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1.  Fast Vibrational Imaging of Single Cells and Tissues by Stimulated Raman Scattering Microscopy 
Accounts of Chemical Research  2014;47(8):2282-2290.
Conspectus
Traditionally, molecules are analyzed in a test tube. Taking biochemistry as an example, the majority of our knowledge about cellular content comes from analysis of fixed cells or tissue homogenates using tools such as immunoblotting and liquid chromatography–mass spectrometry. These tools can indicate the presence of molecules but do not provide information on their location or interaction with each other in real time, restricting our understanding of the functions of the molecule under study. For real-time imaging of labeled molecules in live cells, fluorescence microscopy is the tool of choice. Fluorescent labels, however, are too bulky for small molecules such as fatty acids, amino acids, and cholesterol. These challenges highlight a critical need for development of chemical imaging platforms that allow in situ or in vivo analysis of molecules. Vibrational spectroscopy based on spontaneous Raman scattering is widely used for label-free analysis of chemical content in cells and tissues. However, the Raman process is a weak effect, limiting its application for fast chemical imaging of a living system.
With high imaging speed and 3D spatial resolution, coherent Raman scattering microscopy is enabling a new approach for real-time vibrational imaging of single cells in a living system. In most experiments, coherent Raman processes involve two excitation fields denoted as pump at ωp and Stokes at ωs. When the beating frequency between the pump and Stokes fields (ωp – ωs) is resonant with a Raman-active molecular vibration, four major coherent Raman scattering processes occur simultaneously, namely, coherent anti-Stokes Raman scattering (CARS) at (ωp – ωs) + ωp, coherent Stokes Raman scattering (CSRS) at ωs – (ωp – ωs), stimulated Raman gain (SRG) at ωs, and stimulated Raman loss (SRL) at ωp. In SRG, the Stokes beam experiences a gain in intensity, whereas in SRL, the pump beam experiences a loss. Both SRG and SRL belong to stimulated Raman scattering (SRS), in which the energy difference between the pump and Stokes fields is transferred to the molecule for vibrational excitation. The SRS signal appears at the same wavelengths as the excitation fields and is commonly extracted through a phase-sensitive detection scheme. The detected intensity change because of a Raman transition is proportional to Im[χ(3)]IpIs, where χ(3) represents the third-order nonlinear susceptibility, Ip and Is stand for the intensity of the pump and Stokes fields.
In this Account, we discuss the most recent advances in the technical development and enabling applications of SRS microscopy. Compared to CARS, the SRS contrast is free of nonresonant background. Moreover, the SRS intensity is linearly proportional to the density of target molecules in focus. For single-frequency imaging, an SRS microscope offers a speed that is ∼1000 times faster than a line-scan Raman microscope and 10 000 times faster than a point-scan Raman microscope. It is important to emphasize that SRS and spontaneous Raman scattering are complementary to each other. Spontaneous Raman spectroscopy covers the entire window of molecular vibrations, which allows extraction of subtleties via multivariate analysis. SRS offers the speed advantage by focusing on either a single Raman band or a defined spectral window of target molecules. Integrating single-frequency SRS imaging and spontaneous Raman spectroscopy on a single platform allows quantitative compositional analysis of objects inside single live cells.
doi:10.1021/ar400331q
PMCID: PMC4139189  PMID: 24871269
2.  Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy 
Science (New York, N.Y.)  2008;322(5909):1857-1861.
Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.
doi:10.1126/science.1165758
PMCID: PMC3576036  PMID: 19095943
3.  Structure Enhancement Relationship of Chemical Penetration Enhancers in Drug Transport across the Stratum Corneum 
Pharmaceutics  2012;4(1):71-92.
The stratum corneum is a major barrier of drug penetration across the skin in transdermal delivery. For effective transdermal drug delivery, skin penetration enhancers are used to overcome this barrier. In the past decades, a number of research studies were conducted to understand the mechanisms of skin penetration enhancers and to develop a structure enhancement relationship. Such understanding allows effective prediction of the effects of skin penetration enhancers, assists topical and transdermal formulation development, and avoids extensive enhancer screening in the transdermal delivery industry. In the past two decades, several hypotheses on chemical enhancer-induced penetration enhancement for transport across the skin lipoidal pathway have been examined based on a systematic approach. Particularly, a hypothesis that skin penetration enhancement is directly related to the concentration of the enhancers in the stratum corneum lipid domain was examined. A direct relationship between skin penetration enhancer potency (based on enhancer aqueous concentration in the diffusion cell chamber) and enhancer n-octanol-water partition coefficient was also established. The nature of the microenvironment of the enhancer site of action in the stratum corneum lipid domain was found to be mimicked by n-octanol. The present paper reviews the work related to these hypotheses and the relationships between skin penetration enhancement and enhancer concentration in the drug delivery media and stratum corneum lipids.
doi:10.3390/pharmaceutics4010071
PMCID: PMC3834896  PMID: 24300181
skin penetration enhancers; transdermal drug delivery; n-octanol-water partition coefficient; penetration enhancement; skin
4.  Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier 
PLoS ONE  2016;11(1):e0146514.
In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers.
doi:10.1371/journal.pone.0146514
PMCID: PMC4709185  PMID: 26751684
5.  In Vitro Cutaneous Application of ISCOMs on Human Skin Enhances Delivery of Hydrophobic Model Compounds Through the Stratum Corneum 
The AAPS Journal  2009;11(4):728-739.
