Reverse Micellar System Research Articles

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w0 (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.

Block-Copolymer Micro-emulsion with Solvent-Induced Segregation

Reverse micelle formation of the poly(ethylene oxide)/poly(propylene oxide) block-copolymer (EO)13(PO)30(EO)13 (L64) in p-xylene was investigated as a function of water content and copolymer content, using small-angle neutron scattering (SANS). PEO/PPO block-copolymers are generally soluble in xylene but without forming aggregates. However, the effective block segregation increases dramatically upon addition of small amounts of water, and micelles form. The SANS data were analyzed using an absolute scale model fitting approach. This way, a detailed quantitative description of the system in terms of unimer concentration, micelle structure, and aggregation number as well as particle-particle interactions can be obtained. This approach throws light on very atypical features of the system as compared to standard amphiphilic systems. Data from samples measured along water-swelling lines with fixed EO/p-xylene-d10 molar ratios show that reverse micelles are formed at the water/EO molar ratio, W0 congruent with 0.2, independent of copolymer concentration. The majority of the block-copolymers are on a free monomer state (unimer state) at this W0. Increasing W0 above 0.2 only has a small effect on the micelle size. However, it does induce a strong increase of the total number of micelles and induce a corresponding decrease of the unimer concentration. On the other hand, increasing the overall copolymer concentration at fixed W0 gives rise to a significant decrease of the micelle size in terms of the micellar aggregation number. This observed behavior is totally different from what is normally observed for binary surfactant-solvent systems and droplet micro-emulsion systems, respectively. We believe that the atypical behavior is a result of the unusually weak segregation in the system, and we are not aware of previous discussions of the phenomenon for reverse micellar systems.

Continuous counter-current purification of glucose-6-phosphate dehydrogenase using liquid–liquid extraction by reverse micelles

This work details the continuous counter-current purification of glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae cells by liquid–liquid extraction using reverse micelles. A biocompatible reverse micellar system consisting of 0.05 M soybean lecithin (zwiterionic surfactants) in isooctane with hexanol was employed to study the influence of different flow-rates on G6PD purification. The results showed that the reverse micellar system was able to remove proteins (impurities) from the cell-free. The enzyme recovery yield varied from 32% to 115% and G6PD purification factor from 1.0 to 2.2, under flow-rates ranging from 1.6:1.6 mL min −1 to 6.0:6.0 mL min −1 for both phases (micellar and aqueous phases). The steady hold-up values demonstrated that the mass transfer capability of this extraction apparatus was practically constant. The continuous counter-current purification system employed proved to be useful not only for purifying G6PD, but also for maintaining the enzyme activity.

Higher order structure of Mucor miehei lipase and micelle size in cetyltrimethylammonium bromide reverse micellar system

The higher order structure of Mucor miehei lipase and micelle size in a cationic cetyltrimethylammonium bromide (CTAB) reverse micellar system was investigated. Circular dichroic (CD) measurement revealed that the lipase far-UV CD spectra changed markedly, going from buffer solution to the reverse micellar solution, and were very similar for any organic solvent used. The ellipticity of the solubilized lipase in the far-UV region markedly decreased with increasing water content ( W 0: molar ratio of water to CTAB), indicating that the secondary structure of lipase changed with the water content. The linear correlation between the W 0 and the micelle size was obtained by measuring dynamic light scattering. From the linear correlation between the micelle size and W 0, the higher order structure of the solubilized lipase appears to be affected directly by the micellar interface. The species and concentration of alcohol as a cosurfactant had an inferior effect on lipase structure. Especially, at ratios of 1-pentanol to CTAB of less than 8, the secondary and tertiary structures of lipase were preserved in the reverse micelles. The CTAB concentration had little effect on the lipase structure in the micelles. The catalytic activity of the lipase solubilized in the CTAB reverse micelles increased with increasing the W 0.

