Reverse micellar entrapment

As mentioned above, water structure in reversed micelles deviates considerably from the structure in the bulk-phase. Therefore, the hydration shell of macromolecules entrapped in reversed micellar systems should be changed and thus also their conformation. According to the results of several authors this is indeed the case. 

In addition to solubilization, entrapment of polymers inside reversed micelles can be achieved by performing in situ suitable polymerization reactions. This methodology has some specific peculiarities, such as easy control of the polymerization degree and synthesis of a distinct variety of polymeric structures. The size and shape of polymers could be modulated by the appropriate selection of the reversed micellar system and of synthesis conditions [31,191]. This kind of control of polymerization could model and/or mimic some aspects of that occurring in biological systems. 

In addition, reversed micelles have been used as scaffolding to immobilize proteins via entrapment into gels. Gel formation is induced through addition of gelatin,22 phenols,222 and phospholipid.2 ° With the exception of gels, development of large-scale reverse micellar enzyme processes are difficult due to the inherent batch nature of the medium and complications for downstream separations induced by the surfactant. See reviews cited above for further detail. 

In the literature the possibilities of practical uses of enzymes entrapped in reverse micellar systems were repeatedly discussed (see, e.g., reviews [7,8] and also topical compilation [62-64]). We will further consider some examples from the most prospective areas fine chemistry, chemical analysis, and supramolecular design. 

In a reverse micellar system containing NaPtCl4 in D2O, AOT, and the substrate, CH/CD exchange occurs under mild conditions (80 °C) regioselective in the methyl group. One of the main fields of application of reverse micelles is the entrapment of enzymes in the water cavity [28]. 

Reverse micellar extraction (RME) has been gaining popularity as an attractive hquid-hquid extraction process [108-111]. This is mainly due to the fact that enzymes can be solubilized in organic solvents with the aid of reverse micellar aggregates [112, 113]. Their inner core contains an aqueous micro-phase, which is able to solubilize polar substances, e.g., hydrophilic enzymes [114]. In many cases not only the enzymes retained their activity in organic environment in some cases they seem to perform even better if they are entrapped into reverse micellar aggregates [111]. One of the remarkable findings that gave this field a major boost is that the solubilization of different proteins into micellar solutions is a selective process [112]. 

The liquid gelators, Span 80-Tween 80 also forms emulsion organogels and emulsion hydrogels by fluid-filled fiber mechanism. It has been reported that Span 80 (sorbitan monooleate) and Tween 80 (polyoxyethylene sorbitan monooleate) mixed in the ratio of 1 2 w/w forms organogel with better firmness and architecture as compared to the other surfactant mixture ratios.When water is added dropwise into the homogenous surfactant mixture and oil, it forms spherical reverse-micellar droplets. These droplets/fibers self-assemble to form three-dimensional architecture to immobilize apolar solvent. " Similarly, in case of hydrogels micellar structures are formed, which entraps the external liquid phase to flow and form hydrogel. 

Water, aqueous solutions and many other strongly hydrophilic substances can be solubilized within the micellar core [19,20], Water solubilization involves hydration of the surfactant headgroup accompanied by an increase in the head-group area, a micellar swelling, a marked increase in the surfactant aggregation number, and, at constant surfactant concentration, a decrease in the number density of reversed micelles [21], A representation of a spherical reverse micelle entrapping a polar solubilizate in the core is shown in Fig. 3. 

Reversed micellar enzymology has been studied by many researchers [1, 2]. The conversion of apolar compounds by enzymes entrapped in aqueous cores of reversed micelles has drawn the most attention. Reversed micellar enzymology requires not only a large-scale operation, but also enzyme and product recoveries for its practical application. There is still a lack of understanding of many aspects of enzyme behavior in reversed micelles. 

Multiple emulsions are usually not empty. Soluble active materials are entrapped during the emulsification in the inner oily phase. Because of the osmotic pressure gradient, the active matter tends to diffuse and migrate from the internal phase to the external interface mostly through a controlled reverse micellar transport mechanism (Figure 7.10a) (Garti and Bisperink, 1998 Garti and Benichou, 2001). The dilemma that researchers were faced with was how to control the diffusion of oil molecules, as well as the emulsifier molecules... 

