Insight into the Effect of Submicellar Concentrations of Sodium Deoxycholate on the Structure, Stability, and Activity of Bovine and Human Serum Albumin: An Interesting Comparison between Single and Double Tryptophan Proteins.

The progressive escalation in the applications of bile salts in diverse fields has triggered research on their interaction with various biological macromolecules, especially with proteins. A proper understanding of the interaction process of bile salts, particularly in the lower concentrations range, with the serum albumin seems important since the normal serum concentration of bile salts is approximately in the micromolar range. The current study deals with a comprehensive and comparative analysis of the interaction of submicellar concentrations of sodium deoxycholate (NaDC) with two homologous transport proteins: bovine serum albumin (BSA) and human serum albumin (HSA). HSA and BSA with one and two tryptophans, respectively, provide the opportunity for an interesting comparison of tryptophan fluorescence behavior on interaction with NaDC. The study suggests a sequential interaction of NaDC in three discrete stages with the two proteins. A detailed study using warfarin and ibuprofen as site markers provides information about the sites of interaction, which is further confirmed by inclusive molecular dynamics simulation analysis. Moreover, the comparison of the thermodynamics and stability of the NaDC-serum albumin complexes confirms the stronger interaction of NaDC with BSA as compared to that with HSA. The differential interaction between the bile salt and the two serum albumins is further established from the difference in the extent of decrease in the esterase-like activity assay of the proteins in the presence of NaDC. Therefore, the present study provides important insight into the effect of submicellar concentrations of NaDC on the structure, stability, and activity of the two homologous serum albumins and thus can contribute not only to the general understanding of the complex nature of serum albumin-bile salt interactions but also to the design of more effective pharmaceutical formulations in the field of drug delivery and biomedical research.

Exploring Steroidal Surfactants as Potential Drug Carriers for an Anticancer Drug Curcumin: An Insight into the Effect of Surfactants' Structure on the Photophysical Properties, Stability, and Activity of Curcumin.

Despite having tremendous medicinal benefits, the practical applications of curcumin are limited, owing to two major challenges: poor aqueous solubility and lack of bioavailability. In this regard, biosurfactant-based micellar systems have surged recently for the development of novel and more effective formulations because of their biological relevance. This study deals with a comprehensive and comparative investigation on the effect of seven structurally different steroidal surfactants on the photophysical properties of curcumin and also evaluates these steroidal surfactants as possible drug delivery media for curcumin. The photophysical properties of curcumin exhibited a strong dependence on the structure of the steroidal surfactant; the extent of excited-state proton transfer between curcumin and the surfactants depends strongly on the type of the side chain in the surfactants, which mostly dictates the photophysics of curcumin in the presence of these structural variants. The solubility of curcumin and its stability at different pHs and temperatures and in the presence of salt are significantly enhanced in the presence of these surfactants. Furthermore, the curcumin-loaded micelles exhibited improved intracellular uptake and cytotoxicity against MCF-7 cancer cells than pristine curcumin. Among these steroidal surfactants, CHAPS, the zwitterionic derivative of cholic acid, was the most efficient one to offer better solubility and stability to curcumin under all conditions, and the death rate of MCF-7 cells by curcumin was found to be the highest in the presence of CHAPS, indicating the enhanced bioavailability of curcumin. Therefore, CHAPS-based colloids are found to be promising candidates as potential drug carriers for curcumin.

Photophysical Properties of Coumarin 1 in Bile Salt Aggregates: An Insight into the Role of Bile Salt Structure on the Aggregation Behavior.

The photophysical behavior of Coumarin 1 (Cou1), a well-known 7-aminocoumarin derivative, is very sensitive to the microenvironment in which it resides. In the present study, the effect of six bile salt variants on the photophysical behavior of Cou1 has been investigated. Dihydroxy (deoxycholates) as well as trihydroxy (cholates) bile salts with conjugated and unconjugated side chains have been chosen to get insight into the role of bile salt structure on the microenvironment of Cou1. Cou1 photophysics was found to be extremely sensitive to the aggregation process of the bile salt variants. The reduced polarity of the micellar environment stabilizes the planar intramolecular charge transferred state of Cou1, resulting in significant modulation in its photophysics in the bile salt media. The changes in the fluorescence parameters such as fluorescence intensity, emission energy, fluorescence quantum yield, anisotropy, and lifetime of Cou1 reveal that there is a distinct difference in the aggregation behavior of deoxycholates from that of cholates. The deoxycholates form micelles more or less critically similar to those of conventional surfactants, whereas the cholates self-assemble rather noncritically over a wide concentration range, thus signifying the vital role of the extra hydroxyl group in the aggregation pattern of trihydroxy bile salts. The conjugated bile salts are found to provide a relatively more compact, rigid, and hydrophobic microenvironment to Cou1 as compared to their unconjugated counterparts. Considering the significant modulation in the photophysical properties of Cou1, it has been employed as a molecular reporter for monitoring the aggregation process of bile salt variants and important information could be obtained about the effect of bile salt structure on the aggregation pattern and also about the micellar properties.

