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1.  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.
PMCID: PMC3576036  PMID: 19095943
2.  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.
PMCID: PMC3834896  PMID: 24300181
skin penetration enhancers; transdermal drug delivery; n-octanol-water partition coefficient; penetration enhancement; skin
3.  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.
PMCID: PMC3560127  PMID: 23378942
Alfuzosin hydrochloride; lipophilicity; taguchi robust design method; terpenes; transdermal permeation
4.  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.
PMCID: PMC2782083  PMID: 19862629
CLSM; penetration; Posintro™; TEM; vaccine
5.  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.
PMCID: PMC3564463  PMID: 23390364
skin permeation; anti-inflammatory activity; skin blanching; TEWL
6.  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
7.  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.
PMCID: PMC3396204  PMID: 22316340
8.  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.
PMCID: PMC3322279  PMID: 22261281
laser; transdermal patch; vaccine; drug; transcutaneous delivery; tape stripping
9.  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.
PMCID: PMC2750685
Spectroscopy; microscopy; imaging; molecular; interfacial; lung; mapping
10.  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.
PMCID: PMC2035950  PMID: 17628164
Antimicrobial pore-forming peptide; Magainin; N-lauroylsarcosine; Stratum corneum; Surfactant chemical enhancer; Transdermal drug delivery.
11.  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.
PMCID: PMC3462359  PMID: 21127249
12.  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
13.  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.
PMCID: PMC3255417  PMID: 22247858
Ethosomes; liposomes; novel drug delivery; penetration enhancer; Percutaneous absorption
14.  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.
PMCID: PMC4122424  PMID: 25114524
ultradeformable liposomes; mechanism of enhanced skin penetration; transmission electron microscopy; confocal laser scanning microscopy
15.  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.
PMCID: PMC3799020  PMID: 24155523
16.  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.
PMCID: PMC4094769  PMID: 24858729
toll-like receptor 2; lipolanthionine peptide; retinoic acid; dimethyl sulfoxide; transdermal delivery system
17.  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.
PMCID: PMC3857079  PMID: 24310593
liposome; quercetin; penetration enhancer; skin permeation; rheology; confocal laser scanning microscopy; bioflavonoid
18.  Solvent effects in permeation assessed in vivo by skin surface biopsy 
BMC Dermatology  2003;3:5.
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.
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 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.
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.
PMCID: PMC317329  PMID: 14680512
19.  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.
PMCID: PMC3637845  PMID: 23256635
Raman spectroscopy; Stimulated Raman scattering microscopy; Raman spectroscopic imaging
20.  In vivo coherent anti-Stokes Raman scattering imaging of sciatic nerve tissue 
Journal of microscopy  2007;225(Pt 2):175-182.
We report in vivo nonlinear optical imaging of mouse sciatic nerve tissue by epidetected coherent anti-Stokes Raman scattering and second harmonic generation microscopy. Following a minimally invasive surgery to open the skin, coherent anti-Stokes Raman scattering imaging of myelinated axons and second harmonic generation imaging of the surrounding collagen fibres were demonstrated with high signal-to-background ratio, three-dimensional spatial resolution, and no need for labelling. The underlying contrast mechanisms of in vivo coherent anti-Stokes Raman scattering were explored by three-dimensional imaging of fat cells that surround the nerve. The epidetected coherent anti-Stokes Raman scattering signals from the nerve tissues were found to arise from interfaces as well as back reflection of forward coherent anti-Stokes Raman scattering.
PMCID: PMC2679530  PMID: 17359252
Coherent anti-Stokes Raman scattering; in vivo imaging; myelin
21.  Effective transcutaneous immunization by antigen-loaded flexible liposome in vivo 
Transcutaneous vaccines have received wide attention due to their easy-to-use, needle-free, noninvasive delivery. However, the novel barrier function of stratum corneum hinders the transport of antigen and adjuvant in transcutaneous immunization. Novel nanoscale delivery systems employing, for example, liposomes and nanoparticles, have been widely investigated to overcome the penetration barrier of stratum corneum for effective transcutaneous immunization.
The objective of this study was to prepare two types of flexible liposomes and determine their efficacies for the transcutaneous delivery of antigen and the subsequent immune response induced in vivo.
Ovalbumin (OVA) liposome-based transcutaneous vaccines were prepared using reverse-phase evaporation and film-dispersion methods. Particle sizes and antigen encapsulating efficiency were then evaluated. After application to bare mouse skin, topical sites were examined for the presence of fluorescence-labeled liposome. The efficacy of the transcutaneously delivered OVA-loaded flexible liposome in activating the immune responses was investigated by detecting serum immunoglobulin G levels. The influence of an adjuvant, imiquimod, in the transcutaneous immunization was also tested.
Two flexible liposomes with well-encapsulated OVA were successfully prepared by film-dispersion or reverse-phase evaporation methods. The sizes of the prepared flexible liposomes ranged from 200 to 400 nm. In vivo, the fluorescence-labeled liposome was detected in hair-follicle ducts, indicating that the flexible liposome can penetrate the skin barrier through the hair follicles. Upon transcutaneous administration, the OVA-encapsulated flexible liposome elicited a strong immune response similar to that of positive control (ie, OVA solution administrated by subcutaneous injection with Al(OH)3 as an adjuvant). Co-administration of imiquimod with the OVA-loaded liposome expressed a significant enhancement on the transcutaneous immune responses.
Results of this study highlight the nanoscale formulation, flexible liposome, as a promising carrier for the transcutaneous delivery of antigen proteins. Imiquimod was shown to be an effective adjuvant as a transcutaneous immunization enhancer with the potential for transcutaneous vaccine development.
