Modeling ciliopathies in patient-derived primary cells.

Ciliopathies comprise a group of inherited diseases caused by mutations in genes encoding proteins that localize to cilia or centrosomes. They afflict multiple organs and are one of the most frequent monogenic causes of kidney failure in adults, adolescents and children. Primary cilia play diverse roles in cell signaling, cell cycle regulation, planar cell polarity and mechanosensing. The use of patient-derived cells possessing endogenous disease causing mutations enables the study of these processes and their dysregulation in disease. Here we describe methods to cultivate patient-derived dermal fibroblast and renal epithelial cells isolated from urine. Fibroblasts are highly robust, long-lived, and easy to culture cells in which ciliary assembly can be easily induced. Similarly, the ability to acquire and culture ciliated renal epithelial cells without patient-invasive-intervention holds great potential to further our understanding of ciliopathies. In addition to monolayer cultures, we also detail the formation of three-dimensional renal-epithelial organoids-so-called tubuloids-that demonstrate epithelial-polarization and transepithelial transport activities like those seen in vivo renal-tubules. These in vitro models are powerful tools to investigate the underlying disease mechanisms of human ciliopathies that can be employed without the need for heavy-handed genetic or molecular manipulations.

Breaking the Barrier - Potent Anti-Inflammatory Activity following Efficient Topical Delivery of Etanercept using Thermoresponsive Nanogels.

Topical administration permits targeted, sustained delivery of therapeutics to human skin. Delivery to the skin, however, is typically limited to lipophilic molecules with molecular weight of < 500 Da, capable of crossing the stratum corneum. Nevertheless, there are indications protein delivery may be possible in barrier deficient skin, a condition found in several inflammatory skin diseases such as psoriasis, using novel nanocarrier systems. METHODS: Water in water thermo-nanoprecipitation; dynamic light scattering; zeta potential measurement; nanoparticle tracking analysis; atomic force microscopy; cryogenic transmission electron microscopy; UV absorption; centrifugal separation membranes; bicinchoninic acid assay; circular dichroism; TNFα binding ELISA; inflammatory skin equivalent construction; human skin biopsies; immunohistochemistry; fluorescence microscopy; western blot; monocyte derived Langerhans cells; ELISA Results: Here, we report the novel synthesis of thermoresponsive nanogels (tNG) and the stable encapsulation of the anti-TNFα fusion protein etanercept (ETR) (~150 kDa) without alteration to its structure, as well as temperature triggered release from the tNGs. Novel tNG synthesis without the use of organic solvents was conducted, permitting in situ encapsulation of protein during assembly, something that holds great promise for easy manufacture and storage. Topical application of ETR loaded tNGs to inflammatory skin equivalents or tape striped human skin resulted in efficient ETR delivery throughout the SC and into the viable epidermis that correlated with clear anti-inflammatory effects. Notably, effective ETR delivery depended on temperature triggered release following topical application. CONCLUSION: Together these results indicate tNGs hold promise as a biocompatible and easy to manufacture vehicle for stable protein encapsulation and topical delivery into barrier-deficient skin.

Enhanced topical delivery of dexamethasone by ß-cyclodextrin decorated thermoresponsive nanogels.

Highly hydrophilic, responsive nanogels are attractive as potential systems for the topical delivery of bioactives encapsulated in their three-dimensional polymeric scaffold. Yet, these drug carrier systems suffer from drawbacks for efficient delivery of hydrophobic drugs. Addressing this, ß-cyclodextrin (ßCD) could be successfully introduced into the drug carrier systems by exploiting its unique affinity toward dexamethasone (DXM) as well as its role as topical penetration enhancer. The properties of ßCD could be combined with those of thermoresponsive nanogels (tNGs) based on dendritic polyglycerol (dPG) as a crosslinker and linear thermoresponsive polyglycerol (tPG) inducing responsiveness to temperature changes. Electron paramagnetic resonance (EPR) studies localized the drug within the hydrophobic cavity of ßCD by differences in its mobility and environmental polarity. In fact, the fabricated carriers combining a particulate delivery system with a conventional penetration enhancer, resulted in an efficient delivery of DXM to the epidermis and the dermis of human skin ex vivo (enhancement compared to commercial DXM cream: ∼2.5 fold in epidermis, ∼30 fold in dermis). Furthermore, DXM encapsulated in ßCD tNGs applied to skin equivalents downregulated the expression of proinflammatory thymic stromal lymphopoietin (TSLP) and outperformed a commercially available DXM cream.

Rescue of mitochondrial function in parkin-mutant fibroblasts using drug loaded PMPC-PDPA polymersomes and tubular polymersomes.

Mutations in parkin cause autosomal recessive Parkinsonism and mitochondrial defects. A recent drug screen identified a class of steroid-like hydrophobic compounds able to rescue mitochondrial function in parkin-mutant fibroblasts. Whilst these possess therapeutic potential, the size and high hydrophobicity of some may limit their ability to penetrate the blood-brain barrier from systemic circulation, something that could be improved by novel drug formulations. In the present study, the steroid-like compounds Ursolic Acid (UA) and Ursocholanic Acid (UCA) were successfully encapsulated within nanoscopic polymersomes formed by poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-di-isopropylamino)ethyl methacrylate) (PMPC-PDPA) and separated into spherical and tubular morphologies to assess the effects of nanoparticle mediated delivery on drug efficacy. Following incubation with either morphology, parkin-mutant fibroblasts demonstrated time and concentration dependent increases in intracellular ATP levels, resembling those resulting from treatment with nascent UA and UCA formulated in 0.1% DMSO, as used in the original drug screen. Empty PMPC-PDPA polymersomes did not alter physiological measures related to mitochondrial function or induce cytotoxicity. In combination with other techniques such as ligand functionalisation, PMPC-PDPA nanoparticles of well-defined morphology may prove a promising platform for tailoring the pharmacokinetic profile and organ specific bio-distribution of highly hydrophobic compounds.