Micellar Water Pool Research Articles

Prototropism and dynamics of an anticancer drug in reverse micelles: Focus on the variation of pH in reverse micelles having w0 ≥ 10

The modulations of the photophysics and dynamics of an archetypal anticancer β-carboline drug, namely, harmane (HM) within AOT reverse micelles (RMs) have been explored. To this end, particular focus is given on a pH-dependent study commensurate with the effect of increasing water content within the RM interior. Our results reveal markedly different photophysical behavior of the drug encapsulated within the RM compared to that in bulk aqueous medium. For example, our results show the presence (or absence) of the cationic (or neutral) emission of HM in RMs in pH values apparently much higher (or lower) than that of pure aqueous medium. The characteristic zwitterionic emission in strongly alkaline bulk aqueous solutions (e.g., pH12.2 and 13.5) is also found to be lacking within the reverse micellar core, even in RMs with w0≥10. These data are further substantiated from the study of the dynamical aspects of the interaction scenario, that is, modulation of the fluorescence decay and rotational relaxation behaviors of HM entrapped within the RMs. Cumulatively, our results indicate the presence of a proton gradient across the reverse micellar water pool in which the interfacial regime appears to be more acidic in comparison to the central core. The results with alkaline solutions (e.g., pH10.0, 12.2 and 13.5) suggest selective compartmentalization of the hydroxide ions leaving the effective pH of the water pool lower than that of the bulk aqueous solution.

Modulation of anionic reverse micellar interface with non-ionic surfactants can regulate enzyme activity within the micellar waterpool

In this contribution, we have studied the effect of replacing anionic surfactant 1,4-bis(2-ethylhexyl)sulphosuccinate (AOT) by nonionic surfactant(s) of varying hydrophilic-lipophilic balance (HLB) on the structure, dynamics and activity of water encapsulated in reverse micelles (RMs) formed in a biocompatible oil isopropyl myristate (IPM). The sizes of the RM droplets are measured using dynamic light scattering (DLS) technique, while the structure of water in the RM waterpool is determined using FTIR spectroscopy. The solvation dynamics in these systems is obtained by time-resolved fluorescence spectroscopy using ammonium 8-anilino-1-naphthalenesulfonate (ANS) as the fluorophore. We spectroscopically measure the enzymatic activity of α-chymotrypsin on the substrate Ala-Ala-Phe-7-amido-4-methyl-coumarin (AMC) in these mixed RM systems. It has been found that the rate could either be enhanced or reduced depending on the HLB of the added Brij(s). Such result could be found useful in modulating the reaction for specific applications.

Nature of CTAB/Water/Chloroform Reverse Micelles at Above- and Subzero Temperatures Studied by NMR and Molecular Dynamics Simulations.

The nature and stability of cetyltrimethylammonium bromide (CTAB) reverse micelles in chloroform formed above the critical micellar concentration at above- and subzero temperatures were examined by NMR and molecular dynamics simulations. The experiments showed that the supercooled micellar water pool becomes unstable upon cooling to relatively high temperatures (253 K), and smaller micelles are formed. Upon freezing at lower temperatures (233 K), micelles become completely frozen and remain intact in the solution. With an average hydrodynamic radius of approximately 1.3 nm, we estimate that the water pool contains approximately 50 water molecules, which is well below the onset of ice crystal formation. To support the experimental results, molecular dynamics simulations were used to model the structure of CTAB/water/chloroform reverse micelles of different sizes. The MD simulations show that the reverse micelles contain a water pool with bromide anions residing on its surface and their shape is nonspherical, especially in the case of larger water pools. Upon fast freezing, the mobility of the water molecules is suppressed, and the pool becomes more spherical.

Solvent dynamics in a reverse micellar water-pool: a spectroscopic investigation of DDAB–cyclohexane–water systems

We have measured the hydrogen bonded structure and sub-ns relaxation dynamics of water molecules encapsulated in the DDAB-cyclohexane (Cy)-water reverse micellar (RM) water-pool dependent on water concentration (w(0) = [water]/[DDAB]) and temperatures. The interfacial film of DDAB-Cy undergoes significant alteration upon addition of water as the microscopic phase changes from cylindrical aggregates to discrete droplets which is in contrast to the conventional RM systems. FTIR spectroscopy in mid-infrared (MIR) and far-infrared (FIR) regions suggests the encapsulated water molecules to undergo a transition with increasing w(0) towards a bulk-like behavior. Time resolved fluorescence spectroscopy using Coumarin-500 as the fluorophore reveals a decrease in solvation time constant with increasing w(0) as well as with increasing temperature, a behavior consistent with conventional RM systems. The temperature dependent relaxation dynamics is found to follow an Arrhenius type behavior with a value for E(act) in the range of 2.5-3 kcal mol(-1) for all the studied systems. Our results show that phase modification has a marginal effect on the relaxation dynamics.