Most of release studies are done in W/OAV multiple-emulsion systems where an active water soluble molecule is present in the inner aqueous phase. Several attempts have been made to explain the transport phenomena of entrapped addenda from the inner to the outer phase of multiple-emulsion droplets. It has been demonstrated that for lipid soluble material dissolved in the oil phase, the release obeys first-order kinetics and is diffusion controlled with excellent accordance to Pick s law. Two mechanisms for the permeation through the oil intermediate phase are well accepted, the first being via the reverse micellar transport (Figure 7.10 ) and the second via diffusion across a very thin lamellae of surfactant phase formed in areas where the oil layer is very thin (Figure 10b). 

At the inner phase, BSA provides a mechanical barrier to the release of small molecules from the internal interface. The release proceeds mainly via reverse micellar transport. The presence of BSA reduces the chance of reverse micelle formation and thus decreases the release rate of entrapped addenda within the emulsion droplets. 

Dynamic light-scattering experiments or the analysis of some physicochemical properties have shown that finite amounts of formamide, A-methylformamide, AA-dimethyl-formamide, ethylene glycol, glycerol, acetonitrile, methanol, and 1,2 propanediol can be entrapped within the micellar core of AOT-reversed micelles [33-36], The encapsulation of formamide and A-methylformamide nanoclusters in AOT-reversed micelles involves a significant breakage of the H-bond network characterizing their structure in the pure state. Moreover, from solvation dynamics measurements it was deduced that the intramicellar formamide is nearly completely immobilized [34,35],... 

Differential scanning calorimetry measurements have shown a marked cooling/heat-ing cycle hysteresis and that water entrapped in AOT-reversed micelles is only partially freezable. Moreover, the freezable fraction displays strong supercooling behavior as an effect of the very small size of the aqueous micellar core. The nonfreezable water fraction has been recognized as the water located at the water/surfactant interface engaged in solvation of the surfactant head groups [97,98]. 

Sometimes, the physicochemical properties of ionic species solubilized in the aqueous core of reversed micelles are different from those in bulk water. Changes in the electronic absorption spectra of ionic species (1 , Co ", Cu " ) entrapped in AOT-reversed micelles have been observed, attributed to changes in the amount of water available for solvation [2,92,134], In particular, it has been observed that at low water concentrations cobalt ions are solubihzed in the micellar core as a tetrahedral complex, whereas with increasing water concentration there is a gradual conversion to an octahedral complex [135],... 

The entrapment of a-chymotrypsin, lysozyme, and myehn in AOT-reversed micelles is accompanied by an increase in the micellar water content and in the size of the micelle. As a consequence of the redistribution of water among reversed micelles, the micellar solution results in being constituted by large protein-containing micelles and small unfilled ones [169],... 

Solutions of surfactant-stabilized nanogels share both the advantage of gels (drastic reduction of molecular diffusion and of internal dynamics of solubilizates entrapped in the micellar aggregates) and of nonviscous liquids (nanogel-containing reversed micelles diffuse and are dispersed in a macroscopicaUy nonviscous medium). Effects on the lifetime of excited species and on the catalytic activity and stability of immobilized enzymes can be expected. 

Solubilization of vinylpyrrolidone, acrylic acid, and A,A -methylene-bis-acrylamide in AOT-reversed micelles allowed the synthesis in situ of a cross-linked polymer with narrow size distribution confined in the micellar domain. These particles displayed high entrapment efficiency of small hydrophilic drugs and have been considered interesting drug delivery systems [239],... 

Upon reaction, the heterogenized catalyst can be easily separated from the reaction mixture by filtration and then recycled. The hydro-phobic substrate is microemulsified in water and subjected to an orga-nometallic catalyst, which is entrapped within a partially hydrophobized sol-gel matrix. The surfactant molecules, which carry the hydrophobic substrate, adsorb/desorb reversibly on the surface of the sol-gel matrix breaking the micellar structure, spilling their substrate load into the porous medium that contains the catalyst. A catalytic reaction then takes place within the ceramic material to form the desired products that are extracted by the desorbing surfactant, carrying the emulsified product back into the solution. 

