Visualization of Epidermal Reservoir Formation from Topical Diclofenac Gels by Raman Spectroscopy.

PURPOSE: This work investigated whether topical pain relief diclofenac gels can form a diclofenac reservoir in the epidermal and dermal layers of human skin. METHODS: Excised human skin samples were treated with three topical diclofenac gels ex vivo and examined using Raman microscopy of transversally microtomed sections. The relative diclofenac concentration in the skin layers was calculated as the ratio of the integrated areas of bands characteristic of diclofenac (~445 cm-1) and skin (Amide I). A customized masking algorithm ensured that only diclofenac-specific signal was mapped in the resulting Raman images. RESULTS: A heterogenous spatial distribution of diclofenac was clearly visible in both the epidermis and the dermis in all samples, with a markedly higher diclofenac relative content and number of pixels above the detection limit in the epidermis compared to the dermis. CONCLUSION: The Raman images evidenced that the studied topical gels deliver diclofenac through the stratum corneum skin barrier and form a drug depot localized in the epidermis. The data are in line with earlier clinical findings that this depot acts like a true reservoir and enables sustained drug release.

FT-IR investigation of Terbinafine interaction with stratum corneum constituents.

Terbinafine (Tbf) is a well-established anti-fungal agent used for management of a variety of dermal conditions including ringworm and athlete's foot. Both the biochemical mechanism of Tbf fungicidal action (based on squalene epoxidase inhibition) and the target region for Tbf in vivo (the stratum corneum (SC)) are well determined. However, the biochemical and pharmacokinetic approaches used to evaluate Tbf biochemistry provide no biophysical information about molecular level physical changes in the SC upon Tbf binding. Such information is necessary for improved drug and formulation design. IR spectroscopic methods were used to evaluate the effects of Tbf on keratin structure in environments commonly used in pharmaceutics to mimic those in vivo. The Amide I and II spectral regions (1500-1700 cm-1) provided an effective means to monitor keratin secondary structure changes, while a Tbf spectral feature near 775 cm-1 provides a measure of relative Tbf levels in skin. Interaction of Tbf with the SC induced substantial ß-sheet formation in the keratin, an effect which was partially reversed both by ethanol washing and by exposure to high relative humidity. The irreversibility suggests the presence of a Tbf reservoir (consistent with kinetic studies), permitting the drug to be released in a controlled manner into the surrounding tissue.

A unique gel matrix moisturizer delivers deep hydration resulting in significant clinical improvement in radiance and texture.

Introduction: As skin ages, it loses its ability to retain moisture and becomes rough and dry. This results in a clinically dull appearance with a loss of radiance, firmness, and suppleness. Symptoms can be improved with use of a moisturizer that builds and maintains skin hydration over time; however, most moisturizers that occlude the skin surface are perceived as heavy and greasy and are not consumer preferred. Methods: A unique, consumer-preferred gel matrix formula was developed by combining liquid crystal structures, which mimic skin barrier lipid assembly, with specific emulsifiers that deliver water deep into skin. Ex vivo studies were conducted to investigate the superior hydrating effects of the gel matrix formula. Confocal Raman microscopy studies assessed the spatial distribution of water in ex vivo skin after application of the gel matrix formula. To determine the effects of the gel matrix formula on dry facial skin, a 12-week clinical study was conducted with subjects with self-perceived skin dryness and dullness. Results: The formulation significantly increased the relative water content throughout epidermal regions, which was not observed with the application of a competitive gel formula. Instrumental measurements assessed improvements in skin surface moisturization and barrier function. Clinical grading showed significant improvements in hydration-related endpoints including radiance, clarity, and texture. Subject self-agree assessment demonstrated that subjects observed improvements in the appearance of their facial skin. Conclusion: These studies demonstrated that the gel matrix formula increased skin water content in deeper layers, and resulted in significant clinical improvements in hydration, barrier function, and clinical appearance of radiance.

Novel confocal Raman microscopy method to investigate hydration mechanisms in human skin.

