Two‐Photon Fluorescent Probes for Metal Ions

Two‐photon microscopy (TPM) has become an indispensible tool in biology and medicine owing to the capability of imaging the intact tissue for a long period of time. To make it a versatile tool in biology, a variety of two‐photon probes for specific applications are needed. In this context, many research groups are developing two‐photon probes for various applications. In this Focus Review, we summarize recent results on model studies and selected examples of two‐photon probes that can detect intracellular free metal ions in live cells and tissues to provide a guideline for the design of useful two‐photon probes for various in vivo imaging applications.     

Improved sample preparation for MALDI–MSI of endogenous compounds in skin tissue sections and mapping of exogenous active compounds subsequent to ex-vivo skin penetration

Localization of endogenous and exogenous compounds directly in tissue sections is a challenging task in skin research. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful label-free technique that enables determination of the distribution of a large range of biomolecules directly in tissue sections. Nevertheless, its application in this field is limited in large part by the low adhesion of skin tissue sections to indium–tin oxide-coated (ITO) glass slides. For the first time corona discharge (CD) treatment was used to modify the glass slide surface for improved adhesion. Localization of endogenous cholesterol sulfate was performed directly in human skin tissue sections. A spatial resolution of approximately 30 μm was sufficient for assignment of mass signals to skin structure morphology. Furthermore, imaging of an exogenous model compound, Nile red, was performed directly in skin tissue sections after ex-vivo penetration into porcine skin, enabling determination of the pathway and depth of penetration. Finally, the ion density map of Nile red was compared with its high resolution fluorescence micrograph. This work provides new insights into the application of MALDI–MSI in skin research.     

Preparation of nanostructure hydroxyapatite scaffold for tissue engineering applications

Hydroxyapatite due to its good biocompatibility and similar chemical composition to the mineral part of bone has found various applications in tissue engineering. Porous hydroxyapatite has high surface area, which leads to excellent osteoconductivity and resorbability, providing fast bone ingrowth. In this study, highly porous body of nanostructure hydroxyapatite was successfully fabricated via gelcasting method. The pure phase of hydroxyapatite was confirmed by X-ray diffraction. The result of scanning electron microscopy analysis showed that the prepared scaffold has highly interconnected spherical pores with a size in the range 100–400 μm. The crystallite size of the hydroxyapatite scaffold was measured in the range 30–42 nm. The mean values of true (total) and apparent (interconnected) porosity were calculated in the range 84–91 and 70–78%, respectively. The maximum values of compressive strength and elastic modulus of the prepared scaffold were found to be about 1.5 MPa and 167 MPa, respectively, which were achieved after sintering at 1,000 °C for 4 h. Transmission electron microscopy analysis showed that the particle sizes are smaller than 80 nm. In vitro test showed good bioactivity of the prepared scaffold. The mentioned properties could make the hydroxyapatite scaffold a good candidate for tissue engineering applications, especially applications that did not need to stand any loading.     

Bicellar systems as modifiers of skin lipid structure

The characterization of different bicellar aggregates and the effects of these systems on the stratum corneum (SC) microstructure have been studied. Dynamic light scattering (DLS) and freeze fracture electron microscopy (FFEM) techniques showed that both of the systems studied, dimyristoyl-phosphatidylcholine/dihexanoyl-phosphocholine (DMPC/DHPC) and dipalmitoyl-phosphocholine (DPPC)/DHPC, were formed by small discoidal aggregates at room temperature (20°C). Treating skin with DMPC/DHPC bicelles does not affect the SC lipid microstructure, whereas bicellar systems formed by DPPC and DHPC can promote the formation of new structures in the SC lipid domains. This indicates the passage of lipids from bicelles through the SC layers and also a possible interaction of these lipids with the SC lipids. Given the absence of surfactant in the bicellar composition and the small size of these structures, the use of these smart nano-systems offers great advantages over other lipid systems for dermatological purposes. Bicelles could be promising applications as drug carriers through the skin. This contribution, based on the new biological use of bicelles, may be useful to scientists engaged in colloid science and offers a new tool for different applications in skin and cosmetic research.     

Flavones from Callus Tissue of <Emphasis Type="Italic">Iris ensata</Emphasis>

Flavonoids uncharacteristic of intact plants were isolated from callus tissue of Iris ensata and were identified as 5-hydroxy-4′-methoxyflavone, 5-hydroxy-3′-methoxyflavone, and 5-hydroxy-2′-methoxyflavone using PMR and mass spectrometry. It was proposed that the lack of growth of callus tissue after changing cultivation conditions was related to the inhibiting effect of these flavones on cell proliferation. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 440–442, September–October, 2005.     