This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOMs) on human skin penetration of model compounds in vitro to evaluate their potential as a delivery system, ultimately for transcutaneous vaccination. Special focus was on elucidating the mechanisms of penetration. Preparation of ISCOMs was done by dialysis and subsequent purification in a sucrose density gradient. The penetration pathways of acridine-labeled ISCOMs were visualized using confocal laser scanning microscopy (CLSM). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes in the skin after application of the ISCOMs with or without hydration. Transcutaneous permeation of the model compound, methyl nicotinate, was evaluated in diffusion cells. The prepared ISCOMs were 42–52 nm in diameter as evaluated by dynamic light scattering with zeta potentials of −33 to −26.1 mV. TEM investigations verified the presence of ISCOM structures. Penetration of acridine into skin was greatly increased by incorporation into ISCOMs as visualized by CLSM. Permeation of methyl nicotinate was enhanced in the presence of ISCOMs. Ultrastructural changes of the intercellular space in the stratum corneum after exposure of ISCOMs were observed on micrographs, especially for hydrated skin. In conclusion, cutaneous application of ISCOMs leads to increased penetration of hydrophobic model compounds through human stratum corneum and thus shows potential as a transcutaneous delivery system. The increased penetration seems to be reflected by a change in the intercellular space between the corneocytes, and the effect is most likely caused by the components of the ISCOMs rather than intact ISCOMs.
doi:10.1208/s12248-009-9149-5
PMCID: PMC2782083  PMID: 19862629
CLSM; penetration; Posintro™; TEM; vaccine
6.  Terpenes: Effect of lipophilicity in enhancing transdermal delivery of alfuzosin hydrochloride 
Transdermal drug delivery has attracted much attention as an alternative to intravenous and oral methods of delivery. But the main barrier is stratum corneum. Terpenes classes of chemical enhancers are used in transdermal formulations for facilitating penetration of drugs. The aim of the study is to evaluate terpenes as skin penetration enhancers and correlate its relationship with permeation and lipophilicity. In this study, alfuzosin hydrochloride (AH) hydrogels were prepared with terpenes using Taguchi orthogonal array experimental design. The formulations contained one of eight terpenes, based on their lipophilicity (log P 2.13-5.36). The percutaneous permeation was studied in rat skin using diffusion cell technique. Flux, cumulative amount, lag time and skin content of AH were measured over 24 hours and compared with control gels. Nerolidol with highest lipophilicity (log P 5.36 ± 0.38) showed highest cumulative amount (Q24) of 647.29 ± 18.76 μg/cm2 and fluxrateof 28.16 ± 0.64 μg/cm2/hour. It showed decreased lag time of 0.76 ± 0.15 hours. Fenchone (2.5%) (log P 2.13 ± 0.30) produced the longest lag time 4.8 ± 0.20 hours. The rank order of enhancement effect was shown as nerolidol > farnesol > limonene > linalool > geraniol > carvone > fenchone > menthol. Lowest skin content was seen with carvone. Increase in lipophilicity of terpenes showed increase in flux, cumulative amount (Q24), and enhancement ratio which was significant with P < 0.000. But lag time was decreased and no correlation was found between lipophilicity and skin content. Histological studies showed changes in dermis which can be attributed to disruption of lipid packing of stratum corneum due to effect of nerolidol within lipid lamellae. It was found that small alcoholic terpenes with high degree of unsaturation enhance permeation of hydrophilic drugs, liquid terpenes enhance better than solid terpenes and terpenes with high lipophilicity are good penetration enhancers.
doi:10.4103/2231-4040.104712
PMCID: PMC3560127  PMID: 23378942
Alfuzosin hydrochloride; lipophilicity; taguchi robust design method; terpenes; transdermal permeation
7.  Water-filtered infrared-A (wIRA) can act as a penetration enhancer for topically applied substances 
Background: Water-filtered infrared-A (wIRA) irradiation has been shown to enhance penetration of clinically used topically applied substances in humans through investigation of functional effects of penetrated substances like vasoconstriction by cortisone.
Aim of the study: Investigation of the influence of wIRA irradiation on the dermatopharmacokinetics of topically applied substances by use of optical methods, especially to localize penetrating substances, in a prospective randomised controlled study in humans.
Methods: The penetration profiles of the hydrophilic dye fluorescein and the lipophilic dye curcumin in separate standard water-in-oil emulsions were determined on the inner forearm of test persons by tape stripping in combination with spectroscopic measurements. Additionally, the penetration was investigated in vivo by laser scanning microscopy. Transepidermal water loss, hydration of the epidermis, and surface temperature were determined. Three different procedures (modes A, B, C) were used in a randomised order on three separate days of investigation in each of 12 test persons. In mode A, the two dyes were applied on different skin areas without water-filtered infrared-A (wIRA) irradiation. In mode B, the skin surface was irradiated with wIRA over 30 min before application of the two dyes (Hydrosun® radiator type 501, 10 mm water cuvette, orange filter OG590, water-filtered spectrum: 590–1400 nm with dominant amount of wIRA). In mode C, the two dyes were applied and immediately afterwards the skin was irradiated with wIRA over 30 min. In all modes, tape stripping started 30 min after application of the formulations. Main variable of interest was the ratio of the amount of the dye in the deeper (second) 10% of the stratum corneum to the amount of the dye in the upper 10% of the stratum corneum.
