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.

Microemulsions as Potential Carriers of Nisin: Effect of Composition on Structure and Efficacy.

Water-in-oil (W/O) microemulsions based on either refined olive oil (ROO) or sunflower oil (SO), distilled monoglycerides (DMG), and ethanol were used as nisin carriers in order to ensure its effectiveness as a biopreservative. This work presents experimental evidence on the effects of ethanol concentration, hydration, the nature of oil, and the addition of nisin on the nanostructure of the proposed inverse microemulsions as revealed by electrical conductivity measurements, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and electron paramagnetic resonance (EPR) spectroscopy. Modeling of representative SAXS profiles was applied to gain further insight into the effects of ethanol and solubilized water content on the inverse swollen micelles' size and morphology. With increasing ethanol content, the overall size of the inverse micelles decreased, whereas hydration resulted in an increase in the micellar size due to the penetration of water into the hydrophilic core of the inverse swollen micelles (hydration-induced swelling behavior). The dynamic properties of the surfactant monolayer were also affected by the nature of the used vegetable oil, the ethanol content, and the presence of the bioactive molecule, as evidenced by EPR spin probing experiments. According to simulation on the experimental spectra, two populations of spin probes at different polarities were revealed. The antimicrobial effect of the encapsulated nisin was evaluated using the well diffusion assay (WDA) technique against Lactococccus lactis. It was found that this encapsulated bacteriocin induced an inhibition of the microorganism growth. The effect was more pronounced at higher ethanol concentrations, but no significant difference was observed between the two used vegetable oils (ROO and SO).

Biocompatible colloidal dispersions as potential formulations of natural pyrethrins: a structural and efficacy study.

Biocompatible colloidal dispersions of the micro- and nanoemulsion type based on lemon oil terpenes, polysorbates, water, and glycerol were used for the formulation of pyrethrins, botanical insecticides derived from the white pyrethrum daisy, Tanacetum cinerariifolium. The proposed formulation is based on pyrethrin-containing water-in-oil (W/O) microemulsions that could be diluted in one step with an aqueous phase to obtain kinetically stable oil-in-water (O/W) nanoemulsions. Structural characteristics of the micro- and nanoemulsions were evaluated by electron paramagnetic resonance (EPR) spectroscopy, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and electrical conductivity. Dynamic properties of the surfactant monolayer as evidenced by EPR measurements were affected by the water content, the surfactant, and also the presence of the biocide. DLS and SAXS experiments of the nanoemulsions indicated the existence of two populations of oil droplets dispersed in the aqueous phase, globular droplets of 36-37 nm in diameter, and also larger droplets with diameters >150 nm. All of the applied techniques for structural determination revealed the participation of the biocide in the nanostructure. The insecticidal effect of the encapsulated natural pyrethrin was evaluated in laboratory bioassays upon a target-insect pest, the cotton aphid Aphis gossypii Glover (Hemiptera: Aphididae) in eggplant, and was found to be increased compared to the commercial pyrethrin formulation.

Encapsulation of cannabidiol in oil-in-water nanoemulsions and nanoemulsion-filled hydrogels: A structure and biological assessment study.

HYPOTHESIS: Lipophilic cannabidiol can be solubilized in oil-in water nanoemulsions, which can then be impregnated into chitosan hydrogels forming another colloidal system that will facilitate cannabidiol's release. The delivery from both systems was compared, alongside structural and biological studies, to clarify the effect of the two carriers' structure on the release and toxicity of the systems. EXPERIMENTS: Oil-in-water nanoemulsions (NEs) and the respective nanoemulsion-filled chitosan hydrogels (NE/HGs) were formulated as carriers of cannabidiol (CBD). Size, polydispersity and stability of the NEs were evaluated and then membrane dynamics, shape and structure of both systems were investigated with EPR spin probing, SAXS and microscopy. Biocompatibility of the colloidal delivery systems was evaluated through cytotoxicity tests over normal human skin fibroblasts. An ex vivo permeation protocol using porcine ear skin was implemented to assess the release of CBD and its penetration through the skin. FINDINGS: Incorporation of the NEs in chitosan hydrogels does not significantly affect their structural properties as evidenced through SAXS, EPR and confocal microscopy. These findings indicate the successful development of a novel nanocarrier that preserves the NE structure with the CBD remaining encapsulated in the oil core while providing new rheological properties advantageous over NEs. Moreover, NE/HGs proved to be more efficient as a carrier for the release of CBD. Cell viability assessment revealed high biocompatibility of the proposed colloids.

