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Hypothesis: pH-responsive nanocarriers have the potential to provide targeted delivery of antimicrobial peptides (AMPs) to sites of bacterial infection with typically abnormal pH levels in the body. However, the local pH of the infected sites varies substantially among different infection-related diseases, calling for the development of delivery systems capable of targeting local pathological conditions in an adjustable pH range. Experiments: In this study, a highly versatile pH-responsive nanocarrier platform, based on dispersions of oleic acid (OA) and glycerol monooleate (GMO) self-assemblies with the human cathelicidin AMP LL-37, was designed and characterized. Findings: A detailed pH-composition phase diagram was constructed from small angle X-ray scattering and cryogenic transmission electron microscopy data. In addition, the protonation state and apparent pKa of OA embedded in these nano-self-assemblies were investigated by electrophoretic mobility measurements at different pHs and found to be strongly dependent on nanocarrier composition. By varying composition of these nanocarriers, the apparent pKa of embedded OA molecules could be tuned from 7.8 to 6.3, shifting the range of nanocarriers' pH-response. The study advances our fundamental understanding of self-assembly and pH-responsiveness in lipid-peptide systems containing monounsaturated long-chain fatty acids. The results may guide the future design of highly adaptable nanocarriers for patient-optimized pH-targeted AMP delivery.

Pluronic F127-stabilized non-lamellar liquid crystalline aqueous nanodispersions are promising injectable platforms for drug and contrast agent delivery. These nanodispersions, however, trigger complement activation in the human blood, where the extent of complement activation and opsonization processes may compromise their biological performance and safety. Here, we introduce a broad family of nanodispersions from glycerol monooleate (GMO) and oleic acid (OA) in different weight ratios, and stabilized with a plethora of nonionic methoxypoly(ethylene glycol) (mPEG)-lipids of different PEG chain length and variable lipid moiety (monounsaturated or saturated diglycerides or D-α-tocopheryl succinate). Through an integrated biophysical approach involving dynamic light scattering, synchrotron small-angle scattering, and cryo-transmission electron microscopy, we examine the impact of nonionic mPEG-lipid stabilization on size, internal self-assembled architecture, and gross morphological characteristics of nanodispersions. The results show how the nonionic mPEG-lipid type and concentration, and dependent on GMO/OA weight ratio, can variably modulate the internal architectures of nanoparticles. Assessment of complement profiling from selected nanodispersions with diverse structural heterogeneity further suggests a variable modulatory role for the lipid type of the nonionic mPEG-lipid in the extent of complement activation, which span from no activation to moderate to high levels. We comment on plausible mechanisms driving the observed complement activation variability and discuss the potential utility of these nanodispersions for future development of injectable nanopharmaceuticals.

The aim of this work was to develop novel hybrid nanocarriers made of liposomes coated with chitosan (CS) cross-linked with sodium tripolyphosphate (TPP), named CS-TPP/liposomes, for fish-purified antioxidant peptide fractionated by RP-HPLC (F5). The produced nanodispersion (peptide-loaded CS-TPP/liposomes) was characterized by dynamic light scattering (DLS), high-resolution transmission electron microscopy (HRTEM), Fourier transform infrared (FT-IR) spectroscopy, and differential scanning calorimetry (DSC). The mean nanoparticle sizes of these uncoated and CS-TPP coated liposomes were found to be 409.2 and 687.4 nm, respectively, and the obtained polydispersity index (PDI) was ≤0.2. HRTEM revealed the spherical shape and smooth surface of the produced peptide-loaded nanovesicles. FT-IR spectroscopy revealed that the liposomes have been successfully coated by CS-TPP. In addition, DSC findings indicated enhanced electrostatic interactions among CS and lecithin molecules, leading to an increase in the melting (Tm) and decomposition temperature (Td) in peptide-loaded CS-TPP/liposomes hybrid system. Furthermore, the antioxidant activity of the loaded fish-purified antioxidant peptide to CS-TPP/liposomes was retained as confirmed by the employed free radical scavenging assay. The proposed hybrid system represents a promising idea for the delivery of protein hydrolysate and peptide fractions thereof in nutraceutical and functional foods applications.

Synthetic mycobacterial cord factor analogues, e.g., trehalose 6,6'-dibehenate (TDB), are highly promising adjuvants due to their strong immunopotentiating capabilities, but their biophysical properties have remained poorly characterized. Here, we report the synthesis of an array of synthetic TDB analogues varying in acyl chain length, degree of acylation, and headgroup display, which was subjected to biophysical characterization of neat nondispersed self-assembled nanostructures in excess buffer and as aqueous dispersions with cationic dimethyldioctadecylammonium (DDA) bromide. The array comprised trehalose mono- (TMX) and diester (TDX) analogues with symmetrically shortened acyl chains [denoted by X: arachidate (A), stearate (S), palmitate (P), myristate (Myr), and laurate (L)] and an analogue with a short hydrophilic polyethylene glycol (PEG) linker inserted between the trehalose headgroup of TDS and the acyl chains (PEG-TDS). All dispersions were liposomes, but in contrast to the colloidally stable and highly cationic TDX-containing liposomes, the zeta-potential was significantly reduced for DDA/TMX and DDA/PEG-TDS liposomes, suggesting a charge-shielding effect, which compromises the colloidal stability. An increased d-spacing was observed for the lamellar phase of neat TDB analogues in excess buffer (TDSTMSPEG-TDS), confirming that the charge shielding is caused by an extended molecular configuration of the more flexible headgroup. Differential scanning calorimetry showed highly cooperative phase transitions for all tested dispersions albeit the monoesters destabilized the lipid bilayers. Langmuir experiments demonstrated that incorporation of TDXs and PEG-TDS stabilized DDA monolayers due to improved hydrogen bonding and reduced intermolecular repulsions. In conclusion, data suggest that the DDA/TDS dispersions exhibit favorable physicochemical properties rendering these DDA/TDS liposomes an attractive vaccine adjuvant, and they emphasize that not only the receptor binding and immune activation but also the biophysical properties of immunopotentiator formulations should be collectively considered when designing adjuvants with optimal safety, efficacy, and storage stability.

