Polyelectrolyte-coated nanocapsules containing cyclosporine A protect neuronal-like cells against oxidative stress-induced cell damage

https://doi.org/10.1016/j.colsurfa.2018.07.005Get rights and content

Abstract

Experimental data have demonstrated the neuroprotective potential of cyclosporine A (CsA), however, its clinical usage as neuroprotectant is limited due to poor blood-brain barrier permeability and potential peripheral and central toxicity. In order to overcome these limitations, the nanoparticulate CsA delivery technology has been recently proposed to improve CsA immunosuppressive potential, however, its neuroprotective action has not been sufficiently studied. Thus, in the present study two types of (bio)polyelectrolyte-coated nanocapsules containing CsA (AOT/PLL-CsA and AOT/PLL/PGA-CsA) were synthesized using a nanoemulsification technique and the layer-by-layer (LbL) saturation method. By using human neuroblastoma SH-SY5Y cells, we demonstrated that CsA alone at a concentration above 0.1 μM had a cell-damaging effect. CsA at lower concentrations (0.1–10 nM) was devoid of toxicity and reduced the hydrogen peroxide (H2O2)-induced cell damage, whereas its higher contents (0.5 and 1 μM) increased oxidative stress-induced cell death. Further studies showed that both forms of nanoencapsulated CsA were biocompatible and protected SH-SY5Y cells against the H2O2-induced damage in the range of concentrations 0.04–0.16 μM, therefore, the proposed nanoformulations for CsA delivery are suitable to reduce its cytotoxic effects and enhance the neuroprotective activity against oxidative stress.

Introduction

An elevated intracellular oxidative stress is observed in various neurodegenerative conditions, including Alzheimer's, Parkinson's, and Huntington's diseases [[1], [2], [3], [4]], however, the question on its role as a contributor or a consequence of neurodegeneration remains still open. Reactive oxygen species (ROS) including superoxide anion (O2radical dot), hydroxyl radical (HOradical dot) and peroxides (H2O2, R-OOH, R-OOradical dot) are primarily produced by an electron transport chain. However, there are also other sources of ROS, such as mitochondrial (monoamine oxidase A and B) or cytosolic (NADPH oxidase, xanthine oxidase) enzymes [1]. The cellular redox balance is maintained by enzymatic (superoxide dismutase, glutathione peroxidase, catalase) and non-enzymatic (glutathione, vitamin E) antioxidant systems [1,2,5]. Mitochondrial dysfunctions and lower efficiency of endogenous antioxidants, which not rarely appear during neurodegeneration, are claimed to be a leading cause of elevated oxidative stress that by direct (oxidation of lipids, proteins, and nucleic acids) or indirect (induction of pro- and inhibition of anti-apoptotic factors) mechanisms evokes neuronal cell demise [4,6,7]. Critical mitochondrial targets which could be affected by elevated intracellular ROS or Ca2+ include the mitochondrial permeability transition (MPT) pore [8,9]. Since this pore opening under pathological situations causes the release of various pro-death signals (e.g., cytochrome c, SMAC/DIABLO, apoptosis-inducing factor (AIF)), its specific inhibition could be a promising protective strategy. Many years of research have shown that inhibition of the MPT channel could be strongly neuroprotective during acute CNS pathologies (traumatic brain injury (TBI), spinal cord traumatic injury (SCI) or ischemia), however, in recent years studies have shown that it could also be true for the age-related neurodegenerative diseases (Alzheimer’s and Parkinson’s diseases) [[8], [9], [10], [11]].

