The in vivo and in vitro interactions of elastic and rigid vesicles with human skin

Elastic vesicles are the most novel development in vesicular systems design for dermal and transdermal drug delivery. However, interactions between these vesicles and human skin are not yet fully understood. In this study, the in vivo and in vitro interactions between elastic-, rigid vesicles and micelles with human skin were investigated. Vesicle and micelle solutions were applied onto human skin in vitro and in vivo. Subsequently, a series of tape strippings were performed, which were visualised by freeze fracture electron microscopy (FFEM). The results showed no ultrastructural changes in skin treated with rigid vesicles. Skin treated with elastic vesicles, however, showed a fast partitioning of intact vesicles into the deeper layers of the stratum corneum (SC), where they accumulated in channel-like regions. Only little vesicle material was found in the deepest layers of the SC, suggesting that the partitioning of intact vesicles from the SC into the viable epidermis is unlikely to happen. Treatment with micelles resulted in rough, irregular fracture planes. Similar results were obtained in vitro and in vivo, indicating an excellent in vitro/in vivo correlation. These results support the hypothesis that elastic vesicles have superior characteristics to rigid vesicles for the interaction with human skin. Elastic vesicles and micelles demonstrated very different interactions with human skin and hence probably also have different mechanisms of action for the enhancement of drug transport.     

Interactions Between non-ionic Surfactant Vesicles and human stratum corneum in vitro.

Abstract

The effects of non-ionic surfactant vesicles (NSVs) on human skin in vitro were studied in relation to the physico-chemical properties of the vesicles. The interactions between NSVs and skin were visualized using both freeze fracture electron microscopy and confocal laser scanning microscopy. The physico-chemical properties of the NSVs were varied in a systematic way, using a broad series of polyoxyethylene monoalkyl ether type surfactants (C_nEO_m). The number of oxyethylene units (m) was varied between 3, 7 and 10, and the number of carbon atoms (n) was either 12 or 18. Both the effects of liquid state vesicles composed of ^C12^Eo3,7 ^and C9=9^EO10 surfactants and gel state vesicles (C18EO37) were investigated. After the application of the NS V suspension on the stratum corneum surface two essentially different types of vesicle-skin interactions were visualized. Firstly, an interfacial interaction, involving the adsorption of vesicles, and the deposition of bilayer sheets on the outermost layers of the stratum corneum observed for all NSV formulations tested in this study. Secondly, effects on the ultrastructure of the stratum corneum were observed: the appearance of water pools observed for the liquid state vesicles only, and ultrastructural changes of the intercellular lipid domains are induced only observed after treatment with C₁₂EO₃ NSVs. Neither changes in the ultrastructure of the viable epidermis, nor changes in deeper skin layers were observed.     

The nanoscopic molecular pathway through human skin

BackgroundKnowledge regarding the barrier properties of human skin is important for understanding skin pathology, developing of transdermal drug delivery systems and computational skin absorption models; however, the molecular pathways through human skin remains to be fully investigated on a nanoscopic level. In particular the nanoscopic pathway of molecules passing the intercellular lipid bilayers separating the corneocytes in the stratum corneum (SC) is not fully elucidated.MethodsUsing stimulated emission depletion microscopy (STED) and Förster resonance energy transfer (FRET) the molecular pathways through the SC, the main barrier of the skin, are determined for lipophilic and water-soluble molecules at a nanoscopic resolution.ResultsUsing STED and confocal microscopy, water-soluble dyes, were observed to be present in both the corneocytes and in the intercellular lipid matrix, whereas the lipophilic dyes were predominately in the intercellular lipid bilayers. FRET was observed in the SC between the lipophilic and water-soluble dyes, the existence of a minimum possible distance between acceptor and donor molecules of 4.0 ± 0.1 nm was found.ConclusionsThe results indicate that lipophilic molecules penetrate the stratum corneum via the intercellular lipids bilayers separating the corneocytes in the SC, while the more water-soluble molecules penetrate the stratum corneum via the transcellular route through the corneocytes and intercellular lipid bilayers via the polar head groups of lipid molecules in the bilayers.General significanceKnowledge of the nanoscopic molecular pathways through human skin will help understand the skin barrier function and will be of use for computational skin absorption models and transdermal drug delivery strategies.     

