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

Abstract

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.

Keywords: Anisotropic nanoparticle; Mitochondria; PMPC-PDPA; Parkin; Parkinson’s disease; Polymersome.

Fig. 1

UA and UCA loaded nanoparticles. From left to right, TEM images of polymersome and tube fractions (scale bars = 500 nm, mol/mol cargo:copolymer ratios indicated), DLS auto-correlation functions and estimated size distribution by intensity from (a) empty, (b) UA and (c) UCA loaded nanoparticles.

Fig. 2

PMPC₂₅-PDPA₆₅ nanoparticles of mixed tubular and polymersome morphologies enter, and cause no apparent toxicity to, parkin-mutant fibroblasts. (a) Percentage LDH release, (b) cellular ATP levels and (c) MMP were assessed in a set of age matched parkin-mutant (black) and control (grey) fibroblasts following 48 h incubation with PMPC₂₅-PDPA₆₅ of nanoparticles of mixed morphology (error bars = SD, n = 3). The uptake kinetics of Rh.6G labelled PMPC₂₅-PDPA₆₅ nanoparticles of mixed morphology was assessed by flow cytometry at concentrations of (d) 0.1 (inset shows results on a reduced MFI scale), (e) 0.5 and (f) 1 mg/mL (mean MFIs from three sets of age matched parkin-mutant and control fibroblasts shown, error bars = SD, n = 3).

Fig. 3

UA and UCA encapsulated within PMPC₂₅-PDPA₆₅ polymersomes (dotted lines) and tubes (dashed lines), elicit dose dependent increases in fibroblast ATP levels similar to when dissolved in 0.1% DMSO (solid lines). Control (grey) and matched parkin-mutant fibroblasts (black) were incubated with formulations of UA for (a) 24 and (b) 48 h, as well as UCA formulations for (c) 24 and (d) 48 h (error bars = SD, n = 3).