Mechanism of protein extraction from the solid state by water-in-oil microemulsions

The extraction of solid-phase alpha-chymotrypsin, bovine serum albumin (BSA), and lysozyme by water-in-oil microemulsion (w/o-ME) solution containing Aerosol-OT (AOT) was thoroughly examined as a means to maximize protein solubilization in organic solvent media. Protein extraction occurred simultaneously with the adsorption of water and AOT by the solid protein. Water and AOT were desorbed at nearly equal rates, suggesting that both materials were desorbed together as micreomulsions. The solubilization of protein increased linearly with the ratio of solid protein to extractant solution except at a high value of the ratio, where most protein-containing microemulsions were desorbed. Based on our results, a mechanistic model was developed to describe the solid-phase extraction procedure. First, microemulsions are desorbed from solution by the solid protein, resulting in the formation of a solid protein-AOT-water aggregate. Second, when a protein in the solid phase binds to a sufficient number of microemulsions, the resulting aggregate's increased hydrophobicity drives its solubilization into lipophilic solvent. Third, through the exchange of materials between the solubilized precipitate and the remaining microemulsions, protein-containing w/o-MEs are formed. The presence of adsorption is further indicated by an isotherm existing between the water, AOT, and protein content of the resulting solid phase for each protein. The driving force behind adsorption is either AOT-protein interactions or the protein's affinity for microemulsion-encapsulated water, depending on the properties of the protein and the size of the microemulsions, in agreement with the model of P. L. Luisi [Chimia, 44: 270-282 (1990)]. The second step of our model is mass transfer limited for the extraction of solid alpha-chymotrypsin and BSA. The extraction of solid lysozyme was limited by the occurrence of an irreversible precipitation process. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 583-593, 1997.     

Structure-Activity Relationship of a U-Type Antimicrobial Microemulsion System

The structure-activity relationship of a U-type antimicrobial microemulsion system containing glycerol monolaurate and ethanol at a 1∶1 mass ratio as oil phase and Tween 20 as surfactant were investigated along a water dilution line at a ratio of 80∶20 mass% surfactant/oil phase, based on a pseudo-ternary phase diagram. The differential scanning calorimetry results showed that in the region of up to 33% water, all water molecules are confined to the hydrophilic core of the reverse micelles, leading to the formation of w/o microemulsion. As the water content increases, the water gains mobility, and transforms into bicontinuous in the region of 33–39% water, and finally the microemulsion become o/w in the region of above 39% water. The microstructure characterization was confirmed by the dynamic light scattering measurements and freeze-fracture transmission electron microscope observation. The antimicrobial activity assay using kinetics of killing analysis demonstrated that the microemulsions in w/o regions exhibited relatively high antimicrobial activity against Escherichia coli and Staphylococcus aureus due to the antimicrobial oil phase as the continuous phase, while the antimicrobial activity started to decrease when the microemulsions entered the bicontinuous region, and decreased rapidly as the water content increased in the o/w region, as a result of the dilution of antimicrobial oil droplets in the aqueous continuous phase.     

Proteins in membrane mimetic systems. Insertion of myelin basic protein into microemulsion droplets.

The insertion of myelin basic protein into microemulsion droplets of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) has been studied by quasi-elastic light scattering. Measurements were made at both low and high molar ratios of water to surfactant, as a function of protein occupancy. The hydrodynamic radii of filled and empty droplets were experimentally evaluated. These were compared to values calculated using a water shell model of protein encapsulation, and excellent agreement was obtained. At low molar ratio of water to surfactant (w0 = 5.6), the hydrodynamic radius of filled droplets is significantly larger than the radius of empty ones. Under these conditions, about three empty (water-filled) droplets are required to build up a droplet of sufficient size to accommodate a single protein molecule. At maximum solubilization, which occurs at w0 = 5.6, a small fraction of droplets are found containing protein aggregates. In contrast, results at high values of w0 (22.4) reveal radii for empty and occupied droplets of comparable dimension, and the absence of aggregates. The results are discussed in terms of the model and the mechanism of interaction of this protein with the aqueous interfaces provided by these membrane-mimetic systems.     

