Christian Zoschke

Penetration of normal, damaged and diseased skin — An in vitro study on dendritic core–multishell nanotransporters

A growing intended or accidental exposure to nanoparticles asks for the elucidation of potential toxicity linked to the penetration of normal and lesional skin. We studied the skin penetration of dye-tagged dendritic core-multishell (CMS) nanotransporters and of Nile red loaded CMS nanotransporters using fluorescence microscopy. Normal and stripped human skin ex vivo as well as normal reconstructed human skin and in vitro skin disease models served as test platforms. Nile red was delivered rapidly into the viable epidermis and dermis of normal skin, whereas the highly flexible CMS nanotransporters remained solely in the stratum corneum after 6h but penetrated into deeper skin layers after 24h exposure. Fluorescence lifetime imaging microscopy proved a stable dye-tag and revealed striking nanotransporter-skin interactions. The viable layers of stripped skin were penetrated more efficiently by dye-tagged CMS nanotransporters and the cargo compared to normal skin. Normal reconstructed human skin reflected the penetration of Nile red and CMS nanotransporters in human skin and both, the non-hyperkeratotic non-melanoma skin cancer and hyperkeratotic peeling skin disease models come along with altered absorption in the skin diseases.

SLN for topical application in skin diseases--characterization of drug-carrier and carrier-target interactions.

The modes of drug-particle interactions considerably influence drug delivery by nanoparticulate carrier systems and drug penetration into the skin. The exact mechanism of the drug loading and its release are still ambiguous. Therefore, the loading process, the interaction of the agent and the lipid matrix of solid lipid nanoparticles (SLNs) as well as the uptake of the loaded agent by skin lipids were analysed by electron spin resonance (ESR) and parelectric spectroscopy (PS) using spin probes (TEMPO, TEMPOL, and CAT-1) as model drugs differing in their lipophilicity. The spin probes were closely attached to the particles lipid surface (TEMPO) or located in the layers of the surfactant (CAT-1), respectively. Furthermore, two distinct sub-compartments on the SLN were found. To simulate the processes at the phase boundary SLN dispersion/skin, skin lipid mixtures were prepared and the transfer process of the spin labels was followed by ESR tomography. Transfer rates were related to the lipophilicity of the spin probe, the lipid mixture and the applied pharmaceutical formulation, SLN dispersion and aqueous solution, respectively. In particular, SLN accelerated in particular the distribution of the lipophilic agents.

The barrier function of organotypic non-melanoma skin cancer models

Non-melanoma skin cancer (NMSC) is the most frequent human cancer with continuously rising incidences worldwide. Herein, we investigated the molecular basis for the impaired skin barrier function of organotypic NMSC models. We unraveled disturbed epidermal differentiation by reflectance confocal microscopy and histopathological evaluation. While the presence of claudin-4 and occludin were distinctly reduced, zonula occludens protein-1 was more wide-spread, and claudin-1 was heterogeneously distributed within the NMSC models compared with normal reconstructed human skin. Moreover, the cancer altered stratum corneum lipid packing and profile with decreased cholesterol content, increased phospholipid amount, and altered ceramide subclasses. These alterations contributed to increased surface pH and to 1.5 to 2.6-fold enhanced caffeine permeability of the NMSC models. Three topical applications of ingenol mebutate gel (0.015%) caused abundant epidermal cell necrosis, decreased Ki-67 indices, and increased lactate dehydrogenase activity. Taken together, our study provides new biological insights into the microenvironment of organotypic NMSC models, improves the understanding of the disease model by revealing causes for impaired skin barrier function in NMSC models at the molecular level, and fosters human cell-based approaches in preclinical drug evaluation.

Improving Topical Non-Melanoma Skin Cancer Treatment: In vitro Efficacy of a Novel Guanosine-Analog Phosphonate

Actinic keratosis, a frequent carcinoma in situ of non-melanoma skin cancer (NMSC), can transform into life-threatening cutaneous squamous cell carcinoma. Current treatment is limited due to low complete clearance rates and asks for novel therapeutic concepts; the novel purine nucleotide analogue OxBu may be an option. In order to enhance skin penetration, solid lipid nanoparticles (SLN, 136-156 nm) were produced with an OxBu entrapment efficiency of 96.5 ± 0.1%. For improved preclinical evaluation, we combined tissue engineering with clinically used keratin-18 quantification. Three doses of 10-3 mol/l OxBu, dissolved in phosphate-buffered saline as well as loaded to SLN, were effective on reconstructed NMSC. Tumour response and apoptosis induction were evaluated by an increase in caspase-cleaved fragment of keratin-18, caspase-7 activation as well as by reduced expression of matrix metallopeptidase-2 and Ki-67. OxBu efficacy was superior to equimolar 5-fluorouracil solution, and thus the drug should be subjected to the next step in preclinical evaluation.

Free Energy Simulations of Cargo-Carrier Interactions for Core-MultishellNanotransporters

Dendritic core-multishell (CMS) nanotransporters are composed of three parts: a polyglycerol amine core covalently linked to an inner alkyl shell and an outer polyethylene glycol shell. Aiming to unravel the preferred localization of the guest molecule within the locally well-tolerated delivery system, we transferred molecular dynamics simulations to CMS nanotransporters and verified the results with experimental data. Differences in free energy of the planar, nonpolar, and lipophilic Nile red (log P 3.4) indicated a preferential location within the inner CMS nanotransporter shell. Differences in free energy of the globular, polar and hydrophilic morphine (log P ≤ 0.8) predicted poor loading which has been verified. Replacing the outer CMS nanotransporter shell by glutamate or aspartate results in electrostatic forcemediated morphine attachment. Thus, the investigation of larger molecular systems consisting of many similar building blocks becomes feasible with our approach. In conclusion, the computational approach based on differences in free energy may improve the design of tailor-made CMS nanotransporters and enhance drug development.

