Reverse Iontophoretic Extraction of Skin Cancer-Related Biomarkers.

Non-invasive methods for early diagnosis of skin cancer are highly valued. One possible approach is to monitor relevant biomarkers such as tryptophan (Trp) and kynurenine (Kyn), on the skin surface. The primary aim of this in vitro investigation was, therefore, to examine whether reverse iontophoresis (RI) can enhance the extraction of Trp and Kyn, and to demonstrate how the Trp/Kyn ratio acquired from the skin surface reflects that in the epidermal tissue. The study also explored whether the pH of the receiver medium impacted on extraction efficiency, and assessed the suitability of a bicontinuous cubic liquid crystal as an alternative to a simple buffer solution for this purpose. RI substantially enhanced the extraction of Trp and Kyn, in particular towards the cathode. The Trp/Kyn ratio obtained on the surface matched that in the viable skin. Increasing the receiver solution pH from 4 to 9 improved extraction of both analytes, but did not significantly change the Trp/Kyn ratio. RI extraction of Trp and Kyn into the cubic liquid crystal was comparable to that achieved with simple aqueous receiver solutions. We conclude that RI offers a potential for non-invasive sampling of low-molecular weight biomarkers and further investigations in vivo are therefore warranted.

Mechanism of human nail poration by high-repetition-rate, femtosecond laser ablation.

Optical poration, or drilling, of the human nail has the potential to drastically improve transungual drug delivery. However, this approach is accompanied by thermal damage to the nail tissue surrounding the laser radiation-created pore. In this paper, fluorescence microscopy has been employed to quantitatively evaluate thermal damage to the nail induced by laser ablation with 80 MHz, nanojoule, femtosecond pulses delivered via a hollow-core fibre. An empirical relation has been established between the intensity of the resulting fluorescence signal and temperature to which the nail was exposed. Using this relationship, detailed temperature maps have been created of the areas surrounding the pores, enabling the mechanism of poration to be better understood. It was deduced that plasma-mediated ablation is primarily responsible for nail tissue ablation at the centre of the pore, while cumulative photothermal processes dominate at the pore edges. It is concluded, furthermore, that temperature mapping represents a useful new tool with which to optimise the process of nail poration. The method is potentially generic and may be applicable to other biological materials.

Reverse Iontophoretic Extraction of Metabolites from Living Plants and their Identification by Ion-chromatography Coupled to High Resolution Mass Spectrometry.

INTRODUCTION: The identification and characterisation of cellular metabolites has now become an important strategy to obtain insight into functional plant biology. However, the extraction of metabolites for identification and analysis is challenging and, at the present time, usually requires destruction of the plant. OBJECTIVE: To detect different plant metabolites in living plants with no pre-treatment using the combination of iontophoresis and ion-chromatography with mass spectrometry detection. METHODOLOGY: In this work, the simple and non-destructive method of reverse iontophoresis has been used to extract in situ multiple plant metabolites from intact Ocimum basilicum leaves. Subsequently, the analysis of these metabolites has been performed with ion chromatography coupled directly to high resolution mass spectrometric detection (IC-MS). RESULTS: The application of reverse iontophoresis to living plant samples has avoided the need for complex pre-treatments. With this approach, no less than 24 compounds, including organic acids and sugars as well as adenosine triphosphate (ATP) were successfully detected. CONCLUSION: The research demonstrates that it is feasible to monitor, therefore, a number of important plant metabolites using a simple, relatively fast and non-destructive approach. Copyright © 2016 John Wiley & Sons, Ltd.

Modelling drug flux through microporated skin.

A simple mathematical equation has been developed to predict drug flux through microporated skin. The theoretical model is based on an approach applied previously to water evaporation through leaf stomata. Pore density, pore radius and drug molecular weight are key model parameters. The predictions of the model were compared with results derived from a simple, intuitive method using porated area alone to estimate the flux enhancement. It is shown that the new approach predicts significantly higher fluxes than the intuitive analysis, with transport being proportional to the total pore perimeter rather than area as intuitively anticipated. Predicted fluxes were in good general agreement with experimental data on drug delivery from the literature, and were quantitatively closer to the measured values than those derived from the intuitive, area-based approach.

