Human Skin Drug Metabolism: Relationships between Methyl Salicylate Metabolism and Esterase Activities in IVPT Skin Membranes.

The presence of esterase enzymes in human skin and their role in drug metabolism has been reported, but their distribution in the various skin layers and the relative contributions of those layers to metabolism is poorly defined. To gain further insight into esterase distribution, we performed in vitro skin permeation of a commercial 28.3% methyl salicylate (MeSA) cream (Metsal™) in Franz diffusion cells, using a range of human skin membranes, all from the same donor. The membranes were viable epidermis separated by a dispase II enzymatic method, heat separated epidermis, dermatomed skin, and dermis separated by a dispase II enzymatic method. Methyl salicylate and its metabolite, salicylic acid (SA), were measured by high-performance liquid chromatography. Alpha naphthyl acetate and Hematoxylin and Eosin staining provided qualitative estimations of esterase distribution in these membranes. The permeation of methyl salicylate after 24 h was similar across all membranes. Salicylic acid formation and permeation were found to be similar in dermatomed skin and dermis, suggesting dermal esterase activity. These results were supported by the staining studies, which showed strong esterase activity in the dermal-epidermal junction region of the dermis. In contrast with high staining of esterase activity in the stratum corneum and viable epidermis, minimal stained and functional esterase activity was found in heat-separated and dispase II-prepared epidermal membranes. The results are consistent with dispase II digesting hemidesmosomes, penetrating the epidermis, and affecting epidermal esterases but not those in the dermis. Accordingly, whilst the resulting dispase II-generated dermal membranes may be used for in vitro permeation tests (IVPT) involving esterase-based metabolic studies, the dispase II-generated epidermal membranes are not suitable for this purpose.

Using in vivo multiphoton fluorescence lifetime imaging to unravel disease-specific changes in the liver redox state.

Multiphoton fluorescence lifetime microscopy has revolutionized studies of pathophysiological and xenobiotic dynamics, enabling the spatial and temporal quantification of these processes in intact organs in vivo. We have previously used multiphoton fluorescence lifetime microscopy to characterise the morphology and amplitude weighted mean fluorescence lifetime of the endogenous fluorescent metabolic cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) of mouse livers in vivo following induction of various disease states. Here, we extend the characterisation of liver disease models by using nonlinear regression to estimate the unbound, bound fluorescence lifetimes for NAD(P)H, flavin adenine dinucleotide (FAD), along with metabolic ratios and examine the impact of using multiple segmentation methods. We found that NAD(P)H amplitude ratio, and fluorescence lifetime redox ratio can be used as discriminators of diseased liver from normal liver. The redox ratio provided a sensitive measure of the changes in hepatic fibrosis and biliary fibrosis. Hepatocellular carcinoma was associated with an increase in spatial heterogeneity and redox ratio coupled with a decrease in mean fluorescence lifetime. We conclude that multiphoton fluorescence lifetime microscopy parameters and metabolic ratios provided insights into the in vivo redox state of diseased compared to normal liver that were not apparent from a global, mean fluorescence lifetime measurement alone.

Mechanistic Evaluation of Enhanced Curcumin Delivery through Human Skin In Vitro from Optimised Nanoemulsion Formulations Fabricated with Different Penetration Enhancers.

