Development of a 3D pigmented skin model to evaluate RNAi-induced depigmentation.

Because current skin whitening agents often have insufficient efficacy and side effects, we aim to develop effective and safe therapeutics using RNA interference (RNAi). We established a pigmented human-reconstructed skin model as a first step in the development of novel siRNA-based depigmenting agents. Histological characterization revealed that our model had a similar morphology as normal human skin, expressed keratinocyte differentiation as well as basement membrane markers, and showed a high degree of pigmentation. The utility of the model to study RNAi-induced depigmentation was validated by incorporation of melanocytes transfected with siRNA against tyrosinase, a key enzyme in skin pigmentation. This resulted in a strong reduction in pigmentation and inhibition of melanin transfer proving that siRNA-mediated gene silencing in melanocytes worked successfully in our model. Therefore, this self-made 3D skin model will be a useful and easy tool to validate the whitening potential of candidate genes with a presumed function in melanin synthesis or transfer.

Identification of miR-145 as a key regulator of the pigmentary process.

The current treatments for hyperpigmentation are often associated with a lack of efficacy and adverse side effects. We hypothesized that microRNA (miRNA)-based treatments may offer an attractive alternative by specifically targeting key genes in melanogenesis. The aim of this study was to identify miRNAs interfering with the pigmentary process and to assess their functional role. miRNA profiling was performed on mouse melanocytes after three consecutive treatments involving forskolin and solar-simulated UV (ssUV) irradiation. Sixteen miRNAs were identified as differentially expressed in treated melan-a cells versus untreated cells. Remarkably, a 15-fold downregulation of miR-145 was detected. Overexpression or downregulation of miR-145 in melan-a cells revealed reduced or increased expression of Sox9, Mitf, Tyr, Trp1, Myo5a, Rab27a, and Fscn1, respectively. Moreover, a luciferase reporter assay demonstrated direct targeting of Myo5a by miR-145 in mouse and human melanocytes. Immunofluorescence tagging of melanosomes in miR-145-transfected human melanocytes displayed perinuclear accumulation of melanosomes with additional hypopigmentation of harvested cell pellets. In conclusion, this study has established an miRNA signature associated with forskolin and ssUV treatment. The significant down- or upregulation of major pigmentation genes, after modulating miR-145 expression, suggests a key role for miR-145 in regulating melanogenesis.

Three-dimensional skin models as tools for transdermal drug delivery: challenges and limitations.

INTRODUCTION: Transdermal drug delivery has several known advantages over the oral route and hypodermic injections. The number of drugs that can be taken up transdermally is, however, limited owing to the innate barrier function of the skin. New transdermal drug candidates need to be tested extensively before being used on humans. In this regard, in vitro permeation methods are highly important to predict in vivo permeation of drugs. AREAS COVERED: This review illustrates how different types of reconstructed skin models are being used as alternatives to human and pig skin for in vitro permeation testing of drugs. Insights into how various factors (including the physicochemical nature of molecules and formulations) or skin properties might affect the permeability of drugs in reconstructed skin models are provided. Also, opportunities and pitfalls of reconstructed skin models are highlighted. EXPERT OPINION: Many studies have revealed that the permeability of reconstructed skin models is much higher compared with human excised skin. This is in accordance with the incomplete barrier found in these models. Nevertheless, the reconstructed skin models available today are useful tools for estimating the rank order of percutaneous absorption of a series of compounds with different physicochemical properties. A major challenge in the further development of reconstructed skin models for drug delivery studies is to obtain a barrier function similar to in vivo skin. Whether this goal will be achieved in the near future is uncertain and will be, in the authors' opinion, a very difficult task.

Lipid-mediated gene delivery to the skin.

Cutaneous gene delivery methods have been developed over the past decades as therapeutic strategies for the treatment of a variety of skin disorders. Both viral and non-viral techniques have been frequently described. Mainly due to safety concerns, the application of viral methods is being questioned and non-viral alternatives are gaining major interest. Lipid-based vesicles for the delivery of plasmid DNA by topical application onto the skin hold great potential and have been investigated thoroughly. Here, we give an overview of the different lipid vesicles that have been described in literature. Next to the conventional phospholipid liposomes, new generation liposomes like niosomes and Transfersomes® have been developed for enhanced (trans)dermal delivery. In addition, we draw attention to other lipid-based delivery systems, that could not be classified into one of these categories. Clearly, lipid-based delivery vehicles demonstrate very promising results for DNA delivery into and through the skin, especially for cutaneous vaccination purposes. Apart from simple topical application onto the skin, liposomes have also been described in combination with delivery enhancing techniques. Here we describe this combined approach for some specific skin disorders.

Knockdown of myosin Va isoforms by RNAi as a tool to block melanosome transport in primary human melanocytes.

The movement of melanosomes, dense melanin-containing organelles, within human melanocytes is actin-dependent and mediated through the formation of a Rab27a-Slac2-a-myosin Va (MyoVa) protein complex. We previously showed that only the melanocyte-specific exon F isoforms of MyoVa are involved in melanosome transport to the dendrite extremities. Here, we investigate siRNA to downregulate the exon F-containing isoforms of MyoVa in primary human melanocytes. Efficient and specific knockdown of the MyoVa exon F isofoms were shown at both mRNA and protein levels. Further, a stable shRNA against the MyoVa exon F isoforms was prepared by using a lentiviral system to improve and confirm the silencing effect in hard-to-transfect melanocyte cells. Immunofluorescence microscopy shows that knockdown of the exon F isoforms results in blockade of intramelanocytic melanosome transport due to the inability to form the Rab27a-Slac2-a-MyoVa tripartite complex. Interestingly, the observed phenotypic effect (that is, perinuclear accumulation of melanosomes) is the same as that seen in melanocytes from patients with human Griscelli syndrome causing abnormal pigmentation. We conclude that our siRNA-based strategy provides a previously unreported tool to block the intracellular melanosome movement in primary human melanocytes and may become an innovative drug to treat hyperpigmentation.