Epithelial mesenchymal transition induced nuclear localization of the extracellular matrix protein Fibronectin.

Fibronectin (FN), an extracellular matrix (ECM) glycoprotein, is a well-known marker for Epithelial Mesenchymal Transition (EMT). In the ECM, FN has been shown to form long fibrils and play critical roles in regulating cellular attachment and migration during EMT associated with physiological processes such as embryonic development, wound healing as well as pathological processes such as tissue fibrosis and cancer. Subsequently, the cytokine, Transforming Growth Factor ß (TGFß), an inducer of EMT, was found to induce FN expression in a c-Jun N-terminal kinase (JNK) dependent manner. Moreover, extracellular FN, by itself, was also shown to induce EMT in breast epithelial cells in serum-free condition. Collectively, all the literature published so far has shown and established the role of extracellular FN during EMT. In this report, we have shown that EMT induced entry of FN into the nucleus of mouse breast epithelial cells. To our knowledge, this is the first report showing nuclear localization of the extracellular matrix protein Fibronectin during EMT and thereby suggests a possible nuclear function for the ECM protein.

1,2,3-Triazoles: Controlled Switches in Logic Gate Applications.

A 1,2,3-triazole-based chemosensor is used for selective switching in logic gate operations through colorimetric and fluorometric response mechanisms. The molecular probe synthesized via "click chemistry" resulted in a non-fluorescent 1,4-diaryl-1,2,3-triazole with a phenol moiety (PTP). However, upon sensing fluoride, it TURNS ON the molecule's fluorescence. The TURN-OFF order occurs through fluorescence quenching of the sensor when metal ions, e.g., Cu2+, and Zn2+, are added to the PTP-fluoride ensemble. A detailed characterization using Nuclear Magnetic Resonance (NMR) spectroscopy in a sequential titration study substantiated the photophysical characteristics of PTP through UV-Vis absorption and fluorescence profiles. A combination of fluorescence OFF-ON-OFF sequences provides evidence of 1,2,3-triazoles being controlled switches applicable to multimodal logic operations. The "INH" gate was constructed based on the fluorescence output of PTP when the inputs are F- and Zn2+. The "IMP" and "OR" gates were created on the colorimetric output responses using the probe's absorption with multiple inputs (F- and Zn2+ or Cu2+). The PTP sensor is the best example of the "Write-Read-Erase-Read" mimic.

Rhodamine-Conjugated Anti-Stokes Gold Nanoparticles with Higher ROS Quantum Yield as Theranostic Probe to Arrest Cancer and MDR Bacteria.

Photodynamic therapy (PDT) has recently become significant as a clinical modality for cancer therapy and multidrug-resistant (MDR) infections, replacing conventional chemotherapy and radiation therapy protocols. PDT involves the excitation of certain nontoxic molecules called photosensitizers (PS), applying a specific wavelength of light to generate reactive oxygen species (ROS) to treat cancer cells and other pathogens. Rhodamine 6G (R6G) is a well-known laser dye with poor aqueous solubility, and lower sensitivity poses an issue in using PS for PDT. Nanocarrier systems are needed to deliver R6G to cancer targets since PDT requires a higher accumulation of PS. It was found that R6G-conjugated gold nanoparticles (AuNP) have a higher ROS quantum yield of 0.92 compared to 0.3 in an aqueous R6G solution, increasing their potency as PS. Cytotoxicity assessment on A549 cells and antibacterial assay on MDR Pseudomonas aeruginosa collected from a sewage treatment plant are the evidence to support efficient PDT. In addition to their enhanced quantum yields, the decorated particles are effective in generating fluorescent signals that can be used for cellular imaging and real-time optical imaging, and the presence of AuNP is a valuable addition to CT imaging. Furthermore, the fabricated particle exhibits anti-Stokes properties, which makes it suitable for use as a background-free biological imaging agent. As a result, R6G-conjugated AuNP is an effective theranostic agent that prevents the progression of cancer and MDR bacteria, along with contrasting abilities in medical imaging with minimal toxicity observed in in vitro and in vivo assays using zebrafish embryos.

Exploring the Effects of Various Polymeric Backbones on the Performance of a Hydroxyaromatic 1,2,3-Triazole Anion Sensor.