The Impact of the Oil Phase Selection on Physicochemical Properties, Long-Term Stability, In Vitro Performance and Injectability of Curcumin-Loaded PEGylated Nanoemulsions.

A nanotechnology-based approach to drug delivery presents one of the biggest trends in biomedical science that can provide increased active concentration, bioavailability, and safety compared to conventional drug-delivery systems. Nanoemulsions stand out amongst other nanocarriers for being biodegradable, biocompatible, and relatively easy to manufacture. For improved drug-delivery properties, longer circulation for the nanoemulsion droplets should be provided, to allow the active to reach the target site. One of the strategies used for this purpose is PEGylation. The aim of this research was assessing the impact of the oil phase selection, soybean or fish oil mixtures with medium chain triglycerides, on the physicochemical characteristics and injectability of curcumin-loaded PEGylated nanoemulsions. Electron paramagnetic resonance spectroscopy demonstrated the structural impact of the oil phase on the stabilizing layer of nanoemulsions, with a more pronounced stabilizing effect of curcumin observed in the fish oil nanoemulsion compared to the soybean oil one. The design of the experiment study, employed to simultaneously assess the impact of the oil phase, different PEGylated phospholipids and their concentrations, as well as the presence of curcumin, showed that not only the investigated factors alone, but also their interactions, had a significant influence on the critical quality attributes of the PEGylated nanoemulsions. Detailed physicochemical characterization of the NEs found all formulations were appropriate for parenteral administration and remained stable during two years of storage, with the preserved antioxidant activity demonstrated by DPPH and FRAP assays. In vitro release studies showed a more pronounced release of curcumin from the fish oil NEs compared to that from the soybean oil ones. The innovative in vitro injectability assessment, designed to mimic intravenous application, proved that all formulations tested in selected experimental setting could be employed in prospective in vivo studies. Overall, the current study shows the importance of oil phase selection when formulating PEGylated nanoemulsions.

Influence of nanoreactor environment and substrate location on the activity of horseradish peroxidase in olive oil based water-in-oil microemulsions.

Oxidative enzymatic reactions using horseradish peroxidase (HRP) were carried out in water-in-oil (w/o) microemulsions composed of olive oil/lecithin/1-propanol/water, a model biomimetic system. The substrates used (gallic acid, octyl gallate and 2,2'-azino-bis[3-ethylbenzo-thiazoline-6-sulfonic acid] (ABTS)) have different hydrophobicities and possible locations in the microemulsion system. HRP reactivity with reference to substrate hydrophobicity and structural characteristics of the microemulsions is discussed. The nature of the enzyme microenvironments was examined using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and diffusion NMR (DOSY) methodologies while the location of various enzymatic substrates in the microemulsion phase was assessed by solubility measurements and by taking pressure-area isotherms of mixed monolayers of the substrates with dipalmitoyl-phosphatidylcholine (DPPC), which is a major constituent of lecithin. In contrast to the bulk aqueous phase, in the severely restricted environment of the polar domains of the microemulsion HRP reacted faster with octyl gallate, a substrate that is solubilized at the lipid interfaces. HRP was deactivated in the olive oil microemulsions within a few hours, a phenomenon that has also been observed in other microemulsion systems.

Biological Evaluation of Oil-in-Water Microemulsions as Carriers of Benzothiophene Analogues for Dermal Applications.

During the last decade, many studies have been reported on the design and formulation of novel drug delivery systems proposed for dermal or transdermal administration. The efforts focus on the development of biocompatible nanodispersions that can be delivered to the skin and treat severe skin disorders, including cancer. In this context, oil-in-water (O/W) microemulsions have been developed to encapsulate and deliver lipophilic bioactive molecules for dermal application. An O/W biocompatible microemulsion composed of PBS buffer, Tween 80, and triacetin was assessed for its efficacy as a drug carrier of DPS-2, a lead compound, initially designed in-house to inhibit BRAFV600E oncogenic kinase. The system was evaluated through both in vitro and ex vivo approaches. The cytotoxic effect, in the presence and absence of DPS-2, was examined through the thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay using various cell lines. Further investigation through Western blotting revealed that cells died of necrosis. Porcine ear skin was used as a skin model to evaluate the degree of permeation of DPS-2 through skin and assess its retention. Through the ex vivo experiments, it was clarified that encapsulated DPS-2 was distributed within the full thickness of the stratum corneum (SC) and had a high affinity to hair follicles.