Time-dependent ultrastructural changes to porcine stratum corneum following an electric pulse

The morphological changes to heat-stripped porcine stratum corneum following an electroporating pulse were studied by time-resolved freeze fracture electron microscopy. Pulses at a supra-electroporation threshold of 80 volts and 300 microseconds were applied across the stratum corneum with a pair of copper plate electrodes, which also served as cooling contacts. Multilamellar vesicles of 0.1-5.5 mm in diameter in the intercellular lipid bilayers of the stratum corneum appeared in less than milliseconds after pulsing. Pulsed samples exhibited aggregations of vesicles, whereas only occasional single vesicles were seen in the unpulsed samples. Aggregates form in less than a millisecond and disappear within minutes after the pulse. Their size ranged from 0.3 to 700 mm2. The size of individual vesicles, aggregate density, and size were analyzed as functions of postpulse time. These aggregate formations seem to be a secondary reaction to the pulse-induced skin permeabilization, determined by the resistance drop and recovery after the pulse. Heating the samples to 65 degrees C also caused vesicle aggregates of similar appearance to form, suggesting that these aggregations are related to the heating effect of the pulse. Hydration is thought to play an important role in aggregate formation.     

Formulation and evaluation of ethosomes for transdermal delivery of lamivudine

The purpose of the present research was to investigate the mechanism for improved intercellular and intracellular drug delivery from ethosomes using visualization techniques and cell line study. Ethosomal formulations were prepared using lamivudine as model drug and characterized in vitro, ex vivo and in vivo. Transmission electron microscopy, scanning electron microscopy, and fluorescence microscopy were employed to determine the effect of ethosome on ultrastructure of skin. Cytotoxicity and cellular uptake of ethosome were determined using T-lymphoid cell line (MT-2). The optimized ethosomal formulation showed 25 times higher transdermal flux (68.4 +/- 3.5 microg/cm(2)/h) across the rat skin as compared with that of lamivudine solution (2.8 +/- 0.2 microg/cm(2)/h). Microscopic studies revealed that ethosomes influenced the ultrastructure of stratum corneum. Distinct regions with lamellar stacks derived from vesicles were observed in intercellular region of deeper skin layers. Results of cellular uptake study showed significantly higher intracellular uptake of ethosomes (85.7% +/- 4.5%) as compared with drug solution (24.9% +/- 1.9%). The results of the characterization studies indicate that lipid perturbation along with elasticity of ethosomes vesicles seems to be the main contributor for improved skin permeation.     

Deuterium NMR investigation of polymorphism in stratum corneum lipids

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.     

Evaluation of Meloxicam-Loaded Cationic Transfersomes as Transdermal Drug Delivery Carriers

The aim of this study is to develop meloxicam (MX)-loaded cationic transfersomes as skin delivery carriers and to investigate the influence of formulation factors such as cholesterol and cationic surfactants on the physicochemical properties of transfersomes (i.e., particle size, size distribution, droplet surface charge and morphology), entrapment efficiency, stability of formulations and in vitro skin permeation of MX. The transfersomes displayed a spherical structure. Their size, charge, and entrapment efficiency depended on the composition of cholesterol and cationic surfactants in the formulation. Transfersomes provided greater MX skin permeation than conventional liposomes and MX suspensions. The penetration-enhancing mechanism of skin permeation by the vesicles prepared in this study may be due to the vesicle adsorption to and/or fusion with the stratum corneum. Our results suggest that cationic transfersomes may be promising dermal delivery carriers of MX.     

Elastic vesicles as a tool for dermal and transdermal delivery

The main problem in delivery of drugs across the skin is the barrier function of the skin, which is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid layers, the so-called lipid lamellae. When applying drugs onto the skin, the major penetration pathway is the tortuous intercellular route along the lipid lamellae. In order to increase the number of drugs administered via the transdermal route, novel drug delivery systems have to be designed. Among these systems are iontophoresis, electroporation, microneedles, and vesicular systems.     