Subcellular alterations induced by UV-oxidized low-density lipoproteins in epithelial cells can be counteracted by alpha-tocopherol

Oxidized LDL (ox-LDL) have been involved in the pathogenesis of several human diseases including dermatological pathologies. Oxidative modification of low-density lipoproteins (LDL) is accompanied by both extensive degradation of its polyunsaturated fatty acids and production of lipoperoxides. These highly reactive products induce an intracellular oxidative stress with a variety of cytotoxic effects. In order to evaluate cellular damage induced by oxidative stress in epidermal cells, a human epidermoid carcinoma cell line in culture (A 431) was used as experimental model. Cell treatment with UV-oxidized LDL resulted in cytostatic and cytotoxic effects characterized by morphological and functional alterations: inhibition of cell proliferation, modifications of cytoskeleton network, microtubular derangement, loss of cell–cell and cell–substrate contacts, cell detachment and cell death by apoptosis. The ox-LDL-induced alterations were almost completely prevented by pre-incubating cells with α-tocopherol. The results presented here could be of relevance for a better comprehension of the pathogenic mechanisms of several human diseases, including dermatological pathologies, and could indicate that antioxidants such as α-tocopherol could represent an important therapeutic challenge in the maintenance of cell and tissue homeostasis in the long run.     

A new sample substrate for imaging and correlating organic and trace metal composition in biological cells and tissues

Many disease processes involve alterations in the chemical makeup of tissue. Synchrotron-based infrared (IR) and X-ray fluorescence (XRF) microscopes are becoming increasingly popular tools for imaging the organic and trace metal compositions of biological materials, respectively, without the need for extrinsic labels or stains. Fourier transform infrared microspectroscopy (FTIRM) provides chemical information on the organic components of a material at a diffraction-limited spatial resolution of 2–10 μm in the mid-infrared region. The synchrotron X-ray fluorescence (SXRF) microprobe is a complementary technique used to probe trace element content in the same systems with a similar spatial resolution. However to be most beneficial, it is important to combine the results from both imaging techniques on a single sample, which requires precise overlap of the IR and X-ray images. In this work, we have developed a sample substrate containing a gold grid pattern on its surface, which can be imaged with both the IR and X-ray microscopes. The substrate consists of a low trace element glass slide that has a gold grid patterned on its surface, where the major and minor parts of the grid contain 25 and 12 nm gold, respectively. This grid pattern can be imaged with the IR microscope because the reflectivity of gold differs as a function of thickness. The pattern can also be imaged with the SXRF microprobe because the Au fluorescence intensity changes with gold thickness. The tissue sample is placed on top of the patterned substrate. The grid pattern’s IR reflectivity image and the gold SXRF image are used as fiducial markers for spatially overlapping the IR and SXRF images from the tissue. Results show that IR and X-ray images can be correlated precisely, with a spatial resolution of less than one pixel (i.e., 2–3 microns). The development of this new tool will be presented along with applications to paraffin-embedded metalloprotein crystals, Alzheimer’s disease, and hair composition.     

Application of Nonlinear Optical Microscopy for Imaging Skin†

Recent advances in the use of nonlinear optical microscopy (NLOM) in skin microscopy are presented. Nonresonant spectroscopies including second harmonic generation, coherent anti‐Stokes Raman and two‐photon absorption are described and applications to problems in skin biology are detailed. These nonlinear techniques have several advantages over traditional microscopy methods that rely on one‐photon excitation: intrinsic 3D imaging with <1 μm spatial resolution, decreased photodamage to tissue samples and penetration depths up to 1000 μm with the use of near‐infrared lasers. Thanks to these advantages, nonlinear optical spectroscopy has become a powerful tool to study the physical and biochemical properties of the skin. Structural information can be obtained using the response of endogenous chemical species in the skin, such as collagen or lipids, indicating that optical biopsy may replace current invasive, time‐consuming traditional histology methods. Insertion of specific probe molecules into the skin provides the opportunity to monitor specific biochemical processes such as skin transport, molecular penetration, barrier homeostasis and ultraviolet radiation‐induced reactive oxygen species generation. While the field is quite new, it seems likely that the use of NLOM to probe structure and biochemistry of live skin samples will only continue to grow.     

Quantification of neurotransmitter amino acids by capillary electrophoresis laser-induced fluorescence detection in biological fluids

The role of neurotransmitter amino acids (NAAs) in the functioning of the nervous system has been the focus of increasingly intense research over the past several years. Among the various amino acids that have important roles as neurotransmitters, there are alanine (Ala), glutamic acid (Glu), aspartic acid (Asp), serine (Ser), taurine (Tau) and glycine (Gly). NAAs are present in plasma, cells and—at trace levels—in all biological fluids, but complex components in biological matrices make it difficult to determine them in biological samples. We describe a new capillary electrophoresis (CE) method with laser-induced fluorescence detection by which analytes are resolved in less than 12 minutes in a 18 mmol/L phosphate run buffer at pH 11.6. The use of elevated temperatures during sample derivatization leads to a drastic reduction in the reaction time, down to 20 min, compared to the 6–14 h usually described for reactions between FITC and amino acids at room temperature. In order to demonstrate its wide range of applications, the method was applied to the analysis of NAA in human plasma and in other sample types, such as red blood cells, urine, cultured cells, cerebrospinal fluid, saliva and vitreous humor, thus avoiding the typical limitations of other methods, which are normally suitable for use with only one or two matrix types.     

Stearyl ferulate-based solid lipid nanoparticles for the encapsulation and stabilization of β-carotene and α-tocopherol

UVA exposure induces DNA damage that could result in skin carcinogenesis. Antioxidants are usually employed as protective agents to avoid this problem: in particular, both β-carotene and α-tocopherol can protect the skin against UVA-induced damage. It is well known that the photochemical instability of these compounds has been a limiting factor for their applications to protect skin. In this study, stearyl ferulate-based solid lipid nanoparticles (SF-SLNs), as vehicles for β-carotene and α-tocopherol, were formulated to improve the stability of these compounds. The SF-SLNs were characterized for entrapment efficiency, size and shape together with their cytotoxicity and capability to inhibit lipid peroxidation. After treatment with a pro-oxidant and/or exposition to sunlight the antioxidants entrapped in SF-SLNs were extremely stable. The results highlighted how SF-SLNs represent a suitable vehicle for β-carotene and α-tocopherol stabilizing and protecting them from degradation. A dermatological formulation in order to prevent skin damages is, therefore, suggested.     

MALDI-MS imaging of lipids in ex vivo human skin

Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 μm, using a MALDI quadrupole time-of-flight Q-Star Pulsar-i ^TM (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMS^TM system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations.     

Dispersion of stratum corneum in an aqueous mixed solution of surfactants: the effect of a mixture of sodium dodecyl sulfate and N,N-dimethyldodecylamine oxide

Molecular interactions between sodium dodecyl sulfate (SDS) and N,N-dimethyldodecylamine oxide (C₁₂DMAO), whose mixtures were effective for dispersion of stratum corneum (SC) into intact corneocytes, were studied and found to be strongest at an SDS/C₁₂DMAO molar ratio of 1/3, when dispersion of SC was most effective and the sizes of the mixed micelles were largest.

This dispersion effect was confirmed as being caused by the stronger solubilizing power of molecular complexes formed between SDS and C₁₂DMAO in the binary mixed solutions by using ¹H NMR. The mechanisms for removing intercellular lipids such as ceramides, cholesterol and their derivatives, which play an important role as adhesives among the corneocytes, and for dispersing SC into intact cells were proposed on the basis of supporting data obtained from ¹H-NMR and light scattering measurements.     

Chemical morphology: The chemistry of our shape, <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Our shape is defined and maintained by the connective tissues (skin, tendons, cartilages, blood vessels, etc.) or more precisely by their extracellular matrices. These highly ordered supramolecular organisations are modules of protein fibrils held together by elastic carbohydrate strings. I called these the ‘shape modules.’ The ‘laws.’ which underpin this tissue jigsaw, the changes which come with age and the insight that the concepts give in economically important disorders such as osteoarthrosis begin to provide a new and coherent picture stretching across the animal world and its evolution. This structure/function picture is built on biochemical analyses, which developed into histochemical microscopy and thence into electron histochemistry, and from nuclear magnetic resonance (NMR), molecular modelling and computer simulations on the physicochemical and biomechanical side. As usual, originality bred dissent.     

Elastic properties of chemically modified baker's yeast cells studied by AFM

Chemical pretreatment is widely used to facilitate transformation of living cells when foreign components are introduced into a cell through the cell wall. The influence of appropriate chemicals on the wall properties and mechanism of transformation is still a matter of intensive studies. Saccharomyces cerevisiae cells (also known as baker's yeast) were investigated by atomic force microscopy (AFM). The cell walls were modified by lithium acetate and dithiothreitol. The AFM imaging was performed in liquid water‐based environment. The living cells were fixed by trapping into the holes of a polycarbonate membrane. Mechanical and morphological properties of initial intact cells and treated cells were investigated. The increased stiffness of the chemically treated cells was observed. As deduced from the applied theoretical Hertz‐Sneddon model, the treated cells show completely different response mechanism to applied mechanical pressure in comparison with the intact cells. Also, the increased roughness of the cell wall of the treated yeasts was observed. Copyright © 2011 John Wiley & Sons, Ltd.     