Characterisation of polyphenolic compounds in Clerodendrum petasites S. Moore and their potential for topical delivery through the skin.

ETHNOPHARMACOLOGICAL RELEVANCE: Clerodendrum petasites S. Moore (CP) has been widely prescribed in Thailand and neighbouring countries for both oral and topical administration to treat asthma, fever, cough, vomiting and skin diseases, for at least 30 years. However, the nature of the active species remains poorly characterized and there have been no clinical trials concerning the topical delivery of this medicine. The study aims to characterise polyphenolic compounds in the plant, to predict the feasibility of their topical absorption and to test their ability to penetrate the skin. MATERIALS AND METHODS: Identification and quantification of flavonoids and phenolic acid derivatives in an ethanolic extract of the aerial parts of the plant were carried out using high performance liquid chromatography (HPLC) with photodiode array (PDA) and mass spectrometry (MS) detection. Ambiguous isomeric compounds were distinguished by nuclear magnetic resonance (NMR) spectroscopy. The feasibility of the compounds׳ topical permeability was evaluated by predicting their maximum fluxes from their physicochemical properties. The skin penetration of compounds in the plant extract was measured in vitro over 24h. RESULTS: Vanillic acid, verbascoside, 4-coumaric acid, ferulic acid, nepetin, luteolin, apigenin, naringenin, hispidulin, hesperetin and chrysin, were identified in CP. All compounds except apigenin and hispidulin are reported in this species for the first time. Hispidulin is the predominant compound (1.2% w/w in a dried ethanolic extract) followed by nepetin, verbascoside, vanillic acid, and apigenin. Across mammalian skin, hispidulin was percutaneously absorbed within 3h and vanillic acid and nepetin permeated the skin after 6h. These experimental observations were consistent with the predicted maximum fluxes of these compounds calculated from their physicochemical properties. CONCLUSIONS: Many of the phenolic compounds reported in this study are well-known to possess antimicrobial, anti-inflammatory and anti-oxidant activities. The skin permeation studies reported here support traditional topical uses of the plant in skin treatments and are useful for further topical formulation optimisation.

Evaluation of drug delivery to intact and porated skin by coherent Raman scattering and fluorescence microscopies.

Stimulated Raman scattering microscopy was used to assess the permeation of topically applied drugs and formulation excipients into porcine skin. This chemically selective technique generates high-resolution 3D images, from which semi-quantitative information may be elucidated. Ibuprofen, applied as a close-to-saturated solution in propylene glycol, was directly observed to crystallise in/on the skin, as the co-solvent permeated more rapidly, resulting in precipitation of the drug. Coherent Raman scattering microscopy is also an excellent tool, in conjunction with more conventional confocal fluorescence microscopy, with which to image micro/nanoparticle-based formulations. Specifically, the uptake of particles into thermal ablation transport pathways in the skin has been examined.

Transdermal flux predictions for selected selective oestrogen receptor modulators (SERMs): comparison with experimental results.

The aim of this work was to evaluate the feasibility of delivering transdermally a series of highly lipophilic compounds (log P ~4-7), comprising several selective oestrogen receptor modulators and a modified testosterone (danazol). The maximum fluxes of the drugs were predicted theoretically using the modified Potts & Guy algorithm (to determine the permeability coefficient (kp) from water) and the calculated aqueous solubilities. The correction provided by Cleek & Bunge took into account the contribution of the viable epidermal barrier to the skin permeation of highly lipophilic compounds. Experimental measurements of drug fluxes from saturated hydroalcoholic solutions were determined in vitro through excised pig skin. Overall, the predicted fluxes were in good general agreement (within a factor of 10) with the experimental results. Most of the experimental fluxes were greater than those predicted theoretically suggesting that the 70:30 v/v ethanol-water vehicle employed may have had a modest skin penetration enhancement effect. This investigation shows that the transdermal fluxes of highly lipophilic compounds can be reasonably predicted from first principles provided that the viable epidermis, underlying the stratum corneum, is included as a potentially important contributor to the skin's overall barrier function. Furthermore, the absolute values of the measured fluxes, when considered in parallel with previous clinical studies, indicate that it might be feasible to topically deliver a therapeutically useful amount of some of the compounds considered to treat cancerous breast tissue.

