorescent reporter, gemcitabine (GMC) as a model active drug, and biotin as a cancer-targeting unit. Upon addition of free thiols that are relatively abundant in tumor cells, disulfide bond cleavage occurs as well as active drug GMC release and concomitantly fluorescence intensity increases. Confocal microscopic experiments reveal that 5 is preferentially taken up by A549 cells rather than WI38 cells. Fluorescence-based colocalization studies using lysosome- and endoplasmic reticulum-selective dyes suggest that thiol-induced disulfide cleavage of 5 occur in the lysosome possibly via receptor-mediated endocytosis. The present drug delivery system is a new theranostic agent, wherein both a therapeutic effect and drug uptake can be readily monitored at the subcellular level by two photon fluorescence imaging.

An Activatable Prodrug for the Treatment of Metastatic Tumors

Metastatic cancers have historically been difficult to treat. However, metastatic tumors have been found to have high levels of reactive oxygen species such as hydrogen peroxide (H2O2), supporting the hypothesis that a prodrug could be activated by intracellular H2O2 and lead to a potential antimetastatic therapy. In this study, prodrug 7 was designed to be activated by H2O2-mediated boronate oxidation, resulting in activation of the fluorophore for detection and release of the therapeutic agent, SN-38. Drug release from prodrug 7 was investigated by monitoring fluorescence after addition of H2O2 to the cancer cells. Prodrug 7 activated by H2O2, selectively inhibited tumor cell growth. Furthermore, intratracheally administered prodrug 7 showed effective antitumor activity in a mouse model of metastatic lung disease. Thus, this H2O2-responsive prodrug has therapeutic potential as a novel treatment for metastatic cancer via cellular imaging with fluorescence as well as selective release of the anticancer drug, SN-38.

Fluorescence turn-on sensors for HSO4-.

A coumarin-based derivative (1), a highly selective and sensitive turn-on fluorogenic probe for the detection of HSO4- ions in aqueous solution, has been designed and synthesized. Various spectroscopic and DFT calculations revealed that H-bonding between the phenolic -OH and imine nitrogen of 1 played a crucial role in its high selectivity for HSO4-.

Rationally Designed Fluorescence Turn-On Sensors: A New Design Strategy Based on Orbital Control

Herein, we explore a new strategy in the chemo-sensor field for fluorescence amplification upon binding with metal ions based on controlled participation of the nitrogen lone pair orbital. The basic architecture of the sensor entails a fluorophore, the sp(2) hybridized nitrogen lone pair (-C═N-), and a chelator site referred to as the control part. Though nonplanar and nonfluorescent, compound IC1 achieved pseudo planarity from binding with Zn(2+) as indicated by the increased fluorescence signal. Its other analogue (IC2) is also planar, and unlike IC1-Zn(2+) was fluorescent with a lack of binding affinity to metal ions. The time-dependent density functional theory (TDDFT) calculations revealed that the fluorescence amplification was due to the blocking of the nitrogen lone pair orbital; unlikely geometrical rearrangements were insignificant. This could indicate a breakthrough concept in the future design of fluorescent turn-on sensors.

A fluorescence off–on reporter for real time monitoring of gemcitabine delivery to the cancer cells

We present the design, synthesis, optical properties and in vitro biological assessments of the theranostic prodrug in which a near IR fluorophore is conjugated with a cancer cell-directing biotin unit; further it is linked with the anti-cancer drug gemcitabine via a self-immolative spacer, a disulfide bond. The prodrug is able to monitor drug delivery and cellular imaging.

Cu2+-Responsive Bimodal (Optical/MRI) Contrast Agent for Cellular Imaging

A water-soluble T1 magnetic resonance imaging contrast agent (1) has been synthesized. The bimodal contrast agent 1 responds to the Cu(2+) ion in living cells by enhancing the MRI modality signal whereas the optical signal gradually drops. This dual modality probe response depends on the cellular free copper ions in RAW 264.7 cells even at the micromolar level.

Reversible sol-gel signaling system with epMB for the study of enzyme- and pH-triggered oligonucleotide release from a biotin hydrogel

The biotin-based low molecular weight hydrogel (G1) is able to entrap the epMB and as a consequence, the color of the epMB changes from green to blue; the color change depends on the state of the gelator, i.e. upon proceeding from sol to gel and vice versa.