Bicelles: New Lipid Nanosystems for Dermatological Applications.

Bicelles have emerged as promising membrane models, and due to their attractive combination of lipid composition and physicochemical characteristics, they have become new nanostructures for biomedical research. Depending on the composition, temperature and other experimental factors, these nanosystems exhibit high structural and morphological versatility. Additionally, bicelles are able to modulate the biophysical parameters and barrier function of skin. Given these properties, these nanostructures appear to be smart nanosystems with great potential in biomedicine and dermopharmacy.

Bicellar systems as vehicle for the treatment of impaired skin.

This study assesses the potential usefulness of bicellar systems to retard the penetration of drugs into damaged skin. The active compound used in this study was diclofenac diethylamine (DDEA). Initially, physicochemical characterisation of the DDEA bicellar systems was performed at different temperatures by small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS) and differential scanning calorimetry (DSC) techniques. Subsequently, in vitro percutaneous absorption of bicellar systems into in vitro damaged skin was studied. SAXS results indicated a slight decrease in the width of their bilayers with increasing temperature, with no apparent stacking in those systems. WAXS patterns were compatible with an orthorhombic lateral packing of the nanoaggregates. The thermogram obtained by DSC indicated a decrease in gel-to-liquid crystalline transition temperature (Tm) when the drug was included into bicellar systems. A retardation effect for DDEA was detected by in vitro percutaneous absorption studies when DDEA was vehiculised in the bicellar systems with respect to an aqueous solution of the drug. It seems that the use of bicellar systems as a vehicle for topical application of DDEA on skin with an impaired barrier function may inhibit the penetration of DDEA to the systemic level. Such systems may consequently repair stratum corneum barrier function to some extent. The use of these systems could be considered a new alternative strategy to treat topically pathological skin with different drugs.

Bicellar systems as new delivery strategy for topical application of flufenamic acid.

In this work, bicellar systems, bilayered disc-shaped nanoaggregates formed in water by phospholipids, are proposed as a novel strategy for delivery of the anti-inflammatory flufenamic acid (FFA) to the skin. A comparative percutaneous penetration study of this drug in bicellar systems and other vehicles was conducted. The effects induced on the skin by the application of FFA in the different vehicles were analyzed by attenuated total reflectance-fourier transform infrared (ATR-FTIR). Additionally, using the microscopic technique freeze-substitution transmission electron microscopy (FSTEM) and X-ray scattering technique using synchrotron radiation (SAXS-SR), we studied the possible microstructural and organizational changes that were induced in the stratum corneum (SC) lipids and the collagen of the skin by the application of FFA bicellar systems. Bicellar systems exhibited a retarder effect on the percutaneous absorption of FFA with respect to the other vehicles without promoting disruption in the SC barrier function of the skin. Given that skin disruption is one of the main effects caused by inflammation, prevention of disruption and repair of the skin microstructure should be prioritized in anti-inflammatory formulations.

Bicellar systems as a new colloidal delivery strategy for skin.

The presented work evaluates the use of bicellar systems as new delivery vectors for controlled release of compounds through the skin. Two different active principles were introduced into the bicellar systems: diclofenac diethylamine (DDEA) and flufenamic acid (Ffa). Bicellar systems are discoidal aggregates formed by long and short alkyl chain phospholipids. Characterization of the bicellar systems by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (Cryo-TEM) showed that particle size decreased when DDEA was encapsulated and increased when Ffa was included in the bicellar systems. Percutaneous absorption studies demonstrated a lower penetration of DDEA and Ffa through the skin when the drugs were included in the bicellar systems than when the drugs were applied in an aqueous solution (DDEA) and in an ethanolic solution (Ffa); the reduction in penetration was more pronounced with Ffa. These bicellar systems may have retardant effects on percutaneous absorption, which result in a promising strategy for future drug or cosmetic delivery applications.

Bicellar systems as modifiers of skin lipid structure.

