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At alkaline pH the bacteriorhodopsin mutant D85N, with aspartic acid-85 replaced by asparagine, is in a yellow form ([[lambda].sub.max [double tilde] 405 nm) with a deprotonated Schiff base. This state resembles the M intermediate of the wild-type photocycle. We used time-resolved methods to show that this yellow form of D85N, which has an initially unprotonated Schiff base and which lacks the proton acceptor Asp-85, transports protons in the same direction as wild type when excited by 400-nm flashes. Photoexcitation leads in several milliseconds to the formation of blue (630 nm) and purple (580 nm) intermediates with a protonated Schiff base, which decay in tens of seconds to the initial state (400 nm). Experiments with pH indicator dyes show that at pH 7, 8, and 9, proton uptake occurs in about 5-10 ms and precedes the slow release (seconds). Photovoltage measurements reveal that the direction of proton movement is from the cytoplasmic to the extracellular side with major components on the millisecond and second time scales. The slowest electrical component could be observed in the presence of azide, which accelerates the return of the blue intermediate to the initial yellow state. Transport thus occurs in two steps. In the first step (milliseconds), the Schiff base is protonated by proton uptake from the cytoplasmic side, thereby forming the blue state. From the pH dependence of the amplitudes of the electrical and photocycle signals, we conclude that this reaction proceeds in a similar way as in wild type - i.e., via the internal proton donor Asp-96. In the second step (seconds) the Schiff base deprotonates, releasing the proton to the extracellular side.

Analysis of light induced proton release in pH-indicator dye covalently bound to the surface of the purple membrane at site 72 on the extracellular side of bacteriorhodopsin (br) shows that br-released protons on the extracellular side of the purple membrane are trapped at the surface, where they undergo rapid long-range diffusion. The diffusion protons slowly equilibrate with the bulk phase. Long-range lateral proton diffusing occurs along the purple membrane supporting proton mobility along energy-transducing membranes.

The integrity of the protective mucus layer as a primary defense against pathogen invasion and microbial leakage into the intestinal epithelium can be compromised by the effects of antibiotics on the commensal microbiome. Changes in mucus integrity directly affect the solvent viscosity in the immediate vicinity of the mucin network, that is, the nanoviscosity, which in turn affects both biochemical reactions and selective transport. To assess mucus nanoviscosity, a reliable readout via the viscosity-dependent fluorescence lifetime of the molecular rotor dye cyanine 3 is established and nanoviscosities from porcine and murine ex vivo mucus are determined. To account for different mucin concentrations due to the removal of digestive residues during mucus collection, the power law dependence of mucin concentration on viscosity is used. The impact of antibiotics combinations (meropenem/vancomycin, gentamycin/ampicillin) on ex vivo intestinal mucus nanoviscosity is presented. The significant increase in viscosity of murine intestinal mucus after treatment suggests an effect of antibiotics on the microbiota that affects mucus integrity. This method will be a useful tool to assess how drugs, directly or indirectly, affect mucus integrity. Additionally, the method can be utilized to analyze the role of mucus nanoviscosity in health and disease, as well as in drug development., (© 2024 The Author(s). Macromolecular Bioscience published by Wiley‐VCH GmbH.)

Mucus is a complex biological hydrogel that acts as a barrier for almost everything entering or exiting the body. It is therefore of emerging interest for biomedical and pharmaceutical applications. Besides water, the most abundant components are the large and densely glycosylated mucins, glycoproteins of up to 20 MDa and carbohydrate content of up to 80 wt%. Here, we designed and explored a library of glycosylated peptides to deconstruct the complexity of mucus. Using the well-characterized hFF03 coiled-coil system as a hydrogel-forming peptide scaffold, we systematically probed the contribution of single glycans to the secondary structure as well as the formation and viscoelastic properties of the resulting hydrogels. We show that glycan-decoration does not affect α-helix and coiled-coil formation while it alters gel stiffness. By using oscillatory macrorheology, dynamic light scattering microrheology, and fluorescence lifetime-based nanorheology, we characterized the glycopeptide materials over several length scales. Molecular simulations revealed that the glycosylated linker may extend into the solvent, but more frequently interacts with the peptide, thereby likely modifying the stability of the self-assembled fibers. This systematic study highlights the interplay between glycan structure and hydrogel properties and may guide the development of synthetic mucus mimetics., (© 2024 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