Acid-Base Behavior of Rhodamine B in a Reversed Micellar Medium of Cetyltrimethylammonium Chloride in 1-Hexanol-Cyclohexane/Water

The acid-base behavior of rhodamine B (RB) in reversed micellar solutions of cetyltrimethylammonium chloride (CTAC) in 1-hexanol-cyclohexane/water was investigated through absorption and fluorescence measurements under varying the hydrogen-ion concentration in a water pool, the mole fraction of 1-hexanol in a bulk solvent and the water to surfactant molar concentration ratio. RB exhibited equilibria between the cationic, zwitterion and lactone forms of the dye as a cause of the spectral changes, indicating a change in the RB distribution among the reversed micellar water pool, interface and bulk solvent. Furthermore, RB was used as a probe to develop a method for determining the critical micelle concentration (CMC) in CTAC solutions. Abrupt variations in the intensities and wavelengths of the absorption and emission maxima of RB were observed at the CMC. The standard free energy of micellization was also evaluated from the CMC data.

Do Probe Molecules Influence Water in Confinement?

The water inside reverse micelles can differ dramatically from bulk water. Some changes in properties can be attributed to the interaction of water molecules with the micellar interface, forming a layer of shell water inside the reverse micelle. The work reported here monitors changes in intramicellar water through chemical shifts and signal line widths in 51V NMR spectra of a large polyoxometalate probe, decavanadate, and from infrared spectroscopy of isotopically labeled water, to obtain information on the water in the water pool in AOT reverse micelles formed in isooctane. The studies reveal several things about the reverse micellar water pool. First, in agreement with our previous measurements, the proton equilibrium of the decavanadate solubilized within the reverse micelles differs from that in bulk aqueous solution, indicating a more basic environment compared to the starting stock solutions from which the reverse micelles were formed. Below a certain size, reverse micelles do not form when the polyoxometalate is present; this indicates that the polyanionic probe requires a layer of water to solvate it in addition to the water that solvates the surfactant headgroups. Finally, the polyoxometalate probe appears to perturb the water hydrogen-bonding network in a fashion similar to that in the interior surface of the reverse micelles. These measurements demonstrate the dramatic differences possible for water environments in confined spaces.

Reverse micelle‐based chemiluminescence system for quinine sulphate

A new chemiluminescence (CL) method is proposed for the determination of quinine sulphate, which is based on the dichloromethane solvent extraction of the ion-pair complex of tetrachloroaurate (III) with quinine sulphate, and luminol CL detection in a reversed micellar medium formed by the cation surfactant cetyltrimethylammonium bromide in a dichloromethane-cyclohexane (3:7, v/v)-water (0.35 mol/L Na2CO3 buffer solution, pH 11.5). The ion-pair complex of tetrachloroaurate (III) with quinine sulphate produced an analytical CL signal when it entered the reversed micellar water pool. In optimum conditions, CL intensities are proportional to the concentrations of the studied drug over the range 0.015-10 microg/mL, with a detection limit of 1.5 ng/mL. The relative standard deviation (RSD) is 1.38% for 2.5 microg/mL quinine sulphate (n=11). The method has been applied to the determination of the studied drug in biological fluids, with satisfactory results.