Solubilization of Tea Seed Oil in a Food-Grade Water-Dilutable Microemulsion

Food-grade microemulsions containing oleic acid, ethanol, Tween 20, and water were formulated as a carrier system for tea seed oil (Camellia oleifera Abel.). The effect of ethanol on the phase behavior of the microemulsion system was clearly reflected in pseudo-ternary diagrams. The solubilization capacity and solubilization efficiency of tea seed oil dispersions were measured along the dilution line at a 70/30 surfactant/oil mass ratio with Tween 20 as the surfactant and oleic acid and ethanol (1:3, w/w) as the oil phase. The dispersed phase of the microemulsion (1.5% weight ratio of tea seed oil to the total amount of oil, surfactant, and tea seed oil) could be fully diluted with water without phase separation. Differential scanning calorimetry and viscosity measurements indicated that both the carrier and solubilized systems underwent a similar microstructure transition upon dilution. The dispersion phases gradually inverted from the water-in-oil phase (< 35% water) to the bicontinuous phase (40–45% water) and finally to the oil-in-water phase (> 45% water) along the dilution line.     

Studies on the catalytic behaviour of a cholinesterase-like abzyme in an AOT microemulsion system

The hydrolytic activity of a monoclonal catalytic antibody (9A8) (abzyme) with acetylcholinesterase-like activity was investigated in water-in-oil (w/o) microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane, using p- and o-nitrophenylacetate (p-and o-NPA) as substrates. The dependence of the abzyme hydrolytic activity on the molar ratio of water to surfactant (w(o)) showed a bell-shaped curve, presenting a maximum at w(o)=11.1. An increase of the AOT concentration at constant w(o), resulted in a decrease of the catalytic activity suggesting a possible inhibition effect of the surfactant. The incorporation of the abzyme into the reverse micelle system caused a blue shift of the fluorescence emission maximum by a magnitude of 7-10 nm depending on the w(o) value. This result indicates that the antibody molecule, or a large part of it, is located in the aqueous microphase of the system. Kinetic studies showed that the hydrolysis of p-and o-NPA in microemulsion system as well as in aqueous solution follows Michaelis-Menten kinetics. The catalytic efficiency (k(cat)/K(m)) in w/o microemulsion was significant lower than in aqueous solution.     

Development and antifungal evaluation of a food-grade U-type microemulsion

Aims: Food-grade microemulsions have been of increasing interest to researchers as potential delivery systems for bioactive compounds. However, food-grade microemulsions are difficult to formulate and no microemulsion has been documented for antifungal purpose. The physicochemical characterization of a food-grade glycerol monolaurate (GML)/ethanol (EtOH)/Tween 80/potassium sorbate (PS)/water microemulsion system and the antifungal activities against Aspergillus niger and Penicillium italicum have been studied in this paper. Methods and Results: The influence of EtOH and PS on oil solubilization capability was clearly reflected in the phase behaviour of U-type microemulsion systems. One dilution-stable formulation ME (GML/EtOH/Tween 80/PS/water = 3 : 3 : 3·5 : 10·5 : 16) was selected. After 4 days of incubation, ME showed 80%A. niger growth inhibition at 0·2% and 72%P. italicum growth inhibition at 0·1%, respectively, and a delay of conidiation of 2 days compared with the control. In the antifungal activities of the microemulsion, GML and PS made major contributions with similar antifungal activities at a GML/PS weight ratio of 1: 3·5. Conclusions: Food-grade dilution-stable microemulsions prepared with GML as oil phase for antifungal purpose are feasible and solubilization of a hydrotrope contributes to the formation of microemulsions and enhanced antifungal activities. Significance and Impact of the Study: The present report represents the first to develop a food-grade microemulsion system for antifugal purpose.     

Evaluation of steady-state kinetic parameters for enzymes solubilized in water-in-oil microemulsion systems.