We show that covalent labelling of sialic acids on live cell surfaces or mucin increases the fluorescence of the fluorescence molecular rotors (FMRs) CCVJ, Cy3 and thioazole orange, enabling wash-free imaging of cell surfaces. Dual labelling with an FMR and an environmentally insensitive dye allows detection of changes that occur, for example, when cross-linking is altered.

In this chapter, the visualization of nanocarriers and drugs in cells and tissue is reviewed. This topic is tightly connected to modern drug delivery, which relies on nanoscopic drug formulation approaches and the ability to probe nanoparticulate systems selectively in cells and tissue using advanced spectroscopic and microscopic techniques. We first give an overview of the breadth of this research field. Then, we mainly focus on topical drug delivery to the skin and discuss selected visualization techniques from spectromicroscopy, such as scanning transmission X-ray microscopy and fluorescence lifetime imaging. These techniques rely on the sensitive and quantitative detection of the topically applied drug delivery systems and active substances, either by exploiting their molecular properties or by introducing environmentally sensitive probes that facilitate their detection., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Phytochromes are bistable red/far-red light-responsive photoreceptor proteins found in plants, fungi, and bacteria. Light-activation of the prototypical phytochrome Cph1 from the cyanobacterium Synechocystis sp. PCC 6803 allows photoisomerization of the bilin chromophore in the photosensory module and a subsequent series of intermediate states leading from the red absorbing Pr to the far-red-absorbing Pfr state. We show here via osmotic and hydrostatic pressure-based measurements that hydration of the photoreceptor modulates the photoconversion kinetics in a controlled manner. While small osmolytes like sucrose accelerate Pfr formation, large polymer osmolytes like PEG 4000 delay the formation of Pfr. Thus, we hypothesize that an influx of mobile water into the photosensory domain is necessary for proceeding to the Pfr state. We suggest that protein hydration changes are a molecular event that occurs during photoconversion to Pfr, in addition to light activation, ultrafast electric field changes, photoisomerization, proton release and uptake, and the major conformational change leading to signal transmission, or simultaneously with one of these events. Moreover, we discuss this finding in light of the use of Cph1-PGP as a hydration sensor, e.g., for the characterization of novel hydrogel biomaterials.

The rapid development of microscopic techniques over the past decades enables the establishment of single molecule fluorescence imaging as a powerful tool in biological and biomedical sciences. Single molecule fluorescence imaging allows to study the chemical, physicochemical, and biological properties of target molecules or particles by tracking their molecular position in the biological environment and determining their dynamic behavior. However, the precise determination of particle distribution and diffusivities is often challenging due to high molecule/particle densities, fast diffusion, and photobleaching/blinking of the fluorophore. A novel, accurate, and fast statistical analysis tool, Diffusion Analysis of NAnoscopic Ensembles (DANAE), that solves all these obstacles is introduced. DANAE requires no approximations or any a priori input regarding unknown system-inherent parameters, such as background distributions; a requirement that is vitally important when studying the behavior of molecules/particles in living cells. The superiority of DANAE with various data from simulations is demonstrated. As experimental applications of DANAE, membrane receptor diffusion in its natural membrane environment, and cargo mobility/distribution within nanostructured lipid nanoparticles are presented. Finally, the method is extended to two-color channel fluorescence microscopy., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Phytochromes are biological red/far-red light sensors found in many organisms. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY, PGP) and the C-terminal transmitter (output) module. We recently showed a direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains of the prototypical phytochrome Cph1 PGP. These results suggested that the transient phycocyanobilin (PCB) chromophore deprotonation is closely associated with a higher protein mobility both in proximal and distal protein sites, implying a causal relationship that might be important for the global large-scale protein rearrangements. Here, we investigate the prototypical biliverdin (BV)-binding phytochrome Agp1. The structural changes at various positions in Agp1 PGP were investigated as a function of pH using picosecond time-resolved fluorescence anisotropy and site-directed fluorescence labeling of cysteine variants of Agp1 PGP. We show that the direct correlation of chromophore deprotonation with pH-dependent conformational changes does not occur in Agp1. Together with the absence of long-range effects between the PHY domain and chromophore p K a , in contrast to the findings in Cph1, our results imply phytochrome species-specific correlations between transient chromophore deprotonation and intramolecular signal transduction.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity., (© 2022. The Author(s).)