In a liquid/liquid biphasic system (Figure 9.1a), the enzyme is in the aqueous phase, whereas the hydrophobic compounds are in the organic phase. In pure organic solvent (Figure 9.1b) a solid enzyme preparation is suspended in the solvent, making it a liquid/solid biphasic system. In a micellar system, the enzyme is entrapped in a hydrated reverse micelle within a homogeneous organic solvent... 

The structure of the AOT micellar system, as well as the state of water entrapped inside swollen micelles, have been characterized using different techniques, such as photon correlation spectroscopy (25), positron annihilation (26), NMR (27, 28), fluorescence (29-32) and more recently small angle neutron scattering (33). The existence of reversed micelles has been demonstrated in the domain of concentrations explored by protein extraction experiments. Their size (proportional to the molar ratio of water to surfactant known as wo), shape and aggregation number have been determined. Furthermore, the micelle size distribution is believed to be relatively monodisperse. 

FIG. 5 Regulation of relative maximal reaction rates (F/Kopt) for different enz5mes solubilized in AOT reverse micelles by variation of the degree of surfactant hydration (water-to-AOT molar ratio). The inset shows the correlation between the radius of an entrapped enzyme (fp) and corresponding optimal aqueous micellar cavity (v). (For details see Ref. 40.)... 

These assumptions are confirmed by experiments with the spin-labeled active center of a-chymotrypsin [41] (Fig. 6). As seen from the data in Fig. 6, the enzyme entrapped in reverse micelles is located in the medium with decreased polarity. In similar polar media of water-organic mixtures, enzyme structures are normally disrupted, and protein denatura-tion and loss of catalytic enzymic activity occur. Yet, in systems of reverse micelles a principally different picture is observed. In optimal enzyme activity conditions the protein becomes tightly fixed by the micellar matrix and its conformational mobility is minimized. 

Reverse micelles are normally used in enzyme-catalyzed reactions. The water in the core of the micelle is called the water pool. At a constant surfactant concentration, the amount of water introduced determines the micellar size. The nature of the entrapped water in reverse micelles has been a subject of considerable debate. At low amounts of water, it is thought that most of it is bound, leading to low enzyme activity. At higher amounts, the water becomes more free with a resultant increase in enzymatic activity. 

In this chapter I have attempted to present a panoramic view of the contributions of calorimetry to the study of solutions of reversed micelles. In particular, it has been shown that it is possible with calorimetry to obtain information on the energetic state of water and that of other solubilizates within reversed micelles, the complete set of thermodynamic parameters for the solubilization process, and the preferential solubilization site as well as information on the intermicellar interactions and the energetic state of solid nanoparticles entrapped in the micellar core. All these data together with those obtained by other techniques help to better and better define the structural and dynamical picture of solutions of reversed micelles and to exploit their potential technological applications. 

The catalytic behavior and the stability of enzymes in reverse micelles are highly dependent on the composition and the structure of the micioanulsion. The activity of entrapped enzymes strongly depends on the water content, the nature of the organic solvent, as well as the nature and the concentration of surfactant. Various surfactants, including the anionic AOT, the cationic CTAB, nonionics such as Triton, Brij, ethoxylated fatty alcohols, and zwitterionic phospholipids (phosphatidylcholine), were used for the preparation of reverse miceUar systems-containing enzymes (Table 13.1). Most inveshgated systans used AOT as the surfactant because its phase behavior is well understood. The activity of some enzymes has been reported to depend on the surfactant concentration and in some cases it was attributed to the interaction of the enzymes with the miceUar membrane [8,26,27]. Recent developments in this area inclnde the use of modified surfactants or their mixtures with other additives and cosurfactants such as alcohols and sugars or the use of aprotic solvents for the reduction of the ionic interactions between the enzyme molecules and the micellar interface in order to improve the enzyme catalytic behavior and operational stabihty [8,17,28-34]. 

Spectroscopic studies (fluorescence and circular dichroism) indicate that the variation in lipases stability is related to their hydrophobicity and therefore to the different degrees of interactions between the enzymes and the micellar interface [88]. Two important factors contribute to inducing conformational changes on the enzyme molecules in reverse micelles the unusual properties of the encapsulated water and the electrostatic interactions between ionic head groups of the surfactant and the polypeptide chains of the enzyme. Spectroscopic data on the structure of enzymes entrapped in reverse micelles are controversial, as regards the changes in the protein structure upon incorporation into reverse micelles (Table 13.3). 

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