Nutraceutical phycocyanobilin binding to catalase protects the pigment from oxidation without affecting catalytic activity.

Phycocyanobilin is a dark blue linear tetrapyrrole chromophore covalently attached to protein subunits of phycobiliproteins present in the light-harvesting complexes of the cyanobacteria Arthrospira platensis (Spirulina "superfood"). It shows exceptional health-promoting properties and emerging use in various fields of bioscience and industry. This study aims to examine the mutual impact of phycocyanobilin interactions with catalase, a life-essential antioxidant enzyme. Fluorescence quenching experiments demonstrated moderate binding (Ka of 3.9 × 104 M-1 at 25 °C; n = 0.89) (static type), while van't Hoff plot points to an enthalpically driven ligand binding (ΔG = -28.2 kJ mol-1; ΔH = -41.9 kJ mol-1). No significant changes in protein secondary structures (α-helix content ~22%) and thermal protein stability in terms of enzyme tetramer subunits (Tm ~ 64 °C) were detected upon ligand binding. Alterations in the tertiary catalase structure were found without adverse effects on enzyme activity (~2 × 106 IU/mL). The docking study results indicated that the ligand most likely binds to amino acid residues (Asn141, Arg 362, Tyr369 and Asn384) near the cavity between the enzyme homotetramer subunits not related to the active site. Finally, complex formation protects the pigment from free-radical induced oxidation (bleaching), suggesting possible prolongation of its half-life and bioactivity in vivo if bound to catalase.

Development and Study of Nanoemulsions and Nanoemulsion-Based Hydrogels for the Encapsulation of Lipophilic Compounds.

Biocompatible nanoemulsions and nanoemulsion-based hydrogels were formulated for the encapsulation and delivery of vitamin D3 and curcumin. The aforementioned systems were structurally studied applying dynamic light scattering (DLS), electron paramagnetic resonance (EPR) spectroscopy and viscometry. In vitro studies were conducted using Franz diffusion cells to investigate the release of the bioactive compounds from the nanocarriers. The cytotoxicity of the nanoemulsions was investigated using the thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay and RPMI 2650 nasal epithelial cells as in vitro model. DLS measurements showed that vitamin D3 and curcumin addition in the dispersed phase of the nanoemulsions caused an increase in the size of the oil droplets from 78.6 ± 0.2 nm to 83.6 ± 0.3 nm and from 78.6 ± 0.2 nm to 165.6 ± 1.0 nm, respectively. Loaded nanoemulsions, in both cases, were stable for 60 days of storage at 25 °C. EPR spectroscopy revealed participation of vitamin D3 and curcumin in the surfactants monolayer. In vitro release rates of both lipophilic compounds from the nanoemulsions were comparable to the corresponding ones from the nanoemulsion-based hydrogels. The developed o/w nanoemulsions did not exhibit cytotoxic effect up to the concentration threshold of 1 mg/mL in the cell culture medium.

Physicochemical Characteristics of Four Limonene-Based Nanoemulsions and Their Larvicidal Properties against Two Mosquito Species, Aedes albopictus and Culex pipiens molestus.

Negative impacts on the environment from the continuous use of synthetic insecticides against mosquitoes has driven research towards more ecofriendly products. Phytochemicals, classified as low-risk substances, have been recognized as potential larvicides of mosquitoes; however, problems related to water solubility and stability are limiting factors for their use in mosquito control programs in the field. In this context, many researchers have focused on formulating essential oils in nanoemulsions, exploiting innovative nanotechnology. In the current study, we prepared 4 (R)-(+)-limonene oil-in-water nanoemulsions using low and high energy methods, and we evaluated their physicochemical characteristics (e.g., viscosity, stability, mean droplet diameter, polydispersity index) and their bioactivity against larvae of two mosquito species of great medical importance, namely, Cx. pipiens molestus and Ae. albopictus. According to the dose-response bioassays with the limonene-based nanoemulsions and pure limonene (dissolved in organic solvent), the tested nanoformulations improved the activity of limonene against Ae. albopictus larvae, while the performance of limonene was either the same or better than limonene against Cx. pipiens molestus, depending on the applied system. Overall, we achieved the production of limonene-based delivery nanosystems, with sufficient lethal properties against mosquito larvae to consider them promising larvicidal formulations applicable to mosquito breeding sites.