Higher order structure of proteins solubilized in AOT reverse micelles

The higher order structure of proteins solubilized in an bis(2-ethylhexyl) sulfosuccinate sodium (AOT) reverse micellar system was investigated. From circular dichroic (CD) measurement, CD spectra of cytochrome c, which is solubilized at the interface of reverse micelles, markedly changed on going from buffer solution to the reverse micellar solution, and the ellipticity values in the far- and near-UV regions decreased with decreasing the water content ( W 0: molar ratio of water to AOT), indicating that the secondary and tertiary structures of cytochrome c changed with the water content. The ellipticity of ribonuclease A, which is solubilized in the center of micellar water pool, in the near-UV region was dependent on W 0 and became minimum when W 0 of ca. 8 while the ellipticity in the far-UV region was almost constant, indicating that the tertiary structure of ribonuclease A was affected by the water content, but the secondary structure was conserved. The degree of curvature of the micellar interface appears to influence the protein structure because the reverse micelle size is linearly proportional to the W 0 value. As evidence of this, when the micelle size was comparable to the protein's dimensions, the structures were more affected by the water content. Judging from the dependence of the factor influencing the protein structure on the protein species, the location of solubilized protein in reverse micelles is significantly related to whether the protein structure in the system is affected by the micellar interface. In the cases of cytochrome c and lysozyme, the ellipticity against W 0 was dependent on the AOT concentration. In contrast, ribonuclease A gave very similar ellipticity values whatever the AOT concentration. In the n-hexane micellar system, cytochrome c exhibited lower ellipticity values and ribonuclease A in the lower W 0 range ( W 0 < ca. 8) higher ellipticity values. These results indicated that the interaction between the protein and the micellar interface is a dominant factor influencing the protein structure in reverse micelles, and that it is governed by the location of solubilized proteins and the state of the micellar interface.

Ion-pair complex-based solvent extraction combined with chemiluminescence determination of chlorpromazine hydrochloride with luminol in reverse micelles

A new chemiluminescence (CL) method is proposed for the determination of chlorpromazine hydrochloride, which is based on the dichloromethane solvent extraction of ion-pair complex of tetrachloroaurate(III) with chlorpromazine hydrochloride and luminol chemiluminescence detection in a reversed micellar medium formed by the cation surfactant cetyltrimethylammonium bromide in a dichloromethane–cyclohexane (1:1 V/V)–water (0.3 mol/L Na 2CO 3 buffer solution with the pH of 11.5). The ion-pair complex of tetrachloroaurate(III) with chlorpromazine hydrochloride produced an analytical chemiluminescence signal when it entered the reversed micellar water pool. In the optimum conditions, CL intensities are proportional to concentrations of the studied drug over the range 0.05 ∼ 10 μg/mL with a detection limit (DL) of 6 ng/mL. The relative standard deviation (R.S.D.) is 2.6% for 1.25 μg/mL chlorpromazine hydrochloride ( n = 11). R.S.D. (precision) of inter-day and intra-day is less than 6%, and accuracy of inter-day and intra-day is satisfactory. The method has been applied to the determination of studied drug in pharmaceutical preparations and biological fluids with satisfactory results.

Positive cooperativity in substrate binding of human prostatic acid phosphatase entrapped in AOT–isooctane–water reverse micelles

The kinetics of 1-naphthyl phosphate and phenyl phosphate hydrolysis, catalyzed by human prostatic acid phosphatase (PAP) entrapped in AOT–isooctane–water reverse micelles, has been studied over surfactant hydration degree ( w 0) range 5 to 35. Continuous spectrophotometric acid phosphatase assays, previously prepared, were employed. PAP was catalytically active over the whole w 0 studied range. In order to determine steady-state reaction constants the experimental data were fitted to Hill rate equation. Positive cooperativity in substrate binding was observed, as it was earlier found in aqueous solutions. The extent of cooperativity (expressed as the value of the Hill cooperation coefficient h) increased from 1 to 4, when the micellar water-pool size was growing, at fixed enzyme concentration. In the plots of catalytic activity ( k cat) versus w 0, the maxima have been found at w 0=10 (pH 5.6) and 23 (pH 3.8). It is suggested that catalytically active monomeric and dimeric PAP forms are entrapped in reverse micelles of w 0=10 and 23, respectively.

Effect of the Media on the Quantum Yield of Singlet Oxygen (O2(1Δg)) Production by 9H‐Fluoren‐9‐one: Microheterogeneous Systems

AbstractThe quantum yield (ΦΔ) of singlet oxygen (O2(1Δg) production by 9H‐fluoren‐9‐one (FLU) is very sensitive to the nature of the solvent (0.02 in a highly polar and protic solvent, such as MeOH, to 1.0 in apolar solvents). This high sensitivity has been used for probing the interaction of FLU with micellar media and microemulsions based on anionic (sodium dodecyl sulfate, SDS; bis‐(2‐ethylhexyl)sodium sulfosuccinate, AOT), cationic (cetyltrimethylammonium chloride, CTAC) and nonionic (Triton X‐100, TX) surfactants. Values of ΦΔ of FLU vary in a wide range (0.05–1.0) in both microheterogeneous media and neat solvent, and provide information on the microenvironment of FLU, i.e., on its localization within organized media. In ionic and nonionic micellar media, as well as in four‐component microemulsions, FLU is, to various extents, exposed to solvation by the polar and protic components of the microheterogeneous systems (water and/or butan‐1‐ol) in the micellar interfacial region (ΦΔ=0.05–0.30). In contrast, in AOT reverse micelles (consisting of AOT as surfactant, cyclohexane as hydrophobic component, and water), FLU is located in the hydrophobic continuous pseudophase, and is totally separated from the micellar water pools (ΦΔ≈1.0).

