Molecular Mobility in Keratin-Rich Materials Monitored by Nuclear Magnetic Resonance: A Tool for the Evaluation of Structure-Giving Properties.

Keratins are structural proteins that are abundant in human skin, nails, and hair, where they provide mechanical strength. In the present study, we investigate the molecular mobilities and structures of three keratin-rich materials with clearly different mechanical properties: nails, stratum corneum (upper layer of epidermis), and keratinocytes (from lower layer of epidermis). We use solid-state NMR on natural-abundance C to characterize small changes in molecular dynamics in these biological materials with close to atomistic resolution.

Membrane permeability based on mesh analysis.

The main function of a membrane is to control the exchange of matter between the surrounding regions. As such, accurate modeling of membranes is important to properly describe their properties. In many cases in both biological systems and technical applications, the membranes are composite structures where transport properties may vary between the different sub-regions of the membrane.

Contribution of autofluorescence from intracellular proteins in multiphoton fluorescence lifetime imaging.

Multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) is extensively proposed as a non-invasive optical method to study tissue metabolism. The approach is based on recording changes in the fluorescence lifetime attributed to metabolic co-enzymes, of which nicotinamide adenine dinucleotide (NADH) is of major importance. However, intrinsic tissue fluorescence is complex.

Can mesoporous nanoparticles promote bioavailability of topical pharmaceutics?

When applied to skin, particulate matter has been shown to accumulate in hair follicles. In addition to follicles, the skin topography also incorporates trench-like furrows where particles potentially can accumulate; however, the furrows have not been as thoroughly investigated in a drug delivery perspective. Depending on body site, the combined follicle orifices cover up to 10% of the skin surface, while furrows can easily cover 20%, reaching depths exceeding 25 µm.

Increased antibiotic efficacy and noninvasive monitoring of Staphylococcus epidermidis biofilms using per-cysteamine-substituted γ-cyclodextrin - A delivery effect validated by fluorescence microscopy.

Limited and poor delivery of antibiotics is cited as one reason for the difficulty in treating antibiotic-resistant biofilms associated with chronic infections. We investigate the effectiveness of a positively charged, single isomer cyclodextrin derivative, octakis[6-(2-aminoethylthio)-6-deoxy]-γ-CD (γCys) to improve the delivery of antibiotics to biofilms. Using multiphoton laser scanning microscopy complemented with super-resolution fluorescence microscopy, we showed that γCys tagged with fluorescein (FITC) is uniformly distributed throughout live S.

Monitoring calcium-induced epidermal differentiation in vitro using multiphoton microscopy.

Significance: Research in tissue engineering and in vitro organ formation has recently intensified. To assess tissue morphology, the method of choice today is restricted primarily to histology. Thus novel tools are required to enable noninvasive, and preferably label-free, three-dimensional imaging that is more compatible with futuristic organ-on-a-chip models.

Report on fluorescence lifetime imaging using multiphoton laser scanning microscopy targeting sentinel lymph node diagnostics.

Significance: Sentinel lymph node (SLN) biopsy is an important method for metastasis staging in, e.g., patients with malignant melanoma.

A phototherapeutic fluorescent β-cyclodextrin branched polymer delivering nitric oxide.

We report herein on a novel water-soluble β-cyclodextrin-branched polymer covalently integrating a fluorescein moiety and a nitric oxide (NO) photodonor within its macromolecular skeleton. Photoexcitation with visible light induces the parallel activation of the two chromophores, which results in the green fluorescence emission suitable for imaging accompanied by NO release for therapy. In fact, this polymer internalizes in squamous carcinoma cancer cells in vitro, visualized by fluorescence microscopy, and induces cell mortality as result of the NO photo-decaging.

A Three-Color Fluorescent Supramolecular Nanoassembly of Phototherapeutics Activable by Two-Photon Excitation with Near-Infrared Light.

A supramolecular nanoassembly, of about 30 nm in diameter, that consists of a green-fluorescent, β-cyclodextrin-based, branched polymer co-encapsulating a red-emitting singlet oxygen ( O ) photosensitizer and a nitric oxide (NO) photoreleaser, which comprises a blue fluorescent reporter, is here reported. The system exhibits "five-in-one" photofunctionalities. All components can be simultaneously excited in the phototherapeutic window with two-photons by using near-infrared light at 740 nm and despite their close proximity, behave as independent units.

