Theranostic approach to specifically targeting the interloop region of BCL2 i-motif DNA by crystal violet.

Ligands that recognise specific i-motif DNAs are helpful in cancer diagnostics and therapeutics, as i-motif formation can cause cancer. Although the loop regions of i-motifs are promising targets for ligands, the interaction between a ligand and the loop regions based on sequence information remains unexplored. Herein, we investigated the loop regions of various i-motif DNAs to determine whether these regions specifically interact with fluorescent ligands. Crystal violet (CV), a triphenylmethane dye, exhibited strong fluorescence with the i-motif derived from the promoter region of the human BCL2 gene in a sequence- and structure-specific manner. Our systematic sequence analysis indicated that CV was bound to the site formed by the first and third loops through inter-loop interactions between the guanine bases present in these loops. As the structural stability of the BCL2 i-motif was unaffected by CV, the local stabilisation of the loops by CV could inhibit the interaction of transcription factors with these loops, repressing the BCL2 expression of MCF-7 cells. Our finding suggests that the loops of the i-motif can act as a novel platform for the specific binding of small molecules; thus, they could be utilised for the theranostics of diseases associated with i-motif DNAs.

Exploration of Self-Aggregation of Coumarin 7 and Coumarin 30 in Water: Role of ß-Cyclodextrin as a Modulator.

Steady-state and time-resolved spectroscopic studies demonstrate that two members of the coumarin class of dyes, coumarin 7 (C7) and coumarin 30 (C30), undergo self-aggregation in water. The development of hypsochromically shifted new absorption bands in addition to the existing monomer bands with an increase in concentration of the dyes in an aqueous medium suggests that the aggregates are of H-type. An absorption-based kinetic study reveals that the rate of aggregation of C30 is an order of magnitude faster than that of C7. Second-order rate kinetics, as obtained from the half-life (t1/2) data, implies that the aggregates are dimeric in nature. Observations of isosbestic points in area-normalized absorption spectra (ANAS) and isoemissive points in area-normalized fluorescence excitation spectra (ANFES) and time-resolved area-normalized emission spectra (TRANES) establish that ground-state monomer ⇌ dimer equilibria for both of the systems are preserved in the photoexcited state. The present study further establishes that ß-cyclodextrin is the most efficient of the three common cyclodextrins in shifting the equilibria toward the monomer by encapsulating the monomers within its cavity, making ß-CD a convenient modulator to control the self-aggregation process. Dynamic light scattering (DLS), quantum chemical calculations, and molecular docking studies provide further support to our propositions.

Hydroxyl Group-Directed Solvation of Excited-State Intramolecular Proton Transfer Probes in Water: A Demonstration from the Fluorescence Anisotropy of Hydroxyflavones.

Formation of a probe-solvent network resulting in unusually high fluorescence anisotropy (FA) of an excited-state intramolecular proton transfer (ESIPT) probe, 3-hydroxyflavone (3HF), in water prompted us to explore the solvation patterns on its 7-hydroxy (7HF) and 6-hydroxy (6HF) positional analogues. In the present study, it was observed that 7HF exhibits a lower FA than 3HF does in water, implying that the volume of the 7HF-water cluster is less than that of the 3HF-water cluster. Experimental and computational results led us to propose that 7HF forms its water cluster at the molecular periphery in contrast to the projected-out structure in case of the 3HF-water cluster. Density functional theory (DFT)-based quantum chemical calculations provide an approach for the differential solvation patterns of 3HF and 7HF. 6HF, a non-ESIPT probe, exhibits very low FA in water compared with both 3HF and 7HF. This study demonstrates that proper positioning of the hydroxyl group and its participation in the extended π-conjugation within the molecule dictate the formation of the solvated cluster endorsing directed solvation.

Switching from endogenous to exogenous delivery of a model drug to DNA through micellar engineering.

A potential strategy has been demonstrated, for the first time, for switching the mode of delivery of drugs or small molecular systems from endogenous to exogenous, simply by engineering the chain length of micellar carriers. Ethidium bromide (EB) is exploited as the model drug which has been successfully delivered to natural DNA through endogenous and exogenous modes by tuning the chain length of anionic sodium n-alkyl sulfate micelles. ß-cyclodextrin (ß-CD) is exploited as an extrinsic stimulant for the exogenous delivery of EB to DNA. Multi-spectroscopic techniques involving absorption, emission, fluorescence anisotropy, fluorescence decay analysis, circular dichroism, DNA helix melting etc. have established tuning of the delivery mode between endogenous and exogenous. Differential binding affinity of the model drug with different micelles of varying chain length relative to that with DNA is capitalized to make the switching feasible. Although endogenous mode avoids external stimulant and associated problems, a regulation of the stimulant concentration makes the other mode controllable and quantitative. With appropriate choice of carrier micelle and modulation of this developed strategy can radically change the therapeutic research enabling one to take a control over the drug delivery mode to exploit the advantage of one or the other selectively, whenever required.

Origin of Unusually High Fluorescence Anisotropy of 3-Hydroxyflavone in Water: Formation of Probe-Solvent Cage-like Cluster.

