Topical delivery of cosmetics and drugs. Molecular aspects of percutaneous absorption and delivery

ARTICLE

Auteur(s) : Matthias Förster, Marie-Alexandrine Bolzinger, Hatem Fessi, Stephanie Briançon

Université de Lyon, F-69008 Lyon, France; Université Lyon 1, ISPB, Faculté de Pharmacie
CNRS, UMR 5007, LAGEP, F-69622, Villeurbanne, France

accepté le 11 Février 2009

Topical delivery is, and has been for many thousands of years, a route of cosmetic and drug delivery. In this review we are concerned with modern medical cosmetology, which is part of modern dermatology [1]. In the cosmetic field the active substance must reach a skin compartment to exert its effect because the skin is itself the target. Conversely, in the pharmaceutical field, using patch systems, the skin is often the main barrier to cross in order to deliver hormones and analgesics to the systemic circulation [2]. The transdermal route of administration avoids hepatic first-pass metabolism and allows sustained drug release into the systemic circulation. A drug applied in a vehicle on the skin surface penetrates into the skin by a passive mechanism according to Fick’s Laws, depending on its molar mass and physicochemical properties. In the case of large molecules such as proteins, active mechanisms such as electroporation or iontophoresis have been developed to overcome the barrier. This review focuses on passive diffusion through the skin (the more common process) but some examples of active diffusion will also be considered.

In both cosmetic and drug delivery, despite the high potential of the skin, extensive work has been done to develop new carrier systems because of the high barrier function of the outermost layer of the skin, the stratum corneum. Actually the skin is a heterogeneous membrane; lipophilic on its surface and hydrophilic in its deeper layers. The stratum corneum is a highly resistant barrier which limits the penetration of drugs into the skin because its structure contributes to its function both as a barrier to water loss and as a barrier against the external environment. The skin’s barrier function is therefore important in considering both the transdermal delivery of drugs and in making a risk assessment following dermal exposure to chemicals. The major challenge for dermal or transdermal delivery is to “tune” the vehicle in which the drug is entrapped in order to reach its target site i.e. the skin surface, the skin compartments or the systemic circulation.

Today it is even possible to build three dimensional skin equivalent models by in vitro tissue engineering which are more and more complete and similar to the physiological skin [3]. Auxenfans et al. showed that their models can be used for better understanding the mechanisms of action of active substances and to test the innocuity as well as the efficacy of finished products.

In both pharmaceutical and cosmetic fields, new strategies have therefore been developed to provide an increase in drug penetration and of skin targeting without inducing skin damage. The optimum development of a drug delivery system for the topical route requires percutaneous absorption studies in order to establish the extent of drug repartitioning in the skin in relation to its formulation. Percutaneous absorption describes the entering of a drug into the skin from a drug-loaded formulation. This penetration is called permeation if the drug reaches the systemic circulation. The percutaneous absorption of a broad range of drugs has been extensively reported in the literature, allowing the development of predictive models consistent with the transport mechanisms. The degree of penetration and/or permeation depends not only on the drug itself but also on the vehicle in which it is formulated and on the interaction between the vehicle and the skin.

This review follows the pattern: a) a consideration and discussion of skin barrier function in relation to skin structure; b) a description of the modelling of the drug permeation process relating the physicochemical properties of drugs to a mathematical model and c) a consideration of strategies to enhance or slow down skin permeation. Throughout this review the word “drug” is applied both to pharmaceutical drugs and to cosmetic agents. For each case considered it will be stated whether it is a cosmetic or a pharmaceutical example.

Skin barrier function

The role of the stratum corneum

The stratum corneum represents the outermost part of the skin and is the main skin barrier. The horny layer consists of dead cells (in general 15 to 20 layers) named corneocytes. The corneocytes are filled with keratins and embedded in a complex matrix of organized lipid bilayers. The stratum corneum is lipophilic and contains 13% of water.

The hydrophilic properties of skin increase from the surface as its depth increases. The viable epidermis, represented respectively by the stratum granulosum, the stratum spinosum and the stratum germinativum, is significantly hydrophilic (> 50%). In the dermis the water content reaches 70%, favouring hydrophilic drug uptake. Therefore research interest is focused on the stratum corneum lipid matrix and on water diffusion through it. Knowing the structure and properties of this compartment at the molecular level is essential for studying drug penetration through the stratum corneum and for the development of new dermal drug delivery systems. This barrier is lipophilic and therefore imposes drug limitations on the type of permeant that can cross it.