Effects of iontophoresis, hydration, and permeation enhancers on human nail plate: infrared and impedance spectroscopy assessment.

PURPOSE: To investigate whether permeation enhancement techniques affect the nail plate. METHODS: Infrared and impedance spectroscopies examined the effects of hydration, iontophoresis and N-acetyl-L-cysteine on the human nail. RESULTS: While significant shifts to higher wavenumbers were observed for the symmetric and asymmetric -CH2 stretching vibrations these changes were essentially the same for the three treatments suggesting they were principally due to hydration alone. Spectral changes associated with amide bonds from nail protein were particularly evident post-treatment with N-acetyl-L-cysteine. The alternating current conductivity and permittivity of the nail, particularly at low frequencies, increased with hydration. Iontophoresis increased the low frequency ac conductivity of the nail but had less effect on the nail capacitance/permittivity. Further, the effects seemed to return gradually to baseline after termination of current passage. Treatment with N-acetyl-L-cysteine produced a greater perturbation, leading to increased low-frequency conductivity and a shift of the frequency-dependent conductivity region to a higher frequency. CONCLUSIONS: Overall, the effects of iontophoresis on infrared and impedance spectroscopic profiles of the nail were attributable simply to increased hydration and similar to those observed after skin iontophoresis. In contrast, both spectroscopy techniques indicated that N-acetyl-L-cysteine disrupted nail structure in line with the enhancer's known effect on keratin.

Novel beads made of alpha-cyclodextrin and oil for topical delivery of a lipophilic drug.

PURPOSE: To investigate the potential of a novel lipid carrier, comprising beads of alpha-cyclodextrin and soybean oil, for topical drug delivery. Adapalene was chosen as a model drug to explore the ability of the beads to encapsulate and release a highly lipophilic compound. MATERIALS AND METHODS: Adapalene-loaded beads were prepared and characterised. Skin tolerance to unloaded beads was tested on human volunteers, while drug release and delivery into stratum corneum, was evaluated in pig skin ex vivo. RESULTS: The preparation and physical characteristics of the beads were not dependent on whether adapalene had been previously dissolved or dispersed in soybean oil. Drug encapsulation efficiency was high (>96%) and drug loading on the order of a therapeutic level could be achieved in freeze-dried beads prepared from an oily dispersion of adapalene. After application to human skin, unloaded beads induced no adverse reaction and were better tolerated than an alcoholic gel. Tape-stripping the stratum corneum from treated pig skin showed that adapalene release and penetration from the beads was comparable to that from gel and cream formulations available on the market. CONCLUSION: These novel beads may offer a well-tolerated and efficient system for the encapsulation and topical delivery of lipophilic drugs.

In vivo methods for the assessment of topical drug bioavailability.

This paper reviews some current methods for the in vivo assessment of local cutaneous bioavailability in humans after topical drug application. After an introduction discussing the importance of local drug bioavailability assessment and the limitations of model-based predictions, the focus turns to the relevance of experimental studies. The available techniques are then reviewed in detail, with particular emphasis on the tape stripping and microdialysis methodologies. Other less developed techniques, including the skin biopsy, suction blister, follicle removal and confocal Raman spectroscopy techniques are also described.

Ex vivo evaluation of bioadhesive films for buccal delivery of fentanyl.

