Efficacy of topically applied rapamycin-loaded redox-sensitive nanocarriers in a human skin/T cell co-culture model.

Rapamycin, also known as Sirolimus, is a promising anti-proliferative drug, but its therapeutic use for the topical treatment of inflammatory, hyperproliferative skin disorders is limited by insufficient penetration rates due to its high molecular weight (MW of 914.172 g/mol) and high lipophilicity. We have shown that core multi-shell (CMS) nanocarriers sensitive to oxidative environment can improve drug delivery to the skin. In this study, we investigated the mTOR inhibitory activity of these oxidation-sensitive CMS (osCMS) nanocarrier formulations in an inflammatory ex vivo human skin model. In this model, features of inflamed skin were introduced by treating the ex vivo tissue with low-dose serine protease (SP) and lipopolysaccharide (LPS), while phorbol 12-myristate 13-acetate and ionomycin were used to stimulate IL-17A production in the co-cultured SeAx cells. Furthermore, we tried to elucidate the effects of rapamycin on single cell populations isolated from skin (keratinocytes, fibroblast) as well as on SeAx cells. Further, we measured possible effects of the rapamycin formulations on dendritic cell (DC) migration and activation. The inflammatory skin model enabled the assessment of biological readouts at both the tissue and T cell level. All investigated formulations successfully delivered rapamycin across the skin as revealed by reduced IL-17A levels. Nevertheless, only the osCMS formulations reached higher anti-inflammatory effects in the skin compared to the control formulations with a significant downregulation of mTOR activity. These results indicate that osCMS formulations could help to establish rapamycin, or even other drugs with similar physico-chemical properties, in topical anti-inflammatory therapy.

Biodistribution and intracellular localization of hyaluronan and its nanogels. A strategy to target intracellular S. aureus in persistent skin infections.

Intracellular pathogens are a critical challenge for antimicrobial therapies. Staphylococcus aureus (S. aureus) causes approximately 85% of all skin and soft tissue infections in humans worldwide and more than 30% of patients develop chronic or recurrent infections within three months, even after appropriate antibacterial therapies. S. aureus is also one of the most common bacteria found in chronic wounds. Recent evidences suggest that S. aureus is able to persist within phagolysosomes of skin cells (i.e. keratinocytes, phagocytic cells), being protected from both the immune system and a number of antimicrobials. To overcome these limits, nano-formulations that enable targeted therapies against intracellular S. aureus might be developed. Herein, the biodistribution and intracellular localisation of hyaluronan (HA) and HA-based nanoparticles (nanogels, NHs) are investigated, both after intravenous (i.v.) injections (in mice) and topical administrations (in ex vivo human skin). Results indicate HA and NHs accumulate especially in skin and liver of mice after i.v. injection. After topical application on human skin explants, no penetration of both HA and NHs was detected in skin with intact stratum corneum. By contrast, in barrier-disrupted human skin (with partial removal and loosening of stratum corneum), HA and NHs penetrate to the viable epidermis and are taken up by keratinocytes. In mechanically produced wounds (skin without epidermis) they accumulate in wound tissue and are taken up by dermis cells, e.g. fibroblasts and phagocytic cells. Interestingly, in all cases, the cellular uptake is CD44-mediated. In vitro studies confirmed that after CD44-mediated uptake, both HA and NHs accumulate in lysosomes of dermal fibroblasts and macrophages, as previously reported for keratinocytes. Finally, the colocalisation between intracellular S. aureus and HA or NHs is demonstrated, in macrophages. Altogether, for the first time, these results strongly suggest that HA and HA-based NHs can provide a targeted therapy to intracellular S. aureus, in persistent skin or wound infections.

Soft X-ray microscopy for probing of topical tacrolimus delivery via micelles.