The immunosuppressive drug cyclosporine A (CsA), widely used in transplant medicine and autoimmune diseases (e.g., rheumatoid arthritis, psoriasis), has been shown to act as a blocker of the MPT channel by binding to the cyclophilin D (CypD) [12,13]. The neuroprotective effects of this drug have been demonstrated in various cellular and animal models of neuronal injury as well as in clinical trials (Phase II) for traumatic brain injury (TBI) [13]. However, the usage of CsA as a neuroprotective drug is limited by its poor biopharmaceutical properties (low aqueous solubility, low permeability through the biological membranes due to high molecular weight and an extensive pre-systemic metabolism) and by possible peripheral (nephrotoxicity and hepatotoxicity) and central toxicity effects of high doses of this drug needed to achieve therapeutic efficacy [[14], [15], [16], [17], [18]]. In respect to neuroprotection, many new approaches have been tested to improve CsA neuroprotective properties and eliminate toxic and immunosuppressive impacts (e.g., CsA analogs, CsA in low doses plus BBB disrupting agents) [13,16,19]. Recently, the nanoparticulate CsA delivery technology was highly improved, and it is believed to have a higher efficiency and potentially reduced toxicity [[20], [21], [22]]. Among the proposed various nanoparticulate delivery systems designed mainly to improve CsA immunosuppressive activity, there have been: PLGA (poly(lactic-co-glycolic acid)), PEG-PLGA (PEG-ylated poly(lactic-co-glycolic acid)), DL-PLGA (poly(dl-(lactic-co-glycolic acid)), IBCA (poly(isobutyl-2-cyanoacrylate)), PCL (poly-E-caprolactone) or lipid nanoparticles [[22], [23], [24], [25], [26], [27]]. Moreover, nanoparticulate CsA showed the promise for the efficient ocular drug delivery for the treatment of the dry eye disease [16,28,29]. CsA nanoformulations were also tested in relation to cardioprotection [30]. Recently, it has been shown that polyethylene glycol (PEG)-transactivating-transduction protein (TAT)-modified, CsA-loaded cationic multifunctional polymeric liposome-poly(lactic-co-glycolic acid) (PLGA) core/shell nanoparticles (PLGA/CsA NPs) were neuroprotective in animal spinal cord injury (SCI) model [31].

With the aim to extend a neuroprotective portfolio of CsA nanoformulations, in the present study we tested the neuroprotective potential of two types of polymer-encapsulated CsA in a neuronal cell model of oxidative stress. For this purpose, we used human neuroblastoma SH-SY5Y cells, which are commonly applied in neuroprotection and neurotoxicity studies [[32], [33], [34], [35]]. Taking into account the possibility that neuronal differentiation of SH-SY5Y cells (e.g., by treatment with retinoic acid) could mask the neuroprotective effects of tested compounds [32,[36], [37], [38], [39]] we performed experiments in undifferentiated cells. The hydrogen peroxide (H2O2), a widely used exogenous oxidative stress inducer in neuroprotection screening assays [33,[41], [42], [43], [44], [45]], was chosen to induce SH-SY5Y cell damage.

Section snippets

Chemicals for nanocapsules’ synthesis

Cyclosporine A (CsA; cat. No C3662), docusate sodium salt (AOT), Poly(l-lysine hydrobromide) (PLL) and Poly(l-glutamic acid) sodium salt (PGA) were obtained from Sigma Aldrich. All materials were used as purchased without further purification. The ultrapure water was obtained using the Millipore Direct-Q5 UV purification system.

Materials for cell culture and cytotoxicity tests

Dulbecco’s Modified Eagle Medium (DMEM), Trypsin/EDTA solution, fetal bovine serum (FBS) and supplement N2 were from Gibco. The Cytotoxicity Detection Kit (LDH release

Characterization of nanocapsules

The values of the zeta potential of the synthesized AOT/PLL and AOT/PLL/PGA nanocapsules were 43 and −41 mV, respectively. The values of the average hydrodynamic diameter of AOT/PLL and AOT/PLL/PGA (Fig. 1A) nanocapsules were 157.5 and 160 nm, respectively. The values of the polydispersity index of AOT/PLL and AOT/PLL/PGA nanocapsules were 0.224 and 0.117, respectively. Representative SEM image of AOT/PLL/PGA nanocapsules is shown in Fig. 1B. There were no significant changes in the

Conclusions

Although various nanoparticle systems for CsA delivery have been proposed in recent years, they have been mainly focused on improving CsA pharmacokinetic (increased bioavailability, limited toxicity) and immunosuppressive properties [[22], [23], [24], [25], [26], [27]]. In the present study, we proposed two forms of nanoparticles (AOT/PLL and AOT/PLL/PGA) for CsA delivery and showed their biocompatibility and slightly increased (AOT/PