ABSTRACT
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.
Subject(s)
Biopsy/methods , Microscopy, Confocal/methods , Oxidative Stress , Sunscreening Agents/toxicity , Humans , Metal Nanoparticles/chemistry , Proof of Concept Study , Zinc Oxide/administration & dosageABSTRACT
Melanoma incidence continues to increase across many populations globally and there is significant mortality associated with advanced disease. However, if detected early, patients have a very promising prognosis. The methods that have been utilized for early detection include clinician and patient skin examinations, dermoscopy (static and sequential imaging), and total body photography via 2D imaging. Total body photography has recently witnessed an evolution from 2D imaging with the ability to now create a 3D representation of the patient linked with dermoscopy images of individual lesions. 3D total body photography is a particularly beneficial screening tool for patients at high risk due to their personal or family history or those with multiple dysplastic naevi-the latter can make monitoring especially difficult without the assistance of technology. In this perspective, we discuss clinical examples utilizing 3D total body photography, associated advantages and limitations, and future directions of the technology. The optimal system for melanoma screening should improve diagnostic accuracy, be time and cost efficient, and accessible to patients across all demographic and socioeconomic groups. 3D total body photography has the potential to address these criteria and, most importantly, optimize crucial early detection.
ABSTRACT
The bone marrow is a unique microenvironment where blood cells are produced and released into the circulation. At the top of the blood cell lineage are the hematopoietic stem cells (HSC), which are thought to reside in close association with the bone marrow vascular endothelial cells (Morrison and Scadden, Nature 505:327-334, 2014). Recent efforts at characterizing the HSC niche have prompted us to make close examinations of two distinct types of blood vessel in the bone marrow, the arteriolar vessels originating from arteries and sinusoidal vessels connected to veins. We found the two vessel types to exhibit different vascular permeabilites, hemodynamics, cell trafficking behaviors, and oxygen content (Itkin et al., Nature 532:323-328, 2016; Spencer et al., Nature 508:269-273, 2014). Here, we describe a method to quantitatively measure the permeability and hemodynamics of arterioles and sinusoids in murine calvarial bone marrow using intravital microscopy.
Subject(s)
Arterioles/cytology , Bone Marrow/growth & development , Capillaries/cytology , Capillary Permeability , Hematopoietic Stem Cells/cytology , Hemodynamics , Intravital Microscopy/methods , Animals , Arterioles/metabolism , Bone Marrow/metabolism , Capillaries/metabolism , Cell Movement , Hematopoietic Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.
Subject(s)
Intravital Microscopy/methods , Osteocytes/metabolism , Skull/cytology , Animals , Histone Deacetylases/genetics , Mice , Mice, KnockoutABSTRACT
Identification of appropriate reference genes (RGs) is critical to accurate data interpretation in quantitative real-time PCR (qPCR) experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq) to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer.
ABSTRACT
To develop novel methods for vaccine delivery, the skin is viewed as a high potential target, due to the abundance of immune cells that reside therein. One method, the use of dissolving microneedle technologies, has the potential to achieve this, with a range of formulations now being employed. Within this paper we assemble a range of methods (including FT-FIR using synchrotron radiation, nanoindentation and skin delivery assays) to systematically examine the effect of key bulking agents/excipients - sugars/polyols - on the material form, structure, strength, failure properties, diffusion and dissolution for dissolving microdevices. We investigated concentrations of mannitol, sucrose, trehalose and sorbitol from 1:1 to 30:1 with carboxymethylcellulose (CMC), although mannitol did not form our micro-structures so was discounted early in the study. The other formulations showed a variety of crystalline (sorbitol) and amorphous (sucrose, trehalose) structures, when investigated using Fourier transform far infra-red (FT-FIR) with synchrotron radiation. The crystalline structures had a higher elastic modulus than the amorphous formulations (8-12GPa compared to 0.05-11GPa), with sorbitol formulations showing a bimodal distribution of results including both amorphous and crystalline behaviour. In skin, diffusion properties were similar among all formulations with dissolution occurring within 5s for our small projection array structures (~100µm in length). Overall, slight variations in formulation can significantly change the ability of our projections to perform their required function, making the choice of bulking/vaccine stabilising agents of great importance for these devices.