Mechanisms of Penetration Enhancement and Transport Utilizing Skin Keratine Liposomes for the Topical Delivery of Licochalcone A

Keratin liposomes have emerged as a useful topical drug delivery system given theirenhanced ability to penetrate the skin, making them ideal as topical drug vehicles. However, the mechanisms of the drug penetration enhancement of keratin liposomes have not been clearly elucidated. Therefore, licochalcone A(LA)-loaded skin keratin liposomes (LALs) were prepared to investigate their mechanisms of penetration enhancement on the skin and inB16F10 cells. Skin deposition studies, differential scanning calorimetry (DSC), attenuated total reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and skin distribution and intracellular distribution studies were carried out to demonstrate the drug enhancement mechanisms of LALs. We found that the optimal application of LALs enhanced drug permeation via alterations in the components, structure, and thermodynamic properties of the stratum corneum (SC), that is, by enhancing the lipid fluidization, altering the skin keratin, and changing the thermodynamic properties of the SC. Moreover, hair follicles were the main penetration pathways for the LA delivery, which occurred in a time-dependent manner. In the B16F10 cells, the skin keratin liposomes effectively delivered LA into the cytoplasm without cytotoxicity. Thus, LAL nanoparticles are promising topical drug delivery systems for pharmaceutical and cosmetic applications.     

Phytochemical analysis of young fustic (Cotinus coggygria heartwood) and identification of isolated colourants in historical textiles

Young fustic (Cotinus coggygria Scop.; Anacardiaceae) has been used as a dyestuff since antiquity. Phytochemical investigation of the methanol extract of the heartwood has led to the isolation and structure elucidation by nuclear magnetic resonance and mass spectrometry (MS) of 3′,4′,6-trihydroxyaurone (sulfuretin) and 3′,4′,7-trihydroxyflavonol (fisetin) as well as 3′,4′,7-trihydroxyflavanol (fustin), 3′,4′,5,7-tetrahydroxyflavonol (quercetin), 3′,4′,5,7-tetrahydroxyflavanol (taxifolin), 4′,7-dihydroxyflavanol, 3′,4′,7-trihydroxyflavanone (butin), 4′,7-dihydroxyflavanone (liquiritigenin), trans-2′,3,4,4′-tetrahydroxychalcone (butein), 4′,5,7-trihydroxyflavanone and trans-2′,4,4′-trihydroxychalcone (isoliquiritigenin). The isolated compounds were used as reference materials for the development of a high-performance liquid chromatography–diode array detector–MS method, which was then applied to analyse (1) fresh silk samples dyed with young fustic, (2) dyed silk subjected to artificially accelerated light ageing and (3) historical silk micro-samples, extracted from ecclesiastical post-Byzantine garments (fifteenth to eighteenth century), which belong to monasteries of Mount Athos. Sulfuretin and fisetin, which are usually used as markers for the identification of the yellow dye and, for the first time, some of the aforementioned flavonoid components of young fustic were identified in the historical extracts. Furthermore, preliminary experiments suggested that although the amounts of the dye components decrease with light ageing, the relative ratio of fisetin and sulfuretin, after a first step of ageing, seems to be almost unaffected by such degradation processes raised by light. The effect of the latter on the morphology of the dyed silk fibres is briefly investigated by scanning electron microscopy.     

Structural and biological evaluation of a multifunctional SWCNT-AgNPs-DNA/PVA bio-nanofilm