Owing to their unique structural features, non-lamellar liquid crystalline nanoparticles comprising cubosomes and hexosomes are attracting increasing attention as versatile investigative drug carriers. Background: Depending on their physiochemical characteristics, drug molecules on entrapment can modulate and reorganize structural features of cubosomes and hexosomes. Therefore, it is important to assess the effect of guest molecules on broader biophysical characteristics of non-lamellar liquid crystalline nanoparticles, since drug-induced architectural, morphological, and size modifications can affect the biological performance of cubosomes and hexosomes. Methods: We report on alterations in morphological, structural, and size characteristics of nanodispersions composed from binary mixtures of glycerol monooleate and vitamin E on thymoquinone (a molecule with wide therapeutic potentials) loading. Results: Thymoquinone loading was associated with a slight increase in the mean hydrodynamic nanoparticle size and led to structural transitions from an internal biphasic feature of coexisting inverse cubic Fd3m and hexagonal (H2) phases to an internal inverse cubic Fd3m phase (micellar cubosomes) or an internal inverse micellar (L2) phase (emulsified microemulsions, EMEs). We further report on the presence of &ldquo, flower-like&rdquo, vesicular populations in both native and drug-loaded nanodispersions. Conclusions: These nanodispersions have the potential to accommodate thymoquinone and may be considered as promising platforms for the development of thymoquinone nanomedicines.

Improvement of pain management strategies after arthroscopic surgery by multimodal analgesia may include the use of long-acting amide local anesthetics. Among these anesthetics, the low molecular weight local anesthetic agent bupivacaine (BUP) is attractive for use in postoperative pain management. However, it has a relatively short duration of action and imposes a higher risk of systemic toxicity at relatively large bolus doses. Bupivacaine encapsulation in lipid-based delivery systems is an attractive strategy for prolonging its local anaesthetic effect and reducing the associated undesirable systemic side effects. Here, we discuss the potential development of liquid crystalline nanocarriers for delivering BUP by using a binary lipid mixture of citrem and soy phosphatidylcholine (SPC) at different weight ratios. The produced safe-by-design family of citrem/SPC nanoparticles is attractive for use in the development of nanocarriers owing to the previously reported hemocompatibility. BUP encapsulation efficiency (EE), depending on the lipid composition, was in the range of 65–77%. In this study, nanoparticle tracking analysis (NTA) and synchrotron small-angle X-ray scattering (SAXS) were employed to gain insight into the effect of BUP solubilization and lipid composition on the size and structural characteristics of the produced citrem/SPC nanodispersions. BUP loading led to a slight change in the mean sizes (diameters) and size distributions of citrem/SPC nanoparticles. However, we found that BUP accommodation into the self-assembled interiors of nanoparticles, triggers significant structural alterations in BUP concentration- and lipid composition-dependent manners, which involve vesicle–cubosome and vesicle–hexosome transitions. The structural tunability of citrem/SPC nanoparticles and the implications for potential applications in intra-articular BUP delivery are discussed.

This article introduces a simple microfluidic device that can be combined with synchrotron small-angle X-ray scattering (SAXS) for monitoring dynamic structural transitions. The microfluidic device is a thiol-ene-based system equipped with 125 μm-thick polystyrene windows, which are suitable for X-ray experiments. The device was prepared by soft lithography using elastomeric molds followed by a simple UV-initiated curing step to polymerize the chip material and simultaneously seal the device with the polystyrene windows. The microfluidic device was successfully used to explore the dynamics of the structural transitions of phytantriol/dioleoylphosphatidylglycerol-based cubosomes on exposure to a buffer containing calcium ions. The resulting SAXS data were resolved in the time frame between 0.5 and 5.5 s, and a calcium-triggered structural transition from an internal inverted-type cubic phase of symmetry Im3m to an internal inverted-type cubic phase of symmetry Pn3m was detected. The combination of microfluidics with X-ray techniques opens the door to the investigation of early dynamic structural transitions, which is not possible with conventional techniques such as glass flow cells. The combination of microfluidics with X-ray techniques can be used for investigating protein unfolding, for monitoring the formation of nanoparticles in real time, and for other biomedical and pharmaceutical investigations. A combination of microfluidics with X-ray techniques has been used to perform dynamic structural studies on nanoparticulate formulations. (Less)

Non-lamellar liquid crystalline aqueous nanodispersions, known also as ISAsomes (internally self-assembled 'somes' or nanoparticles), are gaining increasing interest in drug solubilisation and bio-imaging, but they often exhibit poor hemocompatibility and induce cytotoxicity. This limits their applications in intravenous drug delivery and targeting. Using a binary mixture of citrem and soy phosphatidylcholine (SPC) at different weight ratios, we describe a library of colloidally stable aqueous and hemocompatible nanodispersions of diverse nanoarchitectures (internal self-assembled nanostructures). This engineered library is structurally stable in human plasma as well as being hemocompatible (non-hemolytic, and poor activator of the complement system). By varying citrem to lipid weight ratio, the nanodispersion susceptibility to macrophage uptake could also be modulated. Finally, the formation of nanodispersions comprising internally V2 (inverse bicontinuous cubic) and H2 (inverse hexagonal) nanoarchitectures was achieved without the use of an organic solvent, a secondary emulsifier, or high-energy input. The tunable binary citrem/SPC nanoplatform holds promise for future development of hemocompatible and immune-safe nanopharmaceuticals.

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).

There is a need for bio-predictive and well-characterized in vitro release models in the development of intra-articular depot formulations. Here, the commercially-available Scissor system, a membrane-based two-compartment release testing instrument, was applied to characterize the transport and release of the drug diclofenac employing conditions intended to mimic transport in the synovial joint. The fate of hyaluronic acid and human serum albumin, the main bio-relevant components incorporated in the system, was investigated. A promising strategy for providing sustained drug release upon intra-articular administration are lipid-based preformulations forming non-lamellar liquid crystalline phases in situ. The usefulness of the Scissor system for investigating the initial drug release from these delivery systems was evaluated. The diclofenac release rate upon injection of an aqueous solution was influenced by the composition of the injection site matrix, i.e. the hyaluronic acid content. Hyaluronic acid and human serum albumin were found to escape from the donor compartment into the acceptor medium through the employed polycarbonate membrane. Sustained diclofenac release was obtained by formation of highly viscous liquid crystalline phases upon injection of the lipid-based preformulations. The study shows the feasibility and potential of the Scissor system for testing initial release of intra-articular depot formulations of low-molecular-weight drug compounds.