Topical Delivery of Aceclofenac from Lecithin Organogels: Preformulation Study

The purpose of this research is to evaluate the suitability of lecithin organogels containing aceclofenac for topical application. The present article focuses on the preformulation part of the whole research work. Thin layer chromatography was carried out to determine lecithin's purity. The excipients for formulating lecithin organogel were screened. Lecithin organogels are thermo reversible in nature and hence gelation temperature study was carried out to determine the temperature where Sol-Gel and Gel-Sol transformation takes place. Partition coefficient of the drug was estimated. Drug solubility in plain oil and organogel containing reverse micelles was estimated. Effect of water added on the properties of lecithin organogels such as X-ray diffraction pattern, conductivity and viscosity were determined. Microscopy of the gel sample has been carried out at different magnifications. The pseudo ternary phase diagram has been constructed to determine the organogel existence region. The permeation study of aceclofenac from different concentrations of lecithin organogels [200 mM, 300 mM and 400 mM] has been determined using cellulose acetate membrane (0.45 micro) and excised rat skin. Lecithin organogel in ethyl oleate has desired stability and consistency. A single spot on the TLC plate confirms the purity of soy lecithin to be used in organogel formation. Aceclofenac solubility was found to be more in lecithin/oil reverse micellar system as compared to its solubility in oil. The X-ray diffraction pattern confirms the incorporation of water in micellar gel network. The physical properties of organogels are affected by water incorporated and concentration of gelator. The permeation of aceclofenac through artificial membrane and excised rat skin demonstrated the same trend and were in the following order 200 mM>300 mM>400 mM. The results showed that organogel exhibits useful pharmaceutical properties.

Oxidation of Alcohol by Lipopathic Cr(VI): A Mechanistic Study

The oxidation kinetics of various aliphatic primary and secondary alcohols having varied hydrocarbon chain length were studied using cetyltrimethylammonium dichromate (CTADC) in dichloromethane (DCM) in the presence of acetic acid and in the presence of a cationic surfactant. The rate of the reaction is highly sensitive to the change in [CTADC], [alcohol], [acid], [surfactant], polarity of the solvents, and reaction temperature. A Michaelis-Menten type kinetics was observed with respect to substrate. The chemical nature of the intermediate and the reaction mechanism were proposed on the basis of (i) observed rate constant dependencies on the reactants, that is, fractional order with respect to alcohol and acid and a negative order with respect to oxidant, (ii) high negative entropy change, (iii) inverse solvent kinetic isotope effect, k(H2O)/k(D2O) = 0.76, (iv) low primary kinetic isotope effect, kH/kD = 2.81, and (v) the k(obs) dependencies on solvent polarity parameters. The observed experimental data suggested the self-aggregation of CTADC giving rise to a reverse micellar system akin to an enzymatic environment, and the proposed mechanism involves the following: (i) formation of a complex between alcohol and the protonated dichromate in a rapid equilibrium, equilibrium constant K = 5.13 (+/-0.07) dm(3) mol(-1), and (ii) rate determining decomposition (k(2) = (7.6 +/- 0.7) x 10(-3) s(-1)) of the ester intermediate to the corresponding carbonyl compound. The effect of [surfactant] on the rate constant and the correlation of solvent parameters with the rate constants support the contribution of hydrophobic environment to the reaction mechanism.

Using reverse micelles as microreactor for hydrogen production by coupled systems of Nostoc/R. palustris and Anabaena/R. palustris

Uptake hydrogenase negative mutants of bloom forming cyanobacteria (Nostoc and Anabaena) and the fermentative bacteria Rhodopseudomonas palustris P4 were used together for producing hydrogen within the reverse micelles fabricated by N-ethyl hexyl sodium sulfosuccinate (AOT) in isooctane and cetyl trimethyl ammonium bromide (CTAB) in benzene. The rate of H2 production in AOT/isooctane reverse micellar system was found to be more promising in comparison to the CTAB/Benzene reverse micellar entrapment. After mutagenesis in 2.0% (v/v) ethyl methane sulphonate (EMS) mutants of Nostoc and Anabaena were selected on BG-11 plates (containing 2% agar) and then used for analysis of produced hydrogen. In comparison to the unmutated Nostoc with R. palustris (within AOT/isooctane) the coupled system of mutated Nostoc and R. palustris produced H2 by 3.9-fold higher rate, which is 8.6 mmol H2/h/mg protein. Whereas, mutated Anabaena coupled with R. palustris produced 4.8 times higher hydrogen production within (AOT)/isooctane reverse micelles in comparison to the unmutated Anabaena with R. palustris. Effect of nitrogen to carbon ratio (N/C) on hydrogen production was studied and Anabaena/R. palustris and Nostoc/R. palustris systems were, respectively, found to generate 11.2 and 9.8 mmol H2/h/mg protein continuously for 3 days. Effects of temperature and light intensity were also investigated and we found that 32°C temperature and 1,000 Lux light intensity are the optimum values in these systems. Addition of sodium dithionite also resulted in further enhancement of the rate and duration of hydrogen production in both (mutated Nostoc/R. palustris and mutated Anabaena/R.␣palustris) systems.