A bio-nanofilm consisting of a tetrad nanomaterial (nanotubes, nanoparticles, DNA, polymer) was fabricated utilizing in situ reduction and noncovalent interactions and it displayed effective antibacterial activity and biocompatibility. This bio-nanofilm was composed of homogenous silver nanoparticles (AgNPs) coated on single-walled carbon nanotubes (SWCNTs), which were later hybridized with DNA and stabilized in poly(vinyl alcohol) (PVA) in the presence of a surfactant with the aid of ultrasonication. Electron microscopy and bio-AFM (atomic force microscopy) images were used to assess the morphology of the nanocomposite (NC) structure. Functionalization and fabrication were examined using FT–Raman spectroscopy by analyzing the functional changes in the bio-nanofilm before and after fabrication. UV–visible spectroscopy and X-ray powder diffraction (XRD) confirmed that AgNPs were present in the final NC on the basis of its surface plasmon resonance (370 nm) and crystal planes. Thermal gravimetric analysis was used to measure the percentage weight loss of SWCNT (17.5%) and final SWCNT-AgNPs-DNA/PVA (47.7%). The antimicrobial efficiency of the bio-nanofilm was evaluated against major pathogenic organisms. Bactericidal ratios, zone of inhibition, and minimum inhibitory concentration were examined against gram positive and gram negative bacteria. A preliminary cytotoxicity analysis was conducted using A549 lung cancer cells and IMR-90 fibroblast cells. Confocal laser microscopy, bio-AFM, and field emission scanning electron microscopy (FE-SEM) images demonstrated that the NCs were successfully taken up by the cells. These combined results indicate that this bio-nanofilm was biocompatible and displayed antimicrobial activity. Thus, this novel bio-nanofilm holds great promise for use as a multifunctional tool in burn therapy, tissue engineering, and other biomedical applications.     

Stability‐indicating analytical method of quantifying spironolactone and canrenone in dermatological formulations and iontophoretic skin permeation experiments

A simple, stability‐indicating, chromatographic method of quantifying spironolactone (SPI) and its metabolite, canrenone (CAN), in the presence of excipients typical in dermatological formulations and skin matrices in studies of passive and iontophoretic permeation was proposed and validated here. SPI and CAN were separated using a reversed‐phase column with a mobile phase of methanol–water (60:40, v/v) at a flow rate of 1.0 mL/min. Data were collected with a UV detector at 238 and 280 nm, with retention times of 6.2 and 7.9 min for SPI and CAN, respectively. The method was precise, accurate and linear (r² > 0.99) in a concentration range of 1–30 μg/mL, and recovery rates of SPI and CAN from the different skin layers exceeded 85%. The method was not only sensitive (LOD of 0.05 and 0.375 μg/mL and LOQ of 0.157 and 1.139 μg/mL for SPI and CAN, respectively) but also selective against skin matrices and highly representative components of topical formulations. The method moreover demonstrated SPI's degradation in iontophoresis by applying Pt–AgCl electrodes and its continued drug stability using Ag–AgCl electrodes. Altogether, the method proved valuable for quantifying SPI and CAN and may be applied in developing and controlling the quality of dermatological products.     

Keratinocyte stem cells and the targets for nonmelanoma skin cancer

The mammalian skin is a complex dynamic organ composed of thin multilayered epidermis and a thick underlying connective tissue layer dermis. The epidermis undergoes continuous renewal throughout life. The stems cells uniquely express particular surface markers utilized for their identification, isolation and localization in specific niches in epidermis as well as hair follicles (HFs). The two stage skin carcinogenesis model involves stepwise accumulation of genetic alterations and ultimately leading to malignancy. Whereas early research on skin carcinogenesis focused on the molecular nature of carcinogens and tumor promoters, more recent studies have focused on the identification of the target cells and tumor promoting cells for both chemical and physical carcinogens and promoters. Recent studies support the hypothesis that keratinocyte stem cells are the targets in skin carcinogenesis. In this review, we discuss briefly the localization of stem cells in the epidermis and HFs, and review the possibility that skin papillomas and carcinomas are derived from stem cells, as well as from other cells in the cutaneous epithelium whose stem cell properties are not well known.     

Inductively coupled plasma mass spectrometric analysis of the total amount of platinum in DNA extracts from peripheral blood mononuclear cells and tissue from patients treated with cisplatin

We present a highly sensitive method for the determination of platinum (Pt) in DNA extracts of peripheral blood mononuclear cells (PBMCs) and tissue samples from patients treated with cisplatin. The method is based on the measurement of Pt by inductively coupled plasma mass spectrometry (ICP-MS) and allows quantification of Pt-DNA adducts in PBMCs isolated from 10 mL blood and 1 mg tissue. The lower limit of quantification is 0.75 pg Pt or 7.5 fg Pt μg⁻¹ DNA when using 100 μg DNA. The method proved to be accurate and precise. The results obtained using the ICP-MS method were in good agreement with results from the alternative ³²P-postlabelling assay. The ICP-MS method was, however, more sensitive and proved to be less laborious. The advantages of the presented ICP-MS technique were demonstrated by the analysis of PBMCs, normal gastric tissue, and gastric tumour tissue of patients treated with cisplatin.