The attractiveness of new omega-3 (ω-3) polyunsaturated fatty acid (PUFA) monoglycerides (MAGs) lies in the amphiphilic nature and the beneficial health effects as PUFA precursors in various disorders including cancer, pulmonary hypertension, and inflammatory diseases. For exploring the potential therapeutic applications of these new amphiphilic lipids, particularly as main lipid constituents in the development of nanocarriers for delivery of drugs and PUFAs, it is of paramount importance to gain insight into their self-assembly behavior on exposure to excess water. This work describes the structural characteristics of self-assemblies based on two newly synthesized MAGs, namely docosahexaenoic acid (MAG-DHA) and docosapentaenoic acid (MAG-DPA) monoglycerides, on exposure to excess water. We found that both lipids tend to form a dominant inverse hexagonal (H2) phase in excess water at 25 °C and a temperature-triggered structural transition to an inverse micellar solution (L2 phase) is detected similar to that recently reported (A. Yaghmur et al., Langmuir, 2017, 33, 14045-14057) for eicosapentaenoic acid monoglyceride (MAG-EPA). An experimental SAXS structural evaluation study on the temperature-dependent behavior of these new monoglycerides is provided, and the effects of unsaturation degree and fatty acyl chain length on the self-assembled structural features in excess water and on the H2-L2 phase transition temperature are discussed. In addition, hexosomes stabilized by using the triblock copolymer F127 and the food-grade emulsifier citrem were investigated to gain insights into the effects of stabilizer and temperature on the internal nanostructure. These nanoparticles are attractive for use in the development of nanocarriers for delivering drugs and/or nutritional compounds as the beneficial health effects of ω-3 PUFA monoglycerides can be combined with those of loaded therapeutic agents or nutraceuticals.

Cisplatin ( cis-diamminedichloroplatinum(II)) is among the most potent cytotoxic agents used in cancer chemotherapy. The encapsulation of cisplatin in lipid-based drug carriers has been challenging owing to its low solubility in both aqueous and lipid phases. Here, we investigated cisplatin encapsulation in nonlamellar liquid-crystalline (LC) nanodispersions formed from a ternary mixture of phytantriol (PHYT), vitamin E (Vit E), and an anionic phospholipid [either phosphatidylglycerol (DSPG) or phosphatidylserine (DPPS)]. We show an increase in cisplatin encapsulation efficiency (EE) in nanodispersions containing 1.5-4 wt % phospholipid. The EE was highest in DPPS-containing nanodispersions (53-98%) compared to DSPG-containing counterparts (25-40%) under similar experimental conditions. Through structural and morphological characterizations involving synchrotron small-angle X-ray scattering and cryogenic transmission electron microscopy, we further show that varying the phospholipid content of cisplatin-free nanodispersions triggers an internal phase transition from a neat hexagonal (H

In the field of drug delivery to the articular cartilage, it is advantageous to apply artificial tissue models as surrogates of cartilage for investigating drug transport and release properties. In this study, artificial cartilage models consisting of 0.5% (w/v) agarose gel containing 0.5% (w/v) chondroitin sulfate or 0.5% (w/v) hyaluronic acid were developed, and their rheological and morphological properties were characterized. UV imaging was utilized to quantify the transport properties of the following four model compounds in the agarose gel and in the developed artificial cartilage models: H-Ala-β-naphthylamide, H-Lys-Lys-β-naphthylamide, lysozyme, and α-lactalbumin. The obtained results showed that the incorporation of the polyelectrolytes chondroitin sulfate or hyaluronic acid into agarose gel induced a significant reduction in the apparent diffusivities of the cationic model compounds as compared to the pure agarose gel. The decrease in apparent diffusivity of the cationic compounds was not caused by a change in the gel structure since a similar reduction in apparent diffusivity was not observed for the net negatively charged protein α-lactalbumin. The apparent diffusivity of the cationic compounds in the negatively charged hydrogels was highly dependent on the ionic strength, pointing out the importance of electrostatic interactions between the diffusant and the polyelectrolytes. Solution based affinity studies between the model compounds and the two investigated polyelectrolytes further confirmed the electrostatic nature of their interactions. The results obtained from the UV imaging diffusion studies are important for understanding the effect of drug physicochemical properties on the transport in articular cartilage. The extracted information may be useful in the development of hydrogels for in vitro release testing having features resembling the articular cartilage.

Food-grade W/O microemulsions based on lecithin, caprylic/capric triglycerides, isopropyl myristate, alcohols and water were formulated and structurally characterized to be used as potential carrie ...

The delivery of poorly water-soluble antimicrobial peptides (AMPs) that are sensitive to degradation is a major challenge in the pharmaceutical field. In this study, we design and characterize a pH-sensitive nanocarrier with the potential for delivery of AMPs and their protection from degradation. These nanobiointerfaces are prepared through the self-assembly of oleic acid (OA) with the human cathelicidin LL-37 in excess water. Advanced experimental methods including synchrotron small angle X-ray scattering, cryogenic transmission electron microscopy, and dynamic light scattering were used to characterize the OA/LL-37 self-assemblies and their structural alterations in response to changes in pH and composition. Experimental findings reveal colloidal transformations from normal emulsions via micellar cubosomes and hexosomes to vesicles upon increasing the pH from 6.0 to 8.0 at a LL-37 content around 10 wt% relative to OA. Increasing the LL-37 content to 30 wt% in OA led to diminishing of micellar cubosomes and hexosomes in this narrow pH range, favoring the formation of micelles and vesicles of various shapes and sizes. Upon increasing the pH, with the strongest effect around pH 7.5, charge repulsions among the gradually deprotonating carboxylic groups of OA modified the geometric packing of the molecules, significantly affecting the nanostructure. These detailed insights into the formation of this unique family of nanobiointerfaces and their tunable structural features may contribute to the rational design of pH-responsive antimicrobial systems for the delivery of peptides, particularly poorly water-soluble AMPs.

Recent studies demonstrated the potential therapeutic use of newly synthesized omega-3 (ω-3) polyunsaturated fatty acid (PUFA) monoglycerides owing to their beneficial health effects in various disorders including cancer and inflammation diseases. To date, the research was mainly focused on exploring the biological effects of these functional lipids. However, to the best of our knowledge, there is no report on the hydration-mediated self assembly of these lipids that leads to the formation of nanostructures, which are attractive for use as vehicles for the delivery of drugs and functional foods. In the present study, we investigated the temperature-composition phase behaviour of eicosapentaenoic acid monoglyceride (MAG-EPA), which is one of the most investigated ω-3 PUFA monoglycerides, during a heating–cooling cycle in the temperature range of 5–60 °C. Experimental synchrotron small-angle X-ray scattering (SAXS) evidence on the formation of a dominant inverse hexagonal (H2) lyotropic liquid crystalline p...

Lipid exchange among citrem nanoparticles and an ethanol micellar solution containing soy phosphatidylcholine was investigated in situ by coupling small angle X-ray scattering with a microfluidic device. The produced soy phosphatidylcholine/citrem nanoparticles have great potential in the development of hemocompatible nanocarriers for drug delivery.