Cu-catalyzed 1,3-dipolar cycloaddition of ethyl 2-azidoacetate to iodobuta-1,3-diynes and subsequent Sonogashira cross-coupling were used to synthesize a large series of new triazole-based push-pull chromophores: 4,5-bis(arylethynyl)-1 H -1,2,3-triazoles. The study of their optical properties revealed that all molecules have fluorescence properties, the Stokes shift values of which exceed 150 nm. The fluorescent properties of triazoles are easily adjustable depending on the nature of the substituents attached to aryl rings of the arylethynyl moieties at the C4 and C5 atoms of the triazole core. The possibility of 4,5-bis(arylethynyl)-1,2,3-triazoles' application for labeling was demonstrated using proteins and the HEK293 cell line. The results of an MTT test on two distinct cell lines, HEK293 and HeLa, revealed the low cytotoxicity of 4,5-bis(arylethynyl)triazoles, which makes them promising fluorescent tags for labeling and tracking biomolecules.

Chronic inflammation is one of the hallmarks of chronic wounds and is tightly coupled to immune regulation. The dysregulation of the immune system leads to continuing inflammation and impaired wound healing and, subsequently, to chronic skin wounds. In this review, we discuss the role of the immune system, the involvement of inflammatory mediators and reactive oxygen species, the complication of bacterial infections in chronic wound healing, and the still-underexplored potential of natural bioactive compounds in wound treatment. We focus on natural compounds with antioxidant, anti-inflammatory, and antibacterial activities and their mechanisms of action, as well as on recent wound treatments and therapeutic advancements capitalizing on nanotechnology or new biomaterial platforms.

Modern biology investigations on phytochromes as near-infrared fluorescent pigments pave the way for the development of new biosensors, as well as for optogenetics and in vivo imaging tools. Recently, near-infrared fluorescent proteins (NIR-FPs) engineered from biliverdin-binding bacteriophytochromes and cyanobacteriochromes, and from phycocyanobilin-binding cyanobacterial phytochromes have become promising probes for fluorescence microscopy and in vivo imaging. However, current NIR-FPs typically suffer from low fluorescence quantum yields and short fluorescence lifetimes. Here, we applied the rational approach of combining mutations known to enhance fluorescence in the cyanobacterial phytochrome Cph1 to derive a series of highly fluorescent variants with fluorescence quantum yield exceeding 15%. These variants were characterised by biochemical and spectroscopic methods, including time-resolved fluorescence spectroscopy. We show that these new NIR-FPs exhibit high fluorescence quantum yields and long fluorescence lifetimes, contributing to their bright fluorescence, and provide fluorescence lifetime imaging measurements in E.coli cells., (© 2022. The Author(s).)

Simultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance (EPR) spectroscopy for quantification are complementary. Their combination in a multiplexed approach promises a successful but ambitious strategy because of spin label-mediated fluorescence quenching. Here, we solved this problem and present the molecular design of a dual label (DL) compound comprising a highly fluorescent dye together with an EPR spin probe, which also renders the fluorescence lifetime to be concentration sensitive. The DL can easily be coupled to the biomolecule of choice, enabling in vivo and in vitro applications. This novel approach paves the way for elegant studies ranging from fundamental biological investigations to preclinical drug research, as shown in proof-of-principle penetration experiments in human skin ex vivo., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Pharmacotherapy of head and neck squamous cell carcinoma (HNSCC) often fails due to the development of chemoresistance and severe systemic side effects of current regimens limiting dose escalation. Preclinical models comprising all major elements of treatment resistance are urgently needed for the development of new strategies to overcome these limitations. For model establishment, we used tumor cells from patient-derived HNSCC xenografts or cell lines (SCC-25, UM-SCC-22B) and characterized the model phenotype. Docetaxel and cetuximab were selected for comparative analysis of drug-related effects at topical and systemic administration. Cetuximab cell binding was mapped by cluster-based fluorescence lifetime imaging microscopy.The tumor oral mucosa (TOM) models displayed unstructured, hyper-proliferative, and pleomorphic cell layers, reflecting well the original tumor morphology and grading. Dose- and time-dependent effects of docetaxel on tumor size, apoptosis, hypoxia, and interleukin-6 release were observed. Although the spectrum of effects was comparable, significantly lower doses were required to achieve similar docetaxel-induced changes at topical compared to systemic application. Despite displaying anti-proliferative effects in monolayer cultures, cetuximab treatment showed only minor effects in TOM models. This was not due to inefficient cetuximab uptake or target cell binding but likely mediated by microenvironmental components.We developed multi-layered HNSCC models, closely reflecting tumor morphology and displaying complex interactions between the tumor and its microenvironment. Topical application of docetaxel emerged as promising option for HNSCC treatment. Aside from the development of novel strategies for topical drug delivery, our tumor model might help to better understand key regulators of drug-tumor-interactions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Fluorescence microscopy is widely used for high content screening in 2D cell cultures and 3D models. In particular, 3D tissue models are gaining major relevance in modern drug development. Enabling direct multiparametric evaluation of complex samples, fluorescence lifetime imaging (FLIM) adds a further level to intensity imaging by the sensitivity of the fluorescence lifetime to the microenvironment. However, the use of FLIM is limited amongst others by the acquisition of sufficient photon numbers without phototoxic effects in live cells. Herein, we developed a new cluster-based analysis method to enhance insight, and significantly speed up analysis and measurement time for the accurate translation of fluorescence lifetime information into pharmacological pathways. Methods : We applied a fluorescently-labeled dendritic core-multishell nanocarrier and its cargo Bodipy as molecules of interest (MOI) to human cells and reconstructed human tissue. Following the sensitivity and specificity assessment of the fitting-free Cluster-FLIM analysis of data in silico and in vitro , we evaluated the dynamics of cellular molecule uptake and intracellular interactions. For 3D live tissue investigations, we applied multiphoton (mp) FLIM. Owing to Cluster-FLIM's statistics-based fitting-free analysis, we utilized this approach for automatization. Results : To discriminate the fluorescence lifetime signatures of 5 different fluorescence species in a single color channel, the Cluster-FLIM method requires only 170, respectively, 90 counts per pixel to obtain 95% sensitivity (hit rate) and 95% specificity (correct rejection rate). Cluster-FLIM revealed cellular interactions of MOIs, representing their spatiotemporal intracellular fate. In a setting of an automated workflow, the assessment of lysosomal trapping of the MOI revealed relevant differences between normal and tumor cells, as well as between 2D and 3D models. Conclusion : The automated Cluster-FLIM tool is fitting-free, providing images with enhanced information, contrast, and spatial resolution at short exposure times and low fluorophore concentrations. Thereby, Cluster-FLIM increases the applicability of FLIM in high content analysis of target molecules in drug development and beyond., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes, including Cph1 from the cyanobacterium Synechocystis 6803, act as photochemical switches that interconvert between stable red (Pr)- and metastable far-red (Pfr)-absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr state, involving pH titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain), and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and in distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.

Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain of mitochondria and many aerobic prokaryotes that function as a redox-coupled proton pump, catalyzes the reduction of molecular oxygen to water. As part of the respiratory chain, CcO contributes to the proton motive force driving ATP synthesis. While many aspects of the enzyme's catalytic mechanisms have been established, a clear picture of the proton exit pathway(s) remains elusive. Here, we aim to gain insight into the molecular mechanisms of CcO through the development of a new homologous mutagenesis/expression system in Paracoccus denitrificans, which allows mutagenesis of CcO subunits 1, 2, and 3. Our system provides true single thiol-reactive CcO variants in a three-subunit base variant with unique labeling sites for the covalent attachment of reporter groups sensitive to nanoenvironmental factors like protonation, polarity, and hydration. To this end, we exchanged six residues on both membrane sides of CcO for cysteines. We show redox-dependent wetting changes at the proton uptake channel and increased polarity at the proton exit side of CcO upon electronation. We suggest an electronation-dependent conformational change to play a role in proton exit from CcO., (© 2019 Federation of European Biochemical Societies.)

Biological membrane fluidity and thus the local viscosity in lipid membranes are of vital importance for many life processes and implicated in various diseases. Here, we introduce a novel viscosity sensor design for lipid membranes based on a reporting nanoparticle, a sulfated dendritic polyglycerol (dPGS), conjugated to a fluorescent molecular rotor, indocarbocyanine (ICC). We show that dPGS-ICC provides high affinity to lipid bilayers, enabling viscosity sensing in the lipid tail region. The systematic characterization of viscosity- and temperature-dependent photoisomerization properties of ICC and dPGS-ICC allowed us to determine membrane viscosities in different model systems and in living cells using fluorescence lifetime imaging (FLIM). dPGS-ICC distinguishes between ordered lipids and the onset of membrane defects in small unilamellar single lipid vesicles and is highly sensitive in the fluid phase to small changes in viscosity introduced by cholesterol. In microscopy-based viscosity measurements of large multilamellar vesicles, we observed an order of magnitude more viscous environments by dPGS-ICC, lending support to the hypothesis of heterogeneous nanoviscosity environments even in single lipid bilayers. The existence of such complex viscosity structures could explain the large variation in the apparent membrane viscosity values found in the literature, depending on technique and probe, both for model membranes and live cells. In HeLa cells, a tumor-derived cell line, our nanoparticle-based viscosity sensor detects a membrane viscosity of ∼190 cP and is able to discriminate between cell membrane and intracellular vesicle localization. Thus, our results show the versatility of the dPGS-ICC nano-conjugate in physicochemical and biomedical applications by adding a new analytical functionality to its medical properties.