Elastic Vesicles as a Tool for Dermal and Transdermal Delivery

The main problem in delivery of drugs across the skin is the barrier function of the skin, which is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid layers, the so-called lipid lamellae. When applying drugs onto the skin, the major penetration pathway is the tortuous intercellular route along the lipid lamellae. In order to increase the number of drugs administered via the transdermal route, novel drug delivery systems have to be designed. Among these systems are iontophoresis, electroporation, microneedles, and vesicular systems.     

Structure of the skin barrier and its modulation by vesicular formulations

The natural function of the skin is to protect the body from 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. For this reason the organization in the lipid domains is considered to be very important for the skin barrier function. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid phase behavior is different from that of other biological membranes. In stratum corneum crystalline phases are predominantly present, but most probably a subpopulation of lipids forms a liquid phase. Both the crystalline nature and the presence of a 13 nm lamellar phase are considered to be crucial for the skin barrier function. Since it is impossible to selectively extract individual lipid classes from the stratum corneum, the lipid organization has been studied in vitro using isolated lipid mixtures. These studies revealed that mixtures prepared with isolated stratum corneum lipids mimic to a high extent stratum corneum lipid phase behavior. This indicates that proteins do not play an important role in the stratum corneum lipid phase behavior. Furthermore, it was noticed that mixtures prepared only with ceramides and cholesterol already form the 13 nm lamellar phase. In the presence of free fatty acids the lattice density of the structure increases. In stratum corneum the ceramide fraction consists of various ceramide subclasses and the formation of the 13 nm lamellar phase is also affected by the ceramide composition. Particularly the presence of ceramide 1 is crucial. Based on these findings a molecular model has recently been proposed for the organization of the 13 nm lamellar phase, referred to as "the sandwich model", in which crystalline and liquid domains coexist. The major problem for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Therefore, several methods have been assessed to increase the permeation rate of drugs temporarily and locally. 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 vesicles, liquid-state vesicles and elastic vesicles.     

Molecular mechanisms of action of different concentrations of ethanol in water on ordered structures of intercellular lipids and soft keratin in the stratum corneum

Ethanol (EtOH) is one of the bases in topically applied medicines that promote the skin permeation of drugs. Although the effects of EtOH have been attributed to structural modifications in the stratum corneum, the underlying mechanisms, especially the influence of different concentrations of EtOH, have not been examined extensively. Structural modifications in the stratum corneum of hairless mouse due to the application of EtOH/water mixture were herein investigated at the molecular level using synchrotron X-ray diffraction. The results revealed that all EtOH concentrations examined greatly modified the short lamellar structures containing the aqueous layer in intercellular lipids and the structure of keratin fibrils in corneocytes, which can take up hydrophilic compounds. However, the long lamellar and the hydrocarbon-chain packing structures were unaffected by EtOH. Changes to the short lamellar structures were not proportional to the concentration of EtOH. However, the keratin fibril structures changed gradually with increasing EtOH concentration. The X-ray diffraction experiments enabled the effects of different EtOH concentrations on the morphology of the stratum corneum to be assessed by using a number of experimental samples to avoid variations due to individual differences. The results indicated that alterations to the short lamellar structures appeared to be related to the skin permeability of drugs with the application of EtOH/water mixture, and monotonous structural changes in the keratin fibrils with an increase in EtOH concentration may contribute to this permeation as supplement. These results will be useful for the development of new drug formulations containing EtOH.     

Determination of molecular conformation and permeation in skin via IR spectroscopy, microscopy, and imaging

Skin tissue, in addition to its specific use in dermal research, provides an excellent model for developing the techniques of vibrational microscopy and imaging for biomedical applications. In addition to permitting characterization of various regions of skin, the relative paucity of major biological constituents in the stratum corneum (the outermost layer of skin), permits us to image, with microscopic resolution, conformational alterations and concentration variations in both the lipid and protein components. Thus we are able to monitor the effects of exogenous materials such as models for drug delivery agents (liposomes) and permeation enhancers (DMSO) on stratum corneum lipid organization and protein structure. In addition, we are able to monitor protein conformational changes in single corneocytes. The current article demonstrates these procedures, ranging from direct univariate measures of lipid chain conformational disorder, to factor analysis which permits us to image conformational differences between liposomes that have permeated through the stratum corneum from those which have remained on the surface in a reservoir outside the skin.     