Triglycerides in food products such as mayonnaise are a vital source of energy and essential for a complete and healthy diet. Their molecular structures consist of a glycerol backbone esterified with fatty acids on the two outer and the middle positions. During the digestion of triglycerides by pancreatic lipase in the small intestine, the ester bonds on the outer positions are hydrolyzed,leading to amphiphilic monoglycerides and free fatty acids as products. Depending on their chain length and degree of saturation, these products can self-assemble into a variety of structures in excess water. In this study, we report the discovery of highly ordered nanostructures inside of the mayonnaise emulsion droplets during in vitro digestion of mayonnaise under simulated in vivo conditions using timeresolved synchrotron small-angle X-ray scattering. The formation of these structures is most likely linked to their function as a carrier and controlled release system for food nutrients, especially poorly water-soluble components, in the aqueous milieu of the digestive tract. This detailed understanding of nanostructure formation during the digestion of triglyceride-containing food products such as mayonnaise may have fundamental implications for the development of foods with improved nutritional and functional properties. Triglycerides in food products such as mayonnaise are a vital source of energy and essential for a complete and healthy diet. Their molecular structures consist of a glycerol backbone esterified with fatty acids on the two outer and the middle positions. During the digestion of triglycerides by pancreatic lipase in the small intestine, the ester bonds on the outer positions are hydrolyzed, leading to amphiphilic monoglycerides and free fatty acids as products. Depending on their chain length and degree of saturation, these products can self-assemble into a variety of structures in excess water. In this study, we report the discovery of highly ordered nanostructures inside of the mayonnaise emulsion droplets during in vitro digestion of mayonnaise under simulated in vivo conditions using timeresolved synchrotron small-angle X-ray scattering. The formation of these structures is most likely linked to their function as a carrier and controlled release system for food nutrients, especially poorly water-soluble components, in the aqueous milieu of the digestive tract. This detailed understanding of nanostructure formation during the digestion of triglyceride-containing food products such as mayonnaise may have fundamental mplications for the development of foods with improved nutritional and functional properties.

Poly(ethylene glycol)-grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamines (DSPE-mPEGs) are a family of amphiphilic lipopolymers attractive in formulating injectable long-circulating nanoparticulate drug formulations. In addition to long circulating liposomes, there is an interest in developing injectable long-circulating drug nanocarriers based on cubosomes and hexosomes by shielding and coating the dispersed particles enveloping well-defined internal nonlamellar liquid crystalline nanostructures with hydrophilic PEG segments. The present study attempts to shed light on the possible PEGylation of these lipidic nonlamellar liquid crystalline particles by using DSPE-mPEGs with three different block lengths of the hydrophilic PEG segment. The effects of lipid composition, PEG chain length, and temperature on the morphology and internal nanostructure of these self-assembled lipidic aqueous dispersions based on phytantriol (PHYT) were investigated by means of synchrotron small-angle X-ray scattering and Transmission Electron Cryo-Microscopy. The results suggest that the used lipopolymers are incorporated into the water-PHYT interfacial area and induce a significant effect on the internal nanostructures of the dispersed submicrometer-sized particles. The hydrophilic domains of the internal liquid crystalline nanostructures of these aqueous dispersions are functionalized, i.e., the hydrophilic nanochannels of the internal cubic Pn3m and Im3m phases are significantly enlarged in the presence of relatively small amounts of the used DSPE-mPEGs. It is evident that the partial replacement of PHYT by these PEGylated lipids could be an attractive approach for the surface modification of cubosomal and hexosomal particles. These PEGylated nanocarriers are particularly attractive in designing injectable cubosomal and hexosomal nanocarriers for loading drugs and/or imaging probes.

Synthetic analogues of the cell-wall lipid monomycoloyl glycerol (MMG) are promising as next-generation vaccine adjuvants. In the present study, the thermotropic phase behavior of an array of synthetic MMG analogues was examined by using simultaneous small- and wide-angle X-ray scattering under excess water conditions. The MMG analogues differed in the alkyl chain lengths and in the stereochemistry of the polar glycerol headgroup or of the lipid tails (native-like versus alternative compounds). All MMG analogues formed poorly hydrated lamellar phases at low temperatures and inverse hexagonal (H2) phases at higher temperatures prior to melting. MMG analogues with a native-like lipid acid configuration self-assembled into noninterdigitated bilayers whereas the analogues displaying an alternative lipid acid configuration formed interdigitated bilayers in a subgel (Lc′) state. This is in contrast to previously described interdigitated phases for other lipids, which are usually in a gel (Lβ) state. All investi...

The mycobacterial cell-wall lipid monomycoloyl glycerol (MMG) is a potent immunostimulator, and cationic liposomes composed of a shorter synthetic analogue (MMG-1) and dimethyldioctadecylammonium (DDA) bromide represent a promising adjuvant that induces strong antigen-specific Th1 and Th17 responses. In the present study, we investigated the supramolecular structure and in vivo adjuvant activity of dispersions based on binary mixtures of DDA and an array of synthetic MMG-1 analogues (MMG-2/3/5/6) displaying longer (MMG-2) or shorter (MMG-3) alkyl chain lengths, or variations in stereochemistry of the polar headgroup (MMG-5) or of the hydrophobic moiety (MMG-6). Synchrotron small-angle X-ray scattering experiments and cryo transmission electron microscopy revealed that DDA:MMG-1/2/5/6 dispersions consisted of unilamellar and multilamellar vesicles (ULVs/MLVs), whereas a coexistence of both ULVs and hexosomes was observed for DDA:MMG-3, depending on the DDA:MMG molar ratio. The studies also showed that ULVs were formed, regardless of the structural characteristics of the neat MMG analogues in excess buffer [lamellar (MMG-1/2/5) or inverse hexagonal (MMG-3/6) phases]. Immunization of mice with a chlamydia antigen surface-adsorbed to DDA:MMG-1/3/6 dispersions revealed that all tested adjuvants were immunoactive and induced strong Th1 and Th17 responses with a potential for a central effector memory profile. The MMG-1 and MMG-6 analogues were equally immunoactive in vivo upon incorporation into DDA liposomes, despite the reported highly different immunostimulatory properties of the neat analogues in vitro, which were attributed to the different nanostructural characteristics. This clearly demonstrates that optimal formulation and delivery of MMG analogues to the immune system is of major importance and challenges the use of in vitro screening assays with nondispersed compounds to identify potential new vaccine adjuvants.

