Julia C. Schwarz

In vitro vs. in vivo tape stripping: Validation of the porcine ear model and penetration assessment of novel sucrose stearate emulsions

Porcine ear skin is frequently used as a substitute for human skin in dermatological research and is especially useful for tape stripping experiments where the penetration of active substances into the uppermost skin layers is investigated. However, certain differences between the surface properties of these skin types exist, and reports on the comparability of tape stripping data obtained in vitro using porcine ear skin and data obtained in vivo on human forearm skin are scarce. Thus, we performed comparative tape stripping experiments in which the skin penetration of curcumin and fluorescein sodium from conventional microemulsions and hydrogels was investigated. In this context, the skin penetration potential of novel semi-solid macroemulsions and fluid nanoemulsions based on sucrose stearate was evaluated as well. The removed corneocytes were quantified by NIR-densitometry using recent correlation data for human and porcine proteins. The trends observed for the skin penetration into porcine ear skin were highly representative for the in vivo situation on human skin, confirming that the porcine ear is an excellent in vitro model for tape stripping experiments. Moreover, the validity of the NIR-densitometric approach for the quantification of both human and porcine stratum corneum proteins was confirmed in this study for the first time.

Facilitating in vitro Tape Stripping: Application of Infrared Densitometry for Quantification of Porcine Stratum Corneum Proteins

Infrared (IR) densitometry is a highly practical method recently proposed for protein analysis during in vivo tape stripping. However, this method has not yet been validated for the quantification of porcine stratum corneum (SC) proteins. Therefore, the aim of this study was to establish calibration curves for the analysis of adhesive tapes removed from porcine ear skin. To this end, the protein absorption (as determined via IR densitometry) was correlated with the protein content determined with the Micro BCA™ protein assay after extraction of the tapes. The obtained linear regressions confirm that IR densitometry is suitable for the quantification of not only human, but also porcine, SC proteins. The pattern of protein removal observed with porcine skin differs from that of human skin due to more pronounced corneocyte clustering and deep ‘canyons’, which necessitates specific evaluation of porcine skin samples and a working protocol that takes this into account. The presented data will facilitate future analysis of porcine SC proteins during in vitro tape stripping.

Nanocarriers for dermal drug delivery: Influence of preparation method, carrier type and rheological properties

Nanocarriers are highly interesting delivery systems for the dermal application of drugs. Based on a eudermic alkylpolyglycosid nanoemulsions, solid lipid nanoparticles (SLN) and nano-structured lipid carriers (NLC) were prepared by ultrasonic dispersion. The ultrasound preparation technique turned out to be convenient and rapid. For reasons of comparison, nanoemulsions were also prepared by high-pressure homogenisation with highly similar physicochemical properties. Cryo electron microscopy was employed to elucidate the microstructure of the ultrasound-engineered nanocarriers. Furthermore, in vitro skin experiments showed excellent skin permeation and penetration properties for flufenamic acid from all formulations. Moreover, ATR-FTIR studies revealed barrier-restorative properties for NLC and SLN. Furthermore, the rheological characteristics of all nanocarriers were determined. In order to increase the viscosity, three different polymers were employed to also prepare semi-solid NLC drug delivery systems. All of them exhibited comparable skin diffusion properties, but may offer improved dermal applicability.

Ultra-small NLC for improved dermal delivery of coenyzme Q10

Coenzyme Q10 (CoQ10) acts as an antioxidant in the skin and is frequently contained in anti-aging products. In previous studies, it could be shown that nano-structured lipid carriers (NLC) with a size of about 230 nm are beneficial for the dermal delivery of CoQ10. They increased Q10 skin penetration when compared to equally sized nanoemulsion. In this study, ultra-small NLC were prepared with even smaller mean particles sizes of around 80 nm. The influence of this decrease of particle size was investigated in terms of skin permeation and penetration as well as physicochemical stability of the NLC. Improved dermal delivery of CoQ10 by ultra-small NLC could be achieved.

Semi-solid Sucrose Stearate-Based Emulsions as Dermal Drug Delivery Systems.