The characterization of different bicellar aggregates and the effects of these systems on the stratum corneum (SC) microstructure have been studied. Dynamic light scattering (DLS) and freeze fracture electron microscopy (FFEM) techniques showed that both of the systems studied, dimyristoyl-phosphatidylcholine/dihexanoyl-phosphocholine (DMPC/DHPC) and dipalmitoyl-phosphocholine (DPPC)/DHPC, were formed by small discoidal aggregates at room temperature (20°C). Treating skin with DMPC/DHPC bicelles does not affect the SC lipid microstructure, whereas bicellar systems formed by DPPC and DHPC can promote the formation of new structures in the SC lipid domains. This indicates the passage of lipids from bicelles through the SC layers and also a possible interaction of these lipids with the SC lipids. Given the absence of surfactant in the bicellar composition and the small size of these structures, the use of these smart nano-systems offers great advantages over other lipid systems for dermatological purposes. Bicelles could be promising applications as drug carriers through the skin. This contribution, based on the new biological use of bicelles, may be useful to scientists engaged in colloid science and offers a new tool for different applications in skin and cosmetic research.

Bicellar systems for in vitro percutaneous absorption of diclofenac.

This work evaluates the effect of different bicellar systems on the percutaneous absorption of diclofenac diethylamine (DDEA) using two different approaches. In the first case, the drug was included in bicellar systems, which were applied on the skin and, in the second case, the skin was treated by applying bicellar systems without drug before to the application of a DDEA aqueous solution. The characterization of bicellar systems showed that the particle size decreased when DDEA was encapsulated. Percutaneous absorption studies demonstrated a lower penetration of DDEA when the drug was included in bicellar systems than when the drug was applied in an aqueous solution. This effect was possibly due to a certain rigidity of the bicellar systems caused by the incorporation of DDEA. The absorption of DDEA on skin pretreated with bicelles increased compared to the absorption of DDEA on intact skin. Bicelles without DDEA could cause certain disorganization of the SC barrier function, thereby facilitating the percutaneous penetration of DDEA subsequently applied. Thus, depending on their physicochemical parameters and on the application conditions, these systems have potential enhancement or retardant effects on percutaneous absorption that result in an interesting strategy, which may be used in future drug delivery applications.

Liposome formation with wool lipid extracts rich in ceramides.

Internal wool lipids (IWLs) are rich in cholesterol, free fatty acids, cholesteryl sulfate, and, mainly, ceramides. The repairing effect of these lipids structured as liposomes was demonstrated by reinforcing the skin-barrier integrity and increasing the water-holding capacity when applied onto the skin. This work was focused on the formation of liposomes with IWLs rich in ceramides, obtained at pilot plant level with organic solvent extraction by using methanol and acetone. The lipid composition of the two extracts was quantitatively analyzed. IWL extracts containing different amounts of sterol sulfate were used to form liposomes at physiologic p(H). Vesicle size distribution, polydispersity index, and zeta potential of all liposomes were determined to characterize them and to study their stability. The results obtained showed that IWL extract composition, which was different depending on the extraction methodologies used, greatly influences the characteristics of the liposomes formed. Vesicular size and polydispersity index liposomes were smaller when the extract composition contained a higher proportion of either free fatty acids or sterol sulfate. Moreover, liposome stability was improved when some amount of sterol sulfate was added to the composition of methanol and acetone extracts. This natural mixture with keratinaceous origin could have a special interest for cosmetic or dermopharmaceutical companies.

Lipid nanostructures: self-assembly and effect on skin properties.

This work evaluates the relation between the composition and the self-assembly of some lipid aggregates with their effects on the skin. To this end, liposomes, bicelles and micelles formed by dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) were characterized by electron microscopy and dynamic light scattering techniques, and applied on the skin. The results revealed that nanostructures with similar assembly but different composition caused different effects on the skin parameters. In general, samples containing DMPC affected the barrier function to a greater extent than systems containing DPPC. Additionally, our results showed that samples with the same lipid composition but different assembly exerted different effects on the skin. Liposomes decreased or did not modify the transepidermal water loss (TEWL), while bicelles and micelles increased this parameter. Hydration of the skin diminished especially after the application of micellar and bicellar samples. In vitro experiments showed structures like vesicles inside cutaneous SC (stratum corneum) incubated with DPPC/DHPC bicelles. These structures were not detected in SC samples incubated with DMPC/DHPC bicelles probably due to the different thermotropic behavior of DMPC and DPPC at physiological temperatures. Results reported in this work should be considered in terms of design of more efficient and specific skin delivery systems.