Interactions of drug candidates with the biomacromolecules of the synovial fluid affect drug targeting to the articular cartilage as well as clearance from the synovial space upon intra-articular administration. Hyaluronic acid (HA) and human serum albumin (HSA) are two main components existing in the synovial fluid. To this end, we investigated the affinity of seven cationic amino acid and dipeptide β-naphthylamide derivatives towards HA and HSA in order to shed light on possible relationships between physicochemical properties, in particular charge state, and biomacromolecular interactions to increase the joint residence time. Capillary electrophoresis frontal analysis was used for characterization of the binding of the derivatives to hyaluronic acid and HSA at 25 °C in acetate buffer (pH 4.65) and phosphate buffer (pH 7.40), respectively. Linear binding isotherms were observed for the ligand–hyaluronic acid interactions and the obtained binding constants ranged from 43 to 133 M−1. The average fraction of bound ligand towards hyaluronic acid increased with increasing the net charge of the ligands but was less than 67 % for all investigated ligands. The obtained binding constants of the ligands with HSA varied in the range of 103–106 M−1. The interactions of low-molecular weight derivatives with hyaluronic acid were highly dependent on the ligand charge state. This trend was not observed for the interactions with HSA. The obtained affinity data may provide useful information in the design of cartilage adhesive prodrugs with extended residence time in the synovial cavity.

Upon subcutaneous administration, the distribution of drug between the delivery vehicle and the biological tissue critically affects the absorption of drug substances. Utilization of physical models resembling the native tissues appears promising for obtaining a detailed understanding of the performance of drug delivery systems based on in vitro experiments. The objective of this study was to evaluate a UV imaging-based method for real-time characterization of the release and transport of piroxicam in hydrogel-based subcutaneous tissue mimics/surrogates. Piroxicam partitioning from medium chain triglyceride (MCT) into 0.5% (w/v) agarose or 25% (w/v) F127-based hydrogels was investigated by monitoring the concentration profiles of the drug in the gels. The effect of pH on piroxicam distribution and diffusion coefficients was studied. For both hydrogel systems, the diffusion of piroxicam in the gels was not affected significantly by the pH change from 4.0 to 7.4 but a considerable change in the oil-gel distribution coefficients was found (24 and 34 times less at pH 7.4 as compared those observed at pH 4.0 for F127 and agarose gels, respectively). In addition, the release and transport processes of piroxicam upon the injection of aqueous or MCT solutions into an agarose-based hydrogel were investigated by UV imaging. The spatial distribution of piroxicam around the injection site in the gel matrix was monitored in real-time. The disappearance profiles of piroxicam from the injected aqueous solution were obtained. This study shows that the UV imaging methodology has considerable potential for characterizing transport properties in hydrogels, including monitoring the real-time spatial concentration distribution in vitro after administration by injection.

A novel real-time UV imaging approach for non-intrusive investigation of the diffusion and partitioning phenomena occurring during piroxicam release from medium chain triglyceride (MCT) solution into two hydrogel matrices is described. Two binary polymer/buffer gel matrices, 0.5% (w/v) agarose and 25% (w/v) Pluronic F127, were applied as simple models mimicking the subcutaneous tissue. The evolution of the absorbance maps as a function of time provided detailed information on the piroxicam release processes upon the exposure of the gel matrices to MCT. Using calibration curves, the concentration maps of piroxicam in the UV imaging area were determined. Regression of the longitudinal concentration-distance profiles, which were obtained using expressions derived from Fick's second law, provided the diffusivity and the distribution coefficients of piroxicam penetrated into the gels. The obtained MCT-agarose (pH 7.4) distribution coefficient of 1.4 was identical to the MCT-aqueous (pH 7.4) distribution coefficient determined by the shake-flask method whereas that of the MCT-Pluronic F127 system was four times less. The experimental data show that UV imaging may have considerable potential for investigating the transport properties of drug formulations intended for the subcutaneous administration.

In the present study, synchrotron small-angle X-ray scattering (SAXS) combined with a UV light source (in situ SAXS-UV irradiation) was used to determine the structure response of gold nanoparticle (NP)-loaded vesicles. The investigated system consisted of multilamellar vesicles of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) containing hydrophilic gold NPs with a size of 40 A. Our results indicate drastic optothermically induced structural changes in these gold NP-loaded aqueous dispersions, which are accompanied by strong alterations in the phospholipid membrane permeability. A structural mechanism from well-ordered multilamellar vesicles in the fluid lamellar (Lα) phase to an inverted type hexagonal liquid crystalline (H2) phase via the formation of an intermediate phase of uncorrelated fluid bilayers is proposed. In general, these investigations are also crucial for the understanding of the potential of gold NP-loaded model phospholipid systems as efficient drug nanocarriers aiming to impro...

Proteolytically stable α-peptide/β-peptoid peptidomimetics constitute promising cell-penetrating carrier candidates exhibiting superior cellular uptake as compared to commonly used cell-penetrating peptides (CPPs). The aim of the present study was to explore the potential of these peptidomimetics for delivery of small interfering RNA (siRNA) to the cytosol by incorporation of a palmitoylated peptidomimetic construct into a cationic lipid-based nanocarrier system. The optimal construct was selected on the basis of the effect of palmitoylation and the influence of the length of the peptidomimetic on the interaction with model membranes and the cellular uptake. Palmitoylation enhanced the peptidomimetic adsorption to supported lipid bilayers as studied by ellipsometry. However, both palmitoylation and increased peptidomimetic chain length were found to be beneficial in the cellular uptake studies using fluorophore-labeled analogues. Thus, the longer palmitoylated peptidomimetic was chosen for further formulation of siRNA in a dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) nanocarrier system, and the resulting nanoparticles were found to mediate efficient gene silencing in vitro. Cryo-transmission electron microscopy (cryo-TEM) revealed multilamellar, onion-like spherical vesicles, and small-angle X-ray scattering (SAXS) analysis confirmed that the majority of the lipids in the nanocarriers were organized in lamellar structures, yet coexisted with a hexagonal phase, which is important for efficient nanocarrier-mediated endosomal escape of siRNA ensuring cytosolic delivery. The present work is a proof-of-concept for the use of α-peptides/β-peptoid peptidomimetics in an efficient delivery system that may be more generally exploited for the intracellular delivery of biomacromolecular drugs.

This work describes the effect of two different surfactants on the internal nanostructure of the kinetically stabilized isasomes (internally self-assembled particles or "somes"), which are a new family of colloidal particles (cubosomes, hexosomes, micellar cubosomes, and emulsified microemulsions, EME). The stabilization of these systems is performed by using the polymeric stabilizer F127. We demonstrate that the internal structure of these oil-free and oil-loaded dispersed particles can be modulated by varying the lipid composition. To achieve this goal, we replaced part of our primary lipid monolinolein (MLO) with diglycerol monooleate (DGMO) or soybean phosphatidylcholine (PC). We found that DGMO has a counter effect to that of tetradecane (TC) and allows us to tune back the self-assembled nanostructure in the TC-loaded dispersions from H2 (hexosomes) to Im3m (cubosomes). Although TC has a higher impact on confined structures than does DGMO, we demonstrate that the addition of DGMO significantly affects the internal structure of the TC-solubilized dispersions and favors the formation of large water channels. PC can also be used to modify the internal structure for MLO-based systems. It is somehow different from DGMO due to the fact that the fully hydrated Pn3m cubic structure in the presence of PC for the TC-free dispersion is preserved after dispersing. The results also indicate that PC is less effective than DGMO for tuning back the TC-loaded internal structure from H2 to cubic phase, in which it makes the confined structure less ordered. In addition, we found that DGMO has a significant effect on the internal structure of isasomes. It increases the water solubilization capacity for dispersed and nondispersed bulk phases. In contrast to the MLO-based dispersions, the present results indicate that F127 plays an important role in the internal structure of these dispersions due to its penetration into the oil-free cubic phase changing the symmetry from Pn3m to Im3m.