Controlled delivery of ibuprofen from poly(vinyl alcohol)-poly(ethylene glycol) interpenetrating polymeric network hydrogels.

Hydrogels composed of poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) were synthesized using glutaraldehyde as crosslinker and investigated for controlled delivery of the common anti-inflammatory drug, ibuprofen (IBF). To regulate the drug delivery, solid inclusion complexes (ICs) of IBF in ß-cyclodextrin (ß-CD) were prepared and added to the hydrogels. The ICs were prepared by the microwave irradiation method, which is more environmentally benign. The formation of IC was confirmed by various analytical techniques and the synthesized hydrogels were also characterized. Controlled release of drug was achieved from the hydrogels containing the ICs in comparison to the rapid release from hydrogels containing free IBF. The preliminary kinetic analysis emphasized the crucial role of ß-CD in the drug release process that influences the polymer relaxation, thereby leading to prolonged release. The cytotoxicity assay validated the hydrogels as non-toxic in nature and hence can be utilized for controlled delivery of IBF.

Controlled delivery of ibuprofen from poly(vinyl alcohol)-poly (ethylene glycol) interpenetrating polymeric network hydrogels / 药物分析学报

Hydrogels composed of poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) were synthesized using glutaraldehyde as crosslinker and investigated for controlled delivery of the common anti-inflammatory drug, ibuprofen (IBF). To regulate the drug delivery, solid inclusion complexes (ICs) of IBF in β–cyclodextrin (β–CD) were prepared and added to the hydrogels. The ICs were prepared by the microwave irradiation method, which is more environmentally benign. The formation of IC was confirmed by various analytical techniques and the synthesized hydrogels were also characterized. Controlled release of drug was achieved from the hydrogels containing the ICs in comparison to the rapid release from hydrogels containing free IBF. The preliminary kinetic analysis emphasized the crucial role of β–CD in the drug release process that in-fluences the polymer relaxation, thereby leading to prolonged release. The cytotoxicity assay validated the hydrogels as non-toxic in nature and hence can be utilized for controlled delivery of IBF.

Spectroscopic and computational insights into theophylline/ß-cyclodextrin complexation: inclusion accomplished by diverse methods.

Current scenario in asthmatic prevalence worldwide calls for a facile, cost-effective, and energy efficient methodology to formulate the potent bronchodilator, theophylline (THP), into an effective dosage forms. Since the uses of THP are severely impeded by its poor aqueous solubility and low bioavailability, solid inclusion complexes (ICs) of THP in ß-cyclodextrin (ß-CD) were prepared to overcome the limitations. The ICs were developed by conventional methods and also by microwave irradiation method, which is environmentally more benign and requires lesser reaction time. The complexation phenomenon was effectual by the co-precipitation, freeze-drying, and microwave methods as affirmed from various spectroscopic analyses. 1H NMR and molecular docking studies illustrated the total inclusion of THP into ß-CD cavity. Better efficacy of the microwaved product was witnessed in terms of drug content, dissolution, and anti-biofilm activities. Thus microwave irradiation can be utilised as a naive and economical methodology to design ß-CD-THP dosage formulations.

pH-Responsive guar gum hydrogels for controlled delivery of dexamethasone to the intestine.

pH-Responsive hydrogel systems based on guar gum (GG), poly(acrylic acid) (PAA) and ß-cyclodextrin (CD) using a non-toxic crosslinker, tetraethyl orthosilicate for intestinal delivery of dexamethasone (DX) has been reported. Hydrogels of different compositions were synthesized and evaluated for the controlled delivery of DX. The hydrogels exhibited pH-responsive swelling behavior with a maximum swelling around neutral pH. The CD-containing hydrogels deliver the drug at much slower rate in contrast to the ones without CD. Moreover, the drug release rate is found to show a strong dependence on the GG content and the effect of GG is more pronounced in case of the CD containing hydrogels. As the GG content increases, the rate of drug release decreases considerably and the drug release is prolonged. The release studies in the simulated conditions reveal that these hydrogels can be used as delivery vehicles for oral administration of DX targeting the intestine. Cell viability studies reveal the hydrogels to be biocompatible in nature thus validating them as good drug delivery systems.