The penetration of topically applied tacrolimus formulated in micelles into murine skin is reported, measured by X-ray microscopy. Tacrolimus and micelles are probed for the first time by this high spatial resolution technique by element-selective excitation in the C 1s- and O 1s-regimes. This method allows selective detection of the distribution and penetration depth of drugs and carrier molecules into biologic tissues. It is observed that small, but distinct quantities of the drug and micelles, acting as a drug carrier, penetrate the stratum corneum. A comparison is made with the paraffin-based commercial tacrolimus ointment Protopic®, where local drug concentrations show to be low. A slight increase in local drug concentration in the stratum corneum is observed, if tacrolimus is formulated in micelles, as compared to Protopic®. This underscores the importance of the drug formulations for effective drug delivery. Time-resolved penetration shows presence of drug in the stratum corneum 100 min after formulation application, with penetration to deeper skin layers at 1000 min. High resolution micrographs give indications for a penetration pathway along the lipid membranes between corneocytes, but also suggest that the compound may penetrate corneocytes.

Drug delivery across intact and disrupted skin barrier: Identification of cell populations interacting with penetrated thermoresponsive nanogels.

Nanoscaled soft particles, such as nanogels, can be designed to incorporate different types of compounds and release them in a controlled and triggered manner. Thermoresponsive nanogels (tNG), releasing their cargo above a defined temperature, are promising carrier systems for inflammatory skin diseases, where the temperature of diseased skin differs from that of healthy skin areas. In this study a polyglycerol-based tNG with diameter of 156nm was investigated for penetration and release properties upon topical application on ex vivo human skin with intact or disrupted barrier. Furthermore, temperature-triggered effects and the internalization of tNG by skin cells upon translocation to the viable skin layers were analyzed. The investigated tNG were tagged with indodicarbocyanine and loaded with fluorescein, so that fluorescent microscopy and flow cytometry could be used to evaluate simultaneously particle penetration and release of the fluorochrome. Topically applied tNG penetrated into the SC of both intact and disrupted skin explants. Only in barrier-disrupted skin significant amounts of released fluorochrome and tNG penetrated in the epidermis and dermis 2h after topical application. When a thermal trigger was applied by infrared radiation (30s, 3.9mJ/cm2), a significantly higher penetration of tNG in the SC and release of the dye in the epidermis were detected with respect to non-triggered samples. Penetrated tNG particles were internalized by skin cells in both epidermis and dermis. Only few CD1a-positive Langerhans cells associated with tNG were found in the epidermis. However, in the dermis a significant percentage of cells associated with tNG were identified to be antigen presenting cells, i.e. HLA-DR+and CD206+cells. Thus, tNG represent promising carrier systems for the treatment of inflammatory skin diseases, not only because of their improved penetration and controlled release properties, but also because of their ability to effectively reach dermal dendritic cells in barrier-disrupted skin.

Studies for improved understanding of lipid distributions in human skin by combining stimulated and spontaneous Raman microscopy.

Advanced Raman techniques, such as stimulated Raman spectroscopy (SRS), have become a valuable tool for investigations of distributions of substances in biological samples. However, these techniques lack spectral information and are therefore highly affected by cross-sensitivities, which are due to blended Raman bands. One typical example is the symmetric CH2 stretching vibration of lipids, which is blended with the more intense Raman band of proteins. We report in this work an approach to reduce such cross-sensitivities by a factor of 8 in human skin samples. This is accomplished by careful spectral deconvolutions revealing the neat spectra of skin lipids. Extensive Raman studies combining the complementary advantages of fast mapping and scanning, i.e. SRS, as well as spectral information provided by spontaneous Raman spectroscopy, were performed on the same skin regions. In addition, an approach for correcting artifacts is reported, which are due to transmission and reflection geometries in Raman microscopy as well as scattering of radiation from rough and highly structured skin samples. As a result, these developments offer improved results obtained from label-free spectromicroscopy provided by Raman techniques. These yield substance specific information from spectral regimes in which blended bands dominate. This improvement is illustrated by studies on the asymmetric CH2 stretching vibration of lipids, which was previously difficult to identify due to the strong background signal from proteins. The advantage of the correction procedures is demonstrated by higher spatial resolution permitting to perform more detailed investigations on lipids and their composition in skin.