The goals of this work were (i) to develop bioadhesive films for the buccal delivery of fentanyl, and (ii) to evaluate their performance in vitro using the pig esophageal model. Films were made with polyvinylpyrrolidone (PVP) of two different molecular weights: PVP K30 and PVP K90. Delivery of fentanyl was determined across full-thickness mucosa and across heat-separated epithelium (where the permeability barrier was shown to be located). The influence of film pH was investigated, and it was found that fentanyl permeation increased with increasing pH (i.e., when a higher percentage of the unionized fraction of drug was present). However, at the pH values studied, fentanyl was predominantly ionized suggesting that transport pathways offering a hydrophilic, or polar, environment across the mucosa were available. The transport rates achieved from the PVP films providing the highest delivery suggest that a buccal system of only 1-2 cm(2) in surface area could achieve a therapeutic effect equivalent to a 10 cm(2) transdermal patch, with a much shorter lag-time.

Structure-permeation relationships for the non-invasive transdermal delivery of cationic peptides by iontophoresis.

Transdermal iontophoresis enables the controlled, non-invasive administration of peptide therapeutics. The aims of this study were (i) to evaluate the effect of amino acid sequence and the spatial distribution of peptide physicochemical properties on electrotransport, and (ii) to develop a quantitative model to predict peptide transport rates. Experimental results showed that the distribution of molecular properties over the peptide surface significantly affected iontophoretic delivery: different arrangements of the same residues resulted in different transport behavior. Computational studies generated three-dimensional quantitative structure-permeation relationships (3D-QSPR) based on 3D descriptors. The model predicted that iontophoresis was favored by peptide hydrophilicity but hindered by voluminous, localized hydrophobicity. Molecular characteristics that favor electrotransport are the converse of those required for passive diffusion across biological membranes. The data represent the first analysis of peptide electrotransport in terms of the spatial distribution of molecular properties and provide insight into the ab initio prediction of transdermal iontophoretic peptide delivery.

Emerging strategies for the transdermal delivery of peptide and protein drugs.

Transdermal delivery has been at the forefront of research addressing the development of non-invasive methods for the systemic administration of peptide and protein therapeutics generated by the biotechnology revolution. Numerous approaches have been suggested for overcoming the skin's formidable barrier function; whereas certain strategies simply act on the drug formulation or transiently increase the skin permeability, others are designed to bypass or even remove the outermost skin layer. This article reviews the technologies currently under investigation, ranging from those in their early-stage of development, such as laser-assisted delivery to others, where feasibility has already been demonstrated, such as microneedle systems, and finally more mature techniques that have already led to commercialisation (e.g., velocity-based technologies). The principles, mechanisms involved, potential applications, limitations and safety considerations are discussed for each approach, and the most advanced devices in each field are described.

Contributions of electromigration and electroosmosis to peptide iontophoresis across intact and impaired skin.

d-(Arg)-Kyotorphin iontophoresis was investigated across intact and impaired skins in vitro. Iontophoretic flux increased from 68+/-12 to 538+/-116 nmol cm(-2) h(-1) when the peptide concentration in the anodal compartment was raised from 5 to 40 mM. Electromigration was the principal transport mechanism, accounting for approximately 70% of total peptide delivery. Reducing the number of competing ions in the formulation significantly increased iontophoretic flux but did not affect convective solvent flow. The latter was independent of peptide concentration indicating that skin permselectivity was not modified by kyotorphin transport. Total iontophoretic flux was unaffected when the stratum corneum was removed by tape-stripping (146+/-34 versus 150+/-26 nmol cm(-2) h(-1)). However, the contributions of the different transport mechanisms were significantly altered: (i) electromigration decreased, as more of the charge was carried by anions from the sub-dermal milieu; (ii) electroosmosis was absent; and (iii) passive permeation increased significantly. Transport rates across intact and impaired skin barriers were statistically indistinguishable when the donor electrolyte composition was modified; increased competition from anions was mitigated by the decreased Na+ levels in the formulation. Removal of Cl- ions from the receiver phase further increased peptide delivery, and also increased anodal electroosmosis.