逆ミセル有機溶媒における電気的パーコレーション現象とタンパク質の可溶化状態

Electric percolation phenomena of reverse micellar organic phase of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) were investigated. Solubilization of electrolytes into the micellar water pool depressed the percolation between reverse micelles and increased the percolation temperature. In particular, caotropic GuHSCN and strong electrolyte KCl increased the percolation temperature more effectively. Solubilization of low-molecular-weight proteins into the micellar phase induced the percolation processes and lowered the percolation temperature, which was dependent on the protein concentration solubilized. The magnitude order of effectiveness of the protein on the percolation was cytochrome c > lysozyme > ribonuclease A. The efficiency of each protein in promoting percolation corresponded to its local solubilizing state in the micelles. In the cases of solubilizing both the electrolyte and the protein, the percolation processes were mutually affected by each additive. In the reverse micellar systems formed by different organic solvents (C6-10 alkane), the percolation temperature was linearly decreased with molecular volume of the solvent. The shift of the percolation temperature due to protein solubilization (ΔTp) depended upon the solvent molecular volume and the protein species. In the case of lysozyme, ΔTp increased with the molecular volume of solvent, while ΔTp of cytochrome c showed the minimum value at the molecular volume of C8-9 alkane.

Solvent extraction coupled on-line to a reversed micellar mediated chemiluminescence detection system for trace-level determination of atropine.

A fast and sensitive method for the determination of atropine, an alkaloid closely related to cocaine, is proposed. The principles of on-line ion-pair formation of alkaloid-metal complexes and liquid-liquid extraction are applied to the chemiluminescence determination of atropine. On mixing with a reversed micellar medium of cetyltrimethylammonium chloride in dichloromethane-cyclohexane (1:1 v/v)-water (0.3 M Na2CO3) containing luminol, the ion-pair complex of tetrachloroaurate(III) with atropinium produced an analytical chemiluminescence signal when it entered the reversed micellar water pool. Using the reverse-flow injection and chemical conditions optimized for atropine in aqueous samples, a detection limit of 1 ng/mL was achieved and a linear calibration graph was obtained with a wide dynamic range from 10 ng/mL to 100 micrograms/mL. The proposed method is simple and provides a good precision with a relative standard deviation (n = 6) of ca. 3% at the atropine concentration of 100 ng/mL. After a preliminary study involving the potential interference from species of organic, inorganic, and metallic nature, the method was applied to the determination of atropine in artificial urine samples and of atropine and scopolamine in pharmaceutical formulations.

Organized Media as Redox Catalysts

Micellar catalysis for a methylene blue (MB)−ascorbate ion (AA-) redox reaction (activation energy 114 kJ mol-1) has been observed to occur because of the binding of the substrates onto the micellar surface by hydrophobic and electrostatic interaction. This increases the encounter probability, leading to a substantially accelerated reaction. The reported study indicates that the most likely substrate solubilization sites in micelles and reverse micelles are the interfacial and Stern regions. Studies on the effect of various salting-in and salting-out agents, an increase in the hydrophobicity of the dye, and the size of the reverse micellar water pool provide no evidence of deeper penetration into the micellar core. In addition, a fascile reversible electron-transfer reaction for the MB−AA- and MB−BH4- systems has been observed with coinage metal sol particles. Metal sol-induced catalysis occurs due to the availability of a suitable substrate which acts as a mediator for the electron-transfer reaction betw...

Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. Copyright 1998 John Wiley & Sons, Inc.

Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:380–386, 1998.

Preparation and Photocatalytic Reactions of Titanium Dioxide Ultrafine Particles in Reverse Micellar Systems.