Preparation, characterization and in vitro drug release studies of novel polymeric nanoparticles

Polymeric nanoparticles of AADG cross-linked with MBA encapsulating water soluble macromolecules such as FITC-Dextran have been prepared in the reverse micellar system. The particles obtained were of >85 nm in diameter which were highly monodisperse. An optically clear solution was obtained on redispersing these nanoparticles in aqueous buffer. Size and morphology of the particles remains the same on re-dispersing the lyophilized powder of these nanoparticles in aqueous buffer. The size dependency of the particles on the monomer and surfactant concentration was observed. The average size of the nanoparticles as obtained from DLS studies ranges from 74 to 114 nm in case 0.06 M AOT and 62–104 nm in case of 0.1 M AOT concentration. FITC-Dextran was entrapped into nanoparticles with high efficiency (>70%). The pH dependent release of the entrapped molecules from these nanoparticles was also studied. At pH 5.0 solution, ∼43% of FITC-Dx was released and at pH 7.4 it was about 70%.

Monomerization and photodynamic activity of Zn(II) tetraalkyltetrapyridinoporphyrazinium derivatives in AOT reverse micelles

Monomerization and photodynamic activity of cationic Zn(II) tetraalkyltetrapyridinoporphyrazinium derivatives bearing N-alkyl chains of different length (ZnPc 1 R: –CH 3, ZnPc 2 R: –(CH 2) 11CH 3, ZnPc 3, R: –(CH 2) 15CH 3) were evaluated in n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water micellar system. Absorption and fluorescence spectroscopic studies of these sensitizers were analyzed in n-heptane/AOT (0.1 M)/water system by varying the amount of water dispersed in the reverse micelles ( W 0 = [H 2O]/[AOT]). Under these conditions, the absorbance of the monomeric Q-band (∼686 nm) of ZnPcs 1– 3 increases rapidly until W 0 ∼ 30 and then the increase becomes steady. These results were also confirmed by fluorescence studies. Analysis of the Q-band at different AOT concentrations, keeping W 0 = 30 as constant, was carried out to determine the binding constant ( K b) between these sensitizers and AOT reverse micelles. The values of K of 210, 150 and 34 were found for ZnPcs 1, 2 and 3, respectively. Photodynamic activity of these sensitizers was evaluated in AOT system using 9,10-dimethylanthracene (DMA). The photooxidation rate of DMA sensitized by these phthalocyanines follow the order: ZnPc 1 > ZnPc 2 ∼ ZnPc 3. As shown by spectroscopic and photodynamic studies, the AOT reverse micellar system very effectively inhibits the aggregation of phthalocyanines, promoting the formation of photoactive monomeric species.

Effect of aprotic solvents on the enzymatic activity of lipase in AOT reverse micelles

The modification of reverse micellar systems composed of AOT, isooctane, water by the addition of aprotic solvents has been performed. The impact of this change on the activity, stability and kinetics of solubilized Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was investigated. Of seven aprotic solvents tested, dimethyl sulfoxide (DMSO) was found to be most effective. It was found that lipase activity was enhanced by optimizing some relevant parameters, such as water–AOT molar ratio ( W 0), buffer pH and surfactant concentration. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to deduce some kinetic parameters ( V max, K m and K ad), and the values of K m and K ad were significantly reduced by the presence of DMSO. Higher lipase stability was found in AOT reverse micelles with DMSO compared with that in simple AOT systems with half-life of 125 and 33 days, respectively. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were used to elucidate the effects of DMSO on the properties of AOT reverse micelles.

Protein Refolding Mediated by Reverse Micelles of Cibacron Blue F-3GA Modified Nonionic Surfactant

An affinity-based reverse micellar system formulated with nonionic surfactant was applied to the refolding of denatured-reduced lysozyme. The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reverse micelles in n-hexane. The water content of 15 was found optimal for lysozyme refolding in the reverse micellar system of 62.7 mmol/L Span 85 with coupled CB of 0.3 and 0.5 mmol/L. In addition, the operating conditions such as pH and the concentrations of urea and redox reagents were optimized. Under the optimized conditions, complete renaturation of lysozyme at 3-3.5 mg/mL was achieved, whereas dilution refolding in the bulk aqueous phase under the same conditions gave much lower activity recovery. Moreover, the secondary structure of the refolded lysozyme was found to be the same as the native lysozyme. Over 95% of the refolded lysozyme was recovered from CB-Span 85 reverse micelles by a stripping solution of 0.5 mol/L MgCl(2). Thus, the present system is advantageous over the conventional reverse micellar system formed with ionic surfactants in the ease of protein recovery.

