Formulation and Structural Study of a Biocompatible Water-in-Oil Microemulsion as an Appropriate Enzyme Carrier: The Model Case of Horseradish Peroxidase.

A novel biocompatible water-in-oil microemulsion was developed using nonionic surfactants and was investigated as a potential enzyme delivery system for pharmaceutical applications. The system was composed of isopropyl myristate/polysorbate 80 (Tween 80)/distilled monoglycerides/water/propylene glycol (PG), had a low total surfactant concentration (8.3% w/w), and was able to incorporate approximately 3% w/w aqueous phase containing horseradish peroxidase (HRP). Structural and activity aspects of the system were studied using a variety of techniques such as dynamic light scattering (DLS), electron paramagnetic resonance (EPR), and dynamic interfacial tension. The apparent hydrodynamic diameter of the empty droplets was calculated at about 37 nm. Different enzyme concentrations, ranging from 0.01 to 1.39 µM, were used for both DLS and EPR studies to effectively determine the localization of the macromolecule in the microemulsion. According to the results, for high enzyme concentrations, a participation of HRP in the surfactant monolayer of the microemulsion is evident. The number of reverse micelles in the microemulsion was defined by a theoretical model and was used to clarify how the enzyme concentration affects the number of empty and loaded reverse micelles. To assure that the system allows the enzyme to retain its catalytic activity, an oxidative reaction catalyzed by HRP was successfully carried out with the use of the model substrate 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]. The influence of several parameters such as temperature, pH, and PG concentration was examined to optimize the reaction conditions, and a kinetic study was conducted revealing an ordered-Bi-Bi mechanism. Values of all kinetic parameters were determined. The release of the encapsulated enzyme was studied using an adequate receiver phase, revealing the effectiveness of the proposed microemulsion not only as a microreactor but also as a carrier for therapeutic biomolecules.

Nanoencapsulated Lecitase Ultra and Thermomyces lanuginosus Lipase, a Comparative Structural Study.

Two commercially available and widely used enzymes, the parent Thermomyces lanuginosus lipase (TLL) and the shuffled phospholipase A1 Lecitase (Lecitase Ultra), were encapsulated in AOT/isooctane reverse micelles and evaluated regarding their structure and activity. Preparations were also tested as effective biocatalysts. Small-angle X-ray scattering (SAXS), electronic paramagnetic resonance (EPR), and fluorescence spectroscopy were the techniques applied to assess the effects of enzyme incorporation to a reverse micellar nanostructure. SAXS analysis showed that the radius of gyration (Rg) changed from 16 to 38 Å, as the water content (w0) increased. Elongated shapes were more commonly observed than spherical shapes after enzyme encapsulation. EPR studies indicated that enzymes do not participate in the interface, being located in the aqueous center. Fluorescence energy transfer showed that TLL is located in the water core, whereas Lecitase Ultra is closer to the interface. Enzymatic activity toward a standard esterification reaction endured after the enzyme was incorporated into the micelles. The activity of TLL for systems with w0 15 showed the highest conversion yield, 38% in 2 h, while the system with w0 10 showed the highest initial velocity, 0.43 µM/min. This last system had a Rg of 19.3 Å, similar to that of the TLL monomer. Lecitase Ultra showed the highest conversion yields in systems with w0 10, 55% in 2 h. However, the initial rate was much lower than that of TLL, suggesting less affinity for the substrates, which is expected since Lecitase Ultra is a phospholipase. In summary, we here used several spectroscopic and scattering techniques to reveal the shape and stability of TTL and Lecitase Ultra encapsulated systems, which allowed the selection of w0 values to provide optimized enzymatic activity.

Exploring and strengthening the potential of R-phycocyanin from Nori flakes as a food colourant.

This study aimed to purify, characterise and stabilise the natural food colourant, R-phycocyanin (R-PC), from the red algae Porphyra spp. (Nori). We purified R-PC from dried Nori flakes with a high purity ratio (A618/A280 ≥ 3.4) in native form (α-helix content 53%). SAXS measurements revealed that R-PC is trimeric ((αß)3) in solution. The thermal denaturation of α-helix revealed one transition (Tm at 52 °C), while the pH stability study showed R-PC is stable in the pH range 4-8. The thermal treatment of R-PC at 60 °C has detrimental and irreversible effects on R-PC colour and antioxidant capacity (22 % of residual capacity). However, immobilisation of R-PC within calcium alginate beads completely preserves R-PC colour and mainly retains its antioxidant ability (78 % of residual capacity). Results give new insights into the stability of R-PC and preservation of its purple colour and bioactivity by encapsulation in calcium alginate beads.