Increased permeability of reconstructed human epidermis from UVB-irradiated keratinocytes

Graphical abstract Figure. No Caption available. Abstract Extrinsic (photo) aging accelerates chronologically aging in the skin due to cumulative UV irradiation. Despite recent insights into the molecular mechanisms of fibroblast aging, age‐related changes of the skin barrier function have been understudied. In contrast, the constantly increasing subpopulation of aged patients causes a clinical need for effective and safe (dermatological) treatment. Herein, we reconstructed human epidermis from UVB‐irradiated keratinocytes (UVB‐RHE). UVB‐irradiated keratinocytes show higher activity of senescence associated &bgr;‐galactosidase, less cell proliferation, and reduced viability. Higher amounts of &bgr;‐galactosidase are also detectable in UVB‐RHE. Moreover, UVB‐RHE release more interleukin‐1&agr; and ‐8 into the culture medium and present altered differentiation with a thinner stratum corneum compared to normal RHE. For the first time, the permeation of testosterone and caffeine through UVB‐irradiated RHE indicate a clear influence of the UVB stress on the skin barrier function. Impaired barrier function was confirmed by the increased permeation of testosterone and caffeine as well as by the increased penetration of dendritic core‐multishell nanocarriers into the constructs. Taken together, UVB‐RHE emulate hallmarks of skin aging and might contribute to an improved non‐clinical development of medicinal or cosmetic products.

Characterization of reconstructed human skin containing Langerhans cells to monitor molecular events in skin sensitization

Human cell-based approaches to assess defined key events in allergic contact dermatitis (ACD) are well-established, but lack cutaneous penetration and biotransformation as well as cellular cross-talk. Herein, we integrated in vitro-generated immature MUTZ-3-derived Langerhans-like cells (MUTZ-LCs) or monocyte-derived LC-like cells (MoLCs) into reconstructed human skin (RHS), consistent of a stratified epidermis formed by primary keratinocytes on a dermal compartment with collagen-embedded primary fibroblasts. LC-like cells were mainly localized in the epidermal compartment and distributed homogenously in accordance with native human skin. Topical application of the strong contact sensitizer 2,4-dinitrochlorobenzene (DNCB) induced IL-6 and IL-8 secretion in RHS with LC-like cells, whereas no change was observed in reference models. Increased gene expression of CD83, PD-L1, and CXCR4 in the dermal compartment indicated LC maturation. Importantly, exposure to DNCB enhanced mobility of the LC-like cells from epidermal to dermal compartments. In response to the moderate sensitizer isoeugenol and irritant sodium dodecyl sulphate, the obtained response was less pronounced. In summary, we integrated immature and functional MUTZ-LCs and MoLCs into RHS and provide a unique comparative experimental setting to monitor early events during skin sensitization.

Pitfalls in using fluorescence tagging of nanomaterials: Tecto-dendrimers in skin tissue as investigated by cluster-FLIM

Targeted topical application promises high drug concentrations in the skin and low systemic adverse effects. To locate drugs and drug‐delivery systems like nanocarriers, fluorescent dyes are commonly used as drug surrogates or nanocarrier labels in micrographs of tissue sections. Here, we investigate how labeling degree, concentration of fluorophore, and nanocarrier may affect the interpretation of these micrographs. False‐negative penetration results due to inter‐ and intramolecular quenching effects are likely. Using tecto‐dendrimers as an example, we present a detailed analysis of pitfalls in the (semi‐)quantitative evaluation of skin nanocarrier penetration. Fluorescence lifetime imaging microscopy (FLIM) allows distinguishing the target fluorescence of dye‐tagged nanocarriers from skin autofluorescence, providing a highly sensitive tool for clear‐cut localization of the nanocarriers. Cluster‐FLIM images reveal that FITC‐labeled tecto‐dendrimers penetrate the stratum corneum of human skin ex vivo and reconstructed human skin but do not cross the tight junction barrier.

White-Light Supercontinuum Laser-Based Multiple Wavelength Excitation for TCSPC-FLIM of Cutaneous Nanocarrier Uptake

Abstract We report here on a custom-built time-correlated single photon-counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) setup with a continuously tunable white-light supercontinuum laser combined with acousto-optical tunable filters (AOTF) as an excitation source for simultaneous excitation of multiple spectrally separated fluorophores. We characterized the wavelength dependence of the white-light supercontinuum laser pulse properties and demonstrated the performance of the FLIM setup, aiming to show the experimental setup in depth together with a biomedical application. We herein summarize the physical-technical parameters as well as our approach to map the skin uptake of nanocarriers using FLIM with a resolution compared to spectroscopy. As an example, we focus on the penetration study of indocarbocyanine-labeled dendritic core-multishell nanocarriers (CMS-ICC) into reconstructed human epidermis. Unique fluorescence lifetime signatures of indocarbocyanine-labeled nanocarriers indicate nanocarrier-tissue interactions within reconstructed human epidermis, bringing FLIM close to spectroscopic analysis.