Therefore, depending on the lipophilic or hydrophilic properties of a drug, it will accumulate in the stratum corneum (lipophilic substances), or stay on the surface (very hydrophilic drugs) or cross the skin (amphiphilic drugs). The molar mass and the volume of active agent entering the skin are also of some significance.

The intercellular route and the role of the lipid matrix

By means of thin layer chromatography the composition of the lipids was determined as an equimolar mixture of (i) nine ceramide types (40-50% of dry mass) [12], (ii) cholesterol (25 wt%), (iii) free fatty acids (10-15 wt%) and, (iv) about 5% of other lipids such as cholesterol sulphate, cholesterol esters and glucosyl ceramides [13]. The composition and structural details of the different ceramides in pigs, mice and human skin were first determined in the 1980s [14-16] and in a more detailed way in more recent years [12, 17-20].

The lipid arrangement displays a continuous lamellar structure of alternating lipid and aqueous regions, which most effectively hinders the diffusion of both non-polar and polar substances [21]. The first characterization results concerning the structure of the lamellar lipid regions were published in the 1950s and 1960s using X-ray diffraction and indicated a tube-like organization [22, 23]. Using electron microscope and freeze fracture techniques it was shown that the lipids are in a lamellar organization [7, 24-26] localized in the intercellular regions of the stratum corneum. However, the lipid membrane was not visible with a standard electron microscope. This bilayer arrangement constitutes a tortuous diffusion pathway for molecules in the stratum corneum and so is involved in its barrier function [27]. The corneocyte membrane, known as a cornified cell envelope, is composed of a very compact cross-linked protein structure, the corneodesmosome, and reinforces the barrier function [28].

Three main routes have been identified in the stratum corneum: Pathway 1 – the intercellular route (figure 1); Pathway 2 – the intracellular (transcellular) route crossing through successive bilayers and dead cells and Pathway 3 – the route via skin appendages i.e. hair follicles and sweat glands which form shunt pathways through the intact epidermis. Pathway 3 is usually considered of little significance as only 0.1% of the total human skin is occupied by skin appendages [29]. Pathway 2 is the most direct route but requires transport through densely packed keratin-filled corneocytes followed by multiple transfers between the corneocytes and the lipid-filled intercellular areas. A drug passing through this pathway should therefore encounter significant resistance to permeation.

The more common of the two main routes for drug permeation is by intercellular passage between the corneocytes [30]. This was confirmed in vitro by Simonetti and Meuwissen who showed that the penetration pathway of a dye (Nile Red in PEG, PG and DMSO vehicles [31] and FI-DHPE, a fluorescent probe, in liposomes [32]) is principally by traversing the intercellular region in the stratum corneum. The principal barrier to permeation of drugs is therefore the lipid matrix, which constitutes the intercellular pathway. The barrier effects of lipid bilayers have been known since the 1950s. Several studies have shown that a modification of the lipid composition by solvent extraction dramatically increases water permeability [33-36]. For this reason lipid regions are considered to be the major barrier of the skin [37, 38]. These studies were confirmed and extended by Yardley et al. [39]. Wertz et al. wrote a historical review of lipid research up to the 1990s [40].

Understanding the physical structure of a membrane is essential for understanding both its barrier function and the disruption mechanism for that barrier caused by topically applied products and, additionally, for the understanding of several skin diseases.

Recently it has been shown that this epidermal barrier depends not only on the composition of the stratum corneum but also on its structural organization [41-43]. Furthermore its structure is linked to its composition and therefore a small change in the composition of its lipids might have an enormous effect on their physical organization in the liquid crystalline state [44].

More recently still, very detailed studies of lipid organization have been carried out in a synthetic stratum corneum lipid mixture [45, 46]. The conclusion, therefore, from research studies is that the structure of the lipids depends primarily on cholesterol and on the fatty acids that they contain. Fatty acids play an important role in the formation of the lamellar phase because they are one of the principal lipid groups in the stratum corneum. Cholesterol is an omnipresent membrane lipid, and the second component in terms of quantity, in the stratum corneum (~25% of lipid mass). It can either increase the fluidity of membrane domains or make them more rigid, depending on the physical properties of the other lipids and on the relative proportion of cholesterol compared to the other components. The role of cholesterol in the epidermal barrier is probably to provide a degree of fluidity in what would otherwise be a rigid, possibly brittle, membrane system. This may be necessary for the pliability of the skin [47]. More detailed information on the lipid phase structure can be found in the work of Wertz [47] and Bouwstra [48].