Based on the unusually high fluorescence anisotropy (FA) of 3-hydroxyflavone (3HF) in water medium in contrast to the very low FA of its methoxy counterpart (3MF), our proposition invoked formation of an intermolecular hydrogen-bonded cage-like probe-solvent cluster of 3HF in water. In the present work, ab-initio DFT-based quantum chemical calculations have been exploited to provide a foundation for our interpretation. Ground-state optimization of 3HF with varying numbers of water molecules leads to the formation of a cage-like or loop-like probe-water cluster. Our calculations reveal that the structures with four to five water molecules are stabilized to the maximum extent. Classical molecular dynamics simulations reveal that the rotational dynamics of 3HF is much slower in water compared to that in alkane medium, which also goes in favor of the probe-solvent cluster formation in water medium. Apart from the theoretical studies, an indirect experimental approach has been adopted to substantiate formation of the probe-water cluster. The atypical observation of reduced FA of 3HF entrapped in micelles relative to that of the fluorophore in water implies disruption of the probe-water cluster with the addition of micelles, corroborating our original proposition of formation of an intermolecularly hydrogen-bonded 3HF-water cluster.

Aggregation of Nile Red in Water: Prevention through Encapsulation in ß-Cyclodextrin.

The present work, based on various spectroscopic investigations, vividly demonstrates the self-association of Nile red (NR) in aqueous medium. The rapid decrease in the absorbance as well as emission of NR in water bears the signature of the aggregation process. Appearance of a new blue-shifted absorption band in addition to the original one and a drastic decrease in the emission intensity imply that the aggregation is of H-type. Poor solubility of NR in water, hydrophobic interaction, and the planar structure of the dye are ascribed to favor the formation of the aggregate in the aqueous medium. Absorption-based kinetic studies reveal the aggregation process to be second order, thereby establishing the aggregate to be a dimer. Similar kinetic profiles of the absorbance of NR in the presence and absence of light confirm that the aggregation process is not photoassisted. The presence of an isosbestic point in the absorbance spectra and an isoemissive point in the time-resolved area normalized emission spectra bears the evidence of equilibrium between the dimeric and the monomeric species of NR in the ground state as well as in the photoexcited state. Encapsulation of the monomer of NR within the hydrophobic cavity of ß-cyclodextrin is demonstrated to prevent the aggregation process.

Managing efficacy and toxicity of drugs: Targeted delivery and excretion.

Medicine is a natural companion of mankind in the present era for mere survival from the deadly diseases in ever-increasing polluted environments. Hence, in recent years, major focus of pharmaceutical, medicinal and biophysical research has been navigated in exploring and developing new and simple avenues to enhance the efficacy of the administered drugs on one hand and to get rid of, or at least reduce, the toxic side effects of the excess drugs accumulated in human body on the other. A potential approach to amplify the efficacy of the administered drug is to develop proficient targeted drug delivery systems (DDSs). This review provides an essence of some newly developed simple but prospective strategies on enhancing the efficacy of drugs/bioactive molecules exploiting various drug delivery systems like micelles, cyclodextrins, liposomes etc. to serve the purpose of targeted delivery towards DNA, by endogenous and/or exogenous means. Improved bio-availability and solubilization of ionic drugs within the less polar target regions from the bulk aqueous phase has also been achieved through the introduction of some physiologically permissible salts. In the other context, in vitro and in vivo studies demonstrate a simple technique for easy removal of the excess adsorbed drug molecules from the cell membranes/lipid bilayers by exploiting health-amiable supramolecular assemblies. In this review, we summarize the recent experimental findings, mostly from our lab, encompassing the development of simple biocompatible methods to enhance the benevolent role of drugs through their safe, effective and convenient administration. It also presents easy and effective means to remove the excess adsorbed drugs from human body to diminish their malign effects. These prospective approaches of drug delivery and excretion of drug molecules have promising roles to play from both physicochemical and pharmaceutical perspectives, ensuring enhanced bioavailability of drugs as well as disposing of drug-induced adverse side effects.

Endogenous Activation-Induced Delivery of a Bioactive Photosensitizer from a Micellar Carrier to Natural DNA.

The photophysics of phenosafranin (PSF), a member of the photosensitizer phenazinium family, has been explored in nonionic Triton X-165 (TX-165) micelle, calf thymus DNA (ctDNA), and a composite environment consisting of both micelle and DNA, using diverse spectroscopic techniques of both steady-state and time-resolved natures, to divulge the binding interactions of the probe with different hosts. The vivid experimental results demonstrate that PSF binds with both micelle and DNA; however, the binding affinity of the probe is much higher toward the DNA. When micelle-carried PSF comes in proximity to ctDNA, PSF gets released from the micellar environment and intercalates with the DNA base pairs. Endogenous activation, in terms of a higher binding affinity of PSF toward ctDNA relative to that toward the micelle, is ascribed to be responsible for this transfer. Thus, this article demonstrates endogenous transfer of a bioactive molecular probe from a micellar nanocarrier to natural DNA. As the carrier micelle (TX-165) does not perturb the structure of the DNA, this work proposes that it can be used promisingly for the purpose of safe delivery of drugs. The study is expected to stimulate the generation of and/or search for advanced micelle-based carrier systems for delivery of bioactive molecular systems to biological targets like DNA.

Cyclodextrin induced controlled delivery of a biological photosensitizer from a nanocarrier to DNA.

In this article, we have addressed to a demanding physicochemical aspect of therapeutic and drug research. We have reported a simple yet prospective technique that can be exploited for the controlled delivery of drugs and/or bioactive small molecules to the most relevant biomolecular target DNA. Exploiting various steady state and time resolved spectroscopic techniques together with the DNA helix melting study, we have shown that a biologically significant photosensitizer, namely, phenosafranin (PSF), can be quantitatively transferred to the DNA from the micellar nanocarrier made up of sodium tetradecyl sulfate (STS) using the external stimulant ß-cyclodextrin (ß-CD). The complexation property of ß-CD with the nanocarrier (STS) has been utilized for the controlled release of the probe from the micelle to the DNA. Non-toxicity of the stimulant and the noninvasive nature of the carrier towards the target are expected to add to the suitability of this approach from a clinical perspective.