Mild non-ionic sucrose ester surfactants can be employed to produce lipid-based drug delivery systems for dermal application. Moreover, sucrose esters of intermediate lipophilicity such as sucrose stearate S-970 possess a peculiar rheological behavior which can be employed to create highly viscous semi-solid formulations without any further additives. Interestingly, it was possible to develop both viscous macroemulsions and fluid nanoemulsions with the same chemical composition merely by slight alteration of the production process. Optical light microscopy and cryo transmission electron microscopy (TEM) revealed that the sucrose ester led to the formation of an astonishing hydrophilic network at a concentration of only 5% w/w in the macroemulsion system. A small number of more finely structured aggregates composed of surplus surfactant were likewise detected in the nanoemulsions. These discoveries offer interesting possibilities to adapt the low viscosity of fluid O/W nanoemulsions for a more convenient application. Moreover, a simple and rapid production method for skin-friendly creamy O/W emulsions with excellent visual long-term stability is presented. It could be shown by franz-cell diffusion studies and in vitro tape stripping that the microviscosity within the semi-solid formulations was apparently not influenced by their increased macroviscosity: the release of three model drugs was not impaired by the complex network-like internal structure of the macroemulsions. These results indicate that the developed semi-solid emulsions with advantageous application properties are highly suitable for the unhindered delivery of lipophilic drugs despite their comparatively large particle size and high viscosity.

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.

Decrease of liposomal size and retarding effect on fluconazole skin permeation by lysine derivatives.

Liposomes are ideal dermal drug delivery systems because of their ability to alter the biodistribution profile of incorporated drugs. In a novel approach to optimize the liposomal microstructure, lysine derivatives were employed. The effect of the oligopeptides Lys-5 and Lys-7 on the structure as well as on the skin permeation of the antimycotic drug fluconazole in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles was studied using a variety of techniques. It was demonstrated by addition of the shift reagent praseodymium(III)chloride and subsequent (31)P NMR measurements that the liposomes produced consisted mainly of unilamellar vesicles. This was confirmed by cryo-transmission electron microscopy. The addition of Lys-5 and Lys-7 induced a structural change resulting in a decrease in particle size between 10% and 40% and a retarding effect on fluconazole skin permeation.

Comparison of ATR–FTIR spectra of porcine vaginal and buccal mucosa with ear skin and penetration analysis of drug and vehicle components into pig ear

In the present study, porcine buccal and vaginal mucosae were successfully characterised by ATR-FTIR for the first time and compared to porcine ear skin. By analysing typical bands of the spectra, the structure of proteins and the lipid matrix were elucidated. According to the body site, differences in membrane permeability were detected when analysing the CH2-stretching and -scissoring vibrations. The results indicated a higher permeability for porcine vaginal and buccal tissue compared to skin. Furthermore, the influence of a lecithin-based microemulsion on the barrier properties of the above mentioned tissues was investigated by ATR-FTIR; the results revealed structural changes in all tissues. In addition, the ATR-FTIR technique was employed to semi-quantitatively analyse compounds directly on skin. To this end, tape stripping experiments were performed with a deuterated liposomal drug delivery system containing the model drug flufenamic acid. While the amount of penetrated deuterated liposomes was determined directly on skin samples by ATR-FTIR, the drug amount was analysed by HPLC after extraction of the tape strips since higher sensitivity was achieved in this fashion. Thus, it was possible to monitor the skin penetration of drug and vehicle simultaneously. Interestingly, the results indicated an independent drug penetration after release from the liposomal carrier system.

Skin integrity testing and monitoring of in vitro tape stripping by capacitance-based sensor imaging

Despite the frequent use of porcine ear skin for tape‐stripping experiments, the peculiarities of this skin type have not been characterised in detail yet. Thus, different techniques were employed to investigate the skin surface structure of porcine ear skin and the changes in barrier function during in vitro tape stripping. To this end, the potential of capacitance‐based skin hydration imaging as a means of skin quality control was investigated for the first time.

Corneocyte Quantification by NIR Densitometry and UV/Vis Spectroscopy for Human and Porcine Skin and the Role of Skin Cleaning Procedures

Optical methods of corneocyte quantification during tape stripping experiments on the skin are useful tools for the rapid evaluation of the skin penetration potential of dermally applied substances. However, a comparative investigation of the different methods proposed for this task, namely NIR densitometry and UV/Vis spectroscopy, is still missing. Thus, the aim of the present work was to employ these two techniques in comparative tape stripping experiments both in vivo on human forearm skin and in vitro on porcine ear skin. Standard tape stripping experiments were performed in the absence and presence of a marketed formulation containing flufenamic acid as a model drug. In the context of these methodological investigations, different methods of skin cleaning prior to the tape stripping procedure were evaluated to identify the most appropriate working protocol among the approaches proposed in the respective literature. The results showed that the investigated methods of NIR densitometry and UV/Vis spectroscopy deliver highly comparable results. Both optical methods are suitable to determine the skin penetration profiles of active substances during in vivo and in vitro tape stripping, especially if a simple working protocol without any cleaning procedures is maintained.