All free fatty acid chains and amide-linked fatty acids in ceramides are non-branched and have no double bonds. All ceramides and free fatty acids in the stratum corneum are arranged in a bar-like form or a cylindrical form. These structures allow the formation of highly ordered gel phase membrane domains. These gel phase domains are less fluid and so less permeable than typical biological membranes, which are dominated by liquid crystalline phospholipid domains. In contrast to the phospholipids, ceramides cannot form bilayers on their own. Instead they form ordered structures by interaction with other skin lipids.

Several biophysical studies of the structure of the stratum corneum assume the presence and coexistence of liquid crystalline phase domains and of solid or gellified crystalline phase domains in the membrane of the stratum corneum. These conclusions imply low lateral diffusion properties at physiological temperatures. This concept introduced by Forslind [49] was presented as a “mosaic domain” model. Two other models were also suggested for explaining the unique properties of the stratum corneum. Bouwstra presented a model for the existence of fluid phases in the lamellae, which is called the “sandwich model” [50] and Norlen introduced a “single gel phase” model [51] that was, he felt, more consistent with the described properties of the stratum corneum. A review giving more details on this research area has been written by Bouwstra [52].

Percutaneous absorption

Recently, sophisticated formulation strategies have been described in the literature using, for example, liposomes, nanoparticles or microemulsions, in order to enhance drug penetration or to target a particular skin layer. Examples are provided later in this paper.

The mechanisms by which a drug leaves its formulation and penetrates the skin are explained in next paragraphs.

The partition coefficient

Consider a drug formulated in a complex vehicle such as an emulsion, which is applied directly onto the skin. The first contact that the product makes with the skin is with the outermost skin layer, the stratum corneum. The drug therefore has to be absorbed into the stratum corneum from the product. The first critical step determining the skin absorption level of a drug from a topical delivery (for example, from a cream) is partition. Two partitions are involved. The first is partitioning of the drug between two emulsion phases (oil and water) and the second is partitioning of it between the skin and the formulation. Partition between the skin and the formulation is described by the (stratum corneum/formulation) partition coefficient, K_m, for the molecules of the penetrating ingredient. This partition coefficient is defined as:

Equation 1 shows that the partitioning of a drug into the stratum corneum is enhanced when the drug has a higher affinity for the stratum corneum than for the formulation. Lipophilic drugs have a greater affinity for the stratum corneum and therefore tend to accumulate in this layer. The partition coefficient K_oct/water of a drug between octan-1-ol and water (or rather the logarithm of this value, log K_oct/water) is used in pharmaceutics as an in vitro model for the partition coefficient given in Equation 1 [59, 60]. It is considered a good model, representative of the heterogeneous nature of the stratum corneum.

To enhance the partitioning of a drug, the most common way is to change its solubility in the formulation. Changing the solubility or the concentration of dissolved drug in the stratum corneum is rather difficult. The concentration in the stratum corneum can only be increased if the drug leaves the stratum corneum and diffuses into the deeper skin layers.

The permeability coefficient

where P is the permeability coefficient with units of velocity (m.s^-1), K_oct/water the octanol/water partition coefficient, D the diffusion coefficient (m².s^-1) and L the length of the diffusion pathway of the penetrating molecule (m). The exact diffusion pathway of a drug in the skin cannot be determined. In general, the skin thickness is measured and its value is assumed to be the same as the length of the diffusion pathway, L [61]. However its value could be quite different if the intercellular route (pathway 1 in figure 1) through the SC structure was followed.

The determination of the permeability coefficient is rather difficult. Theoretical relationships have been developed taking into consideration only the two reliable parameters: the octan-1-ol/water partition coefficient and the relative molecular mass, M_r [37]. These theoretical relationships were first published in the late 1980s and early 1990s and are based on empirical values. Potts and Guy were amongst the first workers who developed a mathematical relationship [37].

These estimations of skin permeation coefficients are sometimes known as quantitative structure-permeability relationships (QSPeRs or QSPRs). Recent overviews of QSPeRs for permeation into human skin from water have been reported in the literature [62-66].