Polymeric chemosensors are vital sensing tools because of higher sensitivity compared to their monomeric counterparts and tunable mechanical properties. This study focuses on the incorporation of a hydroxyaromatic 1,2,3-triazole sensor, 2-(4-phenyl 1H-1,2,3-triazol-1-yl)phenol (PTP), into polymers. By itself, the triazole has a selective, fluorometric response to the fluoride, acetate, and dihydrogen phosphate anions, and is most responsive to fluoride. Current investigations probe the suitability of various polymeric backbones for the retention and enhancement of the triazole's sensing capabilities. Backbones derived from acrylic acid, methyl methacrylate, divinylbenzene, and styrene were explored. UV-illumination, Nuclear Magnetic Resonance (NMR) titration, and ultraviolet-visible (UV-Vis) absorption and fluorescence spectroscopy studies are used to investigate the performance of newly synthesized polymers and the derivatives of PTP that serve as the polymers' precursors. Among the polymers investigated, copolymers with styrene proved best; these systems retained the sensing capabilities and were amenable to tuning for sensitivity.

Rationally designed phenanthrene derivatized triazole as a dual chemosensor for fluoride and copper recognition.

A 1,2,3-triazole chemosensor containing phenanthrene and phenol moieties (PhTP) was efficiently synthesized via copper (I)-catalyzed azide-alkyne cycloaddition, "click chemistry". PhTP is a dual analyte sensor for fluoride and copper (II) ions in homogeneous medium. Deprotonation of the phenolic OH proton by the fluoride ion is responsible for a change in fluorescence color from blue (PhTP) to yellowish-orange (PhTP-fluoride adduct), while a charge transfer between the triazole nitrogen of the chemosensor and Cu2+ revealed a turn-off fluorescence output. The detection capability of PhTP was analyzed with a series of anions (F-, Cl-, Br-, I-, H2PO4-, ClO4-, OAc-, BF4-) and cations (Fe3+, Fe2+, Cu2+, Ag+, Cr3+, Al3+, Co2+, Ni2+, Cd2+, Zn2+). With anions, competitive fluorescence responses under UV lamp were observed for acetate and dihydrogen phosphate anions, but maximum response from fluoride ion was substantiated from steady state absorption and fluorescence experiments. With cations, PhTP displayed a selective and sensitive recognition towards Cu2+ ion through spectral modulation in absorption spectroscopy and a turn-off fluorescence response. Nuclear magnetic resonance (NMR) spectroscopic titration studies supported the results obtained through photophysical studies and provided evidence for the ion-binding sites on the probe.

Differential Förster resonance energy transfer from the excimers of poly(N-vinylcarbazole) to coumarin 153.

Photophysics of the nonconjugated vinyl polymer poly(N-vinylcarbazole) (PNVC) has been explored in the presence of coumarin 153 (C153) exploiting steady state and time-resolved fluorometric techniques. Dual emission from the two distinct excimers of PNVC adds importance to the study and makes it interesting. The study substantiates the occurrence of Förster resonance energy transfer (FRET) from PNVC to C153. The differential involvement of the two excimers in the energy transfer process has been established. Considering the fact that FRET is a long distance dipole induced phenomenon, this differential effect has been rationalized from a difference in the dipole moments of the two excimers. Determination of the quenching constants reveals an order of magnitude more quenching of the high energy excimer than the low energy one in the presence of the quencher C153.

Spectroscopic exploration of mode of binding of ctDNA with 3-hydroxyflavone: a contrast to the mode of binding with flavonoids having additional hydroxyl groups.

Binding interaction of 3-hydroxyflavone (3HF), a bioactive flavonoid, with calf-thymus DNA (ctDNA) has been explored exploiting various experimental techniques. The dual fluorescence of 3HF resulting from the excited state intramolecular proton transfer (ESIPT) is modified remarkably upon binding with the biomacromolecule. The determined binding constant, fluorescence quenching experiment, circular dichroism (CD) study, comparative binding study with the known intercalative binder ethidium bromide and thermometric experiment relating to the helix melting of ctDNA confirm the groove binding of 3HF with the DNA. This is in contrast to two other members of the flavonoid group, namely, fisetin and quercetin, where the bindings are established to be intercalative. The structural difference of 3HF from the other two probes with respect to the absence/presence of the additional hydroxyl groups is ascribed to be responsible for the difference in the mode of binding.