1. Equations are derived for the steady-state kinetics of substrate conversion by enzymes confined within the water-droplets of water-in-oil microemulsion systems. 2. Water-soluble substrates initially confined within droplets that do not contain enzyme are assumed to be converted into product only after they enter enzyme-containing droplets via the inter-droplet exchange process. 3. Hyperbolic (Michaelis-Menten) kinetics are predicted when the substrate concentration is varied in microemulsions of fixed composition. Both kcat. and Km are predicted to be dependent on the size and concentration of the water-droplets in the microemulsion. 4. The predicted behaviour is shown to be supported by published experimental data. A physical interpretation of the form of the rate equation is presented. 5. The rate equation for an oil-soluble substrate was derived assuming a pseudo-two-phase (oil & water) model for the microemulsion. Both kcat. and Km are shown to be independent of phi aq. Km is larger than the aqueous solution value by a factor approximately equal to the oil/water partition coefficient of the substrate. The validity of the rate equation is confirmed by published data.     

Study of isopropyl myristate microemulsion systems containing cyclodextrins to improve the solubility of 2 model hydrophobic drugs

The objectives of this project were to evaluate the effect of alkanols and cyclodextrins on the phase behavior of an isopropyl myristate microemulsion system and to examine the solubility of model drugs. Triangular phase diagrams were developed for the microemulsion systems using the water titration method, and the solubility values of progesterone and indomethacin were determined using a conventional shake-flask method. The water assimilation capacities were determined to evaluate the effective microemulsion formation in different systems. The alkanols showed higher microemulsion formation rates at higher concentrations. A correlation between the carbon numbers of the alkanol and water assimilation capacity in the microemulsions studied was observed; isobutanol and isopentanol produced the best results. The addition of cyclodextrins showed no effect or had a negative effect on the microemulsion formation based on the type of cyclodextrin used. Isopropyl myristate-based microemulsion systems alone could increase the solubility values of progesterone and indomethacin up to 3300-fold and 500-fold, respectively, compared to those in water. However, the addition of cyclodextrins to the microemulsion systems did not show a synergistic effect in increasing the solubility values of the model drugs. In conclusion, microemulsion systems improve the solubility of progesterone and indomethacin. But the two types of cyclodextrins studied affected isopropyl myristatebased microemulsion systems negatively and did not improve the solubilization of 2 model drugs.     

A New Approach for Increasing Ascorbyl Palmitate Stability by Addition of Non-irritant Co-antioxidant

The aim of this work was to test innovative approach for enhancing ascorbyl palmitate stability in microemulsions for topical application by addition of newly synthesized co-antioxidant 4-(tridecyloxy)benzaldehyde oxime (TDBO) and to investigate its antioxidant activity and finally to evaluate cytotoxicity of TDBO-loaded microemulsions on keratinocyte cells. TDBO significantly increased ascorbyl palmitate stability in oil-dispersed-in-water (o/w) microemulsions, most presumably due to reduction of ascorbyl palmitate radical back to ascorbyl palmitate, since TDBO free-radical scavenging activity was confirmed. Cytotoxicity experiments demonstrated no significant change in cell viability or morphology in the presence of TDBO-loaded microemulsions regarding unloaded microemulsions, although greater cytotoxicity was observed with increased microemulsion concentrations. Therefore, the incorporation of TDBO as non-cytotoxic co-antioxidant in o/w microemulsions is a promising new strategy for enhancing ascorbyl palmitate stability that could be used to support antioxidant network in the skin.     

Solubilization and interaction of α-tocopherol in water-aerosol OT-isooctane systems

Solubilization and interaction of α-tocopherol into bis(2-ethylhexyl)sulphosuc cinate sodium salt microemulsion systems have been studied by temperature dependent phase transition, viscosity and nuclear magnetic resonance studies. Tocopherol being an amphiphilic molecule dissolves into the interfacial surfactant monolayer of the microemul sion droplets. The dissolution leads to an enhancement of the rigidity of the surfactant monolayer as studied by the increase in mixing and phase transition temperatures of the microemulsion droplets. Solubilization of tocopherol into microemulsion droplets causes an increase in the effective size of the droplet and as a consequence, the inter-droplet interactions are also increased. The water binding capacity of the surfactant (bis(2-ethylhexyl)sulphosuccinate sodium salt) is reduced due to solubilization of tocopherol as is evidenced from the downfield shifts of water proton magnetic resonances. In the presence of the dissolved electrolytes into the aqueous core, tocopherol is squeezed out of the microemulsion droplets increasing the membrane fluidity and permeability.     