In reconstructed skin and diffusion cell studies, core-multishell nanocarriers (CMS-NC) showed great potential for drug delivery across the skin barrier. Herein, we investigated penetration, release of dexamethasone (DXM), in excised full-thickness human skin with special focus on hair follicles (HF). Four hours and 16 h after topical application of clinically relevant dosages of 10 μg DXM/cm 2 skin encapsulated in CMS-NC (12 nm diameter, 5.8% loading), presence of DXM in the tissue as assessed by fluorescence microscopy of anti-DXM-stained tissue sections as well as ELISA and HPLC-MS/MS in tissue extracts was enhanced compared to standard LAW-creme but lower compared to DXM aqueous/alcoholic solution. Such enhanced penetration compared to conventional cremes offers high potential for topical therapies, as recurrent applications of corticosteroid solutions face limitations with regard to tolerability and fast drainage. The findings encourage more detailed investigations on where and how the nanocarrier and drug dissociate within the skin and what other factors, e.g. thermodynamic activity, influence the penetration of this formulations. Microscopic studies on the spatial distribution within the skin revealed accumulation in HF and furrows accompanied by limited cellular uptake assessed by flow cytometry (up to 9% of total epidermal cells). FLIM clearly visualized the presence of CMS-NC in the viable epidermis and dermis. When exposed in situ a fraction of up to 25% CD1a + cells were found within the epidermal CMS-NC + population compared to approximately 3% CD1a + /CMS-NC + cells after in vitro exposure in short-term cultures of epidermal cell suspensions. The latter reflects the natural percentage of Langerhans cells (LC) in epidermis suspensions and indicated that CMS-NC were not preferentially internalized by one cell type. The increased CMS-NC + LC proportion after exposure within the tissue is in accordance with the strategic suprabasal LC-localization. More specifically we postulate that the extensive dendrite meshwork, their position around HF orifices and their capacity to modulate tight junctions facilitated a preferential uptake of CMS-NC by LC within the skin. This newly identified aspect of CMS-NC penetration underlines the potential of CMS-NC for dermatotherapy and encourages further investigations of CMS-NC for the delivery of other molecule classes for which intracellular delivery is even more crucial., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nanoparticles hold a great promise in biomedical science. However, due to their unique physical and chemical properties they can lead to overproduction of intracellular reactive oxygen species (ROS). As an important mechanism of nanotoxicity, there is a great need for sensitive and high-throughput adaptable single-cell ROS detection methods. Here, fluorescence lifetime imaging microscopy (FLIM) is employed for single-cell ROS detection (FLIM-ROX) providing increased sensitivity and enabling high-throughput analysis in fixed and live cells. FLIM-ROX owes its sensitivity to the discrimination of autofluorescence from the unique fluorescence lifetime of the ROS reporter dye. The effect of subcytotoxic amounts of cationic gold nanoparticles in J774A.1 cells and primary human macrophages on ROS generation is investigated. FLIM-ROX measures very low ROS levels upon gold nanoparticle exposure, which is undetectable by the conventional method. It is demonstrated that cellular morphology changes, elevated senescence, and DNA damage link the resulting low-level oxidative stress to cellular adverse effects and thus nanotoxicity. Multiphoton FLIM-ROX enables the quantification of spatial ROS distribution in vivo, which is shown for skin tissue as a target for nanoparticle exposure. Thus, this innovative method allows identifying of low-level ROS in vitro and in vivo and, subsequently, promotes understanding of ROS-associated nanotoxicity., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The question whether nanoparticles can cross the skin barrier is highly debated. Even in intact skin rare events of deeper penetration have been reported, but technical limitations and possible artifacts require careful interpretation. In this study, horizontal scanning by 2-photon microscopy (2 PM) of full-thickness human skin samples placed in a lateral position yielded highly informative images for skin penetration studies of fluorescently tagged nanoparticles. Scanning of large fields of view allowed for detailed information on interfollicular and follicular penetration in tissue blocks without damaging the sample. Images in histomorphological correlation showed that 2P-excited fluorescence signals of fluorescently tagged 20 and 200 nm polystyrene nanoparticles preferentially accumulated in the stratum corneum (SC) and in the upper part of vellus hair follicles (HFs). Rare events of deeper penetration in the SC and in the infundibulum of vellus HFs were observed at sites of high focal particle aggregations. Wide-field 2 PM allows for imaging of nanoparticle penetration in large tissue blocks, whereas total internal reflection microscopy (TIRFM) enables selective detection of individual nanoparticles as well as clusters of nanoparticles in the SC and within the epidermal layer directly beneath the SC, thus confirming barrier crossing with high sensitivity., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Owing their unique chemical and physical properties core-multishell (CMS) nanocarriers are thought to underlie their exploitable biomedical use for a topical treatment of skin diseases. This highlights the need to consider not only the efficacy of CMS nanocarriers but also the potentially unpredictable and adverse consequences of their exposure thereto. As CMS nanocarriers are able to penetrate into viable layers of normal and stripped human skin ex vivo as well as in in vitro skin disease models the understanding of nanoparticle crosstalk with components of the immune system requires thorough investigation. Our studies highlight the biocompatible properties of CMS nanocarriers on Langerhans cells of the skin as they did neither induce cytotoxicity and genotoxicity nor cause reactive oxygen species (ROS) or an immunological response. Nevertheless, CMS nanocarriers were efficiently taken up by Langerhans cells via divergent endocytic pathways. Bioimaging of CMS nanocarriers by fluorescence lifetime imaging microscopy (FLIM) and flow cytometry indicated not only a localization within the lysosomes but also an energy-dependent exocytosis of unmodified CMS nanocarriers into the extracellular environment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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., (© 2017 New York Academy of Sciences.)