Subject(s)
Excipients/chemistry , Microinjections , Needles , Vaccines/chemistry , Administration, Cutaneous , Animals , Carboxymethylcellulose Sodium/administration & dosage , Carboxymethylcellulose Sodium/chemistry , Chemistry, Pharmaceutical , Dextrans/administration & dosage , Dextrans/chemistry , Drug Liberation , Excipients/administration & dosage , Female , Mannitol/administration & dosage , Mannitol/chemistry , Mice , Mice, Inbred C57BL , Ovalbumin/administration & dosage , Ovalbumin/chemistry , Rhodamines/administration & dosage , Rhodamines/chemistry , Skin/metabolism , Skin Absorption , Sucrose/administration & dosage , Sucrose/chemistry , Trehalose/administration & dosage , Trehalose/chemistry , Vaccines/administration & dosageABSTRACT
Foroderm is a new cutaneous delivery technology that uses high-aspect ratio, cylindrical silica microparticles, that are massaged into the skin using a 3D-printed microtextured applicator, in order to deliver payloads across the epidermis. Herein we show that this technology is effective for delivery of a non-adjuvanted, inactivated, whole-virus chikungunya virus vaccine in mice, with minimal post-vaccination skin reactions. A single topical Foroderm-based vaccination induced T cell, Th1 cytokine and antibody responses, which provided complete protection against viraemia and disease after challenge with chikungunya virus. Foroderm vaccination was shown to deliver fluorescent, virus-sized beads across the epidermis, with beads subsequently detected in draining lymph nodes. Foroderm vaccination also stimulated the egress of MHC II(+) antigen presenting cells from the skin. Foroderm thus has potential as a simple, cheap, effective, generic, needle-free technology for topical delivery of vaccines.
Subject(s)
Chikungunya Fever/prevention & control , Chikungunya virus/immunology , Drug Delivery Systems , Viral Vaccines/immunology , Administration, Cutaneous , Animals , Antibodies, Viral/blood , Disease Models, Animal , Female , Mice, Inbred Strains , Mice, Nude , T-Lymphocytes/immunology , Treatment Outcome , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunology , Viral Vaccines/administration & dosage , Viremia/prevention & controlABSTRACT
The skin has evolved to resist the penetration of foreign substances and particles. Effective topical drug delivery into and/or through the skin is hindered by these epidermal barriers. A range of physical enhancement methods has been developed to selectively overcome this barrier. This review discusses recent advances in physical drug delivery by broadly separating the techniques into two main areas; indirect and direct approaches. Indirect approaches consist of electrical, vibrational or laser instrumentation that creates pores in the skin followed by application of the drug. Direct approaches consist of mechanical disruption of the epidermis using techniques such as microdermabrasion, biolistic injectors and microneedles. Although, in general, physical techniques are yet to be established in a clinical setting, the potential gains of enhancing delivery of compounds through the skin is of great significance and will no doubt continue to receive much attention.
Subject(s)
Drug Delivery Systems/methods , Pharmaceutical Preparations/administration & dosage , Skin/metabolism , Administration, Cutaneous , Animals , Dermabrasion/instrumentation , Dermabrasion/methods , Dermabrasion/trends , Drug Delivery Systems/instrumentation , Drug Delivery Systems/trends , Electroporation/instrumentation , Electroporation/methods , Electroporation/trends , Equipment Design , Humans , Iontophoresis/instrumentation , Iontophoresis/methods , Iontophoresis/trends , Lasers , Magnets , Needles , Pharmaceutical Preparations/metabolism , Phonophoresis/instrumentation , Phonophoresis/methods , Phonophoresis/trendsABSTRACT
The skin has evolved to resist the penetration of foreign substances and particles. Topical therapeutic and cosmeceutical delivery is a growing field founded on selectively overcoming this barrier. Both the biology of the skin and the nature of the formulation/active ingredient must be aligned for efficient transcutaneous delivery. This review discusses the biological changes in the skin barrier that occur with common dermatological conditions. This context is the foundation for the discussion of formulation strategies to improve penetration profiles of common active ingredients in dermatology. Finally, we compare and contrast those approaches to recent advances described in the research literature with an eye toward the future of topical formulation design.
Subject(s)
Dermatologic Agents/administration & dosage , Drug Delivery Systems , Skin Diseases/drug therapy , Administration, Cutaneous , Animals , Chemistry, Pharmaceutical/methods , Dermatologic Agents/pharmacokinetics , Dermatologic Agents/therapeutic use , Drug Design , Humans , Nanoparticles , Skin/metabolism , Skin/physiopathology , Skin Absorption , Skin Diseases/physiopathologyABSTRACT
Nanodermatology is a rapidly emerging field of study receiving significant interest because of its potential application in the prevention and treatment of skin diseases. However, nanoparticulate penetration into and through the skin is not feasible through topical application alone. Many physical and chemical approaches have been developed to enhance particulate penetration into skin. The most successful have been physical penetration enhancers. We have found that elongated microparticles can significantly improve topical nano- and microsphere delivery in an in vivo porcine model. The delivery efficiency was inversely related to the diameter of the payload. These data support a role for elongated microparticle enhanced delivery of nano- and submicron particulate cosmeceutical or therapeutic applications.