BACKGROUND: Skin hydration is essential for maintaining stratum corneum (SC) flexibility and facilitating maturation events. Moisturizers contain multiple ingredients to maintain and improve skin hydration although a complete understanding of hydration mechanisms is lacking. The ability to differentiate the source of the hydration (water from the environment or deeper skin regions) upon application of product will aid in designing more efficacious formulations. MATERIALS AND METHODS: Novel confocal Raman microscopy (CRM) experiments allow us to investigate mechanisms and levels of hydration in the SC. Using deuterium oxide (D2 O) as a probe permits the differentiation of endogenous water (H2 O) from exogenous D2 O. Following topical application of D2 O, we first compare in vivo skin depth profiles with those obtained using ex vivo skin. Additional ex vivo experiments are conducted to quantify the kinetics of D2 O diffusion in the epidermis by introducing D2 O under the dermis. RESULTS: Relative D2 O depth profiles from in vivo and ex vivo measurements compare well considering procedural and instrumental differences. Additional in vivo experiments where D2 O was applied following topical glycerin application increased the longevity of D2 O in the SC. Reproducible rates of D2 O diffusion as a function of depth have been established for experiments where D2 O is introduced under ex vivo skin. CONCLUSION: Unique information regarding hydration mechanisms are obtained from CRM experiments using D2 O as a probe. The source and relative rates of hydration can be delineated using ex vivo skin with D2 O underneath. One can envision comparing these depth-dependent rates in the presence and absence of topically applied hydrating agents to obtain mechanistic information.

In vitro modeling of unsaturated free fatty acid-mediated tissue impairments seen in acne lesions.

Acne vulgaris is a disease of pilosebaceous units with multifactorial pathogenesis, including hyperkeratinization, increased sebum secretion, and inflammation. Recently, it was suggested that acne subjects may have also impaired skin barrier. We hypothesized that excess unsaturated free fatty acids (UFFA) present in the sebum may cause barrier impairment associated with increased follicular stratum corneum (SC) thickening and inflammation seen in acne. Therefore, epidermal and sebaceous lipid profiles from acne and healthy subjects were analyzed and an in vitro epidermal tissue model was developed to validate this hypothesis. Significantly increased levels of free fatty acids (p < 0.05) were observed in skin lipids of human acne vs. healthy subjects. Exposure of human epidermal equivalents (HEEs) to the UFFA oleic acid (OA), also present in sebum, led to barrier impairment associated with increased SC lipid disorder, increased secretion of interleukin-1α (IL-1α), and excessive SC thickening. Furthermore, the expression of genes encoding for inflammatory cytokines and epidermal differentiation proteins was also increased both in acne lesions and in OA-treated HEEs. Taken together, these data are in agreement with the hypothesis that excess UFFAs in sebum of acne subjects may contribute to impaired skin barrier associated with the increased follicular SC thickness and inflammation seen in acne. Moreover, OA induces similar molecular and phenotypic changes in HEEs as those seen in acne lesions and suggests that an UFFA-treated epidermal tissue model can be used to study the UFFA-mediated pathways involved in the pathogenesis of inflammatory acne and for the development of appropriate therapies.

Lactation-Induced Changes in the Volume of Osteocyte Lacunar-Canalicular Space Alter Mechanical Properties in Cortical Bone Tissue.