Fick’s Diffusion Laws

This law says that the flux (mass.m^-2.s^-1), which is the rate of transfer per unit area of a compound at a given time and position is proportional to the differential concentration change ∂C over the differential distance ∂x. The negative sign indicates that the flow is in the direction of decreasing thermodynamic activity (coefficient of activity multiplied by mole fraction), which can often be represented by concentration. To describe the concentration within a membrane, Fick’s First Law is combined with the differential mass balance existing in a membrane, making several assumptions: the compound is not metabolized, it does not bind with the membrane and its diffusion coefficient does not vary with position or composition [67]. The result is called Fick’s Second Law.

The individual layers of the skin can be treated as pseudo-homogeneous membranes and so Fick’s First Law can be applied to diffusion processes in these layers [68].

The flux at steady state (i.e. when the flux is constant) is given by:

where L is the length of the diffusion pathway of the penetrating molecules (layer thickness) (m), C₁ and C₂ are the mass concentrations (kg.m^-3) of the penetrant in the membrane at the two faces (at x = 0 and x = L) and D is the diffusion coefficient (m².s^-1) (figure 3).

Sink conditions will mean that the concentration at x = L is zero or very small (C₂ = 0). In addition, the concentration of penetrant at x = 0 is in local equilibrium with the concentration of penetrant in the formulation (C₁=K_m⋅C_f in which K_m is the pseudo-homogeneous partition, or distribution coefficient defined by Equation 1 and C_f the concentration in the formulation). Under these conditions, Equation 6 becomes:

Comparing Equation 7 with Equation 1 and replacing K_oct/water by K_m gives Equation 8, an alternative expression for the dermal permeability coefficient, P (m.s^-1) [29].

where J_ss is the steady-state flux of the solute and C_f represents the concentration of the penetrant in the formulation or vehicle when sink conditions apply.

A practical example

Figure 4 shows graphs comparing different formulation systems (a microemulsion, an emulsion and a gel).

These graphs show a diffusion lag time followed by a linear increase. Extrapolation of the linear section to meet the x-axis gives the lag time and the slope yields the pseudo steady-state flux J_SS. The dermal permeability coefficient can be calculated using Equation 8 where C_f is the concentration of the drug in the donor compartment. The lag time reflects a complex sequence of events including the release of the drug from its vehicle, the reorganization of the skin barrier and the diffusion of the drug through this time-varying medium. The time required for the permeation rate across a membrane to reach 95% of its steady-state value is approximately 2.3 times the lag time rising to 99% of the steady state value after approximately 3.2 times the lag time [72, 73].

A problem concerning skin delivery arises from the existence of two parameters that influence the permeability in contrasting ways. In general, firstly, and in accordance with Fick’s First Law, the flux increases when the concentration of the drug in the formulation increases (Equation 7). This can be achieved by increasing the drug solubility in the formulation. Secondly, the flux will also be increased if P is greater i.e. the relative solubility of the drug in the stratum corneum is greater than the solubility of the drug in the formulation. The relative contributions of the velocity of absorption (flux J) and the amount of absorbed active substance cannot be separated as the two parameters have been combined into one equation (Equation 7). The problem therefore remains. Finding the right compromise in each case is the only way to solve this problem. To do so successfully it is necessary to understand as much as possible about each of the parameters which influence the solubility of the active ingredient in the skin and in the formulation.

In the experiment described below, the authors used a constant caffeine concentration. The highest flux obtained with the microemulsion system is probably due to the higher amphiphilic character of the microemulsion system and the high amount of surfactants present which are able to modify the skin barrier. This will be discussed in the next section.

Penetration enhancement techniques

For optimising transdermal delivery of drugs with a molar mass bigger than 500 Da, active mechanisms have to be used. In electroporation, high voltage impulses are applied during short time intervals to create temporary pores on the skin. The driving force for drug permeation is either ion repulsion or electro-osmosis. Sonophoresis uses low frequency ultrasonic energy to disrupt lipid packing in the stratum corneum creating aqueous pores, which improve drug delivery [76, 77]. Other methods include (i) local thermal treatments [78, 79], (ii) the possibility of mechanical perforation of the stratum corneum by high-velocity particles (the so-called ballistic method) [79] and (iii) the use of a micro-needles array, the effects of which include temporary loosening of the barrier properties until the stratum corneum is restored to its usual state by the normal turnover cycle [80-82]. Iontophoresis is an electrically assisted method where the drug has to be used in an ionic form. By applying an external electrical field to the skin the active ingredient will be accelerated and as a result of electromigration and electro-osmotic forces it will be transported into the skin layers. This method is used for proteins and peptides, for example in insulin delivery in diabetes therapy. Insulin is negatively charged and has a relative molar mass of 6000 Da. A number of fundamental in vitro studies have investigated the effect of iontophoretic parameters on insulin delivery [83-88] while pharmacodynamic studies have demonstrated the physiological effect of iontophoretically delivered insulin on blood glucose levels in a variety of small animals, usually mice, rats or rabbits [85, 89, 90]. Kari [91] used cathodal iontophoresis with currents ranging from 0.2 to 0.8 mA (patch area 6.2 cm²) applied for 2 h in delivering insulin to alloxan-diabetic rabbits. The results showed that blood glucose levels decreased as serum insulin concentrations increased. Doubling the iontophoretic current from 0.2 to 0.4 mA produced a 3-fold increase in the serum insulin concentration but a further increase to 0.8 mA had no additional effect. This result was attributed to the generation of hydroxide ions at the electrode surface, which compete in the carrying of the current in the cathodal compartment [91].

