Carl Simonsson

Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy.

The distribution of sulphorhodamine B (SRB), a fluorescent hydrophilic model drug, was investigated in human skin after passive diffusion using four different topical delivery systems. The delivery vehicles applied were two bicontinuous lipid cubic systems, a commercial ointment and water. The lipid cubic systems consisted of either monoolein (MO) or phytantriol (PT) and water. The formulations were applied on full-thickness human skin during 24 h. Thereafter the samples were investigated using two-photon microscopy (TPM). The TPM system consisted of an inverted microscope with a 40x water-immersion objective, laser scan-box, and a pulsed femtosecond titanium:sapphire laser operating at 780 nm. The fluorescence was detected using a 560 nm long-pass filter. Sequential optical sectioning was performed, resulting in images obtained at different tissue depths. TPM revealed that SRB mainly penetrates the skin via the intercellular lipid matrix. Samples exposed to the cubic phases showed a higher accumulation of SRB in micro-fissures, from which a fluorescent network of threadlike structures spread laterally in the tissue. These structures were also detected in some of the ointment samples, but not as frequent. The penetration of SRB into the stratum granulosum was deduced from the fluorescence of SRB present inside polygonal keratinocytes with cell nuclei. Higher SRB fluorescence was obtained in the outermost layer of the epidermis using the bicontinuous cubic phases, compared to when using the reference formulations. Thus, our results suggest that the dominating delivery route using the cubic phases is via micro-fissures caused by microscopic clustering of the keratinocytes in the skin. From these micro-fissures hydrophilic compounds, here modeled by SRB, can diffuse into the surrounding intercellular lipid matrix acting like a source for sustained release.

Caged fluorescent haptens reveal the generation of cryptic epitopes in allergic contact dermatitis.

Allergic contact dermatitis (ACD) is the most prevalent form of human immunotoxicity. It is caused by skin exposure to haptens, i.e., protein-reactive, low-molecular-weight chemical compounds, which form hapten-protein complexes (HPCs) in the skin, triggering the immune system. These immunogenic HPCs are elusive. In this study a series of thiol-reactive caged fluorescent haptens, i.e., bromobimanes, were deployed in combination with two-photon fluorescence microscopy, immunohistochemistry, and proteomics to identify possible hapten targets in proteins in human skin. Key targets found were the basal keratinocytes and the keratins K5 and K14. Particularly, cysteine 54 of K5 was found to be haptenated by the bromobimanes. In addition, elevated levels of anti-keratin antibodies were found in the sera of mice exposed to bromobimanes in vivo. The results indicate a general mechanism in which thiol-reactive haptens generate cryptic epitopes normally concealed from the immune system. In addition, keratinocytes and keratin seem to have an important role in the mechanism behind ACD, which is a subject for further investigations.

Two-photon laser-scanning fluorescence microscopy applied for studies of human skin

Two-photon laser scanning fluorescence microscopy (TPM) has been shown to be advantageous for imaging optically turbid media such as human skin. The ability of performing three-dimensional imaging without presectioning of the samples makes the technique not only suitable for noninvasive diagnostics but also for studies of topical delivery of xenobiotics. Here, TPM is used as a method to visualize both autofluorescent and exogenous fluorophores in skin. Samples exposed to sulforhodamine B have been scanned from two directions to investigate attenuation effects. It is shown that optical effects play a major role. Thus, TPM is excellent for visualizing the localization and distribution of fluorophores in human skin, although quantification might be difficult. Furthermore, an image-analysis algorithm has been implemented to facilitate interpretation of TPM images of autofluorescent features of nonmelanoma skin cancer obtained ex vivo. The algorithm was designed to detect cell nuclei and currently has a sensitivity and specificity of 82% and 78% to single cell nuclei. However, in order to detect multinucleated cells, the algorithm needs further development.

A study of the enhanced sensitizing capacity of a contact allergen in lipid vesicle formulations

The growing focus on nanotechnology and the increased use of nano-sized structures, e.g. vesicles, in topical formulations has led to safety concerns. We have investigated the sensitizing capacity and penetration properties of a fluorescent model compound, rhodamine B isothiocyanate (RBITC), when administered in micro- and nano-scale vesicle formulations. The sensitizing capacity of RBITC was studied using the murine local lymph node assay (LLNA) and the skin penetration properties were compared using diffusion cells in combination with two-photon microscopy (TPM). The lymph node cell proliferation, an indicator of a compounds sensitizing capacity, increased when RBITC was applied in lipid vesicles as compared to an ethanol:water (Et:W) solution. Micro-scale vesicles showed a slightly higher cell proliferative response compared to nano-scale vesicles. TPM imaging revealed that the vesicle formulations improved the skin penetration of RBITC compared to the Et:W solution. A strong fluorescent region in the stratum corneum and upper epidermis implies elevated association of RBITC to these skin layers when formulated in lipid vesicles. In conclusion, the results indicate that there could be an elevated risk of sensitization when haptens are delivered in vehicles containing lipid vesicles. Although the size of the vesicles seems to be of minor importance, further studies are needed before a more generalized conclusion can be drawn. It is likely that the enhanced sensitizing capacity is a consequence of the improved penetration and increased formation of hapten-protein complexes in epidermis when RBITC is delivered in ethosomal formulations.