Results: The penetration profiles of the hydrophilic fluorescein showed in case of pretreatment or treatment with wIRA (modes B and C) an increased penetration depth compared to the non-irradiated skin (mode A): The ratio of the amount of the dye in the deeper (second) 10% of the stratum corneum to the amount of the dye in the upper 10% of the stratum corneum showed medians and interquartile ranges for mode A of 0.017 (0.007/0.050), for mode B of 0.084 (0.021/0.106), for mode C of 0.104 (0.069/0.192) (difference between modes: p=0.0112, significant; comparison mode A with mode C: p<0.01, significant). In contrast to fluorescein, the lipophilic curcumin showed no differences in the penetration kinetics, in reference to whether the skin was irradiated with wIRA or not. These effects were confirmed by laser scanning microscopy. Water-filtered infrared-A irradiation increased the hydration of the stratum corneum: transepidermal water loss rose from approximately 8.8 g m-2 h-1 before wIRA irradiation to 14.2 g m-2 h-1 after wIRA irradiation and skin hydration rose from 67 to 87 relative units. Skin surface temperature increased from 32.8°C before wIRA to 36.4°C after wIRA irradiation.
Discussion: The better penetration of the hydrophilic dye fluorescein after or during skin irradiation (modes B and C) can be explained by increased hydration of the stratum corneum by irradiation with wIRA.
Conclusions: As most topically applied substances for the treatment of patients are mainly hydrophilic, wIRA can be used to improve the penetration of substances before or after application of substances – in the first case even of thermolabile substances – with a broad clinical relevance as a contact free alternative to an occlusive dressing.
PMCID: PMC2703260  PMID: 19675735
water-filtered infrared-A (wIRA); penetration; stratum corneum; skin barrier; penetration enhancer; dermatopharmacokinetics; dye; fluorescein; curcumin; tape stripping method; spectroscopy; laser scanning microscopy; transepidermal water loss (TEWL); hydration of the epidermis; corneometry; skin surface temperature; hydrophilic; lipophilic; occlusive dressing
8.  Enhanced dermal delivery of diflucortolone valerate using lecithin/chitosan nanoparticles: in-vitro and in-vivo evaluations 
The objective of this study was to prepare a suitable formulation for dermal delivery of diflucortolone valerate (DFV) that would maintain the localization in skin layers without any penetration and to optimize efficiency of DFV. Drug-loaded lecithin/chitosan nanoparticles with high entrapment efficiency (86.8%), were successfully prepared by ionic interaction technique. Sustained release of DFV was achieved without any initial burst release. Nanoparticles were also incorporated into chitosan gel at different ratios for preparing a more suitable formulation for topical drug delivery with adequate viscosity. In ex-vivo permeation studies, nanoparticles increased the accumulation of DFV especially in the stratum corneum + epidermis of rat skin without any significant permeation. Retention of DFV from nanoparticle in chitosan gel formulation (0.01%) was twofold higher than commercial cream, although it contained ten times less DFV. Nanoparticles in gel formulations produced significantly higher edema inhibition in rats compared with commercial cream in in-vivo studies. Skin blanching assay using a chromameter showed vasoconstriction similar to that of the commercial product. There were no barrier function changes upon application of nanoparticles. In-vitro and in-vivo results demonstrated that lecithin/chitosan nanoparticles in chitosan gel may be a promising carrier for dermal delivery of DFV in various skin disorders.
doi:10.2147/IJN.S40519
PMCID: PMC3564463  PMID: 23390364
skin permeation; anti-inflammatory activity; skin blanching; TEWL
9.  Quantitative Chemical Imaging with Multiplex Stimulated Raman Scattering Microscopy 
Stimulated Raman scattering (SRS) microscopy is a newly developed label-free chemical imaging technique that overcomes the speed limitation of confocal Raman while avoiding the nonresonant-background problem of coherent anti-Stokes Raman scattering (CARS) microscopy. Previous demonstrations were limited to single Raman band measurement. We present a novel modulation multiplexing approach that allows real-time detection of multiple species using the fast Fourier-transform. We demonstrate quantitative determination of chemical concentration of a ternary mixture. Furthermore, two imaging applications are pursued: (1) quantitative determination of oil content, as well as pigment and protein concentration in microalgae cultures; (2) 3D high resolution imaging of blood, lipids, and protein distribution in ex vivo mouse skin tissue. We believe quantitative multiplex SRS uniquely combines the advantage of fast label-free imaging with the fingerprinting capability of Raman spectroscopy and enables numerous applications lipid biology as well as biomedical imaging.
doi:10.1021/ja210081h
PMCID: PMC3396204  PMID: 22316340
10.  A Time-Encoded Technique for fibre-based hyperspectral broadband stimulated Raman microscopy 
Nature Communications  2015;6:6784.