Influence of Microwave Radiation on the Growth of Gold Nanoparticles and Microporous Zincophosphates in a Reverse Micellar System

The water core of reverse micelles has been extensively used as the site for synthesis of a variety of materials. However, water-in-oil reverse micelles have a limited range of temperatures over which they are stable as a single phase. Directing heat to the water cores, the usual site of synthesis without heating the bulk provides added opportunities for synthesis. Microwave radiation is a method for superheating the water cores. In this study, we use an H2O-sodium bis(2-ethylhexyl) sulfosuccinate (AOT)-heptane reverse micelle system for the synthesis of Au particles by hydrazine reduction of HAuCl4 in the presence and absence of microwave radiation. The duration of the microwave radiation was limited to a 2-min duration at a power of 300 W, thereby ensuring that the reverse micelle phase is maintained during the synthesis. At all hydrazine concentrations studied (0.5-2 M), the presence of microwave radiation led to an increase in the particle size of Au. The second system examined was the growth of microporous zincophosphate-X (ZnPO-X, an analogue of the faujasite structure) synthesized from H2O-dioctyldimethylammonium chloride (DODMAC)-heptane reverse micelle system. Microwave radiation was applied for 1 min at 150 W at various stages of the nucleation and growth process, and did not disrupt the reverse micelle system. Product analysis after 48 h of reaction showed that the 1-min microwave pulse, if applied during the nucleation stage (the first 4 h), promoted the formation of NaZnPO4.H2O over ZnPO-X. The effect of the microwave pulse at the growth stage was to promote the formation of ZnPO-X. Absorption of the microwave radiation by the water core and surrounding polar surfactant molecules leads to a rapid rise in local temperature (predicted to be approximately 150 degrees C/min for the AOT system), increasing the rates of intramicellar reactions.

Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles

The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent mediae, respectively. The influences of the molar ratio of water to surfactant ω 0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show “superactivity” in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of ω 0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at ω 0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.

Molecular Probe Location in Reverse Micelles Determined by NMR Dipolar Interactions

The location and interactions of solutes in microheterogeneous environments, such as reverse micelles, critically influence understanding of many phenomena that utilize probe molecules to characterize properties in chemical, biological, and physical systems. The information gained in such studies depends substantially on the location of the probe used. Often, intuition leads to the assumption that ionic probe molecules reside in the polar water pool of a system. In this work, the location of a charged polar transition metal coordination complex in a reverse micellar system is determined using NMR spectroscopy. Despite the expected Coulomb repulsion between the surfactant headgroups and the negatively charged complex, the complex spends significant time penetrating into the hydrophobic portion of the reverse micellar interface. These results challenge the assumption that ionic probe molecules reside solvated by water in microheterogeneous environments and suggest that probe molecule location be carefully considered before interpreting data from similar systems.

A Monte Carlo Model for the Formation of Core−Shell Nanocrystals in Reverse Micellar Systems

The process of formation of core−shell nanocrystals, using a reverse micellar system, has been modeled using the Monte Carlo technique, and the post-core route for the formation of core−shell nanocrystals has been studied. The model is divided into two parts: (a) the formation of core nanoparticles and their subsequent growth due to coagulation, and (b) the formation of the shell via the ion-displacement mechanism. The model for core nanoparticle formation and subsequent growth includes the phenomena of finite nucleation, growth via intermicellar exchange, and coagulation of nanoparticles after their formation. The growth of the core is taken to be limited by the size of the micellar core. The model explains the possible mechanism of core−shell nanocrystal formation. The simulation results compare well with those observed experimentally.

Degradation patterns of tetracycline antibiotics in reverse micelles and water

The objective of this study was to determine the chemical stability of tetracycline and oxytetracycline hydro-chlorides in reverse micelles. Their reverse micellar solutions were prepared using cetyltrimethylammonium bromide, water and ethyl formate. The aqueous solutions of the tetracycline antibiotics were also prepared for comparison. The reverse micellar and aqueous solutions were stored at 37 degrees C. Samples were analyzed by high performance liquid chromatography. When evaluation was performed on an aqueous tetracycline HCl solution, its half-life was estimated to be 329 h. Its chemical stability was not improved after being dissolved in the reverse micelles, and a similar half-life of 330 h was observed. However, there were noticeable differences between the two systems in terms of degradation kinetics and degradation byproducts. On the other hand, oxytetracycline HCl was unstable in water so that its half-life was only 34 h. Very interestingly, pronounced improvement in stability was attained with the reverse micellar system: upon dissolving in the reverse micelles, its half-life was increased to 2402 h. There were also marked differences in degradation patterns and mechanisms of oxytetracycline HCl in water and the reverse micelles. Our study indicates that the reverse micellar system has potential applications in solubilizing and stabilizing oxytetracycline HCl, thereby contributing to the development of its dosage forms.

