Lecithin-based nanoemulsions of traditional herbal wound healing agents and their effect on human skin cells.

In previous studies, lecithin-based nanoemulsions (NEs) have been shown to be skin friendly drug carrier systems. Due to their nontoxic properties, NEs might also be suitable as wound healing agents. Hence, different O/W NEs based on lecithin Lipoid® S 75 and plant oils or medium chain triglycerides were produced and characterised. Two lipophilic natural wound healing agents, a betulin-enriched extract from birch bark (BET) and a purified spruce balm (PSB), were successfully incorporated and their effects on primary human skin cells were studied in vitro. MTT, BrdU and scratch assays uncovered the positive influence of the drug-loaded NEs on cell viability, proliferation and potential wound closure. Compared to control formulations, the NEs loaded with either BET or PSB led to higher cell viability rates of fibroblasts and keratinocytes. Higher proliferative activity of keratinocytes and fibroblasts was observed after the treatment, which is a prerequisite for wound closure. Indeed, in scratch assays NEs with PSB and notably BET showed significantly ameliorated wound closure rates than the negative control (unloaded NEs) and the positive control (NEs with dexpanthenol). Our findings suggest that BET and PSB are outstanding wound healing drugs and their incorporation into lecithin-based NEs may represent a valid strategy for wound care.

Effects of lecithin-based nanoemulsions on skin: Short-time cytotoxicity MTT and BrdU studies, skin penetration of surfactants and additives and the delivery of curcumin.

Surfactants are important ingredients in pharmaceutical and cosmetic formulations, as in creams, shampoos or shower gels. As conventional emulsifiers such as sodium dodecyl sulfate (SDS) have fallen into disrepute due to their skin irritation potential, the naturally occurring lecithins are being investigated as a potential alternative. Thus, lecithin-based nanoemulsions with and without the drug curcumin, known for its wound healing properties, were produced and characterised in terms of their particle size, polydispersity index (PDI) and zeta potential and compared to SDS-based formulations. In vitro toxicity of the produced blank nanoemulsions was assessed with primary human keratinocytes and fibroblasts using two different cell viability assays (BrdU and EZ4U). Further, we investigated the penetration profiles of the deployed surfactants and oil components using combined ATR-FTIR/tape stripping experiments and confirmed the ability of the lecithin-based nanoemulsions to deliver curcumin into the stratum corneum in tape stripping-UV/Vis experiments. All manufactured nanoemulsions showed droplet sizes under 250 nm with satisfying PDI and zeta potential values. Viability assays with human skin cells clearly indicated that lecithin-based nanoemulsions were superior to SDS-based formulations. ATR-FTIR tests showed that lecithin and oil components remained in the superficial layers of the stratum corneum, suggesting a low risk for skin irritation. Ex vivo tape stripping experiments revealed that the kind of oil used in the nanoemulsion seemed to influence the depth of curcumin penetration into the stratum corneum.

Cytotoxicity of lecithin-based nanoemulsions on human skin cells and ex vivo skin permeation: Comparison to conventional surfactant types.

As constituents of cellular membranes, lecithins feature high biocompatibility and great emulsifying properties due to their amphiphilicity. Additionally, there are expectations that these naturally occurring emulsifying agents can replace other skin damaging emulsifiers like sodium dodecyl sulfate or sodium laureth sulfate. However, cytotoxicity data of lecithin-based formulations on primary human skin cells are scarce. Thus, we developed nanoemulsions with different kinds of surfactants (amphoteric, anionic and non-ionic), studied the skin permeation of a model drug from this formulations employing Franz-type diffusion cells and monitored their cytotoxicity potential on primary human keratinocytes and fibroblasts using a cell proliferation assay. The skin diffusion studies demonstrated that the amphoteric lecithin-based emulsifiers were superior to non-ionic surfactants in terms of skin permeation, but inferior to anionic emulsifiers. Further, we found that the nanoemulsions containing the amphoteric lecithins as emulsifying agents lead to significantly higher viability rates of both epidermal keratinocytes and dermal fibroblasts than the investigated anionic and non-ionic surfactants. This renders them a promising alternative to conventional emulsifiers used in daily products.

Simultaneous penetration monitoring of oil component and active drug from fluorinated nanoemulsions.

In the field of dermal drug delivery, determining the penetration depth of actives is a standard procedure for the development of novel formulations. Regarding the vehicle components, respective penetration studies are rather scarce due to their often challenging analytics. However, an understanding of the interactions between drugs and additives during skin penetrating could help to develop promising drug delivery systems. Thus, the objective of the present study was to simultaneously monitor the skin penetration of the incorporated model drug diclofenac sodium and the semifluorinated oil perfluorohexyloctane (F6H8) from newly developed nanoemulsions. In vitro tape stripping studies were conducted and the tapes were analysed for their content of drug and additive in parallel by HPLC and 19F NMR. The penetration depth and total recovered amount of both substances of interest were successfully determined on each tape strip. The vehicle oil compound F6H8 itself showed a very small skin penetration, while the penetration of diclofenac sodium was consistently about 9- to 10-fold higher. Higher amounts of the oil content led to higher skin penetration of diclofenac sodium and slightly increased oil penetration; this effect might be explained by the increasing occlusion effect caused by increasing amounts of fluorinated oil.