Use of high-pressure freeze fixation and freeze fracture electron microscopy to study the influence of the phospholipid molar ratio in the morphology and alignment of bicelles.

The high-pressure freeze fixation and freeze fracture electron microscopy techniques were combined with the (31)P nuclear magnetic resonance to study the morphological transitions of two different dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine aggregates by the effect of temperature. Through these techniques, the relationship between magnetic alignment and the morphology of alignable and non-alignable aggregates was evaluated. The micrographs related to the non-alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented rounded objects at a temperature below the dimyristoyl-phosphatidilcholine phase transition (T(m)) and, above this temperature an increase of viscosity was followed by the appearance of large elongated aggregates. The micrographs related to the alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented discoidal objects below T(m). Above T(m), when the best alignment was achieved, the images showed large areas of lamellar stacked bilayers and the presence of some multilamellar vesicles. Our results reveal that the composition of the aggregates is a key factor determining the morphological transitions of the bicellar systems. Understanding of the rules governing these transitions is crucial to modulate characteristics of these systems and to adequate them for different applications.

Penetration and growth of DPPC/DHPC bicelles inside the stratum corneum of the skin.

The effect of dipalmitoyl phosphatidylcholine (DPPC)/dihexanoyl phosphatidylcholine (DHPC) bicelles on the microstructure of pig stratum corneum (SC) in vitro was evaluated. The physicochemical characterization of these nanoaggregates revealed small disks with diameters around 15 nm and a thickness of 5.4 nm. Upon dilution, the bicelles grow and transform into vesicles. Cryogenic scanning electron microscopy (cryo-SEM) images of the SC pieces treated with this system showed vesicles of about 200 nm and lamellar-like structures in the intercellular lipid areas. These vesicles probably resulted from the growth and molecular rearrangement of the DPPC/DHPC bicelles after penetrating the SC. The presence of lamellar-like structures is ascribed to the interaction of the lipids from bicelles with the SC lipids. The bicellar system used is suitable to penetrate the skin SC and to reinforce the intercellular lipid areas, constituting a promising tool for skin applications.

Morphological effects of ceramide on DMPC/DHPC bicelles.

Freeze fracture electron microscopy and dynamic light scattering were applied to characterize the morphological changes of DMPC/DHPC bicellar systems induced by the addition of ceramides. The results demonstrate a tendency of the DMPC/DHPC aggregates to evolve forming elongated or tubular structures with the increase of the temperature. At 20 degrees C, low concentrations of ceramide promote the appearance of elongated structures with twisted zones. Higher concentrations of this lipid lead to the formation of liposomes along the elongated structures. The increase of the temperature to 40 degrees C induces the growth of the structures containing low concentrations of ceramide forming branched aggregates. In samples with high amounts of ceramide, the increase of temperature causes phase separation and the formation of a mixed system composed by liposomes and multilamellar tubules. The morphological effects induced by ceramides in this new membrane model give new insights for the role played by this lipid in biological membranes.

Effect of bicellar systems on skin properties.

Bicelles are discoidal aggregates formed by a flat dimyristoyl-glycero-phosphocholine (DMPC) bilayer, stabilized by a rim of dihexanoyl-glycero-phosphocholine (DHPC) in water. Given the structure, composition and the dimensions of these aggregates around 10-50 nm diameter, their use for topical applications is a promising strategy. This work evaluates the effect of DMPC/DHPC bicelles with molar ratio (2/1) on intact skin. Biophysical properties of the skin, such as transepidermal water loss (TEWL), elasticity, skin capacitance and irritation were measured in healthy skin in vivo. To study the effect of the bicellar systems on the microstructure of the stratum corneum (SC) in vitro, pieces of native tissue were treated with the aforementioned bicellar system and evaluated by freeze substitution applied to transmission electron microscopy (FSTEM). Our results show that bicelles increase the TEWL, the skin elastic parameters and, decrease skin hydration without promoting local signs of irritation and without affecting the SC lipid microstructure. Thus, a permeabilizing effect of bicelles on the skin takes place possibly due to the changes in the phase behaviour of the SC lipids by effect of phospholipids from bicelles.