Effect of compressed CO2on the chloroperoxidase catalyzed halogenation of 1,3-dihydroxybenzene in reverse micelles

The effect of compressed CO2 on the specific activity of chloroperoxidase (CPO) to catalyze the chlorination of 1,3-dihydroxybenzene in cetyltrimethylammonium chloride (CTAC)/H2O/octane/pentanol reverse micellar solution was studied. The results show that the specific activity of the enzyme can be enhanced significantly by compressed CO2, and the specific activity can be tuned continuously by changing pressure. The mechanism for the specific activity enhancement of the enzyme by CO2 was also studied. We believe that compressed CO2 can be utilized to tune some other enzyme catalytic reactions in different reverse micellar systems with potential advantages.

Equilibria and kinetics of protein transfer to and from affinity-based reverse micelles of Span 85 modified with Cibacron Blue F-3GA

Sorbitan trioleate was modified with Cibacron Blue F-3GA (CB) to create an affinity surfactant and to form affinity-based reverse micelles in n-hexane. The partitioning equilibria and the extraction kinetics of lysozyme and bovine serum albumin (BSA) were then examined. The solubilization capacity of the reverse micellar system for lysozyme increased linearly with increasing the CB concentration from 0.1 to 0.5 mmol L −1. In contrast, the capacity for BSA at 0.5 mmol L −1 of coupled CB was only about one-fifth that for lysozyme. It indicates a strong steric hindrance effect of the micelles for the high molecular mass protein. The overall volumetric mass transfer coefficient of lysozyme in the forward extraction increased from 0.43 × 10 −3 to 1.25 × 10 −3 s −1 with increasing CB concentration from 0.1 to 0.5 mmol L −1. Due to the high molecular mass of BSA, its volumetric mass transfer coefficient in the forward extraction was only one-sixth that of lysozyme. The ratio of the coefficient in the back extraction to that in the forward extraction was less than 0.03, much lower than those in other micellar systems. It indicates that the interfacial resistance in this system was severer than in others.

Forward and backward extractions of cytochrome c using reverse micellar system of sucrose fatty acid ester

The effects of solvent, pH, and ionic strength on the reverse micellar extraction of cytochrome c have been examined, when sucrose fatty acid esters were employed as surfactants of reverse micelles. The transparent and stable reverse micellar organic phase was formed, when the mixture of isooctane and n-butanol (7:3 v/v) was used as the bulk organic phase. The high forward extraction ratio was obtained under mild alkaline and low ionic strength conditions, while the high backward extraction ratio was obtained for acidic pH values or at high ionic strength. The activity of cytochrome c recovered from the reverse micellar phase was sufficiently retained.

Geometric Constraints at the Surfactant Headgroup: Effect on Lipase Activity in Cationic Reverse Micelles

The primary objective of the present article is to understand how the geometric constraints at the surfactant head affect the lipase activity in the reverse micellar interface. To resolve this issue, surfactants were designed and synthesized, and activity was measured in /water/isooctane/n-hexanol reverse micellar systems at z ([alcohol]/[surfactant])=5.6, pH 6.0 (20 mM phosphate), 25 degrees C across a varying range of W0 ([water]/[surfactant]) using p-nitrophenylalkanoates as the substrate. It was observed that lipase activity increases from surfactants to with the increment in surface area per molecule (Amin) because of the substitution by the bulky tert-butyl group at the polar head. However, the activity was found to be similar for despite an enhancement in the hydrophilic moieties at the interface. This unchanged lipase activity is presumably due to the comparable surface area of to originating from the rigidity at the surfactant head. Noticeably, the enzyme activity improved from with the simultaneous increment of both the hydroxyl group and the flexibility of the headgroup whereas that for increased exclusively with the flexibility of the headgroup. The common parameter in both groups of surfactants and is the flexibility of the headgroup, which possibly enhance Amin and consequently the lipase activity. Thus, the geometric constraints at the surfactant headgroup play a crucial role in modulating the lipase activity profile probably because of the variation in interfacial area.