Natural microemulsions: formulation design and skin interaction.

Microemulsions are thermodynamically stable, colloidal drug delivery systems. This study presents the first substantiated comparison of natural, skin-compatible and biodegradable surfactants in terms of their suitability to form isotropic microemulsions and their skin interaction. Pseudoternery phase diagrams were constructed for lecithin, sucrose laurate and alkylpolyglycoside as single surfactants. Moreover, also mixed surfactant films of lecithin and alkylpolyglycoside as well as lecithin and sucrose laurate were tested. Large isotropic areas could be identified for lecithin, sucrose laurate and lecithin-sucrose laurate. One defined composition was chosen from the pseudoternery phase diagram, prepared with all investigated surfactants and 1:1 surfactant mixtures, respectively, and analysed for their effect on the stratum corneum on a molecular level by ATR-FTIR. Significantly higher frequency values of the symmetric and asymmetric CH(2)-stretching bands compared to the control were recorded for all microemulsions, indicating a hexagonal arrangement of the lipid chains. A similar trend was observed for the lateral packing of the alkyl chains as suggested by the shift of the CH(2)-scissoring bands. Moreover, diffusion cell experiments using porcine skin were performed with the two model drugs flufenamic acid and fluconazole. In both cases, the lecithin-based microemulsions showed the highest permeation rates followed by the alkylpolyglycoside-lecithin microemulsions.

Modification of the conformational skin structure by treatment with liposomal formulations and its correlation to the penetration depth of aciclovir.

The stratum corneum (SC), top layer of the epidermis, is comprised mostly of lipids that are responsible for the permeability properties of the SC and which protect the body from external agents. Changes in these skin microconstituents can be understood by instrumental methods such as attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy. The present work shows that different types of analyzed skin, dermatomed abdominal porcine skin, pig ear skin, and human heat separated skin, influenced both the shape and the intensity of recorded spectra. The typical FTIR spectral bands of the conformation of the lipid aliphatic chains in the skin samples were altered after treatment with pure DPPC liposomes and chitosan (CS) coated DPPC liposomes, but not with aqueous CS-solution. The conformational change could be the reason for the variable permeability of the skin. This was confirmed by tape stripping on pig ear skin (imitating in vivo studies): the amount of aciclovir penetrating from polymer coated and polymer free liposomes was significantly higher under the skin surface in comparison with the aqueous CS-solution. Moreover, the addition of the polymer to liposomes induced a higher skin penetration than pure liposomes. One explanation might be the CS's stronger adhesion to the skin.

Development of sucrose stearate-based nanoemulsions and optimisation through γ-cyclodextrin.

Nanoemulsions aimed at dermal drug delivery are usually stabilised by natural lecithins. However, lecithin has a high tendency towards self-aggregation and is prone to chemical degradation. Therefore, the aim of this study was to develop nanoemulsions with improved structure and long-term stability by employing a natural sucrose ester mixture as sole surfactant. A thorough comparison between the novel sucrose stearate-based nanoemulsions and corresponding lecithin-based nanoemulsions revealed that the sucrose ester is superior in terms of emulsifying efficiency, droplet formation as well as physical and chemical stability. The novel formulations exhibited a remarkably homogeneous structure in cryo TEM investigations, as opposed to the variable structure observed for lecithin-based systems. The in vitro skin permeation rates of lipophilic drugs from sucrose stearate nanoemulsions were comparable to those obtained with their lecithin-based counterparts. Furthermore, it was observed that addition of γ-cyclodextrin led to enhanced skin permeation of the steroidal drug fludrocortisone acetate from 9.99±0.46 to 55.10±3.67 µg cm(-2) after 24 h in the case of sucrose stearate-based systems and from 9.98±0.64 to 98.62±24.89 µg cm(-2) after 24 h in the case of lecithin-based systems. This enhancement effect was significantly stronger in formulations based on lecithin (P<0.05), which indicates that synergistic mechanisms between the surfactant and the cyclodextrin are involved. Cryo TEM images suggest that the cyclodextrin is incorporated into the interfacial film, which might alter drug release rates and improve the droplet microstructure.

Skin-compatible lecithin drug delivery systems for fluconazole: effect of phosphatidylethanolamine and oleic acid on skin permeation.

The purpose of the present study was to evaluate skin-compatible drug delivery systems for fluconazole. Pseudoternary phase diagrams were constructed, composed of different soybean lecithins/oil/isopropanol and water. The role of the various lecithin compositions was expressed in the different resulting isotropic areas. Based on these phase diagrams, two systems were chosen as drug delivery systems for fluconazole. The influence of phosphatidylethanolamine and of the oil component on the skin permeation of fluconazole was investigated. The more phosphatidylethanolamine, the greater was the fluconazole skin permeation, independent of the hydrophilicity of the system. The influence of oleic acid and isopropylmyristate as the oil component was compared and a greater penetration enhancing effect was found for the microemulsion containing oleic acid.