Using the method of homogenization to calculate the effective diffusivity of the stratum corneum with permeable corneocytes

The stratum corneum is the outermost layer of the skin, which acts as a barrier membrane against the penetration of molecules into and out of the body. It has a biphasic structure consisting of keratinized cells (corneocytes) that are embedded in a lipid matrix. The macroscopic transport properties of the stratum corneum are functions of its microstructure and the transport properties of the corneocytes and the lipid matrix, and are of considerable interest in the context of transdermal drug delivery and quantifying exposure to toxins, as well as for determining the relation of skin disorders to disruption of the stratum corneum barrier. Due to the complexity of the tissue and the difference in length scales involved in its microstructure, a direct analysis of the mass transport properties of the stratum corneum is not feasible. In this study, we undertake an approach where the macroscopic diffusion tensor of the stratum corneum is obtained through homogenization using the method of asymptotic expansions. The biphasic structure of the stratum corneum is fully accounted for by allowing the corneocytes to be permeable and considering the partitioning between the corneocytes and the lipid phases. By systematically exploring the effect of permeable corneocytes on the macroscopic transport properties of the stratum corneum, we show that solute properties such as lipophilicity and relative permeabilities in the two phases have large effects on its transdermal diffusion behavior.     

Structure of lamellar lipid domains and corneocyte envelopes of murine stratum corneum. An X-ray diffraction study

The lipid of the outermost layer of the skin is confined largely to the extracellular spaces surrounding the corneocytes of the stratum corneum where it forms a multilamellar adhesive matrix to act as the major permeability barrier of the skin. Knowledge of the molecular architecture of these intercellular domains is important for understanding various skin pathologies and their treatment, percutaneous drug delivery, and the cosmetic maintenance of the skin. We have surveyed by X-ray diffraction the structure of the intercellular domains and the extracted lipids of murine stratum corneum (SC) at 25, 45, and 70 degrees C which are temperatures in the vicinity of known thermal phase transitions [Rehfeld, S. J., & Elias, P. M. (1982) J. Invest. Dermatol. 79, 1-3]. The intercellular domains produce lamellar diffraction patterns with a Bragg spacing of 131 +/- 2 A. Lipid extracted from the SC and dispersed in excess water does not produce a simple lamellar diffraction pattern at any temperature studied, however. This and other facts suggest that another component, probably a protein, must be present to control the architecture of the intercellular lipid domains. We have also obtained diffraction patterns attributable to the protein envelopes of the corneocytes. The patterns suggest a beta-pleated sheet organizational scheme. No diffraction patterns were observed that could be attributed to keratin.     

Avian epidermal differentiation: role of lipids in permeability barrier formation

Though avian skin is known to possess a highly lipogenic epidermis, little is known about its permeability barrier function. We correlated epidermal barrier function, fine structure and lipid biochemistry in the pigeon, Columbia livia, and compared these features with terrestrial mammalian systems. Whereas barrier function, as assessed by transepidermal water loss was not as efficient as in mammals, both groups shared certain morphological features including substantial compartmentalization of lipids in stratum corneum intercellular domains. Avian intercellular lipids derive from extrusion of intracellular non-membrane-bound droplets from lowermost corneocytes, rather than by secretion of lamellar discs from multigranular bodies, as previously reported in some avians, and in mammals. Instead, both the internal lamellae and the limiting membranes of multigranular bodies appear to degenerate, leading to the formation of non-membrane-bound droplets. The lipid content of avian epidermis and stratum corneum demonstrates important similarities to terrestrial mammals, i.e. abundant sphingolipids, a paucity of phospholipids, and abundant neutral lipids, but also certain striking differences, i.e. persistence of glycosphingolipids and triglycerides into the stratum corneum. Thus, avian stratum corneum forms a two-compartment system of lipid-depleted cells embedded in non-polar-lipid enriched intercellular domains, analogous to mammals. But, in contrast to mammals, the highly attenuated corneocytes of avians, which results from a paucity of keratin filaments, produce a 'straws-and-mortar' tissue, rather than the 'bricks-and-mortar' tissue of mammals.     

Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements

The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.     

