Polymersome Mediated Delivery of Mitochondrial therapeutics to parkin Mutant Fibroblasts

Mutations in parkin cause autosomal recessive Parkinsonism and mitochondrial defects. A recent drug screen identified a steroid like class of hydrophobic compounds able rescue mitochondrial function in mutant parkin fibroblasts. These included Ursolic Acid, Ursocholanic Acid, and Ursodeoxycholic Acid. pH-sensitive polymersomes, nanoparticles composed of amphiphilic block co- polymer, have been shown to encapsulate hydrophobic cargoes, enter cells without detriment to viability and release their cargoes therein. PMPC25−PDPA65 nanoparticles successfully encapsulating drugs were made by thin film rehydration, and purified by hollow fibre filtration. High encapsulation efficiencies were revealed by HPLC. Particle characterisation by and Transmission Electron Microscopy revealed a spectrum of morphologies, including spherical particles, branched tubular assemblies, and large high genus lyotropic structures. Morphological fractionation was achieved through stepwise centrifugation, and mass quantification showed drug encapsulation increased the relative proportion of tubular assemblies. Polymersomes were found to enter into parkin mutant fibroblasts without cytotoxic induction, or detriment to mitochondrial function as assessed by LDH release, mitochondrial membrane potential and cellular ATP levels. Drug loaded polymersomes of both spherical and tubular morphology increased cellular ATP levels of parkin mutant fibroblasts, and were found to deliver a fluorescent steroid at least as effectively as DMSO. The results presented here suggest PMPC-PDPA nanoparticles are suitable for use as a therapeutic vector in parkin mutant cells. The production of spherical and tubular nanoparticle morphologies would be of interest to in vivo applications, each being known to show distinct properties affecting nanoparticle distribution within the body

‘Shell shock’ Revisited: An Examination of the Case Records of the National Hospital in London

During the First World War the National Hospital for the Paralysed and Epileptic, in Queen Square, London, then Britain’s leading centre for neurology, took a key role in the treatment and understanding of shell shock. This paper explores the case notes of all 462 servicemen who were admitted with functional neurological disorders between 1914 and 1919. Many of these were severe or chronic cases referred to the National Hospital because of its acknowledged expertise and the resources it could call upon. Biographical data was collected together with accounts of the patient’s military experience, his symptoms, diagnostic interpretations and treatment outcomes. Analysis of the notes showed that motor syndromes (loss of function or hyperkinesias), often combined with somato-sensory loss, were common presentations. Anxiety and depression as well as vegetative symptoms such as sweating, dizziness and palpitations were also prevalent among this patient population. Conversely, psychogenic seizures were reported much less frequently than in comparable accounts from German tertiary referral centres. As the war unfolded the number of physicians who believed that shell shock was primarily an organic disorder fell as research failed to find a pathological basis for its symptoms. However, little agreement existed among the Queen Square doctors about the fundamental nature of the disorder and it was increasingly categorised as functional disorder or hysteria

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 β-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, β-cyclodextrin (βCD) 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 βCD 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 βCD 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 βCD 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