When a drug does not have near-ideal physicochemical properties, formulation studies (passive skin penetration enhancement) become necessary. Formulation is the key to successful topical delivery. As previously mentioned, and in accordance with Fick’s First Law, there are two possibilities of penetration enhancement. Firstly the formulation can change the skin barrier and can therefore influence the Fick’s parameters (diffusion coefficient and partition coefficient). Secondly the formulation can optimize the drug behaviour in the galenic system and therefore also influence the Fick’s parameters.

Modification of the skin barrier

Hydration

Fluhr et al. explain in their review [101] the importance of the hydration of the stratum corneum for normal functioning of the biochemical and biophysical processes in the skin. It has been shown that water acts as a plasticizer for both the corneocyte proteins and the intercellular medium [102]. In dry skin or even in diseases with reduced stratum corneum hydration, e.g. ichthyosis vulgaris and winter xerosis [103], the formation of the rigid cornified envelope (CE) of the corneocytes (corneodesmolysis) is impaired due to the level and activity of transglutaminase (a key enzyme in the cross-linking of CE proteins) [104]. Skin xerosis in patients with end-stage renal disease correlates with reduced levels of endogenous glycerol in the stratum corneum [105]. Thus glycerol plays an important role in sustaining skin hydration. It was confirmed by Breternitz et al. that glycerol exerts its hydrating effect not only on healthy skin but also on subjects with diseased skin, primarily characterized by xerosis and skin barrier impairment [106]. Glycerol has an action on aquaporin-3, a homologous water-transporting protein in many mammalian epithelial, endothelial and other cell types [107]. The effect of glycerol on hydration in the entire stratum corneum was obtained by in vivo Raman microspectroscopy. Chrit et al. revealed an increase in water content and so an increase in skin moisture after application of a glycerol-based cream, which is the most widely used hydrating agent [108].

Barichello et al. [109] studied the transdermal delivery of isosorbide-5-nitrate (ISN) on rat abdominal skin in vitro firstly from liposomal systems and secondly from the drug solution used as control. In both cases studies were carried out using 5% glycerol and without glycerol. The authors showed that the amount of ISN permeated through rat abdominal skin from a liposomal formulation containing 5% glycerol was significantly higher when compared with the amount of ISN permeated from the other formulations (p < 0.001). No significant difference in the permeated ISN mean values was noticed among the other tested formulations. The authors explained that the enhancement effect of glycerol might be due to an increase in stratum corneum hydration.

Lipid disruption/fluidization by chemical penetration enhancers

Surfactants, DMSO, decylmethylsulphoxide and urea can also interact with keratin in the corneocytes [119]. The resulting increase in the diffusion coefficient is caused by binding with keratin filaments after the chemicals have penetrated into the intercellular matrix of the stratum corneum. This results in a disruption of order within the corneocytes. However in many studies it has been shown that there is a close relationship between permeation enhancement and lipid bilayer fluidization and that the lipid lamella of the stratum corneum is the main site of action [7, 26, 41, 120].

Kim et al. used a pore-forming peptide (magainin) under co-enhancement by NLS-ethanol (N-lauroyl sarcosine) to increase skin permeability. They showed a 47-fold increase in penetration of magainin into the stratum corneum, by NLS-ethanol enhancement. The magainin also increased stratum corneum lipid disruption (and especially so in ceramides and cholesterol) and skin permeability. They presented this study as a novel concept, using a first chemical enhancer (NLS-ethanol) to increase penetration of a second chemical enhancer (magainin) into the skin in order to synergistically increase skin permeability of a model drug (fluorescein in their study) [121].