Microstructure and biopharmaceutical performances of curcumin-loaded low-energy nanoemulsions containing eucalyptol and pinene: Terpenes' role overcome penetration enhancement effect?

The objective of this work was to develop low-energy nanoemulsions for enhanced dermal delivery of curcumin, using monoterpene compounds eucalyptol (EUC) and pinene (PIN) as chemical penetration enhancers. Spontaneous emulsification was the preparation method. All formulations contained 10% of the oil phase (medium-chain triglycerides (MCT), or their mixture with EUC or PIN). Formulations were stabilized by the combination of polysorbate 80 and soybean lecithin (surfactant-to-oil-ratio=1). Concentration of curcumin was set to 3 mg/ml. Average droplet diameter of all tested formulations ranged from 102 nm to 132 nm, but the ones containing monoterpenes had significantly smaller size compared to the MCT formulation. Such finding was profoundly studied through electron paramagnetic resonance spectroscopy, which proved that the presence of monoterpenes modified the nanoemulsions' interfacial environment, resulting in droplet size reduction. The release study of curcumin (using Franz cells) demonstrated that the cumulative amount released after 6 h of the experiment was 10.1 ±â€¯0.2% for the MCT nanoemulsions, 13.9 ±â€¯0.1% and 14.0 ±â€¯0.2% for PIN and EUC formulations, respectively. In vivo tape stripping revealed their performances in delivering curcumin into the skin, indicating the following order: EUC>MCT>PIN. The formulation with EUC was clearly the most successful, giving the highest cumulative amount of curcumin that penetrated per surface unit: 34.24±5.68 µg/cm2. The MCT formulation followed (30.62±2.61 µg/cm2) and, finally, the one with PIN (21.61±0.11 µg/cm2). These results corelated with curcumin's solubility in the chosen oils: 4.18±0.02 mg/ml for EUC, 1.67±0.04 mg/ml for MCT and 0.21±0.01 mg/ml for PIN. Probably, higher solubility in the oil phase of the nanoemulsion promoted curcumin's solubility in the superficial skin layers, providing enhanced penetration.

Reverse micelles as nano-carriers of nisin against foodborne pathogens. Part II: The case of essential oils.

During the last years, the food industry is working on the replacement of high energy methodologies with more sustainable techniques for the encapsulation of natural preservatives, in order to enhance their effectiveness as food additives. In the present study, nisin, an antimicrobial agent, was encapsulated in essential oil-containing microemulsions. More specifically, rosemary, thyme, oregano, and dittany essential oil-containing microemulsions were formulated to encapsulate nisin enhancing the system's overall antimicrobial activity. The systems were investigated for the interfacial properties and size of the surfactants' monolayer using electron paramagnetic resonance spectroscopy and dynamic light scattering. Subsequently, nisin-loaded microemulsions were tested for their antimicrobial activity against Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus, using the well diffusion assay. Finally, this technique was validated by a killing assay. Overall, this study provides important information on the antibacterial activity of nisin-loaded nano-carriers enhanced by essential oils, in relation to the microemulsions' structure.

Encapsulation of carotenoids extracted from halophilic Archaea in oil-in-water (O/W) micro- and nano-emulsions.

