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.     

Transdermal drug delivery: penetration enhancement techniques

There is considerable interest in the skin as a site of drug application both for local and systemic effect. However, the skin, in particular the stratum corneum, poses a formidable barrier to drug penetration thereby limiting topical and transdermal bioavailability. Skin penetration enhancement techniques have been developed to improve bioavailability and increase the range of drugs for which topical and transdermal delivery is a viable option. This review describes enhancement techniques based on drug/vehicle optimisation such as drug selection, prodrugs and ion-pairs, supersaturated drug solutions, eutectic systems, complexation, liposomes, vesicles and particles. Enhancement via modification of the stratum corneum by hydration, chemical enhancers acting on the structure of the stratum corneum lipids and keratin, partitioning and solubility effects are also discussed. The mechanism of action of penetration enhancers and retarders and their potential for clinical application is described.     

Influence of nanosized delivery systems with benzyl nicotinate and penetration enhancers on skin oxygenation

Many novel nanosized delivery systems have been designed for topical application of drugs since they can overcome the skin barrier and improve drug bioavailability. The increased absorption is often a consequence of a reversibly disrupted barrier function of the skin by the vehicle itself or by specific ingredients that act as penetration enhancers. This paper reports the effects of two nanosized systems (microemulsion and liposomes), in the presence and absence of penetration enhancers (PE), on the topical delivery of a lipophilic drug in vivo and compares that to classical hydrogel formulation. A vasodilator benzyl nicotinate (BN), which increases the blood flow of the skin, was incorporated into the formulations, and skin oxygenation was followed by electron paramagnetic resonance oximetry. It was found that microemulsions and liposomes (with or without PE) accelerate the rate of BN action when compared to hydrogel. However, incorporation of PE in microemulsion also improves the effectiveness of BN action. To understand why PE enhances the action of BN, its effect on the structure of the stratum corneum was investigated in vitro. The increased fluidity of the stratum corneum lipids provides an explanation for the greater penetration of BN into the skin when the drug and PE are together incorporated into the appropriate formulation.     

Influence of the fluidity of liposome compositions on percutaneous absorption

The penetration into the stratum corneum of fluorescein, as the acid form or as a sodium salt, encapsulated in liposomes formed by liquid- or gel-state phospholipids, with or without cholesterol, was investigated in humans by the stripping method. Liposomes prepared by extrusion were applied to the forearms of healthy human volunteers and 30 min later, strippings were performed. Fluorescein was extracted and determined by spectrofluorimetry. The skin penetration of sodium fluorescein was higher from fluid liposomes (phosphatidylcholine) than from rigid liposomes (hydrogenated phosphatidylcholine), but it was independent of the content of cholesterol. It seems that the liquid-crystalline state of the lipids is the main aspect involved in the fluidity of the liposome bilayer itself as well as in the interaction with the lipids of the stratum corneum. The similar enhanced penetration behavior obtained for unsaturated liposomes containing sodium or acid fluorescein seems to support the hypothesis of a previous destruction of the vesicles during its passage through the lipid intercellular pathway in the stratum corneum.     

Influence of the Fluidity of Liposome Compositions on Percutaneous Absorption

The penetration into the stratum corneum of fluorescein, as the acid form or as a sodium salt, encapsulated in liposomes formed by liquid- or gel-state phospholipids, with or without cholesterol, was investigated in humans by the stripping method. Liposomes prepared by extrusion were applied to the forearms of healthy human volunteers and 30 min later, strippings were performed. Fluorescein was extracted and determined by spectrofluorimetry. The skin penetration of sodium fluorescein was higher from fluid liposomes (phosphatidylcholine) than from rigid liposomes (hydrogenated phosphatidylcholine), but it was independent of the content of cholesterol. It seems that the liquid-crystalline state of the lipids is the main aspect involved in the fluidity of the liposome bilayer itself as well as in the interaction with the lipids of the stratum corneum. The similar enhanced penetration behavior obtained for unsaturated liposomes containing sodium or acid fluorescein seems to support the hypothesis of a previous destruction of the vesicles during its passage through the lipid intercellular pathway in the stratum corneum.     