Raman sensing and microscopy are among the most specific optical technologies to identify the chemical compounds of unknown samples, and to enable label-free biomedical imaging. Here we present a method for stimulated Raman scattering spectroscopy and imaging with a time-encoded (TICO) Raman concept. We use continuous wave, rapidly wavelength-swept probe lasers and combine them with a short-duty-cycle actively modulated pump laser. Hence, we achieve high stimulated Raman gain signal levels, while still benefitting from the narrow linewidth and low noise of continuous wave operation. Our all-fibre TICO-Raman setup uses a Fourier domain mode-locked laser source to achieve a unique combination of high speed, broad spectral coverage (750–3,150 cm−1) and high resolution (0.5 cm−1). The Raman information is directly encoded and acquired in time. We demonstrate quantitative chemical analysis of a solvent mixture and hyperspectral Raman microscopy with molecular contrast of plant cells.
Raman microscopes suffer from the compromise between speed and spectral information and are often unsuited for fibre beam delivery. Karpf et al. overcome these limitations using continuous-wave rapidly wavelength-swept probe lasers and a short-duty-cycle actively modulated pump laser in an all-fibre setup.
doi:10.1038/ncomms7784
PMCID: PMC4410670  PMID: 25881792
11.  Facilitation of transcutaneous drug delivery and vaccine immunization by a safe laser technology 
Journal of Controlled Release  2012;159(1):43-51.
Full-surface laser ablation has been shown to efficiently disrupt stratum corneum and facilitate transcutaneous drug delivery, but it is frequently associated with skin damage that hampers its clinic use. We show here that a safer ablative fractional laser (AFL) can sufficiently deliver not only patch-coated hydrophilic drugs but also protein vaccines. AFL treatment generated an array of self renewable microchannels (MCs) in the skin surface, providing free paths for drug and vaccine delivery into the dermis while sustains integrity of the skin by quick healing of the MCs. AFL was superior to tape stripping in transcutaneous drug and vaccine delivery as a much higher amount of sulforhodamine B (SRB), methylene blue (MB) or a model vaccine ovalbumin (OVA) was recovered from AFL-treated skin than tape stripping-treated skin or control skin after patch application. Following entry into the MCs, the drugs or OVA diffused quickly to the entire dermal tissue via the lateral surface of conical-shaped MCs. In contrast, a majority of the drugs and OVA remained on the skin surface, unable to penetrate into the dermal tissue in untreated control skin or tape stripping-treated skin. Strikingly, OVA delivered through the MCs was efficiently taken up by epidermal Langerhans cells and dermal dendritic cells in the vicinity of the MCs or transported to the draining lymph nodes, leading to a robust immune response, in sharp contrast to a weak, though significant, immune response elicited in tape stripping group or a basal immune response in control groups. These data support strongly that AFL is safe and sufficient for transcutaneous delivery of drugs and vaccines.
doi:10.1016/j.jconrel.2012.01.002
PMCID: PMC3322279  PMID: 22261281
laser; transdermal patch; vaccine; drug; transcutaneous delivery; tape stripping
12.  Transdermal delivery enhanced by magainin pore-forming peptide 
In this study we tested the hypothesis that magainin, a peptide known to form pores in bacterial cell membranes, can increase skin permeability by disrupting stratum corneum lipid structure. We further hypothesized that magainin's enhancement requires co-administration with a surfactant chemical enhancer to increase magainin penetration into the skin. In support of these hypotheses, exposure to a known surfactant chemical enhancer, N-lauroyl sarcosine (NLS), in 50% ethanol solution increased in vitro skin permeability to fluorescein 15 fold and the combination of magainin and NLS-ethanol synergistically increased skin permeability 47 fold. In contrast, skin permeability was unaffected by exposure to magainin without co-enhancement by NLS-ethanol. Furthermore, confocal microscopy showed that magainin in the presence of NLS-ethanol penetrated deeply and extensively into stratum corneum, whereas magainin alone penetrated poorly into the skin. Additional analysis by Fourier-transform infrared spectroscopy, X-ray diffraction, and differential scanning calorimetry showed that NLS-ethanol disrupted stratum corneum lipid structure and that the combination of magainin and NLS-ethanol disrupted stratum corneum lipids even further. Altogether, these data suggest that NLS-ethanol increased magainin penetration into stratum corneum, which further increased stratum corneum lipid disruption and skin permeability. We believe this is the first study to demonstrate the use of a pore-forming peptide to increase skin permeability. This study also introduces the novel concept of using a first chemical enhancer to increase penetration of a second chemical enhancer into the skin to synergistically increase skin permeability to a model drug.
doi:10.1016/j.jconrel.2007.05.031
PMCID: PMC2035950  PMID: 17628164
Antimicrobial pore-forming peptide; Magainin; N-lauroylsarcosine; Stratum corneum; Surfactant chemical enhancer; Transdermal drug delivery.
13.  Nanoethosomes for Dermal Delivery of Lidocaine 
Advanced Pharmaceutical Bulletin  2015;5(4):549-556.
Purpose: It is necessary for local anesthetics to pass through the stratum corneum to provide rapid pain relief. Many techniques have been reported to enhance intradermal penetration of local anesthetics such as vesicular lipid carriers. Ethosomes are lipid vesicles containing phospholipids, ethanol at relatively high concentration. We hypothesized that synergistic effects of phospholipids and high concentration of ethanol in formulation could accelerate penetration of nanoethosomes in deep layers of skin.
Methods: Lidocaine-loaded nanoethosomes were prepared and characterized by size and zeta analyzer, scanning electron microscopy (SEM) and X-ray diffractometer (XRD). Furthermore, encapsulation efficiency (EE), loading capacity (LC), and skin penetration capability were evaluated by in vitro and in vivo experiments.