Subject(s)
Drug Carriers , Fluorescent Dyes/administration & dosage , Nanoparticles , Silicon Dioxide/administration & dosage , Skin Absorption , Skin/metabolism , Administration, Cutaneous , Animals , Chemistry, Pharmaceutical , Female , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Humans , Microscopy, Confocal , Nanomedicine , Particle Size , Silicon Dioxide/chemistry , Silicon Dioxide/metabolism , Swine , Technology, Pharmaceutical/methodsABSTRACT
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.
Subject(s)
Cosmetic Techniques , Microinjections/methods , Peptides/administration & dosage , Skin Physiological Phenomena , Surgery, Plastic/methods , Abdominoplasty , Administration, Cutaneous , Drug Delivery Systems/methods , Humans , MSH Release-Inhibiting Hormone/administration & dosage , Microscopy, Confocal , Oligopeptides/administration & dosage , Optical Imaging , Peptides/therapeutic use , SkinABSTRACT
Delivery of therapeutics into skin is hindered by the epidermal barriers. To overcome these barriers for the treatment of skin diseases, a cutaneous delivery method capable of field treatment using silica-elongated microparticles is developed. The microparticles are massaged into the skin using a 3D-printed microtextured applicator resulting in significant field-directed drug delivery enhancement.
Subject(s)
Drug Carriers/chemistry , Microspheres , Skin/metabolism , Administration, Cutaneous , Fluorescein/administration & dosage , Fluorescein/chemistry , Healthy Volunteers , Humans , In Vitro Techniques , Microscopy, Confocal , Silicon Dioxide/chemistryABSTRACT
Delivery of drugs and biomolecules into skin has significant advantages. To achieve this, herein, a nanomaterial-strengthened dissolving microneedle patch for transdermal delivery is reported. The patch comprises thousands of microneedles, which are composed of dissolving polymers, nanomaterials, and drug/biomolecules in their interior. With the addition of nanomaterials, the mechanical property of generally weak dissolving polymers can be dramatically improved without sacrificing dissolution rate within skin. In this experiments, layered double hydroxides (LDH) nanoparticles are incorporated into sodium carboxymethylcellulose (CMC) to form a nanocomposite. The results show that, by adding 5 wt% of LDH nanoparticles into CMC, the mechanical strength significantly increased. Small and densely packed CMC-LDH microneedles penetrate human and pig skin more reliably than pure CMC ones and attractively the nanocomposite-strengthened microneedles dissolve in skin and release payload within only 1 min. Finally, the application of using the nanocomposite-strengthened microneedle arrays is tested for in vivo vaccine delivery and the results show that significantly stronger antibody response could be induced when compared with subcutaneous injection. These data suggest that nanomaterials could be useful for fabricating densely packed and small polymer microneedles that have robust mechanical properties and rapid dissolution rates and therefore potential use in clinical applications.
Subject(s)
Drug Delivery Systems/instrumentation , Microinjections/instrumentation , Nanocomposites/chemistry , Needles , Administration, Cutaneous , Animals , Carboxymethylcellulose Sodium/chemistry , Humans , Hydroxides/chemistry , Nanocomposites/ultrastructure , Ovalbumin/administration & dosage , Ovalbumin/chemistry , Ovalbumin/immunology , Ovalbumin/pharmacokinetics , SwineABSTRACT
Using a pig ear skin model, it is demonstrated that silica vesicles show higher skin safety compared to dense silica nanoparticles with similar sizes. A hydrophobic UV blocker is efficiently dispersed in silica vesicles in an amorphous state, leading to ultrahigh UV-attenuating efficiency and a sun protection factor of 100 in a sunscreen formulation.
ABSTRACT
The delivery of therapeutics and cosmaceuticals into and/or through the skin is hindered by epidermal barriers. To overcome the skin's barriers we have developed a novel cutaneous delivery method using high aspect ratio elongate microparticles (EMPs). Using ex vivo and in vivo pig skin we assess the penetration and delivery characteristics of the elongate microparticles. With reflectance confocal microscopy we observed that the elongate microparticles successfully penetrated the epidermis and upper dermis. Delivery was then assessed using two different length populations of EMPs, comparing their delivery profile to topical alone using sodium fluorescein and confocal microscopy. We observed a relatively uniform and continuous delivery profile in the EMP treated area within the upper layers of the skin--up to seven times greater than topical alone. Finally, we delivered two therapeutically relevant compounds (Vitamins A and B3), showing enhanced delivery using the EMPs. To our knowledge this is the first report using high aspect ratio elongate microparticles in this manner for enhanced topical delivery to the skin.