Formulation based enhancement

Prodrug

Prodrugs are also used to place charged drugs under the skin. A charge-neutralized complex is formed by adding an oppositely charged species to the charged drug so that the drug can diffuse into the aqueous viable epidermis where the charged parent drug is released by dissociation [126-128]. In each reported case the permeability increase obtained was only two to three-fold. But recently Sarveija et al. reported a 16-fold increase in the steady-state flux of ibuprofen ion pairs across a lipophilic membrane [129].

Eutectic systems

A good example is EMLA^® (AstraZeneca) cream. This is a formulation consisting of a eutectic mixture of the local anaesthetics lignocaine (m.p. 68 °C) and prilocaine (m.p. 16 °C), applied under an occlusive film as the free bases. Together they provide a more effective local anaesthetic effect and consequently prevent the pain associated with needle insertion. The mixture is a 1:1 eutectic mixture (eutectic temperature 18 °C) formulated as an oil-in-water emulsion which maximizes the thermodynamic activity of each component [130]. There are several eutectic systems containing a penetration enhancer as the second component, for example: ibuprofen with terpenes [131], menthol [132] and methyl nicotinate [133], lignocaine with menthol [134] and propranolol with fatty acids [135].

Complexation in cyclodextrins

In cosmetic applications complexation has improved the photostability of sunscreens [137, 138] but its influence on penetration behaviour is a compromise. There is an increasing effect due to better solubility [139-141] but a decreasing effect resulting from using molecules with large relative molar masses (equivalent to more than 1000 Da) [129, 142, 143].

A recent trend is the use of modified cyclodextrin molecules. The most commonly used is hydroxypropylbeta-cyclodextrin (HP-β-CD). It is able to form hydrophilic inclusion complexes with many lipophilic compounds in aqueous solution, which can enhance the aqueous solubilities of lipophilic drugs without changing their intrinsic abilities to permeate lipophilic membranes. An interesting example is sunscreen delivery onto a skin surface. Simeoni et al. have investigated the penetration of oxybenzone, a lipophilic sunscreen agent, on human skin, from HP-β-CD and from SBE-β-CD, a sulfobutylether-β-cyclodextrin [142, 144]. The authors showed that SBE-β-CD had the greater solubilizing activity on oxybenzone, a highly lipophilic sunscreen, (a 1049-fold increase) when compared with the use of HP-β-CD (a 540-fold increase). The sunscreen penetration to the deeper living layers of the skin was remarkably decreased (1.0% and 2.0% of applied dose for epidermis and dermis respectively) compared with the unbound OMC formulation used as control and with OMC loaded HP-β-CD (~5%). This result is interesting because this type of carrier can promote the solubilizing and photostabilizing properties of sunscreen agents while staying on top of the skin where they are intended to act [144].

Even with modified complexes, conflicting results have been found in the literature concerning their effect to promote or decrease skin penetration of drugs. But there still remain the problems of their molar mass and their limited capacity to penetrate into the skin [145].

Chemical potential

where α is the thermodynamic activity of the drug in the formulation and γ is the apparent activity coefficient in the membrane. The dependence of the flux on thermodynamic activity rather than concentration has been demonstrated by Twist and Zatz [146, 147].

Moser [148] has shown the enhancement effect of supersaturated formulations using a lipophilic model compound (a lavendustin derivative, LAP) passing through excised pig skin in vitro. He has also drawn attention to the supersaturation effect, by replacing in Equation 7 the partition coefficient with the relationship given in Equation 1, so that Equation 7 now becomes:

where C_f/C_S,_f is the degree of drug saturation in the formulation. When this latter term is equal to 1 the formulation is saturated and if it is bigger than 1 it is supersaturated. C_f is the drug concentration dissolved in the formulation, C_S,SC is the solubility of the drug in the barrier (stratum corneum) and C_S,f the solubility of the drug in the formulation. Supersaturated solutions can be produced by evaporation of solvent or by a co-solvent technique. This involves mixing two solvents chosen so that the drug is significantly more soluble in one solvent than the other. This supersaturation technique can only be used in certain instances. One of the major drawbacks of this method is that the thermodynamic instability of the supersaturated solutions leads to crystallization of the drug and hence to a decrease of the drug flux.