A fully standardized method of synthesis of gold nanoparticles of desired dimension in the range 15 nm-60 nm.

The citrate reduction method of synthesis of gold nanoparticles (AuNPs) as introduced by Frens has been standardized to enable one to prepare AuNPs of desired dimension by controlling the composition of the reactants. The standardization has been made through characterization of the nanoparticles by UV-vis spectroscopy and from the transmission electron microscopic (TEM) measurements. Linearity of the plot of the plasmon absorption maximum (lambda(max)) of the synthesized AuNPs against their diameter as measured from TEM, as well as the plot of lambda(max) with the fractional concentration of citrate in the reaction mixture provides a convenient and easy route to dictate the size of the synthesized AuNPs from a control on the composition of the reactants. The standardization reveals that a calculated composition of citrate (in terms of fractional concentration) in the reaction mixture produces AuNPs of a desired dimension within the range of 15-60 nm. The diameter of the synthesized gold nanoparticles can be confirmed simply from the UV-vis spectrophotometric technique. This essentially makes the use of costly TEM unnecessary, at least for the primary purposes.

Electrostatic pushing effect: a prospective strategy for enhanced drug delivery.

Contrary to the expected quenching, unprecedented and remarkable enhancement is observed in the fluorescence of an anionic fluorophore, 8-anilino-1-naphthalene sulfonate, with the addition of bromide ion in cationic cetyltrimethylammonium bromide micellar medium. Electrostatic pushing effect of the halide ion on the anionic fluorophore that forces the probe to penetrate further into the micellar interior has been assigned to be responsible for the novel observation. Experiments with other probes and surfactants propose that the electrostatic pushing effect is rather a general phenomenon. While applying to the ionic drugs in real biosystems, potential application of the unorthodox effect remains in enhancing the solubilization of the drugs into the active target region leading to a radical enhancement in the drug efficacy.

Studies of Triton X-165-beta-cyclodextrin interactions using both extrinsic and intrinsic fluorescence.

The interaction of beta-cyclodextrin with the non-ionic micelle-forming surfactant Triton X-165 (TX-165) has been studied using steady state fluorescence and fluorescence anisotropy techniques. Both extrinsic and intrinsic fluorescence have been exploited for the purpose. Phenosafranin (PSF), a cationic phenazinium dye, has been used as the extrinsic probe while fluorescence of TX-165 has served as the intrinsic one. PSF shows discernible interactions with both TX-165 and beta-CD. The experimental results reveal that the extent of interaction of PSF with TX-165 is greater than with beta-CD. However, addition of beta-CD to a micellar solution of TX-165 containing PSF leads to a disruption of the micelles whereby the fluorophore is released from the micellar environment to the bulk aqueous phase. It has been substantiated that an inclusion complex is formed between the non-ionic surfactant and the cyclodextrin. A 1:1 stoichiometry of the TX-165-beta-CD inclusion complex has been proposed. Such a complexation between TX-165 and beta-CD results in an inhibition in the micellization process of TX-165 leading to an enhancement in the apparent CMC value. The inferences are drawn from a series of experiments, viz., binding studies, determination of micropolarity, heavy-ion quenching studies and steady state fluorescence anisotropy experiments monitoring both extrinsic and intrinsic fluorescences.

Differential interaction of beta-cyclodextrin with lipids of varying surface charges: a spectral deciphering using a cationic phenazinium dye.

Interaction of phenosafranin (PSF), a biologically potent cationic dye molecule, has been studied with zwitterionic and anionic lipid membranes of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-alpha-phosphatidylglycerol (DMPG), respectively. The effect of cyclic oligosaccharide, beta-cyclodextrin (beta-CD), on the stability of these probe-bound lipid bilayers has also been investigated exploiting steady state and time-resolved fluorescence, steady state fluorescence anisotropy, and dynamic light scattering techniques. An interpretation of membrane destabilization upon interaction of cyclodextrin with the lipids was drawn exploiting PSF as an extrinsic fluorescent probe. The fluorophore showed discernible interactions with DMPC and DMPG vesicles. Experimental results reveal that the extent of interaction of PSF with DMPG is greater compared to that with DMPC. Addition of beta-CD into the PSF-bound lipids showed a differential effect for the two lipids of varying surface charge characteristics. In the case of DMPC, addition of beta-CD resulted in a preferential interaction of the probe with CD. However, addition of beta-CD to PSF-bound DMPG resulted in the selective interaction of DMPG with the added CD leading to the release of the probe into the bulk aqueous medium.

