Elongated microparticles tuned for targeting hyaluronic acid delivery to specific skin strata.

OBJECTIVE: Microneedle or fractional laser applications are the most common topical delivery enhancement platforms. However, these methods of drug delivery are not skin strata specific. Drug delivery approaches which could target specific stratum of the skin remains a challenge. Elongated microparticles (EMPs) have been used in enhancing drug delivery into the skin. The aim of this study was to evaluate, for the first time, elongated silica microparticles with two different length profiles to enhance delivery of hyaluronic acid into different strata of human skin. METHODS: Two types of EMPs-long (milled EMPs) or short (etched EMPs) length ranges were characterized. A prototypical liquid formulation (Fluorescent hyaluronic acid) with and without EMP enhancement were evaluated for hyaluronic acid delivery in ex-vivo human skin. High performance liquid chromatography, Typhoon fluorescence scanning system, laser scanning confocal microscopy and reflectance confocal microscopy (RCM) were used to validate F-HA stability, visualize fluorescein in the skin, image the depth of F-HA delivery in the skin and define EMP penetration in skin strata, respectively. Statistical analysis was conducted using GraphPad Prism 6 software (GraphPad Software Inc, USA). RESULTS: Fluorescein-hyaluronic acid was stable and EMP enhanced skin penetration. RCM revealed that 'etched EMP' penetrated the skin to the stratum spinosum level. The vast majority (97.8%; p < 0.001) of the etched EMP did not penetrate completely through the viable epidermis and no obvious penetration into the dermis. In contrast, milled EMP showed 41-fold increase in penetration compared to the etched EMP but penetrated beyond the dermoepidermal junction. CONCLUSION: EMPs can enhance delivery of hyaluronic acid. Using EMPs with defined length distributions, which can be tuned for a specific stratum of the skin, can achieve targeted hyaluronic acid delivery.

OBJECTIF: Les microaiguilles ou le laser fractionné sont couramment utilisés pour augmenter l'absorption d'actif appliqué par voie topique. Toutefois, ces approches ne permettent de cibler une strate spécifique de la peau. Ainsi les méthodes permettant de cibler spécifiquement l'épiderme reste un défi. Les microparticules allongées (EMP) ont déjà été utilisé pour augmenter l'absorption cutanée. L'objectif de l'étude est d'évaluer pour la première fois, la capacité de microparticules allongées de silice (de deux longueurs différentes) à délivrer l'acide hyaluronique dans les différentes couches de la peau. MÉTHODES: Deux types d'EMP, longues (EMP broyé) ou courtes (EMP gravé), ont été caractérisées. Une formulation liquide contenant de l'acide hyaluronique marquée avec une sonde fluorescente (F-HA) a été évaluée avec et sans EMP sur peau humaine ex vivo. La chromatographie liquide haute performance, le scanner à fluorescence Typhoon, la microscopie laser confocal à balayage et la microscopie confocale par réflectance (RCM) ont été utilisées respectivement pour contrôler la stabilité de la F-HA, visualiser le signal de la fluorescéine dans la peau, imager l'absorption du F-HA dans la peau en fonction de la profondeur et caractériser la pénétration des EMP. Les analyses statistiques ont été réalisées avec le logiciel GraphPad Prims 6 (GraphPad Software Inc, USA). RÉSULTATS: L'acide hyaluronique marquée avec la fluorescéine est stable et les EMP permettent d'augmenter son absorption cutanée. Le RCM a montré que les EMP gravées pénètrent dans la peau jusqu'au niveau du stratum spinosum. La grande majorité des EMP gravés (97.8% ; p < 0,001) ne pénètre pas complétement dans l'épiderme viable et aucune pénétration mesurable dans le derme. Au contraire, les EMP broyées ont montrées une pénétration 41 fois plus importantes que les EMP gravées et peuvent aller au-delà de la jonction derme-épiderme. CONCLUSION: Les EMP peuvent augmenter l'absorption cutanée de l'acide hyaluronique. En utilisant des EMP de longueur définie et en ajustant celle-ci, il est même possible de cibler spécifiquement une strate cutanée.