Use of microemulsions

Moreover the authors compared the in vitro permeation of microemulsions to EMLA^® cream, a eutectic mixture of lidocaine and prilocaine (2.5%). A superior transdermal permeation coefficient of lidocaine was obtained with EMLA^® compared with microemulsions, which contained between 9 and 27% of lidocaine (saturated conditions in the different microemulsions studied). The authors explained this result by the eutectic composition, which exhibits a unique thermodynamic activity in the formulation. Nevertheless the microemulsion containing 9.1% of lidocaine exhibited an average transdermal flux 50% larger. This could be explained by a larger 4-fold concentration gradient in the microemulsion.

It can be concluded from this study that the drug delivery potential of microemulsions is greatly dependent on the internal structure/fractional composition of the phases.

A correlation between transdermal drug fluxes and their self-diffusion coefficients determined by spin echo NMR spectroscopy has been found. This result has been explained by the increased solubility of the drugs (lidocaine showed a 28-62% increase in solubility and prilocaine hydrochloride an increase of 24-40%), which appeared to be dependent on the drug mobility in the individual vehicles. This result has been supported by other earlier studies [155, 156].

Nanoparticles

Lipid nanoparticles

SLNs possess some advantages when compared with liposomes (which are also lipid carriers but without a solid structure) and emulsions, e.g. protection against chemical degradation of a drug and the modulating capacity of active compound release.

The main disadvantages of SLNs are that during storage the drug entrapped is expulsed due to a change in lipid conformation to a lower energy crystal state. This transformation from polymorphic to a more stable crystalline form stops any guest molecules from being included in the structure.

To overcome this problem NLCs were developed. In these nanocarriers solid and liquid lipids are mixed in such a combination that the particles solidify upon cooling but do not re-crystallize, remaining in an amorphous state. This allows the drug to be accommodated in the particles for a longer time and so will increase the drug loading capacity of the systems [162].

Through the use of solid lipid nanoparticles, an occlusive film can be formed on the skin leading to an increase in skin hydration. However, SLNs have a high surfactant content that causes fast penetration in and through the skin. Often there is no depositing effect in the skin layers and thus they are of interest for fast transdermal delivery of actives [163-166]. Wissing et al. showed a 31% increase in skin hydration after applying an SLN-enriched cream to skin for four weeks [167]. In a recent study, Küchler et al. compared dendritic core-multishell (CMS) nanotransporters (20-30 nm) and SLNs (150-170 nm) loaded with Nile red [168]. They found that SLNs enhanced pig skin penetration 3.8 fold in the stratum corneum and 6.3-fold in the epidermis compared with a reference cream. The potential for CMS use is greater since increased penetrations of 8-fold and 13-fold were obtained; these results can be attributed to the smaller size of CMS transporters.

Jenning et al. studied intensively in vitro penetration with lipid nanoparticles loaded with retinol. They compared the flux of retinol from a lipid particle dispersion with the flux of retinol from an o/w nanoemulsion. The flux of retinol from the nanoemulsion system remained unchanged during 24h but, due to increased order and increased expulsion of drug, the flux from nanoparticle dispersion increased [169, 170].

Lombardi et al. [171] studied the skin penetration profiles of SLNs, NLCs and nanoemulsions (NEs) by using the lipophilic dye Nile red as a model agent. Nile red was incorporated into the lipid matrix or the covering tensed shell. The Nile red concentrations were followed by image analysis of vertical sections of pig skin treated with dye-loaded nanoparticular dispersions and an o/w cream. Using SLN dispersions dye penetration increased about fourfold over the uptake using a cream. The use of NLCs was less effective (< 3-fold increase) and penetration appeared to be even further reduced when applying NEs. In contrast to previous studies with glucocorticoids attached to the surfaces of SLNs, no targeting effect was detected. Therefore drug targeting appears to be more closely related to the mode of interaction of drug and particle than to penetration enhancement [171].

Polymeric nanocarriers

Microencapsulation in such polymers has been extensively applied in the field of protein delivery. The major research challenges in protein delivery include the stabilization of proteins in delivery devices and the design of appropriate protein carriers in order to overcome biological barriers. Moreover, a long lasting immune response could be induced by such systems. For vaccine delivery the immunogenic components must be efficiently delivered in the appropriate compartments of the immune system and especially in the dendritic cells (DCs). Biodegradable polymers such as PLGA are good candidates because they do not induce a strong or durable immune response against themselves. Moreover they are efficiently degraded into non-toxic metabolites [182]. The most important parameters regarding the immune responses are the size of the particles [183] and their surface properties. Using nucleic acids it has been shown that positively charged particles facilitate association with them and, similarly, with negatively charged protein antigens, via ionic cross-linking with the particles.