Results: results showed that the particle size, zeta potential, EE and LC of optimum formulation were 105.4 ± 7.9 nm, -33.6 ± 2.4 mV, 40.14 ± 2.5 %, and 8.02 ± 0.71 respectively. SEM results confirmed the non-aggregated nano-scale size of prepared nanoethosomes. Particle size of ethosomes and EE of Lidocaine were depended on the phospholipid and ethanol concentrations. XRD results demonstrated the drug encapsulation in amorphous status interpreting the achieved high drug EE and LC values. In vitro and in vivo assays confirmed the appropriate skin penetration of Lidocaine with the aid of nanoethosomes and existence of deposition of nanoethosomes in deep skin layers, respectively.
Conclusion: The developed nanoethosomes are proposed as a suitable carrier for topical delivery of anesthetics such as Lidocaine.
doi:10.15171/apb.2015.074
PMCID: PMC4729340  PMID: 26819928
Local anesthetics; Dermal Drug delivery; Ethosome; Lidocaine; Nanoparticle
14.  Video-Rate Molecular Imaging In Vivo with Stimulated Raman Scattering 
Science (New York, N.Y.)  2010;330(6009):1368-1370.
Optical imaging in vivo with molecular specificity is important in biomedicine because of its high spatial resolution and sensitivity compared to MRI. Stimulated Raman scattering (SRS) microscopy allows highly sensitive optical imaging based on vibrational spectroscopy without adding toxic or perturbative labels. However, SRS tissue imaging in living animals and humans has not been feasible because of weak signals from thick tissues and motion blur due to limited acquisition speed. Here we make in vivo SRS imaging possible by significantly enhancing the collection of the backscattered signal and by increasing the imaging speed by three orders of magnitude, to video rate. This allows label-free in vivo imaging of water, lipid and protein in skin and mapping of penetration pathways of topically-applied drugs in mice and humans.
doi:10.1126/science.1197236
PMCID: PMC3462359  PMID: 21127249
15.  A Comprehensive Review of Clocortolone Pivalate 0.1% Cream 
Clocortolone pivalate is a mid-potency topical corticosteroid available as a 0.1% emollient cream approved by the United States Food and Drug Aministration for use in the treatment of corticosteroid-responsive dermatoses. The vehicle is formulated for application to a variety of corticosteroid-responsive skin disorders, including those with inflamed and fissured skin, such as eczematous dermatoses. Hence, the potency of the formulation and its vehicle characteristics are important when treating disorders, such as atopic dermatitis and other eczematous dermatoses, which are prone to cutaneous irritation and skin sensitivity to exogenously applied agents. As both localized and diffuse eczematous dermatoses and seborrheic dermatitis are common in pediatric patients (including infants) as well as in adults, the fact that clocortolone pivalate 0.1% cream has no age restriction related to its use according to United States Food and Drug Aministration-approved product labeling is important to recognize. The chemical structure of clocortolone pivalate is a unique design that provides high lipid solubility. Highly lipophilic topical corticosteroids exhibit augmented penetration through the stratum corneum, which provides higher epidermal concentrations. It has been reported that the structural characteristics of this molecule enhance its potency without increasing the potential for topical corticosteroid-related adverse effects. Clocortolone pivalate 0.1% cream has been studied in randomized, controlled trials of patients with atopic dermatitis and other eczematous dermatoses, psoriasis vulgaris, contact dermatitis, and seborrheic dermatitis. It has been shown to be more effective as monotherapy in the treatment of these corticosteroid-responsive dermatoses than the vehicle. Its efficacy and safety in pediatric patients and patients with facial dermatoses have also been demonstrated. Patients using clocortolone pivalate 0.1% topical cream in clinical trials had a low rate of adverse events, which were primarily minor application-site reactions. Systemic reactions related to the drug were not observed in these trials. Clinical studies of patients with corticosteroid-responsive dermatological conditions have found that clocortolone pivalate 0.1% cream is an effective class 4 topical corticosteroid with a favorable safety profile.
PMCID: PMC3396454  PMID: 22798972
16.  Raman characterization and chemical imaging of biocolloidal self-assemblies, drug delivery systems, and pulmonary inhalation aerosols: A review 
AAPS PharmSciTech  2007;8(4):140-155.
This review presents an introduction to Raman scattering and describes the various Raman spectroscopy, Raman microscopy, and chemical imaging techniques that have demonstrated utility in biocolloidal self-assemblies, pharmaceutical drug delivery systems, and pulmonary research applications. Recent Raman applications to pharmaceutical aerosols in the context of pulmonary inhalation aerosol delivery are discussed. The “molecular fingerprint” insight that Raman applications provide includes molecular structure, drug-carrier/excipient interactions, intramolecular and intermolecular bonding, surface structure, surface and interfacial interactions, and the functional groups involved therein. The molecular, surface, and interfacial properties that Raman characterization can provide are particularly important in respirable pharmaceutical powders, as these particles possess a higher surface-area-to-volume ratio; hence, understanding the nature of these solid surfaces can enable their manipulation and tailoring for functionality at the nanometer level for targeted pulmonary delivery and deposition. Moreover, Raman mapping of aerosols at the micro- and nanometer level of resolution is achievable with new, sophisticated, commercially available Raman microspectroscopy techniques. This noninvasive, highly versatile analytical and imaging technique exhibits vast potential for in vitro and in vivo molecular investigations of pulmonary aerosol delivery, lung deposition, and pulmonary cellular drug uptake and disposition in unfixed living pulmonary cells.