(Hydroxypropyl)methyl Cellulose-Chitosan Film as a Matrix for Lipase Immobilization-Part ΙΙ: Structural Studies.

The present work reports on the structural study of a film made of a hybrid blend of biopolymers used as an enzyme carrier. A cellulose derivative (HPMC) and chitosan (CS) were combined in order to formulate a film on which Mucor miehei lipase was immobilized. The film was successfully used as a biocatalyst; however, little is known about the structure of the system. Therefore, small-angle X-ray scattering, Fourier transform infrared spectroscopy (FTIR), optical microscopy, and scanning electron microscopy (SEM), as well as microindentation measurements, were used to shed light on the structure of the promising biocatalyst. Among the results, intermolecular hydrogen bonds were observed between the amide groups of the two polymers and the lipase. The presence of the enzyme does not seem to affect the mechanical properties of the matrix. The used film after 35 cycles of reaction seemed to be fatigued and had lost part of its humidity, explaining the reduction of the enzyme activity.

Development of a microemulsion for encapsulation and delivery of gallic acid. The role of chitosan.

A novel water-in-oil (W/O) microemulsion based on natural oils, namely extra virgin olive oil (EVOO) and sunflower oil (SO), in the presence of non-ionic surfactants was successfully formulated. The novel microemulsion was used as a carrier for gallic acid (GA) to assure its protection and efficacy upon nasal administration. The work presents evidence that this microemulsion can be used as a nasal formulation for the delivery of polar antioxidants, especially, after incorporation of chitosan (CH) in its aqueous phase. The structure of the system was studied by Small Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS) and Electron Paramagnetic Resonance (EPR) spectroscopy techniques. By the addition of CH, the diameter of the microemulsion remained unaltered at 47 nm whereas after the incorporation of GA, micelles with 51 nm diameter were detected. The dynamic properties of the surfactant monolayer were affected by both the incorporation of CH and GA. Moreover, the antioxidant activity of the latter remained unaltered (99 %). RPMI 2650 cell line was used as the in vitro model for cell viability and for GA nasal epithelial transport studies after microemulsion administration. The results suggested that the nasal epithelial permeation of GA was enhanced, 3 h post administration, by the presence of 0.2 % v/v microemulsion in the culture medium. However, the concentration of the transported antioxidant in the presence of CH was higher indicating the polymer's effect on the transport of the GA. The study revealed that nasal administration of hydrophilic antioxidants could be used as an alternative route besides oral administration.

Structural Study of (Hydroxypropyl)Methyl Cellulose Microemulsion-Based Gels Used for Biocompatible Encapsulations.

(Hydroxypropyl)methyl cellulose (HPMC) can be used to form gels integrating a w/o microemulsion. The formulation in which a microemulsion is mixed with a hydrated HPMC matrix has been successfully used as a carrier of biocompatible ingredients. However, little is known about the structure of these systems. To elucidate this, scanning electron microscopy was used to examine the morphology and the bulk of the microemulsion-based gels (MBGs) and small-angle X-ray scattering to clarify the structure and detect any residual reverse micelles after microemulsion incorporation in the gel. Electron paramagnetic resonance spectroscopy was applied using spin probes to investigate the polar and non-polar areas of the gel. Furthermore, the enzyme-labelling technique was followed to investigate the location of an enzyme in the matrix. A structural model for HPMC matrix is proposed according to which, although a w/o microemulsion is essential to form the final gel, no microemulsion droplets can be detected after incorporation in the gel. Channels are formed by the organic solvent (oil), which are coated by surfactant molecules and a water layer in which the enzyme can be hosted.

Spectroscopic and catalytic studies of lipases in ternary hexane-1-propanol-water surfactantless microemulsion systems.

A series of water-in-oil microemulsion systems formulated without surfactant were used to solubilize lipases from Rhizomucor miehei and Candida antarctica B. The effect of the system's composition on the velocity of enzymic reactions was investigated following a model esterification reaction. The interaction between enzymes and the microemulsion environment was studied by steady state fluorescence spectroscopy. The site of localization of the enzyme within the different microdomains of the dispersed phase was investigated by applying the fluorescence energy transfer technique. To determine the properties of the interface between water and organic solvent of the surfactantless microemulsion systems the Electron Paramagnetic Resonance (EPR) spectroscopic technique was applied. The results indicated that even at low water content, water-rich structures are formed. This was confirmed by conductivity measurements. By the addition of enzyme it was observed that when the aqueous phase of the surfactantless microemulsion systems exceeds 2% (v/v) the enzyme retains its catalytic activity, as it is located within the water pools that protect it from the organic solvent. These confined water phases show a propanol rich interface with hexane and their structure depends on the system's composition.