New arrangement of proteins and lipids in the stratum corneum cornified envelope.

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.

Liposomes as alternative vehicles for sun filter formulations.

The aim of our study was to determine the influence of several types of liposomes with a different lipid composition on the percutaneous absorption of one conventional sun filter with a lipophilic character (ethyl hexyl methoxycinnamate) using both in vitro and in vivo methodologies. Three different liposomes were prepared with unsaturated and saturated phosphatidylcholine (PC, HPC), and with a wool lipid mixture (IWL) with a composition similar to that of the stratum corneum lipids. Results showed that the liquid crystalline state associated with PC liposomes plays a key role in enhancing skin penetration. when liposomes with a composition and structural organization similar to that of the stratum corneum lipids (HPC and IWL) are used, the skin penetration is retarded, suggesting a certain reinforcement of the stratum corneum barrier. These two types of liposomes could be regarded as alternatives to conventional oil/water emulsions in the formulations of lipidic sun filters. Finally, an acceptable correlation was obtained using both in vitro and in vivo methodologies to evaluate the corresponding skin absorption profile.

Influence of chemical and freezing fixation methods in the freeze-fracture of stratum corneum.

A comparison between two fixation techniques for freeze-fracture was established. Stratum corneum (SC) samples from pig epidermis were fixed using high-pressure freezing (HPF) and using plunging in propane freezing; the latter after chemical fixation. Then, frozen samples were freeze-fractured, coated with platinum-carbon, and visualized using a high-resolution low-temperature scanning electron microscope and a transmission electron microscope. Our results indicate that the plane of freeze-fracture was different depending on the fixation and freezing methodology used. In the samples frozen by HPF without chemical fixation, the fracture plane laid mainly between the lipid lamellae. However, when chemical fixation and plunging in propane freezing was used, the fracture plane did not show preference to a specific way. Plunging in propane freezing of chemically fixed samples, on the other hand, provides a more homogeneous fracture behaviour. Thus, depending on the methodology used, we can favour a visualization of either lipid or protein domains of the SC. These results could be very useful in future ultrastructural studies in order to facilitate the microscopic visualization and interpretation of the complex images such as those of SC and even of other samples in which different domains coexist.

Effect of the electrostatic charge on the mechanism inducing liposome solubilization: a kinetic study by synchrotron radiation SAXS.

The anionic surfactant sodium dodecyl sulfate (SDS) was used to induce the initial steps of the solubilization of liposomes. The structural transformations as well as the kinetics associated with this initial period were studied by means of time-resolved small-angle X-ray scattering (SAXS) using a synchrotron radiation source. Neutral and electrically charged (anionic and cationic) liposomes were used to investigate the effect of the electrostatic charges on the kinetics of these initial steps. The mechanism that induces the solubilization process consisted of adsorption of surfactant on the bilayers and desorption of mixed micelles from the liposomes surface to the aqueous medium. In all cases the time needed for desorption of the first mixed micelles was shorter than that for complete adsorption of the surfactant on the liposomes surface. The present work demonstrates that adsorption of the SDS molecules on negatively charged liposomes was slower and release of mixed micelles from the surface of these liposomes was faster than for neutral liposomes. In contrast, in the case of positively charged liposomes, the adsorption and release processes were, respectively, faster and slower than those for neutral vesicles.

Influence of the temperature in the adsorption of sodium dodecyl sulfate on phosphatidylcholine liposomes.