doi:10.1208/pt0804099
PMCID: PMC2750685
Spectroscopy; microscopy; imaging; molecular; interfacial; lung; mapping
17.  The role of hair follicles in the percutaneous absorption of caffeine 
Aims
The skin and its appendages are our protective shield against the environment and are necessary for the maintenance of homeostasis. Hypotheses concerning the penetration of substances into the skin have assumed diffusion through the lipid domains of the stratum corneum. It is believed that while hair follicles represent a weakness in the shield, they play a subordinate role in the percutaneous penetration processes. Previous investigation of follicular penetration has mostly addressed methodical and technical problems. Our study utilized a selective closure technique of hair follicle orifices in vivo, for the comparison of interfollicular and follicular absorption rates of caffeine in humans.
Methods
Every single hair follicle within a delimited area of skin was blocked with a microdrop of a special varnish-wax-mixture in vivo. Caffeine in solution was topically applied and transcutaneous absorption into the blood was measured by a new surface ionization mass spectrometry (SI/MS) technique, which enabled a clear distinction to be made between interfollicular and follicular penetration of a topically applied substance.
Results
Caffeine (3.75 ng ml−1) was detected in blood samples, 5 min after topical application, when the follicles remained open. When the follicles were blocked, caffeine was detectable after 20 min (2.45 ng ml−1). Highest values (11.75 ng caffeine ml−1) were found 1 h after application when the follicles were open.
Conclusions
Our findings demonstrate that hair follicles are considerable weak spots in our protective sheath against certain hydrophilic drugs and may allow a fast delivery of topically applied substances.
What is already known about this subject In recent years, it has been suggested that hair follicles represent important shunt routes into the skin for drugs and chemicals [1–3].In vitro studies have shown the importance of skin appendages for skin penetration by hydrophilic compounds [4]. Investigation of follicular penetration in vivo has been difficult due to the absence of appropriate analytical methods or suitable animal model systems.Recently, a new method was described that quantifies follicular penetration in vivo by using selective closure of hair follicles [5].Caffeine is frequently used in skin penetration experiments as a model for highly water-soluble compounds. Occlusion [6] and skin thickness [7] seem to have little influence on the penetration of caffeine. However, percutaneous absorption rates for caffeine exhibit regional skin differences in humans in vivo[1].
What this study adds The results of the present study demonstrate that a fast drug delivery of caffeine occurs through shunt routes. Therefore, hair follicles are considerable weak spots in our protective sheath against penetration into the body by hydrophilic substances.We showed that there is a quantitative distinction between follicular penetration and interfollicular diffusion of caffeine in vivo.These findings are of importance for the development and optimization of topically applied drugs and cosmetics. In addition, such properties must be considered in the development of skin protection measures.
doi:10.1111/j.1365-2125.2007.03065.x
PMCID: PMC2291387  PMID: 18070215
caffeine; follicular penetration; hair follicle; penetration pathways
18.  Investigation of the mechanism of enhanced skin penetration by ultradeformable liposomes 
This study aimed to determine the mechanism by which ultradeformable liposomes (ULs) with terpenes enhance skin penetration for transdermal drug delivery of fluorescein sodium, using transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Skin treated with ULs containing d-limonene, obtained from in vitro skin penetration studies, was examined via TEM to investigate the effect of ULs on ultrastructural changes of the skin, and to evaluate the mechanism by which ULs enhance skin penetration. The receiver medium collected was analyzed by TEM and CLSM to evaluate the mechanism of the drug carrier system. Our findings revealed that ULs could enhance penetration by denaturing intracellular keratin, degrading corneodesmosomes, and disrupting the intercellular lipid arrangement in the stratum corneum. As inferred from the presence of intact vesicles in the receiver medium, ULs are also able to act as a drug carrier system. CLSM images showed that intact vesicles of ULs might penetrate the skin via a transappendageal pathway, potentially a major route of skin penetration.
doi:10.2147/IJN.S65287
PMCID: PMC4122424  PMID: 25114524
ultradeformable liposomes; mechanism of enhanced skin penetration; transmission electron microscopy; confocal laser scanning microscopy
19.  Therapeutic and cosmeceutical potential of ethosomes: An overview 
The main disadvantage of transdermal drug delivery is the poor penetration of most compounds into the human skin. The main barrier of the skin is located within its uppermost layer, the stratum corneum (SC). Several approaches have been developed to weaken this skin barrier. One of the approaches for increasing the skin penetration of drugs and many cosmetic chemicals is the use of vesicular systems, such as, liposomes and ethosomes. Ethosomes are phospholipid-based elastic nanovesicles containing a high content of ethanol (20–45%). Ethanol is known as an efficient permeation enhancer and has been added in the vesicular systems to prepare elastic nanovesicles. It can interact with the polar head group region of the lipid molecules, resulting in the reduction of the melting point of the stratum corneum lipid, thereby increasing lipid fluidity and cell membrane permeability. The high flexibility of vesicular membranes from the added ethanol permits the elastic vesicles to squeeze themselves through the pores, which are much smaller than their diameters. Ethosomal systems are much more efficient in delivering substances to the skin in the terms of quantity and depth, than either conventional liposomes or hydroalcoholic solutions. The scope of this small review is to introduce the novel concept of ethosomes and to describe some approaches and mechanisms of stimulating topical and transdermal products with ethosomes.
doi:10.4103/0110-5558.72415
PMCID: PMC3255417  PMID: 22247858
Ethosomes; liposomes; novel drug delivery; penetration enhancer; Percutaneous absorption
20.  Novel multi-source phase-controlled radiofrequency technology for non-ablative and micro-ablative treatment of wrinkles, lax skin and acne scars 
Laser Therapy  2011;20(2):139-144.