Influence of the skin barrier on the penetration of topically-applied dexamethasone probed by soft X-ray spectromicroscopy.

The penetration of dexamethasone into human skin ex vivo is reported. X-ray microscopy is used for label-free probing of the drug and quantification of the local drug concentration with a spatial resolution reaching 70±5nm. This is accomplished by selective probing the dexamethasone by X-ray absorption. Varying the penetration time between 10min and 1000min provides detailed information on the penetration process. In addition, the stratum corneum has been damaged by tape-stripping in order to determine the importance of this barrier regarding temporally resolved drug penetration profiles. Dexamethasone concentrations distinctly vary, especially close to the border of the stratum corneum and the viable epidermis, where a local minimum in drug concentration is observed. Furthermore, near the basal membrane the drug concentration strongly drops. High spatial resolution studies along with a de-convolution procedure reveal the spatial distribution of dexamethasone in the interspaces between the corneocytes consisting of stratum corneum lipids. These results on local drug concentrations are interpreted in terms of barriers affecting the drug penetration in human skin.

Core-multishell nanocarriers: Transport and release of dexamethasone probed by soft X-ray spectromicroscopy.

Label-free detection of core-multishell (CMS) nanocarriers and the anti-inflammatory drug dexamethasone is reported. Selective excitation by tunable soft X-rays in the O 1s-regime is used for probing either the CMS nanocarrier or the drug. Furthermore, the drug loading efficiency into CMS nanocarriers is determined by X-ray spectroscopy. The drug-loaded nanocarriers were topically applied to human skin explants providing insights into the penetration and drug release processes. It is shown that the core-multishell nanocarriers remain in the stratum corneum when applied for 100min to 1000min. Dexamethasone, if applied topically to human ex vivo skin explants using different formulations, shows a vehicle-dependent penetration behavior. Highest local drug concentrations are found in the stratum corneum as well as in the viable epidermis. If the drug is loaded to core-multishell nanocarriers, the concentration of the free drug is low in the stratum corneum and is enhanced in the viable epidermis as compared to other drug formulations. The present results provide insights into the penetration of drug nanocarriers as well as the mechanisms of controlled drug release from CMS nanocarriers in human skin. They are also compared to related work using dye-labeled nanocarriers and dyes that were used as model drugs.

Selective Probing of the Penetration of Dexamethasone into Human Skin by Soft X-ray Spectromicroscopy.

Selective probing of dexamethasone in excised human skin using soft X-ray spectromicroscopy provides quantitative concentration profiles as well as two-dimensional drug distribution maps. Element- and site-selective excitation of dexamethasone at the oxygen K-edge with the lateral step width adjusted to 1 µm provides detailed information on the location of the drug in the different skin layers. The key of this work is to probe dexamethasone selectively at the carbonyl site (C3) by the O 1s → π* transition, providing also a most efficient way to quantify the drug concentration as a function of penetration depth in correlation with structural properties of the skin containing carboxyl and amide oxygen sites occurring at higher transition energy than dexamethasone. Following drug exposure for 4 h, the glucocorticoide is located in about equal amounts in the stratum corneum, the outermost horny layer of skin, and in the viable epidermis, whereas in the dermis no dexamethasone is detected. In the stratum corneum, most of the lipophilic drug is found in regions between corneocytes, where epidermal lipids are dominating.

Ultraviolet radiation and nanoparticle induced intracellular free radicals generation measured in human keratinocytes by electron paramagnetic resonance spectroscopy.