Evaluation of an eucalyptus oil containing topical drug delivery system for selected steroid hormones.

In the present study the permeation and the chemical stability of 17-beta-estradiol, progesterone, cyproterone acetate and finasteride incorporated in an eucalyptus oil containing microemulsion system have been investigated. The formulations contained 1% (w/w) of the steroid hormones. Self diffusion coefficients determined by pulsed-field-gradient spin echo NMR spectroscopy were used to characterise the microemulsion. From these results a bicontinuous structure is proposed for the multicomponent system. However a correlation between the self diffusion of the hormones in the vehicle and the transdermal flux was not indicated. Explanations for this were self assembling, formation of aggregates between the components of the microemulsion and drugs and different effects because of different solubility of the drugs. By addition of certain polymers the skin permeation rates could be improved with exception of cyproterone acetate. Beside standard diffusion experiments, the residual drug content in the skin was investigated. Drug stability was monitored by analysing the steroid hormone content in the different formulations over an observation period of 6 weeks and could be improved by polymers. In addition, viscosity measurements were performed. They indicated an influence of the polymers and drugs on the viscosity in all formulations.

Influence of 6-ketocholestanol on skin permeation of 5-aminolevulinic acid and evaluation of chemical stability.

The ability of 6-ketocholestanol to increase the skin permeation of the prodrug aminolevulinic acid (5-ALA) was investigated. 6-Ketocholestanol was incorporated together with 5-ALA in four different formulations. Preparations used were a liquid solution/suspension of 5-ALA in buffer, 5-ALA in phospholipid liposomal formulations with and without gelating agent, and finally, a complex cream formulation also including phospholipids. Standard diffusion experiments of 5-ALA using Franz-type diffusion cells and porcine skin were performed. Drug stability was monitored by analyzing the 5-ALA content in the different formulations over time and viewing the preparation for microbial contamination. The analysis of 5-ALA as a nonfluorescent probe was performed after chemical reaction, leading to a fluorescent derivative. The 5-ALA permeation through porcine skin was increased threefold by 6-ketocholestanol in the cream formulation. The chemical stability of 5-ALA in the tested formulations was in the range of about 33 to 72% after an observation period of 28 days. After that time point microbial stability was no longer evident for formulations 2 and 3. Formulation 1 could be observed until day 34, and only formulation 4 showed a microbial stability over the whole observation period of 42 days.

The use of polymers for dermal and transdermal delivery.

The use of polymers for skin preparations is manifold. Requirements of such polymers are dependent on the formulation types. The most applied polymers on skin belong to various classes, for example to cellulose derivatives, chitosan, carageenan, polyacrylates, polyvinylalcohol, polyvinylpyrrolidone and silicones. They are gelating agents, matrices in patches and wound dressings, anti-nucleants and penetration enhancers. Correlations between commercially available products and results of new scientific investigations are often difficult or not possible, because of the lack of comparative data especially for transdermal patches. Finally, two promising future trends of polymeric systems, gene delivery and tissue engineering, are discussed.

Influence of carrageenan on the rheology and skin permeation of microemulsion formulations.

Three different microemulsions (A-C) and one semisolid preparation D were investigated in terms of viscoelastic properties and skin permeation of the model compound sodium fluorescein. The influence of the polysaccharide carrageenan on these parameters was investigated. Carrageenan is frequently used as food additive and has very interesting properties like good adhesiveness on skin which can be a benefit for topical application. The viscoelastic properties of the preparations (A-D) and of mixtures with carrageenan (A'-D') were characterized by oscillatory measurements. It was possible to adjust the rheologic properties of the formulations and to increase the sodium fluorescein permeation through porcine skin by mixing them with carrageenan gels. Therefore, the presented formulations as well as mixtures with carrageenan might be promising alternative drug carrier systems for topical pharmaceutical as well as cosmetics.

Influence of molecular dipoles on human skin permeability: Use of 6-ketocholestanol to enhance the transdermal delivery of bacitracin.

In the present work, we report the possibility of modifying the electrostatic properties of the skin by treating human epidermis with compounds whose structures possess a large molecular dipole moment. Data are presented showing that such a modification can be used to enhance dermal drug delivery. Inclusion of such compounds in biological membranes affects the so-called membrane dipole potential, an electrical potential originating from molecular dipoles present on the lipid molecules. Modifications in the magnitude of this potential are known to affect the interaction of hydrophobic ions and peptides with model membranes. Using fluorescein-labeled bacitracin and confocal microscopy, we show that the penetration of the antibiotic peptide bacitracin into the epidermis is enhanced when the skin has been pretreated with liposomes loaded with 30 mol % 6-ketocholestanol, a compound known to increase the magnitude of the membrane dipole potential. Studies using the fluorescent indicators fluoresceinphosphatidylethanolamine and 1-(3-sulfonatopropyl)-4-[beta [2-(di-n-octylamino)-6-naphthyl] vinyl] pyridinium betaine show that the interaction of bacitracin with model membranes is also enhanced by the presence of 6-ketocholestanol in the bilayer and offers some indication to the mechanism of penetration enhancement.