The influence of the temperature on the adsorption of monomeric and micellar solutions of the anionic surfactant sodium dodecyl sulfate (SDS) on phosphatidylcholine (PC) liposomes was investigated using the fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS). The number of adsorbed molecules was quantified by measuring changes in the electrostatic potential (Psi(o)) of the liposomes/probe during an incubation with SDS at varying temperatures. At low surfactant concentrations (from 0.05 to 0.25 mM), the increase in temperature reduced the number of surfactant molecules incorporated per vesicle regardless of the incubation time, whereas at high surfactant concentrations (from 0.50 to 1.0 mM) the incubation time has an opposite effect on this process. Thus, after 10s, the surfactant adsorption decreased with temperature, yet it increased progressively with time. The adsorption was linear with temperature below critical micellar concentration (CMC) of SDS and this linear tendency did not change above CMC. This suggests an adsorption of SDS monomers regardless of the surfactant concentration.

Poly(ethyleneglycol) column for the determination of acetaminophen, phenylephrine and chlorpheniramine in pharmaceutical formulations.

New polar reversed-phase stationary phases in HPLC provide specific selectivities which can help to solve traditional chromatographic problems related to the development of chromatographic methods with widely different retention times for the sample components. One such case is the analysis of pharmaceutical formulations against the common cold. Acetaminophen, phenylephrine and chlorpheniramine, compounds with different polarities, are frequently associated in these drugs. An isocratic and rapid HPLC method for the simultaneous determination of the three compounds, acetaminophen, phenylephrine and chlorpheniramine, in capsules as pharmaceutical formulations, including the separation of impurities (4-aminophenol and 4-chloracetanilide) and excipients, has been developed and validated. The final chromatographic conditions employed a Supelco Discovery HS PEG column poly(ethyleneglycol) 15x0.46 cm, 5 microm. The mobile phase was 20 mM phosphate buffer, pH 7.0-acetonitrile (90:10, v/v) at a flow-rate of 1 ml/min. UV detection was performed at 215 nm for all the compounds except acetaminophen, which was measured at 310 nm. Validation parameters permit us to consider this method suitable.

Efficacy of stratum corneum lipid supplementation on human skin.

Recent studies suggest that supplementing intercellular lipids of the stratum corneum in ageing populations or in people with dry skin can stimulate the functioning of the skin. This work lends support to the reinforcement capacity of two different stratum corneum lipid mixtures (synthetic stratum corneum lipid mixtures, SSCL, and internal wool lipid extracts, IWL) formulated as liposomes on healthy skin of two differently aged groups of individuals. Protection of healthy skin against detergent-induced dermatitis was evaluated. Transepidermal water loss and capacitance were used to evaluate the effect of these formulations in in vivo long-term studies. Increase in water-holding capacity is obtained only when the formulations applied are structured as liposomes. This is slightly more pronounced for aged skin. Subsequent SLS exposure reflected the protection of healthy human skin against detergent-induced dermatitis. Slightly better results were obtained with IWL containing a mixture of natural ceramides than with SSCL with only one ceramide present in the formulation. All these results support the beneficial effects of skin lipid supplementation given their resemblance to the lipids in the stratum corneum both in composition and in the structuring of the formulation.

[Detection of the cutaneous melanomas, their metastasis and relapses by scintigraphies with 99MTC-HMPAO]. / Detección de melanomas cutáneos, sus metástasis y recidivas mediante gammagrafías con 99mTc-HMPAO.

A pilot study was carried out in the Nuclear Medicine Department of the National Institute of Oncology and Radiobiology to learn the diagnostic efficacy of the 99mTc-HMPAO scintigraphy in the detection of cutaneous melanoma, distant and local metastases, in patients with a confirmed histological diagnosis. The planar and/or SPECT scintigraphy was performed in 17 patients and revealed 90% sensitivity for the detection of the disease. We calculated the tumor/background index in all the images obtained and found a value greater than 1 in every case. Thus, we can affirm that the 99mTc-HMPAO accumulation in tumors was higher than the background in all cases. We recommend that this procedure be considered in the follow up of these patients as a new diagnostic method which is very useful in the determination of the patient stage and prognostic evaluation.