Spectroscopic investigation of interaction of Nile Blue A, a potent photosensitizer, with bile salts in aqueous medium.

Nile Blue A (NB) is one of the most studied benzophenoxazine dyes, as a potent photosensitizer for photodynamic therapy. The dye when administered intravenously disperses throughout the body by circulating through blood and is taken up by most cells that emphasize its interaction with various biomolecules. Therefore a rational understanding of the interaction of NB with relevant biological and biomimicking systems appears important. The focus of the present work is to investigate the interaction of NB with two bile salts sodium deoxycholate (NaDC) and sodium cholate (NaC) by spectroscopic techniques. The bile salts, in their premicellar concentration range, induce NB dimerization. Both H- and J-dimers are formed, however a major contribution is from the H-dimers. The extent of NB dimerization in NaDC, the dihydroxy bile salt, is higher than that in NaC, the trihydroxy bile salt. The bile salts when present above their micellar concentrations solubilize NB in its monomeric form. NB exhibits stronger binding and partitioning efficiency toward NaDC than NaC micelles at a given temperature. Binding and partitioning of NB to these micelles are spontaneous and exothermic in nature and these are enthalpy-driven processes. The spectral profiles and thermodynamic parameters of NB point toward the dissimilar nature of its environment in the micelles formed by the above two bile salts.

Disaggregation induced solvatochromic switch: a study of dansylated polyglycerol dendrons in binary solvent mixture.

A reversal in solvatochromic behaviour was observed in second and third generation glycerol based dansylated polyether dendrons in water on addition of a second solvent like methanol or acetonitrile. Below a certain percentage of the nonaqueous solvent there is a negative-solvatochromism observed and above that there is a switch to positive-solvatochromism. The negative-solvatochromism is attributed to the progressive disaggregation of the dendron aggregates by the nonaqueous solvent component. Once the disaggregation process is complete, positive-solvatochromism is exhibited by the dendron monomers. Higher the hydrophobicity of the dendron more is the amount of the second solvent required for disaggregation.

Structure of the 2-aminopurine-cytosine base pair formed in the polymerase active site of the RB69 Y567A-DNA polymerase.

The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased "breathing" at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.

Photophysical behaviour of ground state anion and phototautomer of 3-hydroxyflavone in liposome membrane.

A detailed account of the photophysical behaviour of the phototautomer (PT) and the ground state anion (A-) of 3-hydroxyflavone in liposome membrane at various membrane conditions is presented. A quenching study with a hydrophilic quencher Ag+ suggests that the phototautomeric emission generates from the fraction of 3HF that is located at the inner hydrophobic core, whereas the ground state anionic emission is from the fraction that resides near the water-accessible surface site. However, the biexponential nature of fluorescence decays of both the forms indicates that there is local heterogeneity in the distribution. Temperature dependence studies and experiments in the presence of ethanol reveal that, as the membrane becomes more fluid, redistribution of 3HF takes place between the two sites leading to increase in A- population. The temperature dependence of the fluorescence anisotropy change of PT shows good correlation with the phase change and shows a sharp drop at the transition temperature, whereas the corresponding change in the case of A- is gradual.

Use of 2-aminopurine fluorescence to study the role of the beta hairpin in the proofreading pathway catalyzed by the phage T4 and RB69 DNA polymerases.

For DNA polymerases to proofread a misincorporated nucleotide, the terminal 3-4 nucleotides of the primer strand must be separated from the template strand before being bound in the exonuclease active center. Genetic and biochemical studies of the bacteriophage T4 DNA polymerase revealed that a prominent beta-hairpin structure in the exonuclease domain is needed to efficiently form the strand-separated exonuclease complexes. We present here further mutational analysis of the loop region of the T4 DNA polymerase beta-hairpin structure, which provides additional evidence that residues in the loop, namely, Y254 and G255, are important for DNA replication fidelity. The mechanism of strand separation was probed in in vitro reactions using the fluorescence of the base analogue 2-aminopurine (2AP) and mutant RB69 DNA polymerases that have modifications to the beta hairpin, to the exonuclease active site, or to both. We propose from these studies that the beta hairpin in the exonuclease domain of the T4 and RB69 DNA polymerases functions to facilitate strand separation, but residues in the exonuclease active center are required to capture the 3' end of the primer strand following strand separation.

Photophysical properties of Newkome-type dendrimers in aqueous medium.