BACKGROUND: Several nanoparticle-based formulations used in cosmetics and dermatology are exposed to sunlight once applied to the skin. Therefore, it is important to study possible synergistic effects of nanoparticles and ultraviolet radiation. METHODS: Electron paramagnetic resonance spectroscopy (EPR) was used to detect intracellular free radicals induced by ultraviolet B (UVB) radiation and amorphous silica nanoparticle and to evaluate the influence of nanoparticle surface chemistry on particle cytotoxicity toward HaCaT cells. Uncoated titanium dioxide nanoparticles served as positive control. In addition, particle intracellular uptake, viability, and induction of interleukin-6 were measured. RESULTS: We found that photo-activated titanium dioxide particles induced a significant amount of intracellular free radicals. On the contrary, no intracellular free radicals were generated by the investigated silica nanoparticles in the dark as well as under UVB radiation. However, under UVB exposure, the non-functionalized silica nanoparticles altered the release of IL-6. At the same concentrations, the amino-functionalized silica nanoparticles had no influence on UVB-induced IL-6 release. CONCLUSION: EPR spectroscopy is a useful technique to measure nanoparticle-induced intracellular free radicals. Non-toxic concentrations of silica particles enhanced the toxicity of UVB radiation. This synergistic effect was not mediated by particle-generated free radicals and correlated with particle surface charge and intracellular distribution.

Photoprotective properties of the fluorescent europium complex in UV-irradiated skin.

In this study, we compared the UV-protective abilities of the europium complex compared to titanium dioxide, which represents the most common physical filter for ultraviolet light in the broad-band spectral range. The UV absorption and light transformative capacities of the europium complex were evaluated using a spectrometer with a double-integrating sphere showing that the europium complex does not only absorb and reflect UV light, but transforms it into red and infrared light. It was found that the europium complex binds to the surface of Jurkat cells in vitro. Cells incubated with the europium complex showed a significantly higher viability after UVA and UVB irradiation as compared to untreated cells and cells incubated with titanium dioxide pointing out its photoprotective properties. The europium complex and titanium dioxide show similar penetration capacities into the stratum corneum as tested in human and porcine skin using tape stripping analysis. The europium complex has proved to be an efficient UV filter with a low cyto- and phototoxic profile and therefore represents a potential candidate for use in sunscreen formulations.

Water-soluble polymer conjugates of ZnPP for photodynamic tumor therapy.

Zn-protoporphyrin (ZnPP) is a promising candidate for cancer therapy. It is known to inhibit heme-oxygenase-1 (HO-1), resulting in suppressed biliverdin/bilirubin production accompanying lowered antioxidative capacity. As a consequence, a significant suppression of tumor growth in vivo was reported. Recent findings also showed that ZnPP efficiently generated reactive singlet oxygen under illumination of visible light. In the present report, we describe the photosensitizing capabilities of water-soluble polymer conjugates of ZnPP as novel compounds for photodynamic therapy against solid tumors. The polymer conjugation made ZnPP water-soluble, thus possible for injection for its aqueous solution. The cellular uptake and photobiological activity of ZnPP derivatives have been tested using a human T-cell leukemia cell line in vitro and demonstrated most potent phototoxic effects of SMA-ZnPP followed by PEG-ZnPP under aerobic conditions.

Cyclodextrin/chlorophyll a complexes as supramolecular photosensitizers.

The interactions between chlorophyll a, and three cyclodextrins, hydroxypropyl-beta-cyclodextrin heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin and hydroxypropyl-gamma-cyclodextrin, were studied in aqueous solutions by means of absorption, emission and circular dichroism spectroscopy. Nanosecond laser flash photolysis and steady-state singlet oxygen generation experiments were performed to clarify the photoactivity of chlorophyll a in these systems. Moreover the photosensitizing activity of these complexes towards human leukemia T-lymphocytes (Jurkat cells) was tested and compared with that of the free sensitizer, chlorophyll a. The results obtained indicate that each cyclodextrin is able to carry the pigment in monomeric form inside of cells producing singlet oxygen.