Determination of molecular conformation and permeation in skin via IR spectroscopy, microscopy, and imaging

Skin tissue, in addition to its specific use in dermal research, provides an excellent model for developing the techniques of vibrational microscopy and imaging for biomedical applications. In addition to permitting characterization of various regions of skin, the relative paucity of major biological constituents in the stratum corneum (the outermost layer of skin), permits us to image, with microscopic resolution, conformational alterations and concentration variations in both the lipid and protein components. Thus we are able to monitor the effects of exogenous materials such as models for drug delivery agents (liposomes) and permeation enhancers (DMSO) on stratum corneum lipid organization and protein structure. In addition, we are able to monitor protein conformational changes in single corneocytes. The current article demonstrates these procedures, ranging from direct univariate measures of lipid chain conformational disorder, to factor analysis which permits us to image conformational differences between liposomes that have permeated through the stratum corneum from those which have remained on the surface in a reservoir outside the skin.     

Effect of a sake concentrate on the epidermis of aged mice and confirmation of ethyl alpha-D-glucoside as its active component

Generations of Japanese have appreciated the positive effects that sake can have on skin conditions, and studies have shown that concentrated sake suppressed the epidermal barrier disruption caused by ultraviolet B (UVB) irradiation. We investigated the effect of a topical application of a sake concentrate on the murine epidermis and found that the intercellular lipid content in an aged epidermis was significantly increased. Furthermore, the topical application of ethyl alpha-D-glucoside (alpha-EG), a component of sake, brought about a similar improvement in the levels of intercellular lipids. Following on from this, we confirmed that alpha-EG also significantly increased the content of loricrin protein, an indicator of successful corneocyte differentiation, while reducing the number of corneocyte layers in the aged stratum corneum. These results confirmed alpha-EG as the primary active component of the sake concentrate that had a positive effect on the epidermis. alpha-EG increased the intercellular lipid content, accelerated the differentiation of corneocytes, and reduced the thickness, thus improving the functions of the stratum corneum.     

Liposomes as Carriers for Verbascoside: Stability and Skin Permeation Studies

In this study the influence of liposomal incorporation on both the stability and the in vitro (trans) dermal delivery of verbascoside was evaluated. The effect of drug entrapment into vesicles on its radical scavenging activity was also studied. Liposomes were obtained from soy phosphatidylcholine and cholesterol according to the film hydration method. Stability of verbascoside-loaded vesicles was studied over 6 months. Results showed that verbascoside can be incorporated in liposomes (E%?=?57–66%), preventing its degradation. Stability studies (dynamic lager light scattering [DLLS] measurements and transmission electron microscopy [TEM] visualization) pointed out that vesicles were stable for 90 days and neither verbascoside leakage nor vesicle size alteration occurred during this period. The effects of vesicular incorporation on verbascoside diffusion through skin were investigated in vitro using newborn pig skin. Results showed that liposomes promoted drug accumulation into the stratum corneum but they did not give rise to any significant transdermal verbascoside delivery. Finally, results obtained from a 1, 1-diphenyl-2-pierylhydrazyl (DPPH) radical assay demonstrated that liposomes did not interfere with the radical scavenging activity of verbascoside.     

Liposomes as carriers for verbascoside: stability and skin permeation studies

In this study the influence of liposomal incorporation on both the stability and the in vitro (trans) dermal delivery of verbascoside was evaluated. The effect of drug entrapment into vesicles on its radical scavenging activity was also studied. Liposomes were obtained from soy phosphatidylcholine and cholesterol according to the film hydration method. Stability of verbascoside-loaded vesicles was studied over 6 months. Results showed that verbascoside can be incorporated in liposomes (E% = 57-66%), preventing its degradation. Stability studies (dynamic lager light scattering [DLLS] measurements and transmission electron microscopy [TEM] visualization) pointed out that vesicles were stable for 90 days and neither verbascoside leakage nor vesicle size alteration occurred during this period. The effects of vesicular incorporation on verbascoside diffusion through skin were investigated in vitro using newborn pig skin. Results showed that liposomes promoted drug accumulation into the stratum corneum but they did not give rise to any significant transdermal verbascoside delivery. Finally, results obtained from a 1, 1-diphenyl-2-pierylhydrazyl (DPPH) radical assay demonstrated that liposomes did not interfere with the radical scavenging activity of verbascoside.