Kinetic studies of Chromobacterium viscosum lipase in AOT water in oil microemulsions and gelatin microemulsion-based organogels

Kinetic studies have shown that octyl decanoate synthesis by Chromobacterium viscosum (CV) lipase in sodium bis-2-(ethylhexyl) sulfosuccinate (AOT) water in oil (w/o) microemulsions occurs via the nonsequential (ping-pong) bi bi mechanism. There was evidence of single substrate inhibition by decanoic acid at high concentrations. Initial rate data yielded estimates for acid and alcohol Michaelis constants of ca. 10(-1) mol dm(-3) and a maximum rate under saturation conditions of ca. 10(-3) mol dm(-3) s(-1) for a lipase concentration of 0.36 mg cm(-3). CV lipase immobilized in AOT microemulsion-based organogels (MBGs) was also found to catalyze the synthesis of octyl decanoate according to the ping-pong bi bi mechanism. Reaction rates were similar in the free and immobilized systems under comparable conditions. Initial rates at saturating (but noninhibiting) substrate concentrations were first order with respect to CV lipase concentration in both w/o microemulsions and the MBG/oil systems. Gradients yielded an apparent k(cat) = 4.4 x 10(-4) mol g(-1) s(-1) in the case of w/o microemulsions, and 6.1 x 10(-4) mol g(-1) s(-1) for CV lipase immobilized in the MBGs. A third system comprising w/o microemulsions containing substrates and gelatin at concentrations comparable to those employed in the MBG formulations, provided a useful link between the conventional liquid microemulsion medium and the solid organogels. The nongelation of these intermediate systems stems from the early inclusion of substrate during a modified preparative protocol. The presence of substrate appears to prevent the development of a percolated microstructure that is thought to be a prerequisite for MBG formation. FT-NMR was employed as a semicontinuous in situ assay procedure. The apparent activity expressed by CV lipase in compositionally equivalent liquid and solid phase gelatin-containing systems was similar. An apparent activation energy of 24 +/- 2 kJ mol(-1) was determined by (1)H-NMR for esterification in gelatin-containing w/o microemulsions. This value agrees with previous determinations for CV lipase-catalyzed synthesis of octyl decanoate in "conventional" w/o microemulsions and MBG/oil systems. The similarities in lipase behavior are consistent with the claim, based largely on structural measurements, that the physico-chemical properties of the lipase-containing w/o microemulsion are to a large extent preserved on transformation to the daughter organogel. The close agreement of apparrent activation energies suggests that substrate mass transfer is not rate determining in the three studied systems. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54:416-427, 1997.     

Stability of bilirubin oxidase in organic solvent media: a comparative study on two low-water systems

The storage stability of bilirubin oxidase was studied in water-in-oil CTAB microemulsions with a chloroformrich continuous organic phase. The kinetics of the inactivation process were best described by a double exponential equation. Approximately half of enzymatic activity was lost during a "fast" phase with a half life of ca. 50 min, whereas the remaining activity was lost much more slowly (half life ca. 1000 min). Rates of inactivation were not affected significantly by variation of either solvent composition or concentration of water droplets, but inactivation was more rapid when droplet size was very small. Steady-state enzyme kinetics were studied at various stages in the inactivation process, and it was shown that inactivation occurred without change in the K(m) of the enzyme for bilirubin. Stability was also studied in a liquid/solid two-phase system; it was found that the inactivation process in this system; it was found that the inactivation process in this system was best described by a single exponential term. The rate was similar to the "fast" phase rate observed in the water-in-oil microemulsion system. Inactivation of the enzyme slow. Addition of the surfactant CTAB to the aqueous environment increased the rate of inactivation to levels comparable to those of the "slow" phase observed in water-in-oil microemulsions. (c) 1993 Wiley & Sons, Inc.     

Release and recovery of porcine pepsin and bovine chymosin from reverse micelles: a new technique based on isopropyl alcohol addition.