A minimally invasive clinical model to test sunscreen toxicity based on oxidative stress levels using microbiopsy and confocal microscopy - a proof of concept study.

OBJECTIVE: This proof-of-concept study demonstrated that using minimally invasive skin microsampling could enable significantly higher throughput of cosmetic testing in volunteers than conventional biopsy. Nanoparticle sunscreen was used as a model to test toxicity based on oxidative stress using microbiopsy and confocal imaging. METHODS: Six volunteers were recruited for this study (3 males and 3 females). Zinc oxide nanoparticle containing topical formulation was prepared at 10% w/v. Each volunteer had 3 areas of 4 cm2 each mapped on each inner forearm for a total of 6 treatment areas (intact/ tape-stripped and with/without treatment). The topical zinc-nanoparticle formulation was applied directly to volunteer skin (2mg/cm2 ) for 2 hrs. Microbiopsied tissue from each treatment group was stained for reactive oxygen and nitrogen species in addition to mitochondrial superoxide. The stained samples were then imaged using confocal microscopy prior to image analysis. RESULTS: Skin exposed to zinc oxide nanoparticles did not show any significant increases in oxidative stress. Zinc oxide nanoparticle tape-stripped skin resulted in signal significantly lower (P < 0.001) oxidative stress levels than t-butylated hydroxytoluene treated tape-stripped skin for oxidative stress markers. Topically applied zinc oxide nanoparticles had no detectable effect on the oxidative status in volunteer skin. No adverse reactions or effects were observed after all treatments including microbiopsy. CONCLUSION: The data support the hypothesis that microbiopsy is a viable approach to study cosmeceutical- skin interactions in volunteers with capacity for molecular assays and high throughput with very low risk to the volunteer.

OBJECTIFS: Cette étude de validation de concept a démontré que le microprélèvement cutané minimalement invasif pouvait augmenter considérablement la cadence des essais de produits cosmétiques sur des volontaires par rapport à une biopsie conventionnelle. Un écran solaire contenant des nanoparticules a été utilisé comme modèle pour tester la toxicité liée au stress oxydatif à l'aide de la microbiopsie et de l'imagerie confocale. MÉTHODES: Six volontaires ont été recrutés pour cette étude (3 hommes et 3 femmes). Une formulation topique contenant des nanoparticules d'oxyde de zinc a été préparée à 10 % p/v. Chaque volontaire disposait de 3 zones de 4 cm2 situées sur chaque pliure de coude pour un total de 6 zones de traitement (intactes / strippée et avec / sans traitement). La formulation topique contenant des nanoparticules d'oxyde de zinc a été appliquée directement sur la peau des volontaires (2 mg/cm2 ) pendant 2 heures. Les tissus microbiopsiés de chaque groupe de traitement ont été colorés pour détecter des espèces réactives de l'oxygène et de l'azote en plus de la superoxyde mitochondriale. Les échantillons colorés ont ensuite été examinés par microscopie confocale avant l'analyse des images. RÉSULTATS: La peau exposée aux nanoparticules d'oxyde de zinc n'a pas montré de hausse significative de stress oxydatif. La peau strippée traitée aux nanoparticules d'oxyde de zinc a entraîné des niveaux de stress oxydatif nettement inférieurs (p<0,001) comparés à ceux de la peau strippée traitée à l'hydroxytoluène t-butylé en ce que concerne les marqueurs de stress oxydatif. Les nanoparticules d'oxyde de zinc appliquées par voie topique n'ont eu aucun effet détectable sur l'état oxydatif de la peau des volontaires. Aucune réaction ou effet indésirable n'a été observé(e) après tous les traitements, y compris la microbiopsie. CONCLUSION: Les données obtenues étayent l'hypothèse selon laquelle la microbiopsie est une approche viable pour étudier les interactions des produits cosmétiques sur la peau des volontaires, avec la possibilité de réaliser des dosages moléculaires et à haut débit, avec un risque très faible pour les volontaires.

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis.

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

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.