Particles with an internal structure have been found in dilute water solutions of a triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), which has short hydrophilic PEO endblocks compared to the central hydrophobic PPO block (EO5PO68EO5, L121). The properties of the block copolymer particles (i.e., their structure, size, and time stability) have been investigated using cryogenic transmission electron microscopy (cryo-TEM) in combination with dynamic light scattering (DLS) and turbidity measurements. The particles were formed in dilute solutions by quenching the temperature to temperatures where the reversed hexagonal phase is in equilibrium with a solution of unaggregated L121 copolymers (L-1). From the DLS measurements, a mean hydrodynamic radius of 158 mn was extracted. The time-scan turbidity measurements were found to be unchanged for about 46 h. At higher copolymer concentrations, a reversed hexagonal phase (H-2) exists in the L121/water system. SAXS was used to investigate the internal structure of the dispersed L121-based particles containing 15 wt % L121. It was found that the internal structure transforms from H2 to an inverse micellar system (L-2) as the temperature increases from 37 to 70 degrees C. (Less)

In our present study, a model Maillard furfural/cysteine reaction was chosen for exploring and understanding the different aspects that control the reactivity in five-component fully dilutable U-type food-grade oil/water (O/W) microemulsions of R(+)-limonene/ethanol/water/propylene glycol/Tween 60. It was found that these self-assembled nanosized structured systems are capable of enhancing the rate of Maillard reactions in which the selectivity and the reactivity are controlled by the composition of the interface and its curvature. The sulfur-containing flavor formation is very efficient if carried out in those O/W microemulsions (modified oil-swollen nanoreactors) and the reaction yields high amounts of key aroma compounds. The proposed Tween 60-based O/W systems show strong preference for the formation of 2-(2-furanyl)-thiazolidine (the main product). The O/W reaction medium enhances furfural–cysteine reaction rate (microemulsion catalysis) in comparison to the water-based reaction. It was found that the initial reaction rate (V0) is affected by the solubilized aqueous phase content. The furfural–cysteine reaction is affected also by the process conditions (temperature, pH, time of reaction). These results suggest that O/W microemulsions are a promising choice for generation of flavors and food products.

Microemulsions are of growing interest to the food industry as vehicles for delivering and enhancing solubilization of natural food supplements with nutritional and health benefits. The incorporation of molecular phytosterols, cholesterol-lowering agents, in food products is of great interest to the food industry. In this work is demonstrated the use of water dilutable food-grade microemulsions consisting of ethoxylated sorbitan ester (Tween 60), water, R-(+)-limonene, ethanol, and propylene glycol as vehicles for enhancing the phytosterols solubilization. Phytosterols were solubilized up to 12 times more than the dissolution capacity of the oil [R-(+)-limonene] for the same compounds. The solubilization capacity of phytosterols and cholesterol along a dilution line in a pseudo-ternary phase diagram [on this dilution line the weight ratio of R-(+)-limonene/ethanol/Tween 60 is constant at 1:1:3] was correlated to the microstructure transitions along the dilution line. Structural aspects were studied by self-diffusion NMR spectroscopy. The ability of phytosterols to compete with cholesterol for penetration into bile salt micelles in the gut may be limited to rich aqueous systems (O/W microemulsion).

The improved water and oil solubilization in the presence of polyols (propylene glycol, PG, and glycerol, Gly) and short-chain alcohol (ethanol) in U-type nonionic W/O and O/W food microemulsions was investigated. The phase behavior of systems based on Tweens (ethoxylated sorbitan esters) was compared with non-food-grade systems based on C18:1E10 (Brij 96v). Short-chain alcohol (ethanol in food-grade systems) together with polyols (glycerol and propylene glycol) when added to a three component system (oil–surfactant–water) induce the formation of both water-in-oil (W/O) and oil-in-water (O/W) microemulsions. Alcohols and polyols destabilize the liquid crystalline phase and extend the isotropic region to higher surfactant concentrations. The total monophasic area, AT, at R(+)-limonene/ethanol of 1/1 (w/w) and aqueous phase of water/PG of 1/1 (w/w), was 73 and 64% of the total area of the phase diagram for Brij 96v and Tween 60, respectively. The transition from a W/O microemulsion into an O/W microemulsion happens gradually, and continuously without any phase separation. The total monophasic area depends also on the type of the oil, on the composition of the polar and apolar phases, and on the nature of the polyol. The results are discussed in terms of BSO equation, spontaneous curvature, H0, film flexibility, κ and κ , surfactant oil and surfactant cosolvent compatibility and the participation of the polyol at the interface. The difference in temperature sensitivity of PG-based microemulsions vs. temperature sensitivity of Gly-based is demonstrated and explained.

The five-component system is quite unique since it allows formation of reverse micelles with hydrophilic ethoxylated alcohol in the presence of ethanol and it facilitates dilution by water/propylene glycol (1,2-propanediol, PG) aqueous phase, all the way from a water-in-oil (W/O) microemulsion via a bicontinuous phase to an oil-in-water (O/W) microemulsion.

Understanding the delivery dynamics of nucleic acid nanocarriers is fundamental to improve their design for therapeutic applications. We investigated the carrier structure-function relationship of lipid-polymer hybrid nanoparticles (LPNs) consisting of poly(DL-lactic-co-glycolic acid) (PLGA) nanocarriers modified with the cationic lipid dioleoyltrimethyl-ammoniumpropane (DOTAP). A library of siRNA-loaded LPNs was prepared by systematically varying the nitrogen-to-phosphate (N/P) ratio. Atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM) combined with small angle X-ray scattering (SAXS) and confocal laser scanning microscopy (CLSM) studies suggested that the siRNA-loaded LPNs are characterized by a core-shell structure consisting of a PLGA matrix core coated with lamellar DOTAP structures with siRNA localized both in the core and in the shell. Release studies in buffer and serum-containing medium combined with in vitro gene silencing and quantification of intracellular siRNA suggested that this self-assembling core-shell structure influences the siRNA release kinetics and the delivery dynamics. A main delivery mechanism appears to be mediated via the release of transfection-competent siRNA-DOTAP lipoplexes from the LPNs. Based on these results, we suggest a model for the nanostructural characteristics of the LPNs, in which the siRNA is organized in lamellar superficial assemblies and/or as complexes entrapped in the polymeric matrix.

