Pierre Volz

Detecting and Quantifying Biomolecular Interactions of a Dendritic Polyglycerol Sulfate Nanoparticle Using Fluorescence Lifetime Measurements

Interactions of nanoparticles with biomaterials determine the biological activity that is key for the physiological response. Dendritic polyglycerol sulfates (dPGS) were found recently to act as an inhibitor of inflammation by blocking selectins. Systemic application of dPGS would present this nanoparticle to various biological molecules that rapidly adsorb to the nanoparticle surface or lead to adsorption of the nanoparticle to cellular structures such as lipid membranes. In the past, fluorescence lifetime measurements of fluorescently tagged nanoparticles at a molecular and cellular/tissue level have been proven to reveal valuable information on the local nanoparticle environment via characteristic fluorescent lifetime signatures of the nanoparticle bound dye. Here, we established fluorescence lifetime measurements as a tool to determine the binding affinity to fluorescently tagged dPGS (dPGS-ICC; ICC: indocarbocyanine). The binding to a cell adhesion molecule (L-selectin) and a human complement protein (C1q) to dPGS-ICC was evaluated by the concentration dependent change in the unique fluorescence lifetime signature of dPGS-ICC. The apparent binding affinity was found to be in the nanomolar range for both proteins (L-selectin: 87 ± 4 nM and C1q: 42 ± 12 nM). Furthermore, the effect of human serum on the unique fluorescence lifetime signature of dPGS-ICC was measured and found to be different from the interactions with the two proteins and lipid membranes. A comparison between the unique lifetime signatures of dPGS-ICC in different biological environments shows that fluorescence lifetime measurements of unique dPGS-ICC fluorescence lifetime signatures are a versatile tool to probe the microenvironment of dPGS in cells and tissue.

Time-resolved fluorescence microscopy (FLIM) as an analytical tool in skin nanomedicine

Graphical abstract Figure. No Caption available. Abstract The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief, and for monitoring of disease progression. Topical application of drug‐loaded nanoparticles for the treatment of skin disorders is a promising strategy to overcome the stratum corneum, the upper layer of the skin, which represents an effective physical and biochemical barrier. The understanding of drug penetration into skin and enhanced penetration into skin facilitated by nanocarriers requires analytical tools that ideally allow to visualize the skin, its morphology, the drug carriers, drugs, their transport across the skin and possible interactions, as well as effects of the nanocarriers within the different skin layers. Here, we review some recent developments in the field of fluorescence microscopy, namely Fluorescence Lifetime Imaging Microscopy (FLIM)), for improved characterization of nanocarriers, their interactions and penetration into skin. In particular, FLIM allows for the discrimination of target molecules, e.g. fluorescently tagged nanocarriers, against the autofluorescent tissue background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle and its interactions with other biomolecules. Thus, FLIM shows the potential to overcome several limits of intensity based microscopy.

Application of Single Molecule Fluorescence Microscopy to Characterize the Penetration of a Large Amphiphilic Molecule in the Stratum Corneum of Human Skin

We report here on the application of laser-based single molecule total internal reflection fluorescence microscopy (TIRFM) to study the penetration of molecules through the skin. Penetration of topically applied drug molecules is often observed to be limited by the size of the respective drug. However, the molecular mechanisms which govern the penetration of molecules through the outermost layer of the skin are still largely unknown. As a model compound we have chosen a larger amphiphilic molecule (fluorescent dye ATTO-Oxa12) with a molecular weight >700 Da that was applied to excised human skin. ATTO-Oxa12 penetrated through the stratum corneum (SC) into the viable epidermis as revealed by TIRFM of cryosections. Single particle tracking of ATTO-Oxa12 within SC sheets obtained by tape stripping allowed us to gain information on the localization as well as the lateral diffusion dynamics of these molecules. ATTO-Oxa12 appeared to be highly confined in the SC lipid region between (intercellular space) or close to the envelope of the corneocytes. Three main distinct confinement sizes of 52 ± 6, 118 ± 4, and 205 ± 5 nm were determined. We conclude that for this amphiphilic model compound several pathways through the skin exist.

Stratum corneum targeting by dendritic core-multishell-nanocarriers in a mouse model of psoriasis

Inflammatory disorders of the skin pose particular therapeutic challenges due to complex structural and functional alterations of the skin barrier. Penetration of several anti-inflammatory drugs is particularly problematic in psoriasis, a common dermatitis condition with epidermal hyperplasia and hyperkeratosis. Here, we tested in vivo dermal penetration and biological effects of dendritic core-multishell-nanocarriers (CMS) in a murine skin model of psoriasis and compared it to healthy skin. In both groups, CMS exclusively localized to the stratum corneum of the epidermis with only very sporadic uptake by Langerhans cells. Furthermore, penetration into the viable epidermis of nile red as a model for lipophilic compounds was enhanced by CMS. CMS proved fully biocompatible in several in vitro assays and on normal and psoriatic mouse skin. The observations support the concept of CMS as promising candidates for drug delivery in inflammatory hyperkeratotic skin disorders in vivo.