The basic properties of lasers and pulsed light sources limit their ability to deliver high energy to the dermis and subcutaneous tissues without excessive damage to the epidermis. Radiofrequency was shown to penetrate deeper than optical light sources independent of skin color. The early RF-based devices used single source bipolar RF, which is safe but limited in use due to the superficial flow of energy between the two bipolar electrodes. Another type of single source RF employs a single electrode (monopolar) in which the RF energy flows from one electrode on the surface of the skin through the entire body to a plate under the body. Although more effective than bipolar, this devices require intense active cooling of the skin and may be associated with considerable pain and other systemic and local safety concerns. Latest generation of RF technology developed by EndyMed Medical Ltd. (Caesarea, Israel) utilizes simultaneously six or more phase controlled RF generators (3DEEP technology). The multiple electrical fields created by the multiple sources “repel” or “attract” each other, leading to the precise 3 dimensional delivery of RF energy to the dermal and sub-dermal targets minimizing the energy flow through the epidermis without the need for active cooling. Confocal microscopy of the skin has shown that 6 treatment sessions of Multisource RF technology improve skin structure features. The skin after treatment had longer and narrower dermal papilla and denser and finer collagen fiber typical to younger skin as compared to pre treatment skin. Ultrasound of the skin showed after 6 treatment sessions reduction of 10 percent in the thickness of the subcutaneous fat layer. Non ablative facial clinical studies showed a significant reduction of wrinkles after treatment further reduced at 3 months follow-up. Body treatment studies showed a circumference reduction of 2.9 cm immediately after 6 treatments, and 2 cm at 12 months after the end of treatment, proving long term collagen remodeling effect. Clinical studies of the multisource fractional RF application have shown significant effects on wrinkles reduction and deep atrophic acne scars after 1–3 treatment sessions.
doi:10.5978/islsm.20.139
PMCID: PMC3799020  PMID: 24155523
21.  Use of lipolanthionine peptide, a toll-like receptor 2 inhibitor, enhances transdermal delivery efficiency 
Molecular Medicine Reports  2014;10(2):593-598.
The transdermal delivery system (TDS) is able to obtain a systemic therapeutic effect by administration through the skin, which has low side effects and is able to maintain a sustained blood concentration. However, due to the barrier presented by the stratum corneum, numerous drugs have poor percutaneous permeability. Therefore, the improvement of skin permeability is key to TDS. The main method of promoting transdermal absorption is through the usage of penetration enhancers. Dimethyl sulfoxide (DMSO) is a commonly used penetration enhancer, which has anti-inflammatory analgesic effects and is able to penetrate the skin. Retinoic acid (RA) and lipolanthionine peptide (LP) may also benefit the permeation efficiency of TDS. Therefore, the present study examined the function of DMSO, RA and LP as penetration enhancers in TDS. Firstly, the optimum concentration of DMSO was confirmed by detecting the expression of the LacZ gene in vitro. Secondly, different combinations of LP, RA and DMSO were applied to mouse skin to analyze the penetration enhancer combination with the greatest efficacy. All the animals were divided into five groups: The RA + LP + DMSO + pORF-LacZ group, the RA + DMSO + pORF-LacZ group, the LP + DMSO + pORF-LacZ group, the DMSO + pORF-LacZ group and the control group. Skin was soaked in combinations of LP, RA and DMSO for seven days and then the pORF-LacZ plasmids were daubed onto the skin once daily three days. On the 11th day, all the animals were sacrificed by cervical dislocation and the skin and blood samples were collected. The blood samples were used to detect the expression of the LacZ gene by quantitative polymerase chain reaction and the skin samples were used to detect the expression of claudin-4 and zonula occluden-1 (ZO-1) proteins by immunohistochemistry and western blot analysis. The results demonstrated that the combination of LP, RA and DMSO exhibited the greatest transdermal delivery efficiency, which verified that RA and LP were able to increase the penetration effects. Following treatment with LP, the symptoms of dermal edema were relieved and the capillaries contracted, which suggested that LP was a safe and effective penetration enhancer able to reduce the side-effects caused by DMSO. The present study provides a guideline for the synthesis of novel penetration enhancers.