Binding of a cationic phenazinium dye in anionic liposomal membrane: a spectacular modification in the photophysics.

Interaction of a cationic phenazinium dye, phenosafranin (PSF), with the anionic liposomal vesicle/bilayer of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) has been demonstrated using steady state and time resolved fluorescence and fluorescence anisotropy techniques. The charge transfer emission spectrum of PSF shows a dramatic modification in terms of fluorescence yield together with an appreciable hypsochromic shift in the lipid environment. The blue shift indicates a lowering in polarity inside the vesicle as compared to that in bulk water. The fluorescence and fluorescence quenching studies and micropolarity determination reveal that the cationic fluorophore has a profound binding interaction with the anionic DMPG membrane. Anisotropy study indicates the imposition of a motional restriction on the probe inside the bilayer. The electrostatic interaction between the cationic dye and the anionic lipid membrane has been argued to be the reason behind all these observations. The results could be useful in analyzing membrane organization and heterogeneity in natural membranes exploiting PSF or alike compounds as fluorescent probes.

Photophysics and rotational relaxation dynamics of cationic phenazinium dyes in anionic reverse micelles: Effect of methyl substitution.

We present here, a detailed photophysical and rotational relaxation dynamical study of three structurally analogous cationic dyes, namely, phenosafranin (PSF), safranin-T (ST), and safranin-O (SO), carried out in well characterized, monodispersed biomimicking anionic reverse micellar nanocavities composed of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/heptane with increasing water contents. The dyes belong to the phenazinium family and they differ in terms of methyl substitution on the planar phenazinium skeleton. The objective of the present study is to investigate the modification in the photophysical and dynamical behavior of the dyes with the change in the size of the water pool of the reverse micelle and thereby to explore the role of methyl substitution. Steady state and time resolved emission and anisotropy studies have been exploited for the purpose. The dyes are found to exhibit a marked decrease in the fluorescence anisotropy with increasing water/surfactant mole ratio (w), i.e., the water pool size in the reverse micellar core, implying that overall motional restriction experienced by the molecules are decreased with increasing hydration. Some of the depth dependent fluorescence parameters such as fluorescence maximum, fluorescence anisotropy (r) have been monitored for exploring the microenvironment around the probes in the reverse micelles. Fluorescence studies suggest that at low w values, the probes do not penetrate into the reverse micellar core; rather it binds at the interfacial region. Estimates of the micropolarity at the binding sites of the probe molecule have been determined as a function of w. Finally, dynamic studies reveal that both the lifetime and rotational relaxation time decrease with an increase in w for all the three probes, the extent of the decrease being more for PSF than ST and SO. This indicates a stronger binding of the reverse micelle with ST and SO compared to that with PSF which is rationalized in terms of an increase in the hydrophobicity of the former two dyes because of the methyl substitution on the phenazinium moiety.

Excited-state-proton-transfer-triggered fluorescence resonance energy transfer: from 2-naphthylamine to phenosafranin.

Excited-state proton transfer (ESPT) and fluorescence resonance energy transfer (FRET) have been linearly coupled leading to an efficient pH-sensitive energy transfer from 2-naphthylamine (2NA) to a potentially bioactive cationic phenazinium dye, phenosafranin (PSF). The prototropic product produced exclusively from the photoexcited 2NA in the presence of added alkali serves as the donor for the energy transfer process. The energy transfer process is turned on at pH > or = 12, whereas the process is turned off at a pH lower than that. Within the range of pH 12 to 13, the energy transfer efficiency (E) has been shown to follow a linear relation with the solution pH establishing the governing role of pH of the solution on the energy transfer process. The energy transfer follows a long-range dipole-dipole interaction mechanism. The critical energy transfer distance (R0) and the distance between the acceptor and the donor (r) have been determined for the ESPT-promoted FRET process at an optimum pH of 13. The present study involving the coupled processes is simple but has its implication due to its potential to be exploited for designing a pH-sensitive molecular switch.