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2.

A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.

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.

Dendritic Core-Multishell Nanocarriers in Murine Models of Healthy and Atopic Skin

Dendritic hPG-amid-C18-mPEG core-multishell nanocarriers (CMS) represent a novel class of unimolecular micelles that hold great potential as drug transporters, e.g., to facilitate topical therapy in skin diseases. Atopic dermatitis is among the most common inflammatory skin disorders with complex barrier alterations which may affect the efficacy of topical treatment.Here, we tested the penetration behavior and identified target structures of unloaded CMS after topical administration in healthy mice and in mice with oxazolone-induced atopic dermatitis. We further examined whole body distribution and possible systemic side effects after simulating high dosage dermal penetration by subcutaneous injection.Following topical administration, CMS accumulated in the stratum corneum without penetration into deeper viable epidermal layers. The same was observed in atopic dermatitis mice, indicating that barrier alterations in atopic dermatitis had no influence on the penetration of CMS. Following subcutaneous injection, CMS were deposited in the regional lymph nodes as well as in liver, spleen, lung, and kidney. However, in vitro toxicity tests, clinical data, and morphometry-assisted histopathological analyses yielded no evidence of any toxic or otherwise adverse local or systemic effects of CMS, nor did they affect the severity or course of atopic dermatitis.Taken together, CMS accumulate in the stratum corneum in both healthy and inflammatory skin and appear to be highly biocompatible in the mouse even under conditions of atopic dermatitis and thus could potentially serve to create a depot for anti-inflammatory drugs in the skin.

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.

Poly[acrylonitrile-co-(N-vinyl pyrrolidone)] nanoparticles – Composition-dependent skin penetration enhancement of a dye probe and biocompatibility

Graphical abstract Figure. No Caption available. Abstract Nanoparticles can improve topical drug delivery: size, surface properties and flexibility of polymer nanoparticles are defining its interaction with the skin. Only few studies have explored skin penetration for one series of structurally related polymer particles with systematic alteration of material composition. Here, a series of rigid poly[acrylonitrile‐co‐(N‐vinyl pyrrolidone)] model nanoparticles stably loaded with Nile Red or Rhodamin B, respectively, was comprehensively studied for biocompatibility and functionality. Surface properties were altered by varying the molar content of hydrophilic NVP from 0 to 24.1% and particle size ranged from 35 to 244 nm. Whereas irritancy and genotoxicity were not revealed, lipophilic and hydrophilic nanoparticles taken up by keratinocytes affected cell viability. Skin absorption of the particles into viable skin ex vivo was studied using Nile Red as fluorescent probe. Whilst an intact stratum corneum efficiently prevented penetration, almost complete removal of the horny layer allowed nanoparticles of smaller size and hydrophilic particles to penetrate into viable epidermis and dermis. Hence, systematic variations of nanoparticle properties allows gaining insights into critical criteria for biocompatibility and functionality of novel nanocarriers for topical drug delivery and risks associated with environmental exposure.

Effect of drug solubility and lipid carrier on drug release from lipid nanoparticles for dermal delivery.

&NA; Lipid nanoparticles have gained increased interest in the field of dermal products because of various advantages such as improved drug absorption and controlled drug release. The main objective was to investigate the influence of drug solubility and type of lipid carrier on the in vitro drug release. Drugs of different solubilities in the release medium PBS pH 7.4 (dexamethasone: 0.1 mg/ml and diclofenac sodium: 5.0 mg/ml) and three different lipids (in which the drugs had the highest solubility), Gelucire® 50/13 (solid lipid, mp: 50 °C), Witepsol® S55 (solid lipid, mp: 33.5–35.5 °C) and Capryol® 90 (liquid lipid) were chosen. The lipid nanoparticles were prepared by high shear homogenization. All nanosuspensions were in the nanometer range (up to 400 nm) and the drug encapsulation efficiency was between 84% and 95%. The drug release was prolonged over 48 h without an initial burst release and was dependent on the lipid carrier. Formulations containing a higher amount of solid Gelucire® 50/13 released the drugs slower due to the high affinity of the drugs to this lipid product. Inclusion of the liquid lipid Capryol® 90 resulted in a less organized lipidic structures (softer particles) and therefore a faster drug release. Despite its higher water solubility, diclofenac was released slower than dexamethasone because of its higher solubility in the lipid carriers. DSC studies indicated a partial miscibility between the solid lipids and a good miscibility between the solid and liquid lipids. Primary studies using total internal reflection fluorescence (TIRF) microscopy indicated that it is possible to detect individual fluorescently labeled dexamethasone (DXM‐F) molecules dissolved in the liquid lipid Capryol® 90. These studies will allow for the precise determination of the drug distribution within the lipid carrier, and the changes upon drug release. In conclusion, lipid carrier type and drug solubility in the lipid have a large influence on the in vitro drug release from lipid nanoparticles. Graphical abstract Figure. No caption available.