Influence of the level of ceramides on the permeability of stratum corneum lipid liposomes caused by a C12-betaine/sodium dodecyl sulfate mixture.

The sublytic interactions of a mixture of N-dodecyl-N, N-dimethylbetaine dodecyl betaine (C12-Bet)/sodium dodecyl sulfate (SDS) (mole fraction of the zwitterionic surfactant=0.6) with stratum corneum (SC) lipid liposomes varying the proportion of ceramides type III (Cer) were investigated. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients (K) were determined by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6) carboxyfluorescein (CF) released from the interior of vesicles. The fact that the free surfactant mixture concentration was always lower than its critical micelle concentration indicates that permeability changes were ruled by the action of surfactant monomers in all cases. Higher and lower Cer proportions than that of the SC lipids led to a fall and to a rise in the activity of the surfactant mixture on these bilayer structures. However, the surfactant partitioning into liposomes (or affinity with these bilayer structures) increased as the proportion of Cer increased up to the highest value was achieved for a Cer proportion similar to that in the SC lipids (about 40-45%). Thus, at low Cer proportions the ability of the surfactant mixture to alter the permeability of these bilayer structures was higher than that for liposomes approximating the SC lipid composition despite their reduced partitioning into liposomes. These findings are in agreement with the recently reported dependencies of the level of ceramides in skin lipids and function barrier abnormalities and could explain in part these dependencies.     

Mechanism of skin penetration-enhancing effect by laurocapram.

In order to clarify the mechanism of action of laurocapram (Azone) on the skin permeation of drugs, the following experiments were done. First, the effect of Azone on the skin components was compared with that of other penetration enhancers. Azone markedly fluidized liposomal lipids (as a model lipid system) compared with other enhancers. Ethanol extracted large amounts of the stratum corneum lipids, whereas Azone did not. These results suggest that the effect of Azone on the lipids in the stratum corneum is not the same as that of ethanol. In addition, ethanol increased the amount of free sulfhydryl (SH) group of keratin in the stratum corneum, whereas Azone did not directly affect the stratum corneum protein. Azone increased water content in the stratum corneum, as measured by skin conductance. This effect might be a reason for the action of Azone. For further understanding, the enhancing effects of Azone on the skin permeation of several model compounds (alcohols, sugars, and inorganic ions) were compared with the effects of pretreatment with distilled water, which was thought to increase water-holding capacity, and pretreatment with ethanol, which was thought to affect the lipids and protein in the skin barrier (i.e., stratum corneum). Pretreatment with water or ethanol enhanced skin permeation of hydrophilic compounds, whereas they decreased that of octanol, a hydrophobic compound. The tendency of Azone to increase or decrease the skin permeation rate of most compounds was similar to that of pretreatment with water or ethanol. However, the effect of Azone on the skin permeation of inorganic ions was relatively low, whereas that of pretreatment with water or ethanol was high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid nanoparticles for improved topical application of drugs for skin diseases

Due to the lower risk of systemic side effects topical treatment of skin disease appears favourable, yet the stratum corneum counteracts the penetration of xenobiotics into viable skin. Particulate carrier systems may mean an option to improve dermal penetration. Since epidermal lipids are found in high amounts within the penetration barrier, lipid carriers attaching themselves to the skin surface and allowing lipid exchange between the outermost layers of the stratum corneum and the carrier appear promising. Besides liposomes, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been studied intensively. Here we describe the potential of these carrier systems and compare the dermal uptake from SLN and NLC to the one of alternative vehicle systems. A special focus is upon the interactions of active ingredients and the lipid matrix as well as the quantification of dermal penetration.     

