Microheterogeneity and Microviscosity of F127 Micelle: The Counter Effects of Urea and Temperature

F127 is the most widely studied triblock copolymer and due to the presence of very long polypropylene oxide (PPO) and polyethylene oxide (PEO) groups, F127 micelle has different microenvironments clearly separated into core, corona, and peripheral regions. Urea has been known to have adverse effects on the micellar properties and causes demicellization and solvation; on the other hand, rise in temperature causes micellization and solvent evacuation from the core and corona regions. In the present study, we have investigated the microheterogeneity of the core, corona, and peripheral regions of the F127 micelle using red edge excitation shift (REES) at different temperatures and urea concentrations and correlated the effect of both on the micellar system. It was found that the temperature counteracts the effect of urea and also that the counteraction is more prominent in the core region with respect to corona, and the peripheral region is least affected. Also, the core and corona regions are very much heterogeneous, while the peripheral region is more of a homogeneous nature. Using time-resolved fluorescence anisotropy, we found that the microviscosity within the micelles vary in the order of core > corona > peripheral region, and urea has a general tendency to reduce the microviscosity, especially for core and corona regions. On the other hand, rise in temperature initially increases and then decreases the microviscosity throughout, and at elevated temperatures the effect of urea is being dominated by the effect of temperature, thereby establishing the counter effects of temperature and urea on the F127 micellar system

Hydrophobicity Is the Governing Factor in the Interaction of Human Serum Albumin with Bile Salts

The present study demonstrates a detailed characterization of the interaction of a series of bile salts, sodium deoxycholate (NaDC), sodium cholate (NaC), and sodium taurocholate (NaTC), with a model transport protein, human serum albumin (HSA). Here, steady-state and time-resolved fluorescence spectroscopic techniques have been used to characterize the interaction of the bile salts with HSA. The binding isotherms constructed from steady-state fluorescence intensity measurements demonstrate that the interaction of the bile salts with HSA can be characterized by three distinct regions, which were also successfully reproduced from the significant variation of the emission wavelength (λ_em) of the intrinsic tryptophan (Trp) moiety of HSA. The time-resolved fluorescence decay behavior of the Trp residue of HSA was also found to corroborate the steady-state results. The effect of interaction with the bile salts on the native conformation of the protein has been explored in a circular dichroism (CD) study, which reveals a decrease in α-helicity of HSA induced by the bile salts. In accordance with this, the esterase activity of the protein–bile salt aggregates is found to be reduced in comparison to that of the native protein. Our results exclusively highlight the fact that it is the hydrophobic character of the bile salt that governs the extent of interaction with the protein. Isothermal titration calorimetry (ITC) and molecular docking studies further substantiate our other experimental findings

Kinetic Aspects of Enzyme-Mediated Evolution of Highly Luminescent Meta Silver Nanoclusters

The proteolysis of human serum albumin (HSA) templated Ag nanoclusters (NCs) induced by trypsin digestion reveals that photoluminescence (PL) intensity of the blue emitting Ag:HSA NCs exhibits a decremental behavior. The unique aspect of this study is the gradual evolution of a new red emission band characterized by an enhanced quantum yield (QY) of ∼28% and a lifetime ∼80 ns in contrast to the parent blue-emitting Ag:HSA NCs (QY = 16% and lifetime ∼7.4 ns). The decay and growth PL kinetics of Ag NCs upon trypsin digestion have been used to unravel the process of destabilization of the Ag:HSA NCs and the simultaneous formation of the meta AgTp NCs as evidenced from MALDI-TOF spectra. Our fluorescence correlation spectroscopy (FCS) data substantiate the evolution of larger sized Ag NCs due to enzymatic digestion which thereby results in a slower translational diffusion rate as a consequence of their increasing hydrodynamic diameters

Enhanced Binding of Phenosafranin to Triblock Copolymer F127 Induced by Sodium Dodecyl Sulfate: A Mixed Micellar System as an Efficient Drug Delivery Vehicle