Newkome-type first, second and third generation dendrimers, having t-butyl (GB), ethyl (GE) and carboxylic (GA) end groups, were synthesized. A pyrene group, which can act as fluorescent sensor, was attached to the core of the dendrimers and their photophysical properties in aqueous solution were studied. These dendrimers were found to aggregate in aqueous solution, which manifested as an excimer peak in the pyrene emission spectra for the first and second generation dendrimers with ethyl and t-butyl end groups. The excimer peak however was not seen in case of the third generation dendrimer. Dendrimers with carboxylic end groups, did not show the excimer peak in water, which implies the hydrophobic nature of the aggregation. It is observed that the intensity of the excimer peak decreases with the increase in the size of the dendrimer. Lifetime studies carried out on the first and second generation dendrimers showed the formation of excimer species as a risetime in the decay curve. The aggregation of the third generation dendrimer was proposed from the quenching studies using silver ions and CCl(4) as quenchers.

Micellization of bile salts in aqueous medium: a fluorescence study.

Owing to the physiological importance of the micellization process of bile salts, the critical micelle concentration (CMC) becomes a fundamental parameter in the evaluation of their biological activities. The present study suggests fluorescence probing, using 1,6-diphenylhexatriene (DPH), as a simple, convenient, sensitive and economic method for monitoring the micellization process of bile salts in aqueous medium. Three independent parameters: fluorescence intensity, anisotropy and lifetime of DPH have been employed successfully for determining the CMC of two bile salts, sodium deoxycholate (NaDC) and sodium cholate (NaC), in aqueous medium. The CMC values reported by all the above three parameters of DPH are found to be same and it is 16 mM for NaC and 6 mM for NaDC at 25 degrees C in unbuffered solution. The effect of temperature and ionic strength on the micellization process has also been investigated employing DPH as a fluorescent probe. Increasing temperature leads to the formation of fluffier micelles with less rigid interior for both NaC and NaDC. The micelle core of NaC is less perturbed by the presence of NaCl whereas in case of NaDC, the aggregates provide DPH a more nonpolar and rigid environment in presence of NaCl than that in absence of salt.

Photophysical behaviour of 1-(4-N,N-dimethylaminophenylethynyl)pyrene (DMAPEPy) in homogeneous media.

The photophysical behaviour of a new pyrene derivative, 1-(4-N,N-dimethylaminophenylethynyl)pyrene (DMAPEPy), in various solvents has been studied. Due to the presence of an ethynyl link with a cylindrical pi cloud between the donor (N,N-dimethyl group) and the acceptor (pyrene), the molecule shows efficient intramolecular charge transfer, with a high extinction coefficient in all the solvents. There is significant solvatochromism in the fluorescence with a large increase in the Stokes' shift of around 125 nm between n-hexane and acetonitrile. The solvent-dependent spectral data show a good correlation with the Kamlet-Taft solvent polarity parameter (pi*). The plots of Stokes' shifts with E(T)(30) are linear for non-protic solvents and for protic solvents but with different slopes. The fluorescence quantum yields are high for non-polar solvents and decrease as the solvent polarity increases. Unlike the parent molecule pyrene, DMAPEPy shows a short lifetime, which is fairly insensitive to oxygen-induced quenching and is dependent on solvent polarity. The molecule shows high steady-state fluorescence anisotropy, which is very sensitive to the viscosity change of the medium.

Prototropism of 1-hydroxypyrene in liposome suspensions: implications towards fluorescence probing of lipid bilayers in alkaline medium.

The partitioning efficiency of neutral and anionic prototropic forms of 1-hydroxypyrene in liposome suspensions has been studied. The high partition coefficient value of 1-hydroxypyrene indicates an easy incorporation of the molecule into the lipid bilayer. Detailed pH studies indicate that only the neutral form of 1-hydroxypyrene partitions into the membrane and appreciable spectral changes are observed in the pH range of 9.0-11.5 in Tris-NaOH buffer. However, at pH 11 the spectral changes are maximum. The possibility of using 1-hydroxypyrene as a fluorescent molecular probe for lipid bilayer membranes in alkaline media has been examined, by employing fluorescence intensity and fluorescence anisotropy as probe parameters. The neutral form fluorescence intensity as well as fluorescence anisotropy is sensitive to the changes in the membrane properties and is capable of sensing the phase-transition. This is also capable of monitoring the changes in the membrane due to incorporation of cholesterol and the ethanol-induced interdigitation. The time resolved fluorescence data and the quenching experiments show that 1-hydroxypyrene occupies the water inaccessible interior of the liposome. The high anisotropy value of 1-hydroxypyrene in liposome suggests that it resides in a considerably rigid environment and is very sensitive to the temperature-induced changes in the liposome.