Liposomes and niosomes as topical drug delivery systems

The skin acts as a major target as well as a principle barrier for topical/transdermal (TT) drug delivery. The stratum corneum plays a crucial role in barrier function for TT drug delivery. Despite major research and development efforts in TT systems and the advantages of these routes, low stratum corneum permeability limits the usefulness of topical drug delivery. To overcome this, methods have been assessed to increase permeation. One controversial method is the use of vesicular systems, such as liposomes and niosomes, whose effectiveness depends on their physicochemical properties. This review focuses on the effect of liposomes and niosomes on enhancing drug penetration, and defines the effect of composition, size and type of the vesicular system on TT delivery.     

Examination of the mechanism of oleic acid-induced percutaneous penetration enhancement: an ultrastructural study

The epidermal permeability barrier appears to be regulated primarily by the lamellar arrangement of lipid bilayers between coneocytes of the stratum corneum and presents a significant barrier to the transdermal delivery of drugs. The aim of the present study was to investigate the effects of oleic acid on the ultrastructure of stratum corneum lipids in rat skin. Wistar rats were treated topically with 10% oleic acid/propylene glycol for 2 h, the structure of stratum corneum was examined by electron microscopy using osmium tetroxide or ruthenium tetroxide postfixation, and the epidermal barrier function was evaluated in a lanthanum tracer study. Ultrastructural examination revealed that there was a marked alteration in the stratum corneum and the tracer penetrated into the intercellular spaces of the stratum corneum after application of oleic acid. These results suggest that ruthenium tetroxide postfixation is a powerful tool for the study of the stratum corneum lipid structure. Oleic acid might increase the epidermal permeability through a mechanism involving the perturbation of stratum corneum lipid bilayers and lacunae formation to enhance transdermal drug delivery.     

Interaction of lipid nanoparticles with human epidermis and an organotypic cell culture model

Various lipid nanoparticle formulations were investigated with respect to (trans)dermal drug delivery with special regard to the mechanism of their effects on human and an organotypic cell culture epidermis. Potential alterations of stratum corneum lipid domains were studied using fluorescence assays with labeled liposomes and thermal analysis of isolated stratum corneum. Influences on the permeation of corticosterone were investigated and the occlusive properties of the nanoparticles were determined by measurements of the transepidermal water loss (TEWL). The penetration of a fluorescence dye was visualized by fluorescence microscopy of cross sections of human epidermis after incubation with cubic and solid lipid nanoparticles. Corticosterone permeation was limited when applied in matrix-type lipid nanoparticles (fat emulsion, smectic and solid lipid nanoparticles). An adhesion of solid lipid nanoparticles was clearly observed in thermal analysis as reflected by additional phase transitions probably caused by the nanoparticle matrix lipid. However, as for the other matrix-type nanoparticles, no distinct alterations of the phase transitions of the stratum corneum lipids were observed. Cubic nanoparticles led to the most predominant effect on skin permeation where the surface-active matrix lipid may act as penetration enhancer. An alteration of the stratum corneum lipids' thermal behavior as well as an interaction with fluorescence labeled liposomes was observed. Differences observed in permeation studies and thermal analysis of human and cell culture epidermis indicate that surface lipids, which are not present to the same extent in the cell culture model than in human epidermis, seem to play an important role.     

Stratum corneum maturation. A review of neonatal skin function

The importance of the stratum corneum and its barrier function for infants, especially for newborns, is clinically evident. Research regarding the maturation of the stratum corneum in neonates, i.e. when full barrier function is obtained, has produced varying results. Based on transepidermal water loss and percutaneous absorption studies, term infants seem to possess stratum corneum with adult barrier properties. Additionally, postnatal life is thought to accelerate stratum corneum maturation, so that even preterm infants have barrier function similar to term infants at 2-3 weeks of gestational age. However, a look at other parameters, such as skin thickness, skin pH and stratum corneum hydration, shows that neonatal skin is always adjusting to the extrauterine environment in contrast to the steady state of adult skin. This suggests that barrier stabilization may be dependent on achieving a balance between different parameters. However, it is still in question, which parameters, what balance and what timing. This paper provides an up-to-date overview on the neonatal skin barrier based on the review of current literature.     

Effects of cinnamene enhancers on transdermal delivery of ligustrazine hydrochloride.