Effect of charge and molecular weight on transdermal peptide delivery by iontophoresis.

PURPOSE: The study was conducted to investigate the impact of charge and molecular weight (MW) on the iontophoretic delivery of a series of dipeptides. METHODS: Constant current iontophoresis of lysine and 10 variously charged lysine- and tyrosine-containing dipeptides was performed in vitro. RESULTS: Increasing MW was compensated by additional charge; for example, Lys (MW = 147 Da, +1) and H-Lys-Lys-OH (MW = 275 Da, +2) had equivalent steady-state fluxes of 225 +/- 48 and 218 +/- 40 nmol cm(-2) h(-1), respectively. For peptides with similar MW, e.g., H-Tyr-D-Arg-OH (MW = 337 Da, +1) and H-Tyr-D-Arg-NH(2) (MW = 336 Da, +2), the higher valence ion displayed greater flux (150 +/- 26 vs. 237 +/- 35 nmol cm(-2) h(-1)). Hydrolysis of dipeptides with unblocked N-terminal residues, after passage through the stratum corneum, suggested the involvement of aminopeptidases. The iontophoretic flux of zwitterionic dipeptides was less than that of acetaminophen and dependent on pH. CONCLUSIONS: For the series of dipeptides studied, flux is linearly correlated to the charge/MW ratio. Data for zwitterionic peptides indicate that they do not behave as neutral ("charge-less") molecules, but that their iontophoretic transport is dependent on the relative extents of ionization of the constituent ionizable groups, which may also be affected by neighboring amino acids.

Effect of amino acid sequence on transdermal iontophoretic peptide delivery.

The objective of this study was to investigate the effect of amino acid sequence on the transdermal delivery of peptides by iontophoresis. Structurally related, cationic tripeptides based on the residues at positions (i) 6-8 in LHRH (Ac-X-Leu-Arg-NH(2)) and (ii) 3-5 in octreotide (Ac-X-dTrp-Lys-NH(2)) were studied. Iontophoretic transport experiments were conducted using porcine skin in vitro to investigate the dependence of flux on peptide concentration. Co-iontophoresis of acetaminophen enabled deconvolution of the contributions of electromigration (EM) and electroosmosis (EO) and the calculation of an electroosmotic inhibition factor (IF). A two-fold increase in donor peptide concentration increased iontophoretic flux for most peptides, and electroosmotic inhibition for dNal-containing tripeptides. The improvement in transport and the impact on the EM and EO components were peptide-specific. A reduction in the number of competing ions in the formulation significantly increased transport and, specifically, the EM contribution; it also increased IF of compounds with a propensity to interact with the membrane. No monotonic dependence of flux on either molecular weight or lipophilicity was observed. Iontophoretic peptide transport could not be rationalized in terms of either peptide molecular weight or computational 2D estimates of lipophilicity. Data suggest that a more complex three-dimensional approach is required to develop structure permeation relationships governing iontophoretic peptide delivery.

Transdermal iontophoretic delivery of vapreotide acetate across porcine skin in vitro.

PURPOSE: The purpose of this study was to evaluate the feasibility of delivering vapreotide, a somatostatin analogue, by transdermal iontophoresis. METHODS: In vitro experiments were conducted using dermatomed porcine ear skin and heat-separated epidermis. In addition to quantifying vapreotide transport into and across the skin, the effect of peptide delivery on skin permselectivity was also measured. The influence of (1) current density, (2) pre- and post-treatment of the skin, (3) competitive ions, and (4) inclusion of albumin in the receptor on vapreotide delivery were investigated. RESULTS: Epidermis proved to be a better model than dermatomed skin for vapreotide transport studies. Despite the susceptibility of vapreotide to enzymatic degradation, a flux of 1.7 microg/cm2 per hour was achieved after 7 h of constant current iontophoresis (0.15 mA/cm2). Post-iontophoretic extraction revealed that, depending on the experimental conditions, 80-300 microg of peptide were bound to the skin. Vapreotide was found to interact with the skin and displayed a current-dependent inhibition of electroosmosis. However, neither the pre-treatment strategies to saturate the putative binding sites nor the post-treatment protocols to displace the bound peptide were effective. CONCLUSION: Based on the observed transport rate of vapreotide across porcine epidermis and its clinical pharmacokinetics, therapeutic concentrations should be achievable using a 15-cm2 patch.