After complete solubilization by the direct method, porcine pepsin was not released from AOT in isooctane reverse micelles even under aqueous-phase conditions which would not ordinarily allow uptake. Similarly, bovine chymosin, once forward-transferred at a pH below its isoelectric point, was not back-transferred into an aqueous contact phase buffered at a pH value above its isoelectric point. These results show that there is significant hysteresis in the forward- and backward-transfer processes and further imply that kinetics, and not equilibrium, control uptake or release processes for these enzymes. The addition of 10-15% isopropyl alcohol to the aqueous phase increases the rate of protein release dramatically and allows for nearly complete back-transfer of porcine pepsin and 70% back-transfer of bovine chymosin. IPA addition does not destroy the functional integrity of the system since forward transfer of bovine chymosin still occurs at pH values below (but not above) the pI of the protein.     

Squalene epoxide cyclase and microemulsion

Squalene epoxide cyclase was extracted from microsomal preparations of rat liver using anionic, cationic and non-ionic microemulsions. The anionic microemulsion was the best with respect to protein solubilisation, extracted cyclase activity and stability of this activity over time. The activity assay showed cyclase activity to be higher in anionic microemulsion than in buffer in the presence of surfactant. Calcium chloride in the anionic microemulsion had a stabilising effect and less total protein seemed to be extracted.     

Temperature-Sensitive Microemulsion Gel: An Effective Topical Delivery System for Simultaneous Delivery of Vitamins C and E

Microemulsions (ME)—nanostructured systems composed of water, oil, and surfactants—have frequently been used in attempts to increase cutaneous drug delivery. The primary objective addressed in this work has been the development of temperature-sensitive microemulsion gel (called gel-like ME), as an effective and safe delivery system suitable for simultaneous topical application of a hydrophilic vitamin C and a lipophilic vitamin E. By changing water content of liquid o/w ME (o/w ME), a gel-like ME with temperature-sensitive rheological properties was formed. The temperature-driven changes in its microstructure were confirmed by rotational rheometry, viscosity measurements, and droplet size determination. The release studies have shown that the vitamins’ release at skin temperature from gel-like ME were comparable to those from o/w ME and were much faster and more complete than from o/w ME conventionally thickened with polymer (o/w ME carbomer). According to effectiveness in skin delivery of both vitamins, o/w ME was found the most appropriate, followed by gel-like ME and by o/w ME carbomer, indicating that no simple correlation between vitamins release and skin absorption could be found. The cytotoxicity studies revealed good cell viability after exposure to ME and confirmed all tested microemulsions as nonirritant. This work was supported by a grant of Slovenian Research Agency.     

Formulation of Microemulsion Systems for Dermal Delivery of Silymarin

Silymarin is a standardized extract from Silybum marianum seeds, known for its many skin benefits such as antioxidant, anti-inflammatory, and immunomodulatory properties. In this study, the potential of several microemulsion formulations for dermal delivery of silymarin was evaluated. The pseudo-ternary phase diagrams were constructed for the various microemulsion formulations which were prepared using glyceryl monooleate, oleic acid, ethyl oleate, or isopropyl myristate as the oily phase; a mixture of Tween 20®, Labrasol®, or Span 20® with HCO-40® (1:1 ratio) as surfactants; and Transcutol® as a cosurfactant. Oil-in-water microemulsions were selected to incorporate 2% w/w silymarin. After six heating–cooling cycles, physical appearances of all microemulsions were unchanged and no drug precipitation occurred. Chemical stability studies showed that microemulsion containing Labrasol® and isopropyl myristate stored at 40°C for 6 months showed the highest silybin remaining among others. The silybin remainings depended on the type of surfactant and were sequenced in the order of: Labrasol® > Tween 20® > Span 20®. In vitro release studies showed prolonged release for microemulsions when compared to silymarin solution. All release profiles showed the best fits with Higuchi kinetics. Non-occlusive in vitro skin permeation studies showed absence of transdermal delivery of silybin. The percentages of silybin in skin extracts were not significantly different among the different formulations (p > 0.05). Nevertheless, some silybin was detected in the receiver fluid when performing occlusive experiments. Microemulsions containing Labrasol® also were found to enhance silymarin solubility. Other drug delivery systems with occlusive effect could be further developed for dermal delivery of silymarin.Key words: dermal delivery, microemulsion, silybin, silymarin     