Cinnamene compounds, cinnamic acid, cinnamaldehyde and cinnamic alcohol, were employed as enhancers. The effects and mechanisms of penetration promoters on the in vitro percutaneous absorption of ligustrazine hydrochloride across hairless porcine dorsal skin were investigated. Transdermal fluxes of ligustrazine hydrochloride through porcine skin were determined in vitro by Franz-type diffusion cells. The results indicated that the penetration flux of ligustrazine hydrochloride by cinnamic acid was the greatest. Significant statistical differences (P<0.05) were found between cinnamic acid and other promoters. Fourier transform-infrared (FT-IR) were carried out to analyze the effects of enhancers on the biophysical properties of the stratum corneum and the permeation enhancement mechanisms. FT-IR results revealed that the changes of peak shift and peak area due to C-H stretching vibrations in the stratum corneum lipids were associated with the selected enhancers. All of them could perturb and extract the stratum corneum lipids to different extent. Morphological changes of the skin treated with enhancers were monitored by a scanning electron microscope. It was demonstrated that the extraction of the stratum corneum lipids by the enhancers led to the disruption of stratum corneum and the desquamation of stratum corneum flake. Apparent density was newly proposed to estimate the desquamated extent of stratum corneum flake. Correlation analysis revealed that there was a linear relationship between apparent density and decrease in peak area. The results showed that the permeation enhancement mechanisms of cinnamene were pleiotropic ones, including disordering the lipids, extracting the lipids and competitive hydrogen bonding between cinnamene enhancers and amides of ceramide head groups in stratum corneum.     

Skin permeability enhancement by low frequency sonophoresis: lipid extraction and transport pathways

The objective of this study was to shed light on the mechanism(s) by which low-frequency ultrasound (20 KHz) enhances the permeability of the skin. The physical effects on the barrier and the transport pathway, in particular, were examined. The amount of lipid removed from the intercellular domains of the stratum corneum following sonophoresis was determined by infrared spectroscopy. Transport of the fluorescent probes nile red and calcein, under the influence of ultrasound, was evaluated by laser-scanning confocal microscopy. The results were compared with the appropriate passive control data and with data obtained from experiments in which the skin was exposed simply to the thermal effects induced by ultrasound treatment. A significant fraction ( approximately 30%) of the intercellular lipids of the stratum corneum, which are principally responsible for skin barrier function, were removed during the application of low-frequency sonophoresis. Although the confocal images from the nile red experiments were not particularly informative, ultrasound clearly and significantly (again, relative to the corresponding controls) facilitated transport of the hydrophilic calcein via discrete permeabilized regions, whereas other areas of the barrier were apparently unaffected. Lipid removal from the stratum corneum is implicated as a factor contributing the observed permeation enhancement effects of low-frequency ultrasound. However, microscopic observations imply that sonophoresis induces localized (aqueous?) permeation pathways at discrete sites.     

Organization of Stratum Corneum Lipids in Relation to Permeability: Influence of Sodium Lauryl Sulfate and Preheating

The role of the structural organization of intercorneocyte lipids in the barrier function of human stratum corneum was evaluated by treatment with heat and sodium lauryl sulfate. Measurement of transepidermal water loss in treated samples was used to quantify variations in stratum corneum permeability. Thermodynamic transition of lamellar lipids and their degree of organization were evaluated by differential scanning calorimetry and small-angle X-ray diffraction, respectively. Progressively preheating stratum corneum samples from 75°C to 90°C increased stratum corneum permeability to water vapor, while the fusion temperature of lamellar lipids and the intensity of the X-ray diffraction peaks of the polar lipids decreased. Sodium lauryl sulfate induced similar variations of these three parameters. These results support the hypothesis that, in addition to the chemical nature of intercorneocyte lipids, their structural arrangement and thermodynamic properties play an important role in the barrier function of the stratum corneum to water vapor.     