In this study, we explored the interaction of a cationic phenazinium dye, phenosafranin (PSF, here used as a model drug), with pluronic block copolymer F127, both in the presence and in the absence of the anionic surfactant sodium dodecyl sulfate (SDS), which forms mixed micelles with F127. We applied both steady-state and time-resolved spectroscopic techniques, along with isothermal titration calorimetry (ITC), to demonstrate the binding of the probe PSF to both the pluronic and F127/SDS mixed micelles. Dynamic light scattering (DLS) study revealed that, upon interaction with SDS, the hydrodynamic diameter (<i>d</i>_H) of F127 micelles decreased due to the formation of the mixed micelles. The PSF penetrated to the more hydrophobic interior of the mixed micellar system as compared to F127 micelles alone. Micropolarity and fluorescence-quenching experiments revealed that PSF is more deeply seated in the case of F127/SDS mixed micelles. Through a partition coefficient, lifetime measurements, and time-resolved anisotropy experiments, we also established that the partitioning of the probe within the F127 micelles in the presence of SDS is almost double than that in its absence. ITC data corroborates the fact that the binding of PSF is the strongest and most thermodynamically favorable when mixed micelles are formed, which enables our system to serve as an excellent drug delivery vehicle when compared to F127 alone

Inverse Temperature Dependence in Static Quenching versus Calorimetric Exploration: Binding Interaction of Chloramphenicol to β‑Lactoglobulin

The binding interaction between the whey protein bovine β-lactoglobulin (βLG) with the well-known antibiotic chloramphenicol (Clp) is explored by monitoring the intrinsic fluorescence of βLG. Steady-state and time-resolved fluorescence spectral data reveal that quenching of βLG fluorescence proceeds through ground state complex formation, i.e., static quenching mechanism. However, the drug–protein binding constant is found to vary proportionately with temperature. This anomalous result is explained on the basis of the Arrhenius theory which states that the rate constant varies proportionally with temperature. Thermodynamic parameters like Δ<i>H</i>, Δ<i>S</i>, Δ<i>G</i>, and the stoichiometry for the binding interaction have been estimated by isothermal titration calorimetric (ITC) study. Thermodynamic data show that the binding phenomenon is mainly an entropy driven process suggesting the major role of hydrophobic interaction forces in the Clp−βLG binding. Constant pressure heat capacity change (Δ<i>C</i>_p) has been calculated from enthalpy of binding at different temperatures which reveals that hydrophobic interaction is the major operating force. The inverse temperature dependence in static quenching is however resolved from ITC data which show that the binding constant regularly decreases with increase in temperature. The modification of native protein conformation due to binding of drug has been monitored by circular dichroism (CD) spectroscopy. The probable binding location of Clp inside βLG is explored from AutoDock based blind docking simulation

Toggling Between Blue- and Red-Emitting Fluorescent Silver Nanoclusters

A very efficient protocol for synthesizing highly fluorescent, protein-templated silver nanoclusters (Ag/NCs) has been discussed. Two types of Ag/NCs (Ag₉/HSA and Ag₁₄/HSA), although showing significant differences in their photophysical properties, can be interconverted at will, which makes this study unique. The Ag/HSA NCs have been quantified by several spectroscopic techniques, and they find tremendous applications as photoluminescent markers. Besides their rather easy synthetic methodology, our Ag/HSA NCs show two-photon excitation properties that enable them to be used in bioimaging

Interplay of Multiple Interaction Forces: Binding of Norfloxacin to Human Serum Albumin

Herein, the binding interaction of a potential chemotherapeutic antibacterial drug norfloxacin (NOF) with a serum transport protein, human serum albumin (HSA), is investigated. The prototropic transformation of the drug (NOF) is found to be remarkably modified following interaction with the protein as manifested through significant modulations of the photophysics of the drug. The predominant zwitterionic form of NOF in aqueous buffer phase undergoes transformation to the cationic form within the protein-encapsulated state. This implies the possible role of electrostatic interaction force in NOF–HSA binding. This postulate is further substantiated from the effect of ionic strength on the interaction process. To this end, the detailed study of the thermodynamics of the drug–protein interaction process from isothermal titration calorimetric (ITC) experiments is found to unfold the signature of electrostatic as well as hydrophobic interaction forces underlying the binding process. Thus, interplay of more than one interaction forces is argued to be responsible for the overall drug–protein binding. The ITC results reveal an important finding in terms of enthalpy–entropy compensation (EEC) characterizing the NOF–HSA binding. The effect of drug-binding on the native protein conformation has also been evaluated from circular dichroism (CD) spectroscopy which unveils partial rupture of the protein secondary structure. In conjunction to this, the functionality of the native protein (in terms of esterase-like activity) is found to be lowered as a result of binding with NOF. The AutoDock-based docking simulation unravels the probable binding location of NOF within the hydrophilic subdomain IA of HSA. The present program also focuses on exploring the dynamical aspects of the drug–protein interaction scenario. The rotational-relaxation dynamics of the protein-bound drug reveals the not-so-common “dip-and-rise” pattern