Swelling lecithin: cholesterol implants for the controlled release of proteins

This work demonstrated the effect of two salts as potential simple formulation excipients in modifying hydration properties, phase behavior, and protein release from lecithin-based implants. In vitro release of a model protein, bovine serum albumin (BSA), from cylindrical-shaped lecithin and lecithin:cholesterol (1:1 w/w) implants containing 0, 10, or 30% w/w NaCl or CaCl₂ was studied. In the absence of salts, BSA was released from lecithin and lecithin:cholesterol implants with a high monomer content and the release profiles were similar to those previously reported. Cholesterol increased the swelling, induced the formation of myelin structures, and reduced BSA release from the matrices. Addition of the salts to lecithin:cholesterol implants further enhanced the swelling, altered the hydrated morphology, and inhibited protein release. Analyses showed that BSA associated into multimers within these swollen lipid matrices but retained a high degree of protein native structure. Factors that may have contributed to the inhibition of the in vitro release included 1) the swollen multilamellar layers assembled as diffusional barriers, 2) adsorption of BSA onto the hydrated lipid vesicles, and 3) formation of protein aggregates.     

Interactions of native and modified cytochrome C with a negatively charged reverse micellar liquid interface

Native and chemically modified cytochrome C were dissolved in sodium bis(2-ethylhexyl) sulphosuccinate (AOT)-oil-buffer microemulsions. The native cytochrome C contains 19 lysine residues, these groups were modified by 1) acetic anhydride or 2) succinic anhydride. At pH 8.4 the native, acetylated and succinylated proteins carry +8, –3 and –12 elementary charges, respectively. The phase behaviour of the microemulsion systems was found to be highly dependent on the charge of the proteins. Compared to a protein free system the native protein induces a L-2 phase separation at lower temperatures. The acetylated protein has a small effect on the temperature for the phase transition, whereas in the case of succinylated cytochrome C the phase transition takes place at higher temperatures. When dissolved in AOT microemulsions, the native cytochrome C has a perturbed tertiary structure, as indicated by loss of the 695 nm absorption band, while both the modified proteins retain the same optical properties when dissolved in an AOT microemulsion as in a pure buffer solution. The pertubed structure of the native cytochrome C was further investigated by testing the stability of the reduced form of the protein dissolved in the microemulsion media. The native cytochrome is unstable at W > 10, whereas the two modified proteins were found to be stable at all W-values investigated. The average location of the three proteins was determined by pulse radiolysis. The quenching rate constant of the hydrated electron depends upon the location of the probe in the reverse micelle; the succinylated protein is localised in the aqueous core of the reverse micelles, but both the native and the acetylated forms were found to be localised close to or at the AOT interface.     

Beta-hydroxysteroid dehydrogenase: activity in microemulsion and extraction from Pseudomonas testosteroni cells with microemulsion

The stability of purified beta-hydroxysteroid dehydrogenase activity measured as a function of time was good in buffered cationic and non-ionic microemulsions. The use of 1-pentanol and 1-hexanol in place of 1-butanol as cosurfactant gave increased activity and stability. The NAD+ Michaelis constant was 0.22 mM in buffer and 3.5 mM in waterpool concentration in microemulsion. Proteins, among them beta-hydroxysteroid dehydrogenase, were extracted from Pseudomonas testosteroni with cationic microemulsion, thus indicating that microemulsions may be utilized in protein release from cells.     

Fabrication of Transparent Lemon Oil Loaded Microemulsions by Phase Inversion Temperature (PIT) Method: Effect of Oil Phase Composition and Stability after Dilution

This research was addressed to develop transparent microemulsions as delivery system of lemon oil. To this aim, phase inversion temperature (PIT) method was employed. The effect of the surfactant Tween 80 content as well as lipid phase type and concentration (lemon oil, peanut oil and their mixtures) on microemulsion characteristics was studied. Transparent emulsions were obtained up to 1.3 and 7.5% (w/w) of lemon oil and peanut oil, respectively. Only by considering as lipid phase a mixture of lemon oil and peanut oil, it was possible to increase the delivering capacity of emulsions up to 15% of lemon oil (total oil phase 20%). Therefore, blending peanut oil rich in long chain fatty acids with lemon oil expanded the lipid phase loading capacity of microemulsions while maintaining particle size lower than 30 nm and thus system transparency. Microemulsions showed good dilutability in aqueous solutions simulating beverage formulations with different pH values.