PMCID: PMC3252672  PMID: 22228992
flexible liposome; transcutaneous vaccine; immunization enhancement; adjuvant
22.  Polymeric nanoparticles-based topical delivery systems for the treatment of dermatological diseases 
Human skin not only functions as a permeation barrier (mainly due to the stratum corneum layer), but also provides a unique delivery pathway for therapeutic and other active agents. These compounds penetrate via intercellular, intracellular and transappendageal routes, resulting in topical delivery (into skin strata) and transdermal delivery (to subcutaneous tissues and into the systemic circulation). Passive and active permeation enhancement methods have been widely applied to increase the cutaneous penetration.
The pathology, pathogenesis and topical treatment approaches of dermatological diseases, such as psoriasis, contact dermatitis, and skin cancer, are then discussed. Recent literature has demonstrated that nanoparticles-based topical delivery systems can be successful in treating these skin conditions. The studies are reviewed starting with the nanoparticles based on natural polymers specially chitosan, followed by those made of synthetic, degradable (aliphatic polyesters) and non-degradable (polyarylates) polymers; emphasis is given to nanospheres made of polymers derived from naturally occurring metabolites, the tyrosine-derived nanospheres (TyroSpheres™).
In summary, the nanoparticles-based topical delivery systems combine the advantages of both the nano-sized drug carriers and the topical approach, and are promising for the treatment of skin diseases. For the perspectives, the penetration of ultra-small nanoparticles (size smaller than 40 nm) into skin strata, the targeted delivery of the encapsulated drugs to hair follicle stem cells, and the combination of nanoparticles and microneedle array technologies for special applications such as vaccine delivery are discussed.
PMCID: PMC3631287  PMID: 23386536
polymeric nanospheres; topical delivery; psoriasis; skin; tyrosine-derived nanospheres
23.  Hydrogel-forming Microneedle Arrays Exhibit Antimicrobial Properties: Potential for Enhanced Patient Safety 
We describe, for the first time, the microbial characterisation of hydrogel-forming polymeric microneedle arrays and the potential for passage of microorganisms into skin following microneedle penetration. Uniquely, we also present insights into the storage stability of these hydroscopic formulations, from physical and microbiological viewpoints, and examine clinical performance and safety in human volunteers. Experiments employing excised porcine skin and radiolabelled microorganisms showed that microorganisms can penetrate skin beyond the stratum corneum following microneedle puncture. Indeed, the numbers of microorganisms crossing the stratum corneum following microneedle puncture was greater than 105 cfu in each case. However, no microorganisms crossed the epidermal skin. When using a 21G hypodermic needle, more than 104 microorganisms penetrated into the viable tissue and 106 cfu of C. albicans and S. epidermidis completely crossed the epidermal skin in 24 h. The hydrogel-forming materials contained no microorganisms following de-moulding and exhibited no microbial growth during storage, while also maintaining their mechanical strength, apart from when stored at relative humidities of 86%. No microbial penetration through the swelling microneedles was detectable, while human volunteer studies confirmed that skin or systemic infection is highly unlikely when polymeric microneedles are used for transdermal drug delivery. Since no pharmacopoeial standards currently exist for microneedle-based products, the exact requirements for a proprietary product based on hydrogel-forming microneedles are at present unclear. However, we are currently working towards a comprehensive specification set for this microneedle system that may inform future developments in this regard.
PMCID: PMC4119957  PMID: 23644043
Microneedle; microorganism; stability; pharmacopoeial standards
24.  In Vivo Imaging of Human and Mouse Skin with a Handheld Dual-Axis Confocal Fluorescence Microscope 
The Journal of investigative dermatology  2010;131(5):10.1038/jid.2010.401.
Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.
PMCID: PMC3839658  PMID: 21191407
25.  Immunofluorescence Deconvolution Microscopy and Image Reconstruction of Human Defensins in Normal and Burned Skin 
Objective: The aim of this study was visualization and localization of the human antimicrobials human beta defensins 1, 2, and 3, neutrophil defensin alpha (human neutrophil peptide), and the cathelicidin LL-37 in normal and burned skin, and determination of the cell types in which these antimicrobials were localized. Methods: Tissue sections were probed with antimicrobial antibodies, tagged with fluorescently labeled secondary antibodies, and subjected to fluorescence deconvolution microscopy and image reconstruction. Images were generated by stacking multiple-section scans, which were then volume rendered by rotating stacks 360° about an axis, or modeled in 3 dimensions. Results: This technique yields a definitive image, providing a rapid basis for further quantification and manipulation from a full 3-dimensional aspect. In normal skin, human beta defensin-1 was localized to the perinuclear region of keratinocytes; human beta defensin-2 was primarily localized to the stratum germinativum; human beta defensin-3 was found in dendritic cells of the stratum spinosum; human neutrophil peptide was randomly distributed in the papillary dermis; and LL-37 was concentrated in the stratum corneum and along ducts. In burned skin, in which keratinocytes are lost or destroyed, human beta defensin-1 was present in dermal glandular structures including hair shafts; human beta defensin-2 and human beta defensin-3 were found in the remaining keratin layers and glands of the lower dermis; human neutrophil peptide was primarily localized to hair shafts, though visible in residual keratin layers; and LL-37 was evident in very high concentrations in the epithelium of sweat ducts. Conclusion: We conclude via this technique that cells in the lower dermal and subdermal regions of burned skin synthesize antimicrobials after burn injury, and maintain something of a barrier against infection. This methodology is discussed and explained in this article.
PMCID: PMC1501114  PMID: 16921412

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