Argan ( Argania spinosa L.) oil has a high dietetic and culinary value because it consists of high percentage of unsaturated fatty acids, and it is rich in aroma and flavour. The main objectives of this study were to determine the stability of argan oil (55.4% oleic acid and 24.4% linoleic acid) and to compare it to high-oleic olive oil (78.2% oleic acid and 7.9% linoleic acid) and cottonseed oil (19.8% oleic acid and 52.0% linoleic acid) at high temperatures in heating and deep-fat frying conditions. Several quality parameters were tested and compared in time-temperature conditions simulating abuse by heating and deep-fat frying. After frying no change in the contact angle of argan, olive and cottonseed oils was observed, while in other tests (colour index, viscosity, peroxide value, induction period, conjugated dienes content, total polar compounds) the stability of argan and olive oil was better than that of cottonseed oil. Oil uptake of deep-fried potatoes in argan oil was slightly lower than that in olive or cottonseed oil. It was concluded that argan oil can substitute olive or cottonseed oils in deep-fat frying. The aroma, flavour, oxidative stability and the health benefits might ‘compensate’ for the high cost of the oil. Sensory evaluation of fried french potatoes and their storage stability were not studied, and addition work is needed to evaluate the quality and storage stability of french potatoes fried in argan, olive and cottonseed oils.

The regioselective transfer hydrogenation of a carbon−carbon double bond of different chlorobenzylidene ketones is effectuated in ethylene glycol with a very high selectivity in the presence of hom...

The phase behavior of the ternary unsaturated monoglycerides (UMG)-DL-α-tocopheryl acetate-water system has been studied. The effects of lipid composition in both bulk and dispersed lyotropic liquid crystalline phases and microemulsions were investigated. In excess water, progressive addition of DL-α-tocopheryl acetate to a binary UMG mixture results in the following phase sequence: reversed bicontinuous cubic phase, reversed hexagonal (H(II)) phase, and a reversed microemulsion. The action of DL-α-tocopheryl acetate is then compared to that of other lipids such as triolein, limonene, tetradecane, and DL-α-tocopherol. The impact of solubilizing these hydrophobic molecules on the UMG-water phase behavior shows some common features. However, the solubilization of certain molecules, like DL-α-tocopherol, leads to the presence of the reversed micellar cubic phase (space group number 227 and symmetry Fd3m) while the solubilization of others does not. These differences in phase behavior are discussed in terms of physical-chemical characteristics of the added lipid molecule and its interaction with UMG and water. From an applications point of view, phase behavior as a function of the solubilized content of guest molecules (lipid additive in our case) is crucial since macroscopic properties such as molecular release depend strongly on the phase present. The effect of two hydrophilic emulsifiers, used to stabilize the aqueous dispersions of UMG, was studied and compared. Those were Pluronic F127, which is the most commonly used stabilizer for these kinds of inverted type structures, and the partially hydrolyzed emulsifier lecithin (Emultop EP), which is a well accepted food-grade emulsifier. The phase behavior of particles stabilized by the partially hydrolyzed lecithin is similar to that of bulk sample at full hydration, but this emulsifier interacts significantly with the internal structure and affects it much more than F127.

In recent years, there has been a surge of interest in exploring the effect of trans-fatty acids (TFAs) on biological membrane properties. The research studies are motivated by an increasing body of evidence suggesting that the consumption of TFAs increases the risk of developing negative health effects such as coronary heart disease and cancer. The ultimate goal of studying the lipid-fatty acid interactions at the molecular level is to predict the biological role of fatty acids in cells. In this regard, it is interesting to elucidate the effect of loading TFAs and their counterpart cis-fatty acids (CFAs) on the physical properties of lipid model membranes. Here, the present study focuses on discussing the following: (1) the effect of mixing monoelaidin (ME, TFA-containing lipid) with its counterpart monoolein (MO, CFA-containing lipid) on modulating the fully hydrated self-assembled structure, and (2) the influence of solubilizing oleic acid (OA) and its trans counterpart elaidic acid (EA) on the fully hydrated ME system. The ME model membrane was selected due to its sensitivity to variations in lipid composition and temperature. Synchrotron small-angle X-ray scattering (SAXS) was applied for studying the temperature-dependent structural behavior of the fully hydrated ME/MO-based system prepared with an equal ME/MO weight ratio and also for characterizing the fully hydrated OA- and EA-loaded ME systems. Wide-angle X-ray (WAXS) experiments were also performed for characterizing the formed crystalline lamellar phases at ambient temperatures. The results demonstrate the significant influence of the partial replacement of ME by MO on the phase behavior. The addition of MO induces the lamellar-nonlamellar phase transitions at ambient temperatures and promotes the formation of the inverted type hexagonal (H(2)) phase above 72 °C. The fully hydrated ME/EA and ME/OA systems with their rich polymorphism exhibit an interesting temperature-dependent complex behavior. The experimental findings show that the temperature-induced phase transitions are dictated by the solubilized fatty acid concentration and its configuration. Both EA and OA have a significant impact on the fully hydrated ME system. Similar to previous published studies, OA induces a significantly stronger mean negative membrane curvature as compared to EA. The two phase diagrams are discussed in terms of water-lipid and lipid-fatty acid interactions, membrane bending, and lipid packing concepts. A newly observed interesting epitaxial relationship for the lamellar-hexagonal phase transition in the EA-loaded ME system is illustrated and discussed in detail.

Liposomes embedded with gold nanoparticles show light-triggered contents release. We investigated the mechanism of the light-induced changes and functionality of the light-induced release in the cells. The real time small angle X-ray scattering (SAXS) analysis revealed time-dependent phase transitions in distearoylphosphatidylcholine (DSPC)/dipalmitoylphosphatidylcholine (DPPC) liposomes upon heating. Similar changes were observed when gold nanoparticle-embedded liposomes were exposed to the UV light: gold nanoparticles absorb light energy and transfer it to heat, thereby causing lipid phase transition from gel phase to rippled phase, and further to fluid phase. Without UV light exposure the gold nanoparticles did not affect the liposomal bilayer periodicity. The light-triggered release of hydrophilic fluorescent probe (calcein) from the gold nanoparticle-loaded liposomes was demonstrated with fluorescence-activated cell sorting after liposome internalization into the ARPE-19 cells. The liposome formulations did not decrease the cell viability in vitro. In conclusion, the light-triggered release from the liposomes is functional in the cells, and the release is triggered by thermal phase changes in the lipid bilayers.