Biocolloids based on amphiphilic block copolymers as a medium for enzyme encapsulation.

The ability of two biocompatible amphiphilic block copolymers consisting of hydrophilic poly(ethylene oxide) and hydrophobic poly(ε-caprolactone) with different hydrophilic/hydrophobic block ratio to act as stabilizers of water-in-oil (w/o) microemulsions and enzyme encapsulation therein has been tested. Phase diagrams of the two block copolymers in mixtures of chloroform/isopropanol/water were constructed, revealing that the systems can incorporate important amounts of aqueous phase. The w/o microemulsions were then used to encapsulate R. miehei lipase. Empty as well as lipase-loaded systems were characterized by DLS as well as EPR spectroscopy. It was found that the incorporated lipase was preferably localized in the interior of the droplets. The apparent hydrodynamic radii of the droplets were found to vary from 86 to 3000 nm and from 66 to 2140 nm for empty PEO-PCL 30 and PEO-PCL 53 stabilized systems, respectively. In the presence of the lipase, the hydrodynamic radii were considerably decreased. The catalytic activity of the encapsulated lipase was successfully tested via a model esterification reaction. The effect of temperature on the catalytic behavior of the encapsulated R. miehei lipase was investigated, revealing that the initial rate of the esterification reaction depended on the type of the block copolymer used.

Microemulsion-based organogels as matrices for lipase immobilization.

Organogels based on water-in-oil microemulsions can be formed using various natural polymers such as gelatin, agar or cellulose derivatives. Enzymes entrapped in the water core of the microemulsion can keep their activity and enhance their stability within the gel matrix. The importance of the microemulsion based organogels (MBGs) leans on their numerous potential biotechnological applications. An important example is the use of various lipase microemulsion systems for hydrolytic or synthetic reactions. In this review, several MBGs are being evaluated as immobilization matrices for various enzymes. The main subject focuses on the parameters that affect the use of MBGs as media for bioorganic reactions using lipases as catalysts.

Biocompatible microemulsions based on limonene: formulation, structure, and applications.

The preparation of biocompatible (w/o) microemulsions based on R-(+)-limonene, water, and a mixture of lecithin and either 1-propanol or 1,2-propanediol as emulsifiers was considered. The choice of the compositions of the microemulsions used was based on the pseudo-ternary phase diagrams of the four-component system determined at 30 degrees C for different weight ratios of the components. When 1-propanol was considered as co-surfactant, the area of the microemulsion zone was remarkably increased. Interfacial properties and the dynamic structure of the emulsifier's monolayer were studied by electron paramagnetic resonance (EPR) spectroscopy using the spin-labeling technique. The rigidity and polarity of the interface were affected by the nature of the alcohol used as co-surfactant. When 1-propanol was used, the emulsifier's interface was much more flexible, indicating a less tight packing of lecithin molecules than in the case of 1,2-propanediol. In addition, the membrane's polarity was decreased when the diol was added as co-surfactant in the microemulsion system. To evaluate the size of the dispersed aqueous domains as a function of water content and other additives concentration, dynamic light scattering (DLS) measurements were carried out. Radii in the range from 60 to 180 nm were observed when 1-propanol was used as co-surfactant, and the water content varied from 0 to 12% w/w. Electrical conductivity measurements of R-(+)-limonene/lecithin/1-propanol/water microemulsions with increasing weight fractions of water indicated the appearance of a percolation threshold at water content above 4% w/w. Lipase from Rhizomucor miehei was solubilized in the aqueous domains of the biocompatible microemulsions, and the esterification of octanoic, dodecanoic, and hexadecanoic acids with the short-chained alcohols used as co-surfactants for the formulation of microemulsions was studied. The enzyme efficiency was affected by the chain length of the carboxylic acids and the nature of the alcohol. In the case of 1-propanol, a preference for the long-chain carboxylic acids was observed. On the contrary, when 1,2-propanediol was used formulation of the corresponding esters was not observed. This behavior could be possibly attributed to either the specificity of the lipase toward the alcohol employed for the esterification of the acids or the structural changes induced in the system when 1-propanol was replaced by 1,2-propanediol.