Ultrasound-mediated gene delivery: kinetics of plasmid internalization and gene expression.

Sonoporation is an approach that can be used to transfer DNA or drugs into cells. However, very little is known about the mechanism of ultrasound-mediated membrane permeabilization. In this investigation, DNA transport post-sonoporation and the subsequent plasmid internalization and protein expression kinetics have been studied. Using a plasmid encoding for the green fluorescent protein (GFP), labelled or not with an intercalating agent (YOYO-1), it was found that, as compared to lipofection that requires endocytosis, sonoporation allowed a rapid and direct transfer of naked DNA into the cell cytoplasm probably via ultrasound-induced pores in the membrane. The kinetics of protein expression were significantly faster for sonoporation than for lipofection, the mechanism of which requires endocytosis. However, unprotected DNA in the cytoplasm could be degraded by resident cytosolic DNases, thereby decreasing ultrasound-mediated gene delivery efficiency.

Plasma membrane poration induced by ultrasound exposure: implication for drug delivery.

Sonoporation, in the presence of ultrasound contrast agents (UCA), is a technique that permits the transfer of drugs, including genes, into cells. In this study, the size of the pores created by ultrasound application, and the duration of pore opening, have been characterized via indirect molecular probing and microscopic observation. Internalization of molecules with diameters up to 37 nm was efficient and generally well-tolerated; on the other hand, confocal microscopy revealed that 75 nm particles entered only a few cells when sonoporation was applied. In general, the larger the species to internalize, the poorer the transfer. Direct visualization of pores following insonification, using scanning electron microscopy, was hampered by the presence of numerous villi on the surface of the cells employed (MAT B III), and by the short duration of pore opening. Clearer observations of porated regions were possible using red blood cells. This research (i) confirms that sonoporation is a means with which to achieve macromolecule delivery into cells, and (ii) characterizes in some detail the phenomenon of ultrasound induction of transient pores in the cell membrane.

Enhanced delivery of 5-aminolevulinic acid esters by iontophoresis in vitro.

The goals of this study were to quantitatively evaluate the iontophoretic delivery of a homologous series of cationic aminolevulinic acid (ALA) esters and to determine the contributions of electromigration and electroosmosis to their overall electrotransport in vitro. Anodal iontophoretic transport of ALA esters through porcine skin in vitro was followed for 2 h at a constant current of 0.5 mA/cm2. To deduce the mechanism, the concomitant transport of an electroosmotic marker, mannitol, was also assessed. Positively charged ALA esters of moderate lipophilicity showed increased iontophoretic flux through the skin. A more than 50-fold enhancement as compared with the zwitterionic parent ALA was observed for the methyl ester. As the size and lipophilicity of the ester increased, the efficiency of electrotransport decreased. The most lipophilic esters reduced the electroosmotic flow presumably because of the association of these cations with negative charges in the skin. Iontophoresis of methyl-ALA and hexyl-ALA also increased the amount of prodrug delivered into the skin. In summary, significant topical delivery of ALA esters can be achieved by iontophoresis, and transport into and across the skin was greatly enhanced compared with that of ALA itself. It remains to be seen whether this enhanced local bioavailability of the protoporphyrin prodrug can allow improved photodynamic therapy for the treatment of skin cancer.