Curcumin is a natural product with chemopreventive and other properties that are potentially useful in treating skin diseases, including psoriasis and melanoma. However, because of the excellent barrier function of the stratum corneum and the relatively high lipophilicity of curcumin (log P 3.6), skin delivery of curcumin is challenging. We used the principles of a Quality by Design (QbD) approach to develop nanoemulsion formulations containing biocompatible components, including Labrasol and Lecithin as surfactants and Transcutol and ethanol as cosurfactants, to enhance the skin delivery of curcumin. The nanoemulsions were characterised by cryo-SEM, Zeta potential, droplet size, pH, electrical conductivity (EC) and viscosity (η). Physicochemical long-term stability (6 months) was also investigated. The mean droplet sizes as determined by dynamic light scattering (DLS) were in the lower submicron range (20-50 nm) and the average Zeta potential values were low (range: -0.12 to -2.98 mV). Newtonian flow was suggested for the nanoemulsions investigated, with dynamic viscosity of the nanoemulsion formulations ranging from 5.8 to 31 cP. The droplet size of curcumin loaded formulations remained largely constant over a 6-month storage period. The inclusion of terpenes to further enhance skin permeation was also examined. All nanoemulsions significantly enhanced the permeation of curcumin through heat-separated human epidermal membranes, with the greatest effect being a 28-fold increase in maximum flux (Jmax) achieved with a limonene-based nanoemulsion, compared to a 60% ethanol in water control vehicle. The increases in curcumin flux were associated with increased skin diffusivity. In summary, we demonstrated the effectiveness of nanoemulsions for the skin delivery of the lipophilic active compound curcumin, and elucidated the mechanism of permeation enhancement. These formulations show promise as delivery vehicles for curcumin to target psoriasis and skin cancer, and more broadly for other skin delivery applications.

Cellular metabolism and pore lifetime of human skin following microprojection array mediation.

Skin-targeting microscale medical devices are becoming popular for therapeutic delivery and diagnosis. We used cryo-SEM, fluorescence lifetime imaging microscopy (FLIM), autofluorescence imaging microscopy and inflammatory response to study the puncturing and recovery of human skin ex vivo and in vivo after discretised puncturing by a microneedle array (Nanopatch®). Pores induced by the microprojections were found to close by ~25% in diameter within the first 30 min, and almost completely close by ~6 h. FLIM images of ex vivo viable epidermis showed a stable fluorescence lifetime for unpatched areas of ~1000 ps up to 24 h. Only the cells in the immediate puncture zones (in direct contact with projections) showed a reduction in the observed fluorescence lifetimes to between ~518-583 ps. The ratio of free-bound NAD(P)H (α1/α2) in unaffected areas of the viable epidermis was ~2.5-3.0, whereas the ratio at puncture holes was almost double at ~4.2-4.6. An exploratory pilot in vivo study also suggested similar closure rate with histamine administration to the forearms of human volunteers after Nanopatch® treatment, although a prolonged inflammation was observed with Tissue Viability Imaging. Overall, this work shows that the pores created by the microneedle-type medical device, Nanopatch®, are transient, with the skin recovering rapidly within 1-2 days in the epidermis after application.

Optical Characterization of Zinc Pyrithione.

Zinc pyrithione is ubiquitous in commercial products particularly antidandruff shampoos. For the efficacy of zinc pyrithione therapeutic cleansers to be assessed accurately, the distribution of particles on the scalp needs to be visualized. Currently, no technique is available which provides the chemical specificity and sensitivity required. Here, we report application of fluorescence-lifetime imaging microscopy (FLIM) for high-contrast mapping of zinc pyrithione distribution on the scalp. Characterization of the zinc pyrithione emission by using both one-photon excitation at five specific wavelengths and two-photon excitation in the range of 740-820 nm revealed its FLIM fingerprint-a characteristic short average time-weighted emission lifetime of ΤZnPT = 250 ps. Bandpass-filtering FLIM signals at ΤZnPT enabled an efficient discrimination between the zinc pyrithione and major endogenous skin species in comparison with that of the conventional reflectance confocal microscopy. Our findings provide means for in vivo high-sensitivity assaying and high-contrast imaging of zinc pyrithione in biological systems.

Permeation Mechanism of Caffeine and Naproxen through in vitro Human Epidermis: Effect of Vehicles and Penetration Enhancers.