Osteocytes can remove and remodel small amounts of their surrounding bone matrix through osteocytic osteolysis, which results in increased volume occupied by lacunar and canalicular space (LCS). It is well established that cortical bone stiffness and strength are strongly and inversely correlated with vascular porosity, but whether changes in LCS volume caused by osteocytic osteolysis are large enough to affect bone mechanical properties is not known. In the current studies we tested the hypotheses that (1) lactation and postlactation recovery in mice alter the elastic modulus of bone tissue, and (2) such local changes in mechanical properties are related predominantly to alterations in lacunar and canalicular volume rather than bone matrix composition. Mechanical testing was performed using microindentation to measure modulus in regions containing solely osteocytes and no vascular porosity. Lactation caused a significant (∼13%) reduction in bone tissue-level elastic modulus (p < 0.001). After 1 week postweaning (recovery), bone modulus levels returned to control levels and did not change further after 4 weeks of recovery. LCS porosity tracked inversely with changes in cortical bone modulus. Lacunar and canalicular void space increased 7% and 15% with lactation, respectively (p < 0.05), then returned to control levels at 1 week after weaning. Neither bone mineralization (assessed by high-resolution backscattered scanning electron microscopy) nor mineral/matrix ratio or crystallinity (assessed by Raman microspectroscopy) changed with lactation. Thus, changes in bone mechanical properties induced by lactation and recovery appear to depend predominantly on changes in osteocyte LCS dimensions. Moreover, this study demonstrates that tissue-level cortical bone mechanical properties are rapidly and reversibly modulated by osteocytes in response to physiological challenge. These data point to a hitherto unappreciated role for osteocytes in modulating and maintaining local bone mechanical properties. © 2016 American Society for Bone and Mineral Research.

Effects of permeation enhancers on flufenamic acid delivery in Ex vivo human skin by confocal Raman microscopy.

For effective topical delivery, a drug must cross the stratum corneum (SC) barrier into viable tissue. The use of permeation enhancers is a widespread approach for barrier modification. In the current study, flufenamic acid (FluA), a non-steroidal anti-inflammatory drug, is a model agent for investigating the influence of hydrophobic versus hydrophilic enhancers. In separate experiments, FluA in octanol or propylene glycol/ethanol (75/25) is applied to the SC for varying times followed by confocal Raman microscopic mapping of drug and enhancer penetration and spatial distribution. Deuterated versions of the enhancers permit us to spectroscopically distinguish the exogenous chemicals from the endogenous SC lipids without affecting penetration parameters. The FluA pathway is tracked by the CC stretching mode at ∼1618cm(-1). Discrete, small inclusions of both enhancers are observed throughout the SC. High concentrations of FluA are co-localized with octanol domains which appear to provide a pathway to the viable epidermis for the drug. In contrast, FluA concentrates in the upper SC when using the hydrophilic agent and endogenous lipids appear unperturbed in regions outside the enhancer pockets. The ability to examine perturbations to endogenous ultrastructure and molecular structure in skin while tracking penetration pathways provides insight into delivery mechanisms.

P-Doped Porous Carbon as Metal Free Catalysts for Selective Aerobic Oxidation with an Unexpected Mechanism.

An extremely simple and rapid (seconds) approach is reported to directly synthesize gram quantities of P-doped graphitic porous carbon materials with controlled P bond configuration. For the first time, it is demonstrated that the P-doped carbon materials can be used as a selective metal free catalyst for aerobic oxidation reactions. The work function of P-doped carbon materials, its connectivity to the P bond configuration, and the correlation with its catalytic efficiency are studied and established. In direct contrast to N-doped graphene, the P-doped carbon materials with higher work function show high activity in catalytic aerobic oxidation. The selectivity trend for the electron donating and withdrawing properties of the functional groups attached to the aromatic ring of benzyl alcohols is also different from other metal free carbon based catalysts. A unique catalytic mechanism is demonstrated, which differs from both GO and N-doped graphene obtained by high temperature nitrification. The unique and unexpected catalytic pathway endows the P-doped materials with not only good catalytic efficiency but also recyclability. This, combined with a rapid, energy saving approach that permits fabrication on a large scale, suggests that the P-doped porous materials are promising materials for "green catalysis" due to their higher theoretical surface area, sustainability, environmental friendliness, and low cost.

Topically applied ceramide accumulates in skin glyphs.

Ceramides (CERs), structural components of the stratum corneum (SC), impart essential barrier properties to this thin outer layer of the epidermis. Variations in CER species within this layer have been linked to several skin diseases. A recent proliferation of CER-containing topical skin-care products warrants the elucidation of CER penetration profiles in both healthy and diseased skin. In the current study, the spatial distributions of CER concentration profiles, following topical application of two species of CER, were tracked using infrared imaging. Suspensions of single-chain perdeuterated sphingosine and phytosphingosine CER in oleic acid were applied, in separate experiments, to the surface of healthy intact ex vivo human skin using Franz diffusion cells. Following either a 24- or 48-hour incubation period at 34°C, infrared images were acquired from microtomed skin sections. Both CER species accumulated in glyph regions of the skin and penetrated into the SC, to a limited extent, only in these regions. The concentration profiles observed herein were independent of the CER species and incubation time utilized in the study. As a result, a very heterogeneous, sparse, spatial distribution of CERs in the SC was revealed. In contrast, oleic acid was found to be fairly homogeneously distributed throughout the SC and viable epidermis, albeit at lower concentrations in the latter. A more uniform, lateral distribution of CERs in the SC would likely be important for barrier efficacy or enhancement.

Fatty Acid Chain Length Dependence of Phase Separation Kinetics in Stratum Corneum Models by IR Spectroscopy.

The main barrier to permeability in human skin resides in the stratum corneum (SC), a layered structure consisting of anucleated, flattened cells (corneocytes) embedded in a heterogeneous lamellar lipid matrix. While lipid structures and packing propensities in the SC and in SC models have been extensively investigated, only limited data are available concerning the kinetics and mechanism of formation of lamellar phases and particular lipid packing motifs. In our prior investigation, kinetic IR spectroscopy measurements probed the temporal sequence of phase separation leading to ordered structures in a three component SC model of equimolar structurally heterogeneous ceramide[NS], chain perdeuterated stearic acid, and cholesterol. In the current work, the phase separation kinetic effects of specific fatty acid chain lengths with a synthetic structurally homogeneous ceramide[NS] in similar ternary mixtures are examined. These are compared with a mixture containing ceramide[NS] with an unsaturated acid chain. The kinetic events are sensitive to the difference in chain lengths between the ceramide acid chain and the fatty acid as well as to the presence of unsaturation in the former. The observed kinetic behaviors span a wide range of phase separation times, ranging from the formation of a solid solution stable for at least 200 h, to a system in which an orthorhombic fatty acid structure is essentially completely formed within the time resolution of the experiment (15 min). The data seem to offer some features of a spinodal phase separation at relatively short times. Overall the approach offers a possible means for addressing several unanswered questions pertinent to skin pharmacology, such as the roles of a wide variety of ceramide and fatty acid species and the design of therapeutic interventions for repair of pathological conditions of the SC.

Infrared spectroscopic imaging tracks lateral distribution in human stratum corneum.

PURPOSE: To demonstrate the efficacy of infrared (IR) spectroscopic imaging for evaluation of lateral diffusion in stratum corneum (SC) and for elucidation of intermolecular interactions between exogenous agents and SC constituents. METHODS: In separate experiments, acyl chain perdeuterated oleic acid (OA-d) and deuterated dimethyl sulfoxide (DMSO-d) were applied to the surface of isolated human SC. The lateral distribution of permeant concentrations was monitored using the time-dependence of IR images. Diffusion coefficients (D) were estimated from Fick's second law. Interactions between the exogenous agents and the SC were tracked from changes in CD2 and Amide I stretching frequencies. RESULTS: Networked glyphs served as the major pathway for lateral distribution of OA-d. In glyph-poor regions, D values from 0.3-1 × 10(-8) cm(2)/s bracketed the OA-d data and apparently decreased with time. Although diffusion of DMSO-d is relatively fast compared to our experimental measurement time, the results suggest values of ~10(-7) cm(2)/s. OA-d spectral changes suggest penetration into the ordered lipids of the SC; DMSO-d penetration results in perturbation of SC keratin structure. CONCLUSIONS: IR imaging provides concentration profiles, diffusion coefficients, and unique molecular level information about structural changes in the endogenous SC constituents and exogenous agents upon their mutual interaction. Transport along glyphs is the dominant mode of distribution for OA-d.

Kinetic evidence suggests spinodal phase separation in stratum corneum models by IR spectroscopy.

Although lipid structure in models for the stratum corneum (SC), the main barrier to skin permeability, has been extensively studied, only limited data are extant concerning the kinetic mechanism for the formation of domains, lamellar phases, and lipid packing motifs. Such information would be of substantial interest in the characterization of the effects of disease states which disrupt the barrier. Kinetic IR spectroscopy measurements probed the temporal sequence of molecular events producing ordered structures in a three-component SC model of equimolar ceramide[NS] (cer[NS]), perdeuterated stearic acid-d35 (SA-d35), and cholesterol. Samples, heated above Tm, were quenched to 31 °C, and then spectra were recorded at ∼15 min intervals for a total of 20-150 h. IR provides unique molecular structure information about headgroup H-bonding, lipid packing, and lipid chain order. The following sequence for phase separation was observed: (1) Formation of ceramide amide H-bonds from disordered forms to ordered structures (0.5-4 h); (2) appearance of ordered ceramide chains with some orthorhombically packed structures (0.5-8 h); and (3) phase separation of large orthorhombic domains of SA-d35 (4-10 h). A spinodal decomposition mechanism, defined by continuous composition changes during the phase separation, suggests a qualitative description for these events.

Molecular interactions of plant oil components with stratum corneum lipids correlate with clinical measures of skin barrier function.

Plant-derived oils consisting of triglycerides and small amounts of free fatty acids (FFAs) are commonly used in skincare regimens. FFAs are known to disrupt skin barrier function. The objective of this study was to mechanistically study the effects of FFAs, triglycerides and their mixtures on skin barrier function. The effects of oleic acid (OA), glyceryl trioleate (GT) and OA/GT mixtures on skin barrier were assessed in vivo through measurement of transepidermal water loss (TEWL) and fluorescein dye penetration before and after a single application. OA's effects on stratum corneum (SC) lipid order in vivo were measured with infrared spectroscopy through application of perdeuterated OA (OA-d34 ). Studies of the interaction of OA and GT with skin lipids included imaging the distribution of OA-d34 and GT ex vivo with IR microspectroscopy and thermodynamic analysis of mixtures in aqueous monolayers. The oil mixtures increased both TEWL and fluorescein penetration 24 h after a single application in an OA dose-dependent manner, with the highest increase from treatment with pure OA. OA-d34 penetrated into skin and disordered SC lipids. Furthermore, the ex vivo IR imaging studies showed that OA-d34 permeated to the dermal/epidermal junction while GT remained in the SC. The monolayer experiments showed preferential interspecies interactions between OA and SC lipids, while the mixing between GT and SC lipids was not thermodynamically preferred. The FFA component of plant oils may disrupt skin barrier function. The affinity between plant oil components and SC lipids likely determines the extent of their penetration and clinically measurable effects on skin barrier functions.

Direct production of graphene nanosheets for near infrared photoacoustic imaging.

Hummers method is commonly used for the fabrication of graphene oxide (GO) from graphite particles. The oxidation process also leads to the cutting of graphene sheets into small pieces. From a thermodynamic perspective, it seems improbable that the aggressive, somewhat random oxidative cutting process could directly result in graphene nanosheets without destroying the intrinsic π-conjugated structures and the associated exotic properties of graphene. In Hummers method, both KMnO4 and NO2(+) (nitronium ions) in concentrated H2SO4 solutions act as oxidants via different oxidation mechanisms. From both experimental observations and theoretical calculations, it appears that KMnO4 plays a major role in the observed oxidative cutting and unzipping processes. We find that KMnO4 also limits nitronium oxidative etching of graphene basal planes, therefore slowing down graphene fracturing processes for nanosheet fabrication. By intentionally excluding KMnO4 and exploiting pure nitronium ion oxidation, aided by the unique thermal and kinetic effects induced by microwave heating, we find that graphite particles can be converted into graphene nanosheets with their π-conjugated aromatic structures and properties largely retained. Without the need of any postreduction processes to remove the high concentration of oxygenated groups that results from Hummers GO formation, the graphene nanosheets as-fabricated exhibit strong absorption, which is nearly wavelength-independent in the visible and near-infrared (NIR) regions, an optical property typical for intrinsic graphene sheets. For the first time, we demonstrate that strong photoacoustic signals can be generated from these graphene nanosheets with NIR excitation. The photo-to-acoustic conversion is weakly dependent on the wavelength of the NIR excitation, which is different from all other NIR photoacoustic contrast agents previously reported.