Confined photo-release of nitric oxide with simultaneous two-photon fluorescence tracking in a cellular system.

Nitric oxide (NO) is a key signaling molecule in biological systems. New tools are required to therapeutically modulate NO levels with confined precision. This study explores the photoactivatable properties of an NO releasing compound (CPA), based on cupferron O-alkylated with an anthracene derivative.

Exploring photoinactivation of microbial biofilms using laser scanning microscopy and confined 2-photon excitation.

One pertinent complication in bacterial infection is the growth of biofilms, that is, communities of surface-adhered bacteria resilient to antibiotics. Photodynamic inactivation (PDI) has been proposed as an alternative to antibiotic treatment; however, novel techniques complementing standard efficacy measures are required. Herein, we present an approach employing multiphoton microscopy complemented with Airyscan super-resolution microscopy, to visualize the distribution of curcumin in Staphylococcus epidermidis biofilms.

Monitoring the release of a NO photodonor from polymer nanoparticles via Förster resonance energy transfer and two-photon fluorescence imaging.

Core-shell polymeric nanoparticles (NPs) made of either di-block or tri-block poly-ε-caprolactone and polyethylene glycol copolymers, covalently integrating Rhodamine B in the core or the shell have been prepared and a green fluorescent NO photodonor entrapped therein. One- and two-photon fluorescence experiments demonstrate that effective Förster Resonance Energy Transfer (FRET) occurs exclusively in the di-block NPs having the Rhodamine in the core, accounting for a localization of the NO photoreleaser in the inner part of the polymeric nanocarrier. These di-block NPs are stable in the presence of human serum albumin and their cargo release NO under exclusive excitation with visible light.

Imaging mass spectrometry for novel insights into contact allergy - a proof-of-concept study on nickel.

Background: In spite of extensive regulation to limit exposure, nickel remains the main cause of contact allergy in the general population. More detailed knowledge on the skin uptake of haptens is required. So far, no method exists for the visualization of this clinically relevant hapten and its distribution in the skin.

Delivery of cyclodextrin polymers to bacterial biofilms - An exploratory study using rhodamine labelled cyclodextrins and multiphoton microscopy.

Cyclodextrin (CD) polymers are interesting nanoparticulate systems for pharmaceutical delivery; however, knowledge regarding their applications towards delivery into complex microbial biofilm structures is so far limited. The challenge is to demonstrate penetration and transport through the biofilm and its exopolysaccharide matrix. The ideal functionalization for penetration into mature biofilms is unexplored.

Mesoporous silica particles as a lipophilic drug vehicle investigated by fluorescence lifetime imaging.

Three types of new label-free fluorescent mesoporous silica micro- and nanoparticles were prepared by controlled thermal decomposition of carboamino groups linked on the surface without compromising the drug loading capacity of the silica particles. Clofazimine, a lipophilic antibiotic drug with excellent in vitro activity against mycobacterium tuberculosis, was encapsulated inside these fluorescent particles to obtain multifunctional drug carriers of interest in the field of theranostics. The morphological features together with the photophysical properties of both powders and aqueous suspensions are described.

Peptide Functionalized Gold Nanoparticles as a Stimuli Responsive Contrast Medium in Multiphoton Microscopy.

There is a need for biochemical contrast mediators with high signal-to-noise ratios enabling noninvasive biomedical sensing, for example, for neural sensing and protein-protein interactions, in addition to cancer diagnostics. The translational challenge is to develop a biocompatible approach ensuring high biochemical contrast while avoiding a raise of the background signal. We here present a concept where gold nanoparticles (AuNPs) can be utilized as a stimuli responsive contrast medium by chemically triggering their ability to exhibit multiphoton-induced luminescence (MIL) when performing multiphoton laser scanning microscopy (MPM).

Using QCM-D to study the adhesion of human gingival fibroblasts on implant surfaces.

Sealing the soft tissue-implant interface is one of the key issues in preventing transcutaneous implant-associated infections. A promising surface modification for improving osseointegration and possibly soft tissue integration is to coat the implant surface with hydroxyapatite (HA) nanoparticles. When new implant materials are developed, their ability to facilitate cell attachment and spreading are commonly investigated in vitro to establish their potential for good in vivo performance.