Accumulation of FITC near stratum corneum–visualizing epidermal distribution of a strong sensitizer using two‐photon microscopy

Background: The allergenic potency of a hapten is related to its skin penetration properties, but little is known about the distribution of haptens in the skin following topical application.

The pilosebaceous unit--a phthalate-induced pathway to skin sensitization.

Allergic contact dermatitis (ACD) is caused by low-molecular weight compounds called haptens. It has been shown that the potency of haptens can depend on the formulation in which they are applied on the skin. Specifically the sensitization potency of isothiocyanates, a group of haptens which can be released from e.g. adhesive tapes and neoprene materials, increases with the presence of phthalates; however, the underlying mechanisms are not clear. A better understanding of the mechanisms governing the potency of haptens is important, e.g. to improve the risk assessment and the formulation of chemicals in consumer products. In this study we have explored phthalate-induced effects on the sensitization potency, skin distribution, and reactivity of fluorescent model isothiocyanate haptens using non-invasive two-photon microscopy to provide new insights regarding vehicle effects in ACD. The data presented in this paper indicate that the sensitization potency of isothiocyanates increases when applied in combination with dibutylphthalate due to a specific uptake via the pilosebaceous units. The results highlight the importance of shunt pathways when evaluating the bioavailability of skin sensitizers. The findings also indicate that vehicle-dependent hapten reactivity towards stratum corneum proteins regulates the bioavailability, and thus the potency, of skin sensitizers.

Ethosome Formulations of Known Contact Allergens can Increase their Sensitizing Capacity

Vesicular systems, such as liposomes and ethosomes, are used in cosmetic and pharmaceutical products to encapsulate ingredients, to protect ingredients from degradation, to increase bioavailability, and to improve cosmetic performance. Some reports have suggested that formulation of cosmetic ingredients in vesicular carrier systems may increase their contact allergy elicitation potential in humans. However, no sensitization studies have been published. We formulated two model contact allergens (isoeugenol and dinitrochlorobenzene) in ethosomes and investigated the sensitization response using a modified local lymph node assay (LLNA). The results were compared with those for the same allergens in similar concentrations and vehicles without ethosomes. Both allergens encapsulated in 200-300 nm ethosomes showed increased sensitizing potency in the murine assay compared with the allergens in solution without ethosomes. Empty ethosomes were non-sensitizing according to LLNA. The clinical implications are so far uncertain, but increased allergenicity from ethosome-encapsulated topical product ingredients cannot be excluded.

Two-photon fluorescence correlation microscopy combined with measurements of point spread function; investigations made in human skin.

Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been applied in connection to measurements of the point spread function (PSF) for quantitative analysis of sulphorhodamine B (SRB) in excised human skin. The PSF was measured using subresolution fluorescent beads embedded in the skin specimen. The PSF, measured as full width at half maximum (FWHM) was found to be 0.41 +/- 0.05 microm in the lateral direction, and 1.2 +/- 0.4 microm in the axial direction. The molecular diffusion of SRB inside the skin ranged between 0.5 and 15.0 x 10(-8) cm(2)/s. The diffusion coefficient is not dependent on depths down to 40 microm. The fluorophores were found to accumulate on the upper layers of the skin. This work is the first TPFCS study in human skin. The results show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.

Ethosome formulation of contact allergens may enhance patch test reactions in patients

Background: Ethosomes and liposomes are ultra‐small vesicles capable of encapsulating drugs and cosmetic ingredients for topical use, thereby potentially increasing bioavailability and clinical efficacy. So far, few reports have suggested that formulation of cosmetic ingredients in vesicular carrier systems may increase the allergenicity potential.

Modification and expulsion of keratins by human epidermal keratinocytes upon hapten exposure in vitro.

Allergic contact dermatitis is the most prevalent form of human immunotoxicity. It is caused by reactive low molecular weight chemicals, that is, haptens, coming in contact with the skin where hapten-peptide complexes are formed, activating the immune system. By using sensitizing fluorescent thiol-reactive haptens, that is, bromobimanes, we show how keratinocytes respond to hapten exposure in vitro and reveal, for the first time in a living system, an exact site of haptenation. Rapid internalization and reaction of haptens with keratin filaments were visualized. Subsequently, keratinocytes respond in vitro to hapten exposure by release of membrane blebs, which contain haptenated keratins 5 and 14. Particularly, cysteine 54 of K5 was found to be a specific target. A mechanism is proposed where neoepitopes, otherwise hidden from the immune system, are released after hapten exposure via keratinocyte blebbing. The observed expulsion of modified keratins by keratinocytes in vitro might play a role during hapten sensitization in vivo and should be subject to further investigations.