This study reports the effect of loading four different charged designer lipid-like short anionic and cationic peptide surfactants on the fully hydrated monoolein (MO)-based Pn3m phase (Q(224)). The studied peptide surfactants comprise seven amino acid residues, namely A(6)D, DA(6), A(6)K, and KA(6). D (aspartic acid) bears two negative charges, K (lysine) bears one positive charge, and A (alanine) constitutes the hydrophobic tail. To elucidate the impact of these peptide surfactants, the ternary MO/peptide/water system has been investigated using small-angle X-ray scattering (SAXS), within a certain range of peptide concentrations (R

In our recent work, we reported on the effect of varying temperature and solubilizing tetradecane (TC) on the structural transitions observed in dispersed particles based on the monolinolein (MLO)-water-TC system. At a given temperature, the addition of TC induces a transition of the internal structure from the bicontinuous cubic phase, Pn3m, to the reversed hexagonal, H2, and to the isotropic liquid phase (water-in-oil (W/O) microemulsions). Our present study focuses on the discovery of a Fd3m phase (reversed discontinuous micellar cubic), which is formed in the MLO-water-TC system at a specific TC/MLO weight ratio. It is situated between the H2 and the isotropic liquid phase (W/O microemulsion). Remarkably, it is not found in the absence of TC by increasing the temperature. The Fd3m structure was investigated in detail by means of small-angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). The present work proves that the structural transformation in the dispersed particles from H2 (hexosomes) to the W/O microemulsion system (emulsified microemulsion (EME)) is indirect and it occurs gradually via an emulsified intermediate phase. Specifically, in addition to the nanostructured aqueous dispersions described above, we present new TC-loaded aqueous dispersions with a confined intermediate phase, which is a discontinuous micellar cubic phase of the symmetry Fd3m. We denoted this type of emulsified particles as "micellar cubosomes".

Self-assembled nanostructures, such as inverted type mesophases of the cubic or hexagonal geometry or reverse microemulsion phases, can be dispersed using a polymeric stabilizer, such as the PEO-PPO-PEO triblock copolymer Pluronic F127. The particles, which are described in the present study, are based on monolinolein (MLO)-water mixtures. When adding tetradecane (TC) to the MLO-water-F127 system at constant temperature, the internal nanostructure of the kinetically stabilized particles transforms from a Pn3m (cubosomes) to a H2 (hexosomes) and to a water-in-oil (W/O, L2) microemulsion phase (emulsified microemulsion (EME)). To our knowledge, this is the first time that the formation of stable emulsified microemulsion (EME) systems has been described and proven to exist even at room temperature. The same structural transitions can also be induced by increasing temperature at constant tetradecane content. The internal nanostructure of the emulsified particles is probed using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). At each investigated composition and temperature, the internal structure of the dispersions is observed to be identical to the corresponding structure of the nondispersed, fully hydrated bulk phase. This is clear evidence for the fact that the self-assembled inner particle nanostructure is preserved during the dispersion procedure. In addition, the internal structure of the particles is in thermodynamic equilibrium with the surrounding water phase. The internal structure of the dispersed, kinetically stabilized particles is a "real" and stable self-assembled nanostructure. To emphasize this fact, we denoted this new family of colloidal particles (cubosomes, hexosomes, and EMEs) as "ISASOMES" (internally self-assembled particles or "somes").

Winsor IV water-dilutable food-grade microemulsions based on ethoxylated sorbitan esters, water, (R)-(+)-limonene, ethanol, and propylene glycol have been prepared. Tocopherols and lycopene, two active lipophilic antioxidants, have been solubilized in the water-in-oil, bicontinuous, and oil-in-water microemulsions several times more than the oil (R)-(+)-limonene dissolution capacity. Dilution of the aqueous phase and decrease of the surfactant content strongly affect the solubilization capacity and solubilization efficiency of the microemulsion. Self-diffusion coefficients of the water and the oil in the presence of the solubilizates were correlated to the solubilization capacity, and are indicative of the locus of solubilization of the lycopene in the different microstructures.

Microemulsions based on five-component mixtures for food applications and improved oil solubilization have been studied. The compositions included water, oil phase [such as R(+)-limonene and medium-chain triglycerides (MCT)], short-chain alcohols (such as ethanol), polyols (propylene glycol and glycerol), and several surfactants and their corresponding mixtures (nonionic, such as ethoxylated sorbitan esters, polyglycerol esters, sugar ester, and anionic, such as phosphatidylcholine). The phase behavior of these systems is discussed with respect to the influence of polyols and short-chain alcohols on the degree of solubilization of oils in the aqueous phase. The alcohol and polyols modify the interfacial spontaneous curvature and the flexibility of the surfactant film, enhancing the oil solubilization capacity of the microemulsions. The solubilization of R(+)-limonene was dramatically improved in the presence of the alcohol and polyols, whereas the improvement of solubilization for triglycerides containing MCT was less pronounced. In some systems high oil solubilization was achieved, and some of them can be easily diluted to infinity both with the aqueous phase and with the oil phase. Viscosity measurements along selected dilution lines [characterized by a single continuous microemulsion region starting from a pseudo binary solution (surfactant/oil phase) to the microemulsion (water/polyol corner)] indicate that at a certain composition the system inverts from a W/O to an O/W microemulsion.

The thermal reaction between cysteine and furfural was investigated at 65 degrees C in five-component food grade oil/water (O/W) microemulsions of R-(+)-limonene/ethanol, EtOH/water/propylene glycol, PG/Tween 60 as apart of a systematic study on the generation of aroma compounds by utilizing structured W/O and O/W fluids. The furfural-cysteine reaction led to the formation of unique aroma compounds such as 2-furfurylthiol (FFT), 2-(2-furanyl)thiazolidine (main reaction product), 2-(2-furanyl)thiazoline, and N-(2-mercaptovinyl)-2-(2-furanyl)thiazolidine. These products were determined and characterized by GC-MS. Enhancement in flavor formation is termed "microemulsion catalysis". The chemical reaction occurs preferably at the interfacial film, and therefore a pseudophase model was assumed to explain the enhanced flavor formation. The product internal composition is dictated by process conditions such as temperature, time, pH, and mainly the nature of the interface. Increasing water/PG ratio leads to a dramatic increase in the initial reaction rate (V(0)). V(0) increased linearly as a function of the aqueous phase content, which could be due to the increase in the interfacial concentration of furfural. Microemulsions offer a new reaction medium to produce selective aroma compounds and to optimize their formation.