Interaction of Bile Salts with β‑Cyclodextrins Reveals Nonclassical Hydrophobic Effect and Enthalpy–Entropy Compensation

Herein, we present an endeavor toward exploring the lacuna underlying the host:guest chemistry of inclusion complex formation between bile salt(s) and β-cyclodextrin(s) (βCDs). An extensive thermodynamic investigation based on isothermal titration calorimetry (ITC) demonstrates a dominant contribution from exothermic enthalpy change (Δ<i>H</i> < 0) accompanying the phenomenon of inclusion complex formation, along with a relatively smaller contribution to total free energy change from the entropic component. However, the negative heat capacity change (Δ<i>C</i>_p < 0) displays the hallmark for a pivotal role of hydrophobic effect underlying the interaction. Contrary to the classical hydrophobic effect, such apparently paradoxical thermodynamic signature has been adequately described under the notion of “nonclassical hydrophobic effect”. On the basis of our results, the displacement of disordered water from hydrophobic binding sites has been argued to mark the enthalpic signature and the key role of such interaction forces is further corroborated from enthalpy–entropy compensation behavior showing indication for almost complete compensation. To this end, we have quantified the interaction of two bile salt molecules (namely, sodium deoxycholate and sodium glycocholate) with a series of varying chemical substituents on the host counterpart, namely, βCD, (2-hydroxypropyl)-βCD, and methyl βCD

Binding Interaction of a Prospective Chemotherapeutic Antibacterial Drug with β‑Lactoglobulin: Results and Challenges

This Article reports a detailed characterization of the binding interaction of a potential chemotherapeutic antibacterial drug, norfloxacin (NOF), with the mammalian milk protein β-lactoglobulin (βLG). The thermodynamic parameters, Δ<i>H</i>, Δ<i>S</i>, and Δ<i>G</i>, for the binding phenomenon as-evaluated on the basis of van’t Hoff relationship reveal the predominance of electrostatic/ionic interactions underlying the binding process. However, the drug-induced quenching of the intrinsic tryptophanyl fluorescence of the protein exhibits intriguing characteristics on Stern–Volmer analysis (displays an upward curvature instead of conforming to a linear regression). Thus, an extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to unveil the actual quenching mechanism. The invariance of the fluorescence decay behavior of βLG as a function of the quencher (here NOF) concentration coupled with the commensurate dependence of the drug–protein binding constant (<i>K</i>) on temperature, the drug-induced fluorescence quenching of βLG is argued to proceed through static mechanism. This postulate is aided further support from absorption, fluorescence, and circular dichroism (CD) spectral studies. The present study also throws light on the important issue of drug-induced modification in the native protein conformation on the lexicon of CD, excitation–emission matrix spectroscopic techniques. Concurrently, the drug–protein interaction kinetics and the energy of activation of the process are also explored from stopped-flow fluorescence technique. The probable binding locus of NOF in βLG is investigated from AutoDock-based blind docking simulation

Photostable Copper Nanoclusters: Compatible Förster Resonance Energy-Transfer Assays and a Nanothermometer

To address the concern of material chemists over the issue of stability and photoluminescent (PL) characteristics of Cu nanoclusters (NCs), herein we present an efficient protocol discussing PL Cu NCs (Cu/HSA) having blue emission and high photostability. These PL NCs were illustrated as efficient probes for Förster resonance energy transfer (FRET) with a compatible fluorophore (Coumarin 153). Our spectroscopic results were well complemented by our molecular docking calculations, which also favored our proposed mechanism for Cu NC formation. The beneficial aspect and uniqueness of these nontoxic Cu/HSA NCs highlights their temperature-dependent PL reversibility as well as the reversible FRET with Coumarin 153, which enables them to be used as a nanothermometer and a PL marker for sensitive biological samples