BACKGROUND/AIMS: The mechanisms by which permeation enhancers increase human skin permeation of caffeine and naproxen were assessed in vitro. METHODS: Active compound solubility in the vehicles and in the stratum corneum (SC), active compound flux across epidermal membranes and uptake of active and vehicle components into the SC were measured. The effect of vehicle pH on the permeation of caffeine and naproxen was also determined. RESULTS: Oleic acid and eucalyptol significantly enhanced the skin penetration of caffeine and naproxen, compared to aqueous controls. Naproxen permeation was increased from vehicles with pH presenting more ionized naproxen. Caffeine maximum flux enhancement was associated with an increase in caffeine SC solubility and skin diffusivity, whereas for naproxen a penetration enhancer/vehicle-induced increase in solubility in the SC correlated with an increase in maximum flux. SC solubility was related to experimentally determined active uptake, which was in turn predicted by vehicle uptake and active compound solubility in the vehicle. CONCLUSION: A permeation enhancer-induced alteration in diffusivity, rather than effects on SC solubility, was the main driving force behind increases in permeation flux of the hydrophilic molecule caffeine. For the more the lipophilic molecule naproxen, increased SC solubility drove the increases in permeation flux.

Support for the Safe Use of Zinc Oxide Nanoparticle Sunscreens: Lack of Skin Penetration or Cellular Toxicity after Repeated Application in Volunteers.

Zinc oxide is a widely used broad-spectrum sunscreen, but concerns have been raised about the safety of its nanoparticle (NP) form. We studied the safety of repeated application of agglomerated zinc oxide (ZnO) NPs applied to human volunteers over 5 days by assessing the skin penetration of intact ZnO-NPs and zinc ions and measuring local skin toxicity. Multiphoton tomography with fluorescence lifetime imaging microscopy was used to directly visualize ZnO-NP skin penetration and viable epidermal metabolic changes in human volunteers. The fate of ZnO-NPs was also characterized in excised human skin in vitro. ZnO-NPs accumulated on the skin surface and within the skin furrows but did not enter or cause cellular toxicity in the viable epidermis. Zinc ion concentrations in the viable epidermis of excised human skin were slightly elevated. In conclusion, repeated application of ZnO-NPs to the skin, as used in global sunscreen products, appears to be safe, with no evidence of ZnO-NP penetration into the viable epidermis nor toxicity in the underlying viable epidermis. It was associated with the release and penetration of zinc ions into the skin, but this did not appear to cause local toxicity.

Space- and time-resolved investigation on diffusion kinetics of human skin following macromolecule delivery by microneedle arrays.

Microscale medical devices are being developed for targeted skin delivery of vaccines and the extraction of biomarkers, with the potential to revolutionise healthcare in both developing and developed countries. The effective clinical development of these devices is dependent on understanding the macro-molecular diffusion properties of skin. We hypothesised that diffusion varied according to specific skin layers. Using three different molecular weights of rhodamine dextran (RD) (MW of 70, 500 and 2000 kDa) relevant to the vaccine and therapeutic scales, we deposited molecules to a range of depths (0-300 µm) in ex vivo human skin using the Nanopatch device. We observed significant dissipation of RD as diffusion with 70 and 500 kDa within the 30 min timeframe, which varied with MW and skin layer. Using multiphoton microscopy, image analysis and a Fick's law analysis with 2D cartesian and axisymmetric cylindrical coordinates, we reported experimental trends of epidermal and dermal diffusivity values ranging from 1-8 µm2 s-1 to 1-20 µm2 s-1 respectively, with a significant decrease in the dermal-epidermal junction of 0.7-3 µm2 s-1. In breaching the stratum corneum (SC) and dermal-epidermal junction barriers, we have demonstrated practical application, delivery and targeting of macromolecules to both epidermal and dermal antigen presenting cells, providing a sound knowledge base for future development of skin-targeting clinical technologies in humans.

Using elongated microparticles to enhance tailorable nanoemulsion delivery in excised human skin and volunteers.