Oleic acid disorders stratum corneum lipids in Langmuir monolayers.

Oleic acid (OA) is well-known to affect the function of the skin barrier. In this study, the molecular interactions between OA and model stratum corneum (SC) lipids consisting of ceramide, cholesterol, and palmitic acid (PA) were investigated with Langmuir monolayer and associated techniques. Mixtures with different OA/SC lipid compositions were spread at the air/water interface, and the phase behavior was tracked with surface pressure-molecular area (π-A) isotherms. With increasing OA levels in the monolayer, the films became more fluid and more compressible. The thermodynamic parameters derived from π-A isotherms indicated that there are preferential interactions between OA and SC lipids and that films of their mixtures were thermodynamically stable. The domain structure and lipid conformational order of the monolayers were studied through Brewster angle microscopy (BAM) and infrared reflection absorption spectroscopy (IRRAS), respectively. Results indicate that lower concentrations of OA preferentially mix with and disorder the ceramide-enriched domains, followed by perturbation of the PA-enriched domains and disruption of SC lipid domain separation at higher OA levels.

Fourier transform infrared spectroscopic imaging parameters describing acid phosphate substitution in biologic hydroxyapatite.

Acid phosphate substitution into mineralized tissues is an important determinant of their mechanical properties and their response to treatment. This study identifies and validates Fourier transform infrared spectroscopic imaging (FTIRI) spectral parameters that provide information on the acid phosphate (HPO4) substitution into hydroxyapatite in developing mineralized tissues. Curve fitting and Fourier self-deconvolution were used to identify subband positions in model compounds (with and without HPO4). The intensity of subbands at 1127 and 1110 cm(-1) correlated with the acid phosphate content in these models. Peak height ratios of these subbands to the ν3 vibration at 1096 cm(-1) found in stoichiometric apatite were evaluated in the model compounds and mixtures thereof. FTIRI spectra of bones and teeth at different developmental ages were analyzed using these spectral parameters. Factor analysis (a chemometric technique) was also conducted on the tissue samples and resulted in factor loadings with spectral features corresponding to the HPO4 vibrations described above. Images of both factor correlation coefficients and the peak height ratios 1127/1096 and 1112/1096 cm(-1) demonstrated higher acid phosphate content in younger vs. more mature regions in the same specimen. Maps of the distribution of acid phosphate content will be useful for characterizing the extent of new bone formation, the areas of potential decreased strength, and the effects of therapies such as those used in metabolic bone diseases (osteoporosis, chronic kidney disease) on mineral composition. Because of the wider range of values obtained with the 1127/1096 cm(-1) parameter compared to the 1110/1096 cm(-1) parameter and the smaller scatter in the slope, it is suggested that this ratio should be the parameter of choice.

Infrared and Raman imaging spectroscopy of ex vivo skin.