Deciphering the perturbation of serum albumins by a ketocyanine dye: a spectroscopic approach.

Steady state and time resolved fluorometric and circular dichroism (CD) techniques have been exploited to explore the binding interaction of a ketocyanine dye, namely, 2-[3-(N-methyl-N-phenylamino)-2-propenylidene] indanone (MPAPI) with transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA). The emission spectrum of buffered solution of the dye is found to be perturbed remarkably upon binding with the proteins. An explicit study with respect to modification of fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds with both BSA and HSA; the binding being stronger with the latter. Denaturation and CD studies reveal that stability of the proteins increases upon binding with the dye. The probable binding sites of the dye in the proteinous environments have been assessed from fluorescence resonance energy transfer (FRET) study. The probe is argued to be located in the inter domain cleft region of HSA (near Trp-214). From the similarity in the fluorescence behavior of the dye in BSA and HSA it is inferred that in BSA environment the probe is located near Trp-212 rather than Trp-132.

Photophysics and rotational relaxation dynamics of a beta-carboline based fluorophore in cationic alkyltrimethylammonium bromide micelles.

Photophysics and rotational relaxation dynamics of a beta-carboline analog, 3-acetyl-4-oxo-6,7-dihydro-12H-indolo-[2,3-a] quinolizine (AODIQ) have been investigated in cationic alkyltrimethylammonium bromide (nTAB) micelles using steady-state and time-resolved fluorometric techniques. The study reveals modification of its photophysics by the conjugate effect of polarity and rigidity of the micellar environments with varying alkyl chain lengths of the surfactants. Furthermore, it suggests that the fluorophore resides at the micelle-water interfacial domain. Contrary to the single exponential nature of the fluorescence anisotropy decay of AODIQ in aqueous medium, the decay is found to be biexponential in all the micellar environments studied. The enhancements in the steady-state anisotropy and rotational relaxation time in the micellar media compared to that in pure aqueous solution reflect that the fluorophore resides in a motionally restricted environment introduced by the cationic micelles. The rotational correlation time increases marginally with an increase in the surfactant chain length. The rotational relaxation of AODIQ in the micellar environments has been discussed in the light of the two-step and wobbling in a cone model. The model helps in evaluating different rotational parameters and in ascertaining the location of the fluorophore in the micellar media. This technique provides valuable information regarding the rotational relaxations of the fluorophore within an organized assembly. When the lifetime measurements and orientational relaxation measurements are combined, significant inferences can be made regarding the partitioning of the probe in different regions of the micelles.

Photophysics and rotational dynamics of a beta-carboline analogue in nonionic micelles: effect of variation of length of the headgroup and the tail of the surfactant.

Effect of variation of length of nonionic surfactants in terms of the headgroup as well as the tail part on the photophysical and rotational dynamical properties of a beta-carboline analogue, 3-acetyl-4-oxo-6,7-dihydro-12H-indolo-[2,3-a]quinolizine (AODIQ) has been investigated. Steady-state and time-resolved fluorescence and fluorescence anisotropy have been exploited for the purpose. The experiments revealed modification of the photophysics of AODIQ by the conjugate effect of polarity and rigidity of the micellar environments with varying poly(ethylene oxide) chain length in the case of Triton X series and the alkyl chain length in the case of Tween series surfactants. Fluorometric studies suggest that the fluorophore resides at the micelle-water interface in all these systems. The enhancements in the steady-state anisotropy in all the micellar media compared to those in pure aqueous solution reflect that the fluorophore is located in motionally restricted regions introduced by the nonionic micelles. Contrary to the single exponential nature of the fluorescence anisotropy decay of AODIQ in aqueous medium, they were found to be biexponential in the micellar environments. The rotational relaxation of AODIQ in the micellar environments has been discussed in light of the two-step and wobbling in a cone model. The model helps to evaluate different rotational parameters and to ascertain the location of the fluorophore in the micellar media. The significant feature is that the motional restriction decreases with an increase in the poly(ethylene oxide) chain length while it increases with an increase in the alkyl chain length. The difference in the extent of water penetration due to variation in the thickness of the palisade layer and therefore a variation in the micellar polarity with a variation of the length of poly(ethylene oxide) and alkyl chain has been argued to be responsible.