Carotenoids extracted from halophilc Archaea have potential health benefits. Their poor water-solubility and low bioavailability is a challenge to their incorporation into foods. The aim of this work was the carotenoids encapsulation into two oil-in-water (O/W) dispersions, to increase their use as functional food applications. A nanoemulsion produced by high pressure homogenization and a spontaneously formed microemulsion were conceived. The limonene was the dispersed oil phase, and mixtures of Triton X-100/Tween-80 (3:1) as emulsifiers and of water/glycerol (2:1) as the continuous aqueous phase. The microemulsion monophasic area was determined through the pseudo-ternary phase diagram. Dynamic Light Scattering was used for the structural characterization of the nano- and micro-emulsions in the presence of the carotenoids. Moreover, the radical scavenging activity of the encapsulated carotenoids was examined by Electron Paramagnetic Resonance spectroscopy. The results confirmed the delivery systems design effectiveness to encapsulate and stabilize the carotenoids for food applications.

Nano-formulation enhances insecticidal activity of natural pyrethrins against Aphis gossypii (Hemiptera: Aphididae) and retains their harmless effect to non-target predators.

The insecticidal activity of a new nano-formulated natural pyrethrin was examined on the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), and the predators Coccinella septempunctata L. (Coleoptera: Coccinellidae) and Macrolophus pygmaeus Rambur (Hemiptera: Miridae), in respect with the nano-scale potential to create more effective and environmentally responsible pesticides. Pyrethrin was nano-formulated in two water-in-oil micro-emulsions based on safe biocompatible materials, i.e., lemon oil terpenes as dispersant, polysorbates as stabilizers, and mixtures of water with glycerol as the dispersed aqueous phase. Laboratory bioassays showed a superior insecticidal effect of the pyrethrin micro-emulsions compared to two commercial suspension concentrates of natural pyrethrins against the aphid. The nano-formulated pyrethrins were harmless, in terms of caused mortality and survival time, to L3 larvae and four-instar nymphs of the predators C. septempunctata and M. pygmaeus, respectively. We expect that these results can contribute to the application of nano-technology in optimization of pesticide formulation, with further opportunities in the development of effective plant protection products compatible with integrated pest management practices.

Reverse micelles as nanocarriers of nisin against foodborne pathogens.

Reverse micelles (RMs) as nanocarriers of nisin were optimized for the highest water and bacteriocin content. RMs formulated with either refined olive oil or sunflower oil, distilled monoglycerides, ethanol, and water were effectively designed. Structural characterization of the RMs was assessed using Electron Paramagnetic Resonance Spectroscopy and Small Angle X-ray Scattering in the presence and absence of nisin. No conformational changes occurred in the presence of nisin for the nanocarriers. To assess efficacy of the loaded systems, their antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes was tested in lettuce leaves and minced meat, respectively. Antimicrobial activity was evident in both cases. Interestingly, a synergistic antimicrobial effect was observed in lettuce leaves and to a lesser extent in minced meat between nisin and some of the nanocarriers' constituents (probably ethanol). Our findings suggest complex interactions that take place when RMs are applied in different food matrices.

Oil-In-Water Microemulsions as Hosts for Benzothiophene-Based Cytotoxic Compounds: An Effective Combination.

Targeted delivery of chemotherapeutics in order to overcome side effects and enhance chemosensitivity remains a major issue in cancer research. In this context, biocompatible oil-in-water (O/W) microemulsions were developed as matrices for the encapsulation of DPS-2 a benzothiophene analogue, exhibiting high cytotoxicity in various cancer cell lines, among them the MW 164 skin melanoma and Caco-2 human epithelial colorectal adenocarcinoma cell lines. The microemulsion delivery system was structurally characterized by dynamic light scattering (DLS) and electron paramagnetic resonance (EPR) spectroscopy. The effective release of a lipophilic encapsulated compound was evaluated via confocal microscopy. The cytotoxic effect, in the presence and absence of DPS-2, was examined through the thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay. When encapsulated, DPS-2 was as cytotoxic as when dissolved in dimethyl sulfoxide (DMSO). Hence, the oil cores of O/W microemulsions were proven effective biocompatible carriers of lipophilic bioactive molecules in biological assessment experiments. Further investigation through fluorescence-activated cell sorting (FACS) analysis, comet assay, and Western blotting, revealed that DPS-2, although non-genotoxic, induced S phase delay accompanied by cdc25A degradation and a nonapoptotic cell death in both cell lines, which implies that this benzothiophene analogue is a deoxyribonucleic acid (DNA) replication inhibitor.

Drug nanocarriers for cancer chemotherapy based on microemulsions: The case of Vemurafenib analog PLX4720.