Photocatalytic oxidation of cyclohexane forming the organic phase of reverse micellar systems on in situ prepared TiO2 ultrafine particles has been investigated. The cyclohexane was photo-oxidized on the TiO2 ultrarine particles to form cyclohexanone and cyclohexanol under air-saturated conditions. In the absence of O2, H2 was generated via reduction of water in the micellar waterpools. CO was also generated in the absence of O2 probably via a photocatalytic side reaction of 1-butanol which was added as a solvent of the precursor of TiO2 (titanium tetrabutoxide: TTB) to prepare the particles, 1-butoxyl groups of TTB or 1-butanol formed via hydrolysis of TTB. The molar ratio of water to TTB, Wt (=[H2O]/[TTB]) was a major factor in controlling the photocatalytic reaction behavior of the system. When the irradiation was carried out under air-saturated conditions for 18 h, the consumption rate of O2 increased monotonously as the value of the parameter Wt decreased, while the quantities of cyclohexanone and cyclohexanol formed decreased at Wt Wt range owing to incomplete hydrolysis of TTB molecules.

Solubility Studies and Synthesis of Acpheleunh2in Reversed Micellar Systems

The aim of the work presented was to study the precipitation of the dipeptide derivative AcPheLeuNH2 in order to improve its yield and productivity during enzymatic synthesis catalysed by α-chymotrypsin in tetradecyltrimethylammonium bromide (TTAB)/heptane/octanol reversed micelles. AcPheLeuNH2 solubility in reversed micellar solutions was examined, revealing that a decrease in TTAB concentration, water to surfactant ratio (Wao) and octanol concentration in the organic mixture led to a decrease in solubility. In contrast, no significant influence of buffer molarity and pH was detected. These results were explained by suggesting that the main environment for the solubilization of the dipeptide molecules is the micellar interface rather than the bulk organic phase or the micellar water pool. The synthesis of AcPheLeuNH2 was then carried out using those process conditions that led to the lower solubility values. The activity and stability of α-chymotrypsin, dipeptide yield, selectivity, dipeptide recovery an...

Electron paramagnetic resonance, electron spin echo modulation and electron nuclear double resonance studies on the photoionization of N-alkylphenothiazines in cetyltrimethylammonium bromide–alcohol reverse micelles. Effects of alkyl chain length of alkylphenothiazines, reverse micellar water pool size and cosurfactant alcohol

Neutral N-alkylphenothiazines (PCn, n= 1, 3, 6, 9, 12, 16) are solubilized and photoionized in cetyltrimethylammonium bromide (CTAB)–alcohol reverse micelles at 77 K to investigate the optimum photoionization conditions by varying the cosurfactant alcohol, reverse micellar water pool size and pendant alkyl chain length. The photo-produced radicals are identified and quantified by electron paramagnetic resonance (EPR). The relative locations from deuteriated water at the interface are monitored by deuteron electron spin echo modulation (ESEM) and proton matrix electron nuclear double resonance (ENDOR).The photoyields and deuteron modulation depths of PCn compounds show a U-shaped trend with increasing pendant alkyl chain length which indicates U-shaped conformations for the longer alkyl chains. The photoyields of the PCn compounds decrease with the water to surfactant mole ratio and correlate with decreasing deuteron modulation depths which indicate an increasing distance from the phenothiazine moiety to deuteriated interface water. The deeper penetration is attributed to a decreased packing density of the interface region of the reverse micelles. This is supported by decreasing ENDOR linewidths with increasing water pool size.A change of cosurfactant alcohol from butanol to octanol slightly decreases the photoyield and the deuteron modulation depths. This reflects deeper solubilization and a decreased rate of photoinduced radical conversion.

Effects of average molecular charge on amino acid interfacial partitioning in reversed micelles

The effect of pH on amino acid interfacial partitioning in reversed micelles is reported. A simple thermodynamic analysis of the data allows estimates of the interfacial electrostatic potential in these reversed micelles to be made, from which the effects of external electrolyte type and concentration may be inferred. Good agreement with the predictions of the Poisson-Boltzmann equation is obtained. Inferences can be drawn as to the orientation of the amino acids within the structured interfaces, and it is argued that the amino acid headgroup dipole must be aligned almost normal to the direction of the electric field. Changes in amino acid speciation in reversed micellar systems, coupled with an apparent shift in pH of the external aqueous phase, can be attributed to a concentration of the hydroxonium ion in the reversed micellar water pools, and not to changes in intrinsic dissociation constants. It is demonstrated that species distribution in the AOT Winsor II system closely resembles various chromatographic processes, there being a direct analogy between the chromatographic retention coefficient and the reversed micellar interfacial partition coefficient.