Particle charge is also an important parameter for enhanced delivery in vivo. Kohli et al. have reported that efficient permeation on pig skin was obtained with negatively charged particles ranging in size between 50 and 500 nm [184]. Despite these advances there is little information concerning biological interactions between DCs and particulate carrier systems [182].

PLGA microparticles have also been described as vehicles for topical drug delivery of rhodamine and acyclovir, providing a reservoir system for release in the basal epidermis [180, 185].

Alvarez-Román et al. reported the visualization of skin penetration of nanoparticulate carriers by confocal laser scanning microscopy (CLSM) [186]. In this study, CLSM was used to visualize the distribution of non-biodegradable, fluorescent, polystyrene nanoparticles (diameters 20 and 200 nm) across porcine skin. The surface images revealed that polystyrene nanoparticles and, in particular, the smaller particles, accumulated preferentially in follicular openings, and that this distribution increased in a time-dependent manner. In all cases, the progression of nanoparticules into the skin was impeded by the stratum corneum. The accumulation of such particles in the epidermis is of interest in anti-solar studies. Some research has been focused on the skin delivery of UV filters from polymeric nanoparticles because these substances must accumulate on the skin surface and penetrate as little as possible into the viable skin. Alvarez-Román et al. [187] reported epidermal targeting of octylmethoxycinnamate (OMC) poly-ε-caprolactone nanoparticles. OMC-loaded nanoparticles resulted in better photoprotection thus promoting a partial protection against erythema. These authors suggested that the extent of crystallinity in a polymer may contribute to reflection and scattering of UV radiation on its own thus leading to partial photoprotection [188, 189]. Later, Jimenez et al., who studied the permeation of octylmethoxycinnamate (OMC) from OMC-loaded poly-ε-caprolactone nanoparticles using the Franz cell method in our laboratory, showed quantitatively that the penetration % of OMC in the skin was three to four times lower than from a conventional emulsion and was clearly impeded by the stratum corneum, confirming the results of Alvarez-Román et al. Despite the apparent advantages of polymeric nanoparticles compared with other drug delivery systems, they appear to have been comparatively little studied for drug delivery to the skin.

Liposomes and analogue vehicles

Whilst researchers were reporting mainly localised or, rarely, transdermal effects of liposomes, Cevc and Blume [194] claimed that certain types of lipid vesicles (ultradeformable vesicles) can penetrate intact into the deeper layers of the skin and may progress far enough to reach the systemic circulation, but to do so they must be applied under non-occlusive conditions [192].

Transfersomes^® (IDEA AG, Munich, Germany) contain 10-25% surfactant (sodium cholate) and 3-10% ethanol. Such compositions confer ultradeformability to the transfersomes^® allowing them to squeeze through channels in the stratum corneum that are less than one-tenth the diameter of the transfersomes^® themselves (up to 500 nm). Cevc et al. suggested that the driving force for penetration into the skin is the transdermal gradient caused by the difference in water content between a relatively dehydrated skin surface (approximately 20% water) and an aqueous viable epidermis (close to 100% water) [195-198]. Extraordinary claims have been made for the penetration enhancement ability of transfersomes^®, such as skin transport of 50-80% of the applied dose of transfersome^®-associated insulin [199].

Ethosomes are liposomes with a high ethanol content. The alcohol fluidizes the ethosomal lipids and stratum corneum bilayer lipids thus allowing better penetration [200, 201]. Dayan et al. have compared the flux of trihexyphenidyl hydrochloride (THP HCl) from THP ethosomes and liposomes. Their results indicated that the flux of THP through nude mouse skin from THP ethosomes was, respectively, 87, 51 and 4.5 times higher than from liposomes, phosphate buffer solution and hydroethanolic solution (p < 0.01) [202].

Conclusion

At the same time research is taking place on galenic formulations for topical application to skin. This is currently an exciting and fast moving area. There are numerous formulation parameters and formulation systems, which influence percutaneous penetration. In this review the newest examples have been given and discussed. But the effects of these systems on the skin still cannot be completely explained. The study of microemulsions and nanoparticles is an on-going area of research seeking to optimize and explore their effects. Even after thousands of years the development of new strategies guarantees that topical delivery remains an area of considerable interest.

Acknowledgement

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