Oil-in-water (O/W) microemulsions based on Tween 80 as the emulsifier and triacetin as the dispersed oil phase were formulated to be used as delivery vehicles of Vemurafenib analog PLX4720. PLX4720 is a lipophilic antitumor drug against various cancer types correlated with the BRAFV600E mutation. The limits of the single-phase region corresponding to O/W microemulsions as described by ternary phase diagrams were examined. Droplet size measurements determined by dynamic light scattering (DLS) showed mean droplet diameters equal to 10±0.1nm both in the presence and in absence of the drug. Cryogenic-transmission electron microscopy (Cryo-TEM) images of the microemulsions showed the existence of small structures with uniform size distribution having also average diameters of approximately 10nm. Electron paramagnetic resonance (EPR) spectroscopy applying the spin probing technique confirmed PLX4720 location in the oil cores excluding its participation in the surfactants monolayer. Furthermore, cell viability assays on colon cancer cell lines Colo-205 and HT29 showed that microemulsions did not exhibit any cytotoxicity when added in ratios between 0.005% v/v and 0.2% v/v. When the cells were treated with encapsulated PLX4720 at two different concentrations (0.063 and 0.12µΜ) the same response as when dissolved in classic DMSO was observed.

Effect of oleic acid on the properties of protein adsorbed layers at water/oil interfaces: An EPR study combined with dynamic interfacial tension measurements.

Multiphase food systems consist of complex interfacial layers where surface active molecules complete compete for adsorption and interact with one another affecting the interfacial properties and the behavior of the food systems involved. The present work focuses on the examination of interactions between proteins playing an which play an important role in milk-rich food systems (namely κ-casein and ß-lactoglobulin) and oleic acid (an anionic surfactant abundant in food systems) at the oil/water interface. An interesting feature of this system is that the molecules interacting at the interface originate from different phases and do not transfer/dissolve significantly to the other phase. The systems were examined using Electron Paramagnetic Resonance (EPR) spectroscopy and Dynamic Interfacial Tension measurements (dynamic interfacial tension and dynamic interfacial dilatational rheology). This combination of experimental methods provided the dynamics of adsorption at the interface and the mechanical properties of the interface allowing valuable insight on the interactions of the different molecules. The above information was coupled with direct information on fatty acid mobility in the oil bulk phase and indirect information on the degree of relative fatty acid/protein adsorption at the interface by EPR. Overall a synergistic effect of the protein and fatty acid on decreasing interfacial tension of the oil/water interface was evidenced. The fatty acid interacted differently with the random coil κ-casein and globular ß-lactoglobulin in terms of relative adsorption and in terms on its effect on mechanical properties. Thermal denaturation of ß-lactoglobulin affected the protein's interaction with the fatty acid due to conformational changes and exposure of non-polar sites.

Tacrolimus loaded biocompatible lecithin-based microemulsions with improved skin penetration: Structure characterization and in vitro/in vivo performances.

In order to improve skin penetration of tacrolimus we aimed to develop potentially non-irritant, lecithin-based microemulsions containing ethanol, isopropanol and/or propylene glycol as cosurfactants, varying caprylic/capric triglycerides and propylene glycol monocaprylate as oil phase. The influence of excipients on the size of microemulsion region in pseudo-ternary phase diagrams and their ability to form different types of microemulsions was evaluated. The comprehensive physicochemical characterization of microemulsions and the evaluation of their structure was performed, while the localization of tacrolimus in microemulsions was further investigated using electron paramagnetic resonance spectroscopy. Moreover, stability studies proved no change in tacrolimus content during one year of storage at room temperature. In addition, in vivo skin performance indicated no skin irritation potential of blank microemulsions, whereas in vitro release testing using Franz diffusion cells showed superior release rate of tacrolimus from microemulsions (0.98±0.10 and 0.92±0.11µg/cm2/h for two bicontinuous and 1.00±0.24µg/cm2/h for oil-in-water microemulsion) compared to referent Protopic ointment (0.15±0.08µg/cm2/h). Furthermore, ex vivo penetration assessed through porcine ear skin using tape stripping, confirmed superiority of two microemulsions related to the reference, implying developed microemulsions as promising carriers for dermal delivery of tacrolimus.

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.