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 244nm. 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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 β-galactosidase, less cell proliferation, and reduced viability. Higher amounts of β-galactosidase are also detectable in UVB-RHE. Moreover, UVB-RHE release more interleukin-1α 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., (Copyright © 2016. Published by Elsevier B.V.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Phytochromes are biological red/far-red light sensors found in many organisms. Photoisomerization of the linear methine-bridged tetrapyrrole triggers transient proton translocation events in the chromophore binding pocket (CBP) leading to major conformational changes of the protein matrix that are in turn associated with signaling. By combining pH-dependent resonance Raman and UV-visible absorption spectroscopy, we analyzed protonation-dependent equilibria in the CBP of Cph1 involving the proposed Pr-I and Pr-II substates that prevail below and above pH 7.5, respectively. The protonation pattern and vibrational properties of these states were further characterized by means of hybrid quantum mechanics/molecular mechanics calculations. From this combined experimental-theoretical study, we were able to identify His260 as the key residue controlling pH-dependent equilibria. This residue is not only responsible for the conformational heterogeneity of CBP in the Pr state of prokaryotic phytochromes, discussed extensively in the past, but it constitutes the sink and source of protons in the proton release/uptake mechanism involving the tetrapyrrole chromophore which finally leads to the formation of the Pfr state. Thus, this work provides valuable information that may guide further experiments toward the understanding of the specific role of protons in controlling structure and function of phytochromes in general.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Drug loading capacity in nanostructured lipid carriers (NLC) depends on the formation of nanostructures within the lipid matrix. However, investigation of these nanostructures with sizes below the diffraction limit of visible light is quite challenging. Thus, until now the determination of structures and drug distribution within NLCs was not possible. Therefore, we aimed at developing a method to visualize the nanostructures within the lipid carriers. Model NLCs loaded with a lipophilic fluorescent drug mimetic, ATTO-Oxa12, were produced and investigated by single-molecule tracking and localization-based superresolution microscopy. Results revealed spherical ATTO-Oxa12-filled nanostructures with diameters of ∼70nm and 120-130nm, both smaller than the NLC size (∼160nm). The ATTO-Oxa12 diffusion constant was calculated from the single-molecule traces (D⩾1μm 2 /s) and indicated the distribution of the model drug in the oily component. Together these data suggest the existence of drug-loaded oily nanocompartments, which could fill up to ∼50% of the model NLCs' volume. In conclusion, a novel tool based on single-molecule microscopy is now available that allows for the precise determination of drug distribution and the characterization of lipid nanostructures, information that is paramount for optimizing lipid nanoparticle formulations., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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: 0.1mg/ml and diclofenac sodium: 5.0mg/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 400nm) and the drug encapsulation efficiency was between 84% and 95%. The drug release was prolonged over 48h 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

The topical application of drug-loaded particles has been explored extensively aiming at a dermal, follicular or transdermal drug delivery. This review summarizes the present state of the field of polymeric nanocarriers for skin application, also covering methodologies to clinically characterize their interaction and penetration in skin in vivo. Furthermore, with a focus on a clinical perspective, a number of questions are addressed: How well are existing nanoparticle systems penetrating the skin? Which functions of new carrier concepts may meet the clinical requirements? To which extend will instrumental imaging techniques provide information on the biological functions of nanocarriers? Which issues have to be addressed for translating experimental concepts into a future clinical application?, (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)