Two-photon fluorescence imaging and bimodal phototherapy of epidermal cancer cells with biocompatible self-assembled polymer nanoparticles.

We have developed herein an engineered polymer-based nanoplatform showing the convergence of two-photon fluorescence imaging and bimodal phototherapeutic activity in a single nanostructure. It was achieved through the appropriate choice of three different components: a β-cyclodextrin-based polymer acting as a suitable carrier, a zinc phthalocyanine emitting red fluorescence simultaneously as being a singlet oxygen ((1)O2) photosensitizer, and a tailored nitroaniline derivative, functioning as a nitric oxide (NO) photodonor. The self-assembly of these components results in photoactivable nanoparticles, approximately 35 nm in diameter, coencapsulating a multifunctional cargo, which can be delivered to carcinoma cells.

A polymer-based nanodevice for the photoregulated release of NO with two-photon fluorescence reporting in skin carcinoma cells.

We have developed a multifunctional biocompatible nanoconstruct based on polymeric nanoparticles encapsulating a molecular conjugate, able to photorelease nitric oxide (NO) with a fluorescent reporting function. We demonstrate that two-photon excitation (TPE) using biofriendly NIR 700 nm laser light can be applied for monitoring as well as triggering the release of NO, wherein the uncaging of a strongly fluorescent co-product acts in turn as a TPE fluorescent reporter for the simultaneous NO release from the nanoassembly. This supramolecular nanodevice internalizes in skin carcinoma cells, induces significant cell death upon light excitation and preserves its TPE properties, allowing the nearly instantaneous quantification of the NO photoreleased in cancer cells by two-photon NIR fluorescence microscopy.

Cubic and sponge phases in ether lipid-solvent-water ternary systems: phase behavior and NMR characterization.

The phase behavior of 1-glyceryl monoleyl ether (GME) in mixtures of water and the solvents 1,5-pentanediol (POL) or N-methyl-2-pyrrolidone (NMP) was investigated by ocular inspection, polarization microscopy, and small-angle X-ray diffraction (SAXD). Phase diagrams were constructed based on analyses of more than 200 samples prepared using the two different solvents at 20 °C. The inverse hexagonal phase formed by GME in excess of water was transformed into the cubic and sponge phase with the increasing amount of each solvent.

In vivo study of an instantly formed lipid-water cubic phase formulation for efficient topical delivery of aminolevulinic acid and methyl-aminolevulinate.

We demonstrate a rapidly formed cubic liquid crystalline phase, i.e. typically 1g cubic phase in less than 1 min confirmed by X-ray diffraction, consisting of an ether lipid, 1-glyceryl monooleyl ether (GME), an aprotic solvent (propylene glycol or pentane-1,5-diol) and water.

Poor correlation between spectrophotometric intracutaneous analysis and histopathology in melanoma and nonmelanoma lesions.

Spectrophotometric intracutaneous analysis (SIAscopy) is an imaging technique developed for diagnostics of pigmented skin lesions. By image analysis, the displayed images indicate the potential distribution and position of melanin, blood, and collagen within the lesion. A topographic comparison was performed between SIAscopic findings and histopathology.

Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin.

There is a need for tools enabling quantitative imaging of biological tissue for pharmaceutical applications. In this study, two-photon fluorescence microscopy (TPM) has been combined with fluorescence correlation spectroscopy (FCS), demonstrating proof-of-principle providing quantitative data of fluorophore concentration and diffusion in human skin. Measurements were performed on excised skin exposed to either rhodamine B (RB) or rhodamine B isothiocyanate (RBITC), chosen based on their similarity in fluorescence yield and molecular weight, but difference in chemical reactivity.

Anti-Stokes fluorescence from endogenously formed protoporphyrin IX--implications for clinical multiphoton diagnostics.

Multiphoton imaging based on two-photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce.

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

Allergic contact dermatitis (ACD) is caused by low-molecular weight compounds called haptens. It has been shown that the potency of haptens can depend on the formulation in which they are applied on the skin. Specifically the sensitization potency of isothiocyanates, a group of haptens which can be released from e.