This study demonstrates, for the first time, clinical testing of elongated silica microparticles (EMP) combined with tailorable nanoemulsions (TNE) to enhance topical delivery of hydrophobic drug surrogates. Likewise, this is the first report of 6-carboxyfluorescein (a model molecule for topically delivered hydrophobic drugs) AM1 & DAMP4 (novel short peptide surfactants) used in volunteers. The EMP penetrates through the epidermis and stop at the dermal-epidermal junction (DEJ). TNE are unusually stable and useful because the oil core allows high drug loading levels and the surface properties can be easily controlled. At first, we chose alginate as a crosslinking agent between EMP and TNE. We initially incorporated a fluorescent lipophilic dye, DiI, as a hydrophobic drug surrogate into TNE for visualization with microscopy. We compared four different coating approaches to combine EMP and TNE and tested these formulations in freshly excised human skin. The delivery profile characterisation was imaged by dye- free coherent anti-Stoke Raman scattering (CARS) microscopy to detect the core droplet of TNE that was packed with pharmaceutical grade lipid (glycerol) instead of DiI. These data show the EMP penetrating to the DEJ followed by controlled release of the TNE. Freeze-dried formulations with crosslinking resulted in a sustained release profile, whereas a freeze-dried formulation without crosslinking showed an immediate burst-type release profile. Finally, we tested the crosslinked TNE coated EMP formulation in volunteers using multiphoton microscopy (MPM) and fluorescence-lifetime imaging microscopy (FLIM) to document the penetration depth characteristics. These forms of microscopy have limitations in terms of image acquisition speed and imaging area coverage but can detect fluorescent drug delivery through the superficial skin in volunteers. 6-Carboxyfluorescein was selected as the fluorescent drug surrogate for the volunteer study based on the similarity of size, charge and hydrophobicity characteristics to small therapeutic drugs that are difficult to deliver through skin. The imaging data showed a 6-carboxyfluorescein signal deep in volunteer skin supporting the hypothesis that EMP can indeed enhance the delivery of TNE in human skin. There were no adverse events recorded at the time of the study or after the study, supporting the use of 6-carboxyfluorescein as a safe and detectable drug surrogate for topical drug research. In conclusion, dry formulations, with controllable release profiles can be obtained with TNE coated EMP that can effectively enhance hydrophobic payload delivery deep into the human epidermis.

Removing physiological motion from intravital and clinical functional imaging data.

Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene, a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that Galene is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, Galene can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data.

Using a simple equation to predict the microporation-enhanced transdermal drug flux.

The mathematical model describing drug flux through microporated skin was previously developed. Based on this model, two mathematical equations can be used to predict the microporatio-enhanced transdermal drug flux: the complex primal equation containing a variety of experimentally-determined variables, and the simplified straightforward equation. In this study, experimental transdermal fluxes of three corticosteroids through split-thickness human skin treated with a microneedle roller were measured, and the values of fluxes compared with those predicted using both the more complex and simplified equations. According to the results of the study, both equations demonstrated high accuracy in the prediction of the fluxes of corticosteroids. The simplified equation was validated and confirmed as robust using regression analysis of literature data. Further, its capability and ease of use was exemplified by predicting the flux of methotrexate through the skin microporated with laser and comparing with published experimental data.

A unique iridium(III) complex-based chemosensor for multi-signal detection and multi-channel imaging of hypochlorous acid in liver injury.