doi:10.3892/mmr.2014.2251
PMCID: PMC4094769  PMID: 24858729
toll-like receptor 2; lipolanthionine peptide; retinoic acid; dimethyl sulfoxide; transdermal delivery system
22.  Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes 
Ultradeformable vesicles (UDV) have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE) after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by “transfersomal method”, for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by “ethosomal method”. Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE) > ethosomes (E) ≥ transfersomes (T). This result was consistent with the release and skin penetration profiles for Vitamin E-loaded UDV. However, the releasing results were totally the opposite for caffeine-loaded UDV, which might be explained by the solubility and thermodynamic activity of this active in each formulation instead of the UDV deformability attending to the higher non-incorporated fraction of caffeine. Anyway, a high skin penetration and permeation for all caffeine-loaded UDV were obtained. Transethosomes were more deformable than ethosomes and transfersomes due to the presence of both ethanol and surfactant in their composition. All these UDV were suitable for a deeper skin penetration, especially transethosomes.
doi:10.2147/IJN.S86186
PMCID: PMC4583114  PMID: 26425085
lipid vesicles; topical delivery studies; vitamin E; caffeine
23.  Effect of Penetration Enhancer Containing Vesicles on the Percutaneous Delivery of Quercetin through New Born Pig Skin 
Pharmaceutics  2011;3(3):497-509.
Quercetin (3,3′,4′,5,7-pentahydroxyflavone) exerts multiple pharmacological effects: anti-oxidant activity, induction of apoptosis, modulation of cell cycle, anti-mutagenesis, and anti-inflammatory effect. In topical formulations quercetin inhibits oxidative skin damage and the inflammatory processes induced by solar UV radiation. In this work, quercetin (2 mg/mL) was loaded in vesicular Penetration Enhancer containing Vesicles (PEVs), prepared using a mixture of lipids (Phospholipon® 50, P50) and one of four selected hydrophilic penetration enhancers: Transcutol® P, propylene glycol, polyethylene glycol 400, and Labrasol® at the same concentration (40% of water phase). Photon Correlation Spectroscopy results showed a mean diameter of drug loaded vesicles in the range 80–220 nm. All formulations showed a negative surface charge and incorporation efficiency in the range 48–75%. Transmission Electron Microscopy confirmed that size and morphology varied as a function of the used penetration enhancer. The influence of PEVs on ex vivo quercetin (trans)dermal delivery was evaluated using Franz-type diffusion cells, new born pig skin and Confocal Laser Scanning Microscopy. Results showed that drug delivery is affected by the penetration enhancer used in the PEVs' formulation.
doi:10.3390/pharmaceutics3030497
PMCID: PMC3857079  PMID: 24310593
liposome; quercetin; penetration enhancer; skin permeation; rheology; confocal laser scanning microscopy; bioflavonoid
24.  Solvent effects in permeation assessed in vivo by skin surface biopsy 
BMC Dermatology  2003;3:5.
Background
Transdermal drug delivery has become an important means of drug administration. It presents numerous advantages but it is still limited by the small number of drugs with a suitable profile. The use of solvents that affect the skin barrier function is one of the classic strategies of penetration enhancement. Some of these solvents have well characterised actions on the stratum corneum, but the majority are still selected using empirical criteria. The objective of this work was to conduct a systematic study on the ability to affect skin permeation of solvents commonly used in transdermal formulations. An innovative methodology in this area was employed, consisting of the combination of skin surface biopsy with colorimetry.
Methods
The study compared in vivo differences in the permeation of a hydrophilic (methylene blue) and a lipophilic (Sudan III) dye, after treatment of the skin with different vehicles. Consecutive skin surface biopsies of each site were taken and the cumulative amounts of the dyes in the stripped stratum corneum were measured by reflectance colourimetry.
Results
Results indicate that the amount of methylene blue present in the stratum corneum varied significantly with different skin pre-treatments. Some solvents provided a 1.5 fold penetration enhancement but others decreased by almost half the permeation of the dye. The permeation of Sudan III was less significantly affected by solvent pre-treatment.
Conclusions
This study has only superficially explored the potential of the combination of skin surface biopsy and colourimetry, but the encouraging results obtained confirm that the methodology can be extended to the study of more complex formulations.
doi:10.1186/1471-5945-3-5
PMCID: PMC317329  PMID: 14680512
25.  Fast Hyperspectral Imaging with Stimulated Raman Scattering by Chirped Femtosecond Lasers 
The journal of physical chemistry. B  2013;117(16):4634-4640.
Raman microscopy is a quantitative, label-free and noninvasive optical imaging technique for studying inhomogeneous systems. However, the feebleness of Raman scattering significantly limits the use of Raman microscopy to low time resolutions and primarily static samples. Recent developments in narrowband stimulated Raman Scattering (SRS) microscopy have significantly increased the sensitivity of Raman based label-free chemical imaging by a few orders of magnitude, at the expense of reduced spectroscopic information. Based on a spectral focusing approach, we present a fast SRS hyperspectral imaging system using chirped femtosecond lasers to achieve rapid Raman spectra acquisition while retaining the full speed and image quality of narrowband SRS imaging. We demonstrate that quantitative concentration determination of mixed chemical species can be achieved with sensitivity down to 4 mM. For imaging purposes, an entire Raman spectral data cube can be obtained within a minute. We show that mammalian cells SRS hyperspectral imaging reveals the spatially inhomogeneous distribution of saturated lipids, unsaturated lipids, cholesterol and protein. The combination of fast spectroscopy and label-free chemical imaging will enable new applications in studying biological systems and material systems.
doi:10.1021/jp308938t
PMCID: PMC3637845  PMID: 23256635
Raman spectroscopy; Stimulated Raman scattering microscopy; Raman spectroscopic imaging

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