A Novel in Vitro Percutaneous Penetration Model: Evaluation of Barrier Properties with P-Aminobenzoic Acid and Two of Its Derivatives

Purpose The objective of this study was to evaluate the utility of a stratum corneum substitute (SCS) as a novel in vitro percutaneous penetration model. The SCS consists of synthetic stratum corneum (SC) lipids (cholesterol, free fatty acids, and specific ceramides) applied onto a porous substrate. The composition, organization, and orientation of lipids in the SCS bear high resemblance to that of the intercellular barrier lipids in SC. Methods The barrier integrity of the SCS was evaluated by means of passive diffusion studies, using three model compounds with different lipophilicities. The effects of lipid layer thickness, permeant lipophilicity, and altered lipid composition on the barrier properties were investigated, using isolated human SC as a control sample. Results For all three model compounds, the permeability characteristics of the SCS with a 12-μm-thick lipid layer closely resemble those of human SC. Modification of the lipid composition, generating an SCS that lacks the characteristic long periodicity phase as present in SC, was accompanied by a 2-fold increased permeability. Conclusions The SCS offers an attractive tool to predict solute permeation through human skin. Moreover, as its lipid composition can be modified, they may also serve as a suitable screening model for diseased skin.     

Skin structure and mode of action of vesicles

The natural function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin always have to pass these regions. Therefore, in the first part of this paper, the barrier function has been explained, focusing on the lipid composition and organisation. The major obstacle for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Several methods have been assessed to increase the permeation rate of drugs temporarily. One of the approaches is the application of drugs in formulations containing vesicles. In order to unravel the mechanisms involved in increasing the drug transport across the skin, information on the effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper, the possible interactions between vesicles and skin are described, focusing on differences between the effects of gel-state, liquid-state, and elastic vesicles.     

Investigating the barrier function of skin lipid models with varying compositions

The lipids in the uppermost layer of the skin, the stratum corneum (SC), play an important role in the barrier function. The main lipid classes in stratum corneum are ceramides, cholesterol, and free fatty acids. In previous publications, a lipid model was presented, referred to as the stratum corneum substitute (SCS), that closely mimics the SC lipid organization and SC barrier function. In the present study, we use the SCS to study the effect of changes in lipid organization on the lipid barrier function using benzoic acid as permeation compound. First, in the SCS, we increased the level of one of the three major lipid classes keeping the ratio between the other lipid classes constant. An increased cholesterol level resulted in an increase in phase-separated cholesterol and a reduction in the permeability. An increase in ceramide or free fatty acid level resulted in the formation of additional phases, but had no significant influence on the permeability. We also examined models that mimic selected changes in lipid composition reported for dry or diseased skin. The SCS that mimics the composition in recessive X-linked ichthyosis skin displayed a twofold increase in permeability. This increase is possibly related to the formation of an additional, less ordered phase in this model.     

Evaluation of in-vivo topical anti-inflammatory activity of indometacin from liposomal vesicles

The aim of this study was to evaluate the in-vivo drug release profile of indometacin-loaded liposomes into the skin. Large unilamellar vesicles (LUVs), composed of dipalmitoyl-L-alpha-phosphatidylcholine and cholesterol (9:1), were obtained using the extrusion method and then incorporated in hydrogels (LUV-A and LUV-B). The delivery of indometacin from the liposomal system was evaluated by determining its in-vivo local anti-inflammatory activity after cutaneous application of liposomal gel formulations; the anti-inflammatory activity is directly proportional to the amount of drug that actually crosses the skin. UVB-induced erythema on healthy human volunteers was chosen as the inflammatory model and the extent of erythema was monitored by the non-invasive technique of reflectance spectrophotometry. The results showed that LUV dispersions containing indometacin provided a high percentage of entrapped drug (approximately 84%). Furthermore, in-vivo findings revealed that the anti-inflammatory effect was more prolonged when indometacin was delivered from a liposomal gel formulation rather than from a gel formulation without liposomes. In particular, the indometacin-loaded gel formulation LUV-A showed a sustained effect, probably related to an interaction between LUV lipids and stratum corneum lipid structure. This interaction produces a depot in the stratum corneum that ensures sustained release of the drug to deeper skin layers.