Although hypochlorous acid (HOCl) has long been associated with a number of inflammatory diseases in mammalian bodies, the functions of HOCl in specific organs at abnormal conditions, such as liver injury, remain unclear due to its high reactivity and the lack of effective methods for its detection. Herein, a unique Ir(III) complex-based chemosensor, Ir-Fc, was developed for highly sensitive and selective detection of HOCl. Ir-Fc was designed by incorporating a ferrocene (Fc) quencher to a Ir(III) complex through a HOCl-responsive linker. In the presence of HOCl, the fast cleavage of Fc moiety in less than 1s led to the enhancement of photoluminescence (PL) and electrochemical luminescence (ECL), by which the concentration of HOCl was determined by both PL and ECL analysis. Taking advantages of excellent properties of Ir(III) complexes, optical and electrochemical analyses of the response of Ir-Fc towards HOCl were fully investigated. Followed by the measurements of low cytotoxicity of Ir-Fc by MTT analysis, one-photon (OP), two-photon (TP) and lifetime imaging experiments were conducted to visualise the generation of HOCl in live microphage and HepG2 cells, and in zebrafish and mouse, respectively. Furthermore, the generation and distribution of HOCl in liver cells and liver injury of zebrafish and mouse were investigated. The results demonstrated the applicability of Ir-Fc as an effective chemosensor for imaging of HOCl generation in mitochondria of cells and liver injury in vivo, implying the potential of Ir-Fc for biomedical diagnosis and monitoring applications.

Skin models for the testing of transdermal drugs.

The assessment of percutaneous permeation of molecules is a key step in the evaluation of dermal or transdermal delivery systems. If the drugs are intended for delivery to humans, the most appropriate setting in which to do the assessment is the in vivo human. However, this may not be possible for ethical, practical, or economic reasons, particularly in the early phases of development. It is thus necessary to find alternative methods using accessible and reproducible surrogates for in vivo human skin. A range of models has been developed, including ex vivo human skin, usually obtained from cadavers or plastic surgery patients, ex vivo animal skin, and artificial or reconstructed skin models. Increasingly, largely driven by regulatory authorities and industry, there is a focus on developing standardized techniques and protocols. With this comes the need to demonstrate that the surrogate models produce results that correlate with those from in vivo human studies and that they can be used to show bioequivalence of different topical products. This review discusses the alternative skin models that have been developed as surrogates for normal and diseased skin and examines the concepts of using model systems for in vitro-in vivo correlation and the demonstration of bioequivalence.

Permeation of topically applied Magnesium ions through human skin is facilitated by hair follicles.

Magnesium is an important micronutrient essential for various biological processes and its deficiency has been linked to several inflammatory disorders in humans. Topical magnesium delivery is one of the oldest forms of therapy for skin diseases, for example Dead Sea therapy and Epsom salt baths. Some anecdotal evidence and a few published reports have attributed amelioration of inflammatory skin conditions to the topical application of magnesium. On the other hand, transport of magnesium ions across the protective barrier of skin, the stratum corneum, is contentious. Our primary aim in this study was to estimate the extent of magnesium ion permeation through human skin and the role of hair follicles in facilitating the permeation. Upon topical application of magnesium solution, we found that magnesium penetrates through human stratum corneum and it depends on concentration and time of exposure. We also found that hair follicles make a significant contribution to magnesium penetration.

Effect of flexing and massage on in vivo human skin penetration and toxicity of zinc oxide nanoparticles.

AIM: We assessed the effects of flexing and massage on human skin penetration and toxicity of topically applied coated and uncoated zinc oxide nanoparticles (˜75 nm) in vivo. MATERIALS & METHODS: Noninvasive multiphoton tomography with fluorescence lifetime imaging was used to evaluate the penetration of nanoparticles through the skin barrier and cellular apoptosis in the viable epidermis. RESULTS: All nanoparticles applied to skin with flexing and massage were retained in the stratum corneum or skin furrows. No significant penetration into the viable epidermis was seen and no cellular toxicity was detected. CONCLUSION: Exposure of normal in vivo human skin to these nanoparticles under common in-use conditions of flexing or massage is not associated with significant adverse events.

Synergistic Skin Penetration Enhancer and Nanoemulsion Formulations Promote the Human Epidermal Permeation of Caffeine and Naproxen.