This primer describes and illustrates experimental protocols for both Fourier transform infrared (FTIR) spectroscopic imaging and confocal Raman mapping of ex vivo skin and thereby acquaints the reader with these measurement techniques, including the temporal and spatial limitations associated with each technique. The experimental conditions by which the unique 'molecular histology' information obtained from confocal Raman mapping and infrared spectroscopic mapping of ex vivo skin is generated will be described. Raman and FTIR spectra of tissue, when collected in spatially resolved arrays, permit the generation of 'molecular images' of tissue components and tissue organization without the use of fluorescent labels or chemical stains. To illustrate the molecular information from ex vivo skin that can be spectroscopically imaged with confocal Raman and infrared microspectroscopy, we have collected new data using both techniques and generated spectral images which illustrate the capacity of each technique to provide unique insights into skin histology, biochemistry and biophysics. Understanding the measurement possibilities and specific constraints of both approaches is a prerequisite to their meaningful use as powerful research tools in skin research.

Vibrational spectroscopy and microscopic imaging: novel approaches for comparing barrier physical properties in native and human skin equivalents.

Vibrational spectroscopy and imaging have been used to compare barrier properties in human skin, porcine skin, and two human skin equivalents, Epiderm 200X with an enhanced barrier and Epiderm 200 with a normal barrier. Three structural characterizations were performed. First, chain packing and conformational order were compared in isolated human stratum corneum (SC), isolated porcine SC, and in the Epiderm 200X surface layers. The infrared (IR) spectrum of isolated human SC revealed a large proportion of orthorhombically packed lipid chains at physiological temperatures along with a thermotropic phase transition to a state with hexagonally packed chains. In contrast, the lipid phase at physiological temperatures in both porcine SC and in Epiderm 200X, although dominated by conformationally ordered chains, lacked significant levels of orthorhombic subcell packing. Second, confocal Raman imaging of cholesterol bands showed extensive formation of cholesterol-enriched pockets within the human skin equivalents (HSEs). Finally, IR imaging tracked lipid barrier dimensions as well as the spatial disposition of ordered lipids in human SC and Epiderm 200X. These approaches provide a useful set of experiments for exploring structural differences between excised human skin and HSEs, which in turn may provide a rationale for the functional differences observed among these preparations.

Infrared microscopic imaging of cutaneous wound healing: lipid conformation in the migrating epithelial tongue.

Infrared microscopic imaging has been utilized to analyze for the first time the spatial distribution of lipid structure in an ex vivo human organ culture skin wound healing model. Infrared images were collected at zero, two, four, and six days following wounding. Analysis of lipid infrared spectral properties revealed the presence of a lipid class with disordered chains within and in the vicinity of the migrating epithelial tongue. The presence of lipid ester C=O bands colocalized with the disordered chains provided evidence for the presence of carbonyl-containing lipid species. Gene array data complemented the biophysical studies and provided a biological rationale for the generation of the disordered chain species. This is the first clear observation, to our knowledge, of disordered lipid involvement in cutaneous wound healing. Several possibilities are discussed for the biological relevance of these observations.

Microwave- and nitronium ion-enabled rapid and direct production of highly conductive low-oxygen graphene.

Currently the preferred method for large-scale production of solution-processable graphene is via a nonconductive graphene oxide (GO) pathway, which uncontrollably cuts sheets into small pieces and/or introduces nanometer-sized holes in the basal plane. These structural changes significantly decrease some of graphene's remarkable electrical and mechanical properties. Here, we report an unprecedented fast and scalable approach to avoid these problems and directly produce large, highly conductive graphene sheets. This approach intentionally excludes KMnO(4) from Hummers' methods and exploits aromatic oxidation by nitronium ions combined with the unique properties of microwave heating. This combination promotes rapid and simultaneous oxidation of multiple non-neighboring carbon atoms across an entire graphene sheet, thereby producing only a minimum concentration of oxygen moieties sufficient to enable the separation of graphene sheets. Thus, separated graphene sheets, which are referred to as microwave-enabled low-oxygen graphene, are thermally stable and highly conductive without requiring further reduction. Even in the absence of polymeric or surfactant stabilizers, concentrated dispersions of graphene with clean and well-separated graphene sheets can be obtained in both aqueous and organic solvents. This rapid and scalable approach produces high-quality graphene sheets of low oxygen content, enabling a broad spectrum of applications via low-cost solution processing.