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

Effect of Delivery Platforms Structure on the Epidermal Antigen Transport for Topical Vaccination

Transdermal immunization is highly attractive because of the skin's accessibility and unique immunological characteristics. However, it remains a relatively unexplored route of administration because of the great difficulty of transporting antigens past the outermost layer of skin, the stratum corneum. In this article, the abilities of three poly(N-vinylcaprolactam) (PVCL)-based thermoresponsive assemblies - PVCL hydrogels and nanogels plus novel film forming PVCL/acrylic nanogels - to act as protein delivery systems were investigated. Similar thermal responses were observed in all systems, with transition temperatures close to 32 °C, close to that of the skin surface. The investigated dermal delivery systems showed no evidence of cytotoxicity in human fibroblasts and were able to load and release ovalbumin (OVA), a well-studied antigen, in a temperature-dependent manner in vitro. The penetration of OVA into ex vivo human skin following topical application was evaluated, where enhanced skin delivery was seen for the OVA-loaded PVCL systems relative to administration of the protein alone. The distinct protein release and skin penetration profiles observed for the different PVCL assemblies were here discussed on the basis of their structural differences.Fil: Sonzogni, Ana Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Yealland, Guy. Universidad Libre de Berlin; AlemaniaFil: Kar, Mrityunjoy. Universidad Libre de Berlin; AlemaniaFil: Wedepohl, Stefanie. Universidad Libre de Berlin; AlemaniaFil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: González, Verónica Doris Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Hedtrich, Sarah. Universidad Libre de Berlin; AlemaniaFil: Calderon, Marcelo. Universidad Libre de Berlin; AlemaniaFil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentin

pH-Sensitive Tubular Polymersomes: Formation and Applications in Cellular Delivery

Optimizing the shape of a nanovector influences its interaction with a cell and determines the internalization kinetics. Block copolymer amphiphiles self-assemble into monodisperse structures in aqueous solutions and have been explored extensively as drug delivery vectors. However, the structure of self-assembled block copolymers has mainly been limited to spherical vesicles or spherical and worm-like micelles. Here we show the controlled formation and purification of tubular polymersomes, long cylindrical vesicles. Tubular polymersomes are purified from other structures, and their formation is manipulated by incorporating the biocompatible membrane components cholesterol and phospholipids. Finally we show that these tubular polymersomes have different cellular internalization kinetics compared with spherical polymersomes and can successfully encapsulate and deliver fluorescent bovine serum albumin protein intracellularly

Protein Corona Formation on Colloidal Polymeric Nanoparticles and Polymeric Nanogels: Impact on Cellular Uptake, Toxicity, Immunogenicity, and Drug Release Properties

The adsorption of biomolecules to the surface of nanoparticles (NPs) following administration into biological environments is widely recognized. In particular, the “protein corona” is well understood in terms of formation kinetics and impact upon the biological interactions of NPs. Its presence is an essential consideration in the design of therapeutic NPs. In the present study, the protein coronas of six polymeric nanoparticles of prospective therapeutic use were investigated. These included three colloidal NPssoft core–multishell (CMS) NPs, plus solid cationic Eudragit RS (EGRS), and anionic ethyl cellulose (EC) nanoparticlesand three nanogels (NGs)thermoresponsive dendritic-polyglycerol (dPG) nanogels (NGs) and two amino-functionalized dPG-NGs. Following incubation with human plasma, protein coronas were characterized and their biological interactions compared with pristine NPs. All NPs demonstrated protein adsorption and increased hydrodynamic diameters, although the solid EGRS and EC NPs bound notably more protein than the other tested particles. Shifts toward moderately negative surface charges were also observed for all corona bearing NPs, despite varied zeta potentials in their pristine states. While the uptake and cellular adhesion of the colloidal NPs in primary human keratinocytes and human umbilical vein endothelial cells were significantly decreased when bearing the protein corona, no obvious impact was seen in the NGs. By contrast, corona bearing NGs induced marked increases in cytokine release from primary human macrophages not seen with corona bearing colloidal NPs. Despite this, no apparent enhancement to in vitro toxicity was noted. Finally, drug release from EGRS and EC NPs was assessed, where a decrease was seen in the EGRS NPs alone. Together these results provide a direct comparison of the physical and biological impact the protein corona has on NPs of widely varied character and in particular highlights a distinction between the corona’s effects on NGs and colloidal NPs

Biocompatibility and characterization of polyglycerol-based thermoresponsive nanogels designed as novel drug-delivery systems and their intracellular localization in keratinocytes

<p>Novel nanogels that possess the capacity to change their physico-chemical properties in response to external stimuli are promising drug-delivery candidates for the treatment of severe skin diseases. As thermoresponsive nanogels (tNGs) are capable of enhancing penetration through biological barriers such as the <i>stratum corneum</i> and are taken up by keratinocytes of human skin, potential adverse consequences of their exposure must be elucidated. In this study, tNGs were synthesized from dendritic polyglycerol (dPG) and two thermoresponsive polymers. tNG_dPG_tPG are the combination of dPG with poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)) and tNG_dPG_pNIPAM the one with poly(N-isopropylacrylamide) (pNIPAM). Both thermoresponsive nanogels are able to incorporate high amounts of dexamethasone and tacrolimus, drugs used in the treatment of severe skin diseases. Cellular uptake, intracellular localization and the toxicological properties of the tNGs were comprehensively characterized in primary normal human keratinocytes (NHK) and in spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT). Laser scanning confocal microscopy revealed fluorescently labeled tNGs entered into the cells and localized predominantly within lysosomal compartments. MTT assay, comet assay and carboxy-H2DCFDA assay, demonstrated neither cytotoxic or genotoxic effects, nor any induction of reactive oxygen species of the tNGs in keratinocytes. In addition, both tNGs were devoid of eye irritation potential as shown by bovine corneal opacity and permeability (BCOP) test and red blood cell (RBC) hemolysis assay. Therefore, our study provides evidence that tNGs are locally well tolerated and underlines their potential for cutaneous drug delivery.</p