We examined the extent of skin permeation enhancement of the hydrophilic drug caffeine and lipophilic drug naproxen applied in nanoemulsions incorporating skin penetration enhancers. Infinite doses of fully characterized oil-in-water nanoemulsions containing the skin penetration enhancers oleic acid or eucalyptol as oil phases and caffeine (3%) or naproxen (2%) were applied to human epidermal membranes in Franz diffusion cells, along with aqueous control solutions. Caffeine and naproxen fluxes were determined over 8 h. Solute solubility in the formulations and in the stratum corneum (SC), as well as the uptake of product components into the SC were measured. The nanoemulsions significantly enhanced the skin penetration of caffeine and naproxen, compared to aqueous control solutions. Caffeine maximum flux enhancement was associated with a synergistic increase in both caffeine SC solubility and skin diffusivity, whereas a formulation-increased solubility in the SC was the dominant determinant for increased naproxen fluxes. Enhancements in SC solubility were related to the uptake of the formulation excipients containing the active compounds into the SC. Enhanced skin penetration in these systems is largely driven by uptake of formulation excipients containing the active compounds into the SC with impacts on SC solubility and diffusivity.

Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging.

Conventional histology with light microscopy is essential in the diagnosis of most liver diseases. Recently, a concept of real-time histology with optical biopsy has been advocated. In this study, live mice livers (normal, with fibrosis, steatosis, hepatocellular carcinoma and ischemia-reperfusion injury) were imaged by MPM-FLIM for stain-free real-time histology. The acquired MPM-FLIM images were compared with conventional histological images. MPM-FLIM imaged subsurface cellular and subcellular histopathological hallmarks of live liver in mice models at high resolution. Additional information such as distribution of stellate cell associated autofluorescence and fluorescence lifetime changes was also gathered by MPM-FLIM simultaneously, which cannot be obtained from conventional histology. MPM-FLIM could simultaneously image and quantify the cellular morphology and microenvironment of live livers without conventional biopsy or fluorescent dyes. We anticipate that in the near future MPM-FLIM will be evaluated from bench to bedside, leading to real-time histology and dynamic monitoring of human liver diseases.

Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG.

Modulation of the immune response is an important step in the induction of protective humoral and cellular immunity against pathogens. In this study, we investigated the possibility of using a nanomaterial conjugated with the toll-like receptor (TLR) ligand CpG to modulate the immune response towards the preferred polarity. MgAl-layered double hydroxide (LDH) nanomaterial has a very similar chemical composition to Alum, an FDA approved adjuvant for human vaccination. We used a model antigen, ovalbumin (OVA) to demonstrate that MgAl-LDH had comparable adjuvant activity to Alum, but much weaker inflammation. Conjugation of TLR9 ligand CpG to LDH nanoparticles significantly enhanced the antibody response and promoted a switch from Th2 toward Th1 response, demonstrated by a change in the IgG2a:IgG1 ratio. Moreover, immunization of mice with CpG-OVA-conjugated LDH before challenge with OVA-expressing B16/F10 tumor cells retarded tumor growth. Together, these data indicate that LDH nanomaterial can be used as an immune adjuvant to promote Th1 or Th2 dominant immune responses suitable for vaccination purposes.

Microneedle enhanced delivery of cosmeceutically relevant peptides in human skin.

Peptides and proteins play an important role in skin health and well-being. They are also found to contribute to skin aging and melanogenesis. Microneedles have been shown to substantially enhance skin penetration and may offer an effective means of peptide delivery enhancement. The aim of this investigation was to assess the influence of microneedles on the skin penetration of peptides using fluorescence imaging to determine skin distribution. In particular the effect of peptide chain length (3, 4, 5 amino acid chain length) on passive and MN facilitated skin penetration was investigated. Confocal laser scanning microscopy was used to image fluorescence intensity and the area of penetration of fluorescently tagged peptides. Penetration studies were conducted on excised full thickness human skin in Franz type diffusion cells for 1 and 24 hours. A 2 to 22 fold signal improvement in microneedle enhanced delivery of melanostatin, rigin and pal-KTTKS was observed. To our knowledge this is the first description of microneedle enhanced skin permeation studies on these peptides.