Subject(s)
Drug Delivery Systems/instrumentation , Pharmaceutical Preparations/administration & dosage , Skin/metabolism , Vitamins/administration & dosage , Administration, Cutaneous , Animals , Equipment Design , Needles , Skin/ultrastructure , Skin Absorption , SwineABSTRACT
Skin photoageing results from a combination of factors including ultraviolet (sun) exposure, leading to significant changes in skin morphology and composition. Conventional methods assessing the degree of photoageing, in particular histopathological assessment involve an invasive multistep process. Advances in microscopy have enabled a shift towards non-invasive in vivo microscopy techniques such as reflectance confocal microscopy (RCM) in this context. Computational image analysis of RCM images has the potential to be of use in the non-invasive assessment of photoageing. In this report, we computationally characterized a clinical RCM data set from younger and older Caucasians with varying levels of photoageing. We identified several mathematical relationships that related to the degree of photoageing as assessed by conventional scoring approaches (clinical photography, SCINEXA and RCM). Furthermore, by combining the mathematical features into a single computational assessment score, we observed significant correlations with conventional RCM (P < 0.0001) and the other clinical assessment techniques.
Subject(s)
Image Processing, Computer-Assisted , Microscopy, Confocal/methods , Skin Aging , Skin Neoplasms/diagnosis , Adult , Age Factors , Algorithms , Automation , Female , Humans , Light , Male , Middle Aged , Models, Theoretical , Pattern Recognition, Automated , Skin/pathology , Skin Diseases/diagnosis , White PeopleABSTRACT
AIM: Nanoparticle removal from skin is relevant given the concern over topical nanoparticle toxicity. Zinc oxide nanoparticles (ZnO-NPs) are commonly used in sunscreens and their use is currently debated. This study explores the penetration and removal of ZnO-NPs from injured skin. MATERIALS & METHODS: Ex vivo/in vivo human skin was tape-stripped and/or microneedled followed by ZnO-NP application. After 2 h, treated skin was washed three-times using soapy water. Multiphoton tomography assessed the ZnO-NP signal before and after washing. RESULTS: Washing once removed over 85 and 83% of ZnO-NP signal from ex vivo intact and tape-stripped skin, respectively (p < 0.05) but only 28% (p = 0.5) was removed from puncture sites. A similar trend was found in vivo with removal of 85 and 93% of ZnO-NP signal from intact and tape-stripped skin, respectively (p < 0.05). CONCLUSION: Washing is effective for the removal of ZnO-NPs from superficial layers of intact and tape-stripped skin, but not from puncture wounds.
Subject(s)
Metal Nanoparticles/chemistry , Skin/chemistry , Zinc Oxide/chemistry , Humans , In Vitro TechniquesABSTRACT
We examine by both experimental and computational means the diffusion of macromolecules through the skin strata (both the epidermis and dermis). Using mouse skin as a test case, we present a novel high-resolution technique to characterize the diffusion properties of heterogeneous biomaterials using 3D imaging of fluorescent probes, precisely-deposited in minimally-perturbed in vivo skin layers. We find the diffusivity of the delivered macromolecules (70 kDa and 2 MDa rhodamine-dextrans) low within the packed cellular arrangement of the epidermis, while gradually increasing (by ~an order of magnitude) through the dermis--as pores in the fibrillar network enlarge from the papillary to the reticular dermis. Our experimental and computational approaches for investigating the diffusion through skin strata help in the assessment and optimization of controlled delivery of drugs (e.g. vaccines) to specific sites (e.g. antigen presenting cells).
Subject(s)
Skin Absorption/physiology , Algorithms , Animals , Delayed-Action Preparations , Dermis/metabolism , Dextrans , Diffusion , Drug Delivery Systems , Epidermis/metabolism , Fluorescence , Fluorescent Dyes , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , In Vitro Techniques , Mice , Microscopy, Confocal , RhodaminesABSTRACT
We describe the development of a sub-millimetre skin punch biopsy device for painless and suture-free skin sampling for molecular diagnosis and research. Conventional skin punch biopsies range from 2-4 mm in diameter. Local anaesthesia is required and sutures are usually used to close the wound. Our microbiopsy is 0.50 mm wide and 0.20 mm thick. The microbiopsy device is fabricated from three stacked medical grade stainless steel plates tapered to a point and contains a chamber within the centre plate to collect the skin sample. We observed that the application of this device resulted in a 0.21 ± 0.04 mm wide puncture site in volunteer skin using reflectance confocal microscopy. Histological sections from microbiopsied skin revealed 0.22 ± 0.12 mm wide and 0.26 ± 0.09 mm deep puncture sites. Longitudinal observation in microbiopsied volunteers showed that the wound closed within 1 day and was not visible after 7 days. Reflectance confocal microscope images from these same sites showed the formation of a tiny crust that resolved by 3 weeks and was completely undetectable by the naked eye. The design parameters of the device were optimised for molecular analysis using sampled DNA mass as the primary end point in volunteer studies. Finally, total RNA was characterized. The optimised device extracted 5.9 ± 3.4 ng DNA and 9.0 ± 10.1 ng RNA. We foresee that minimally invasive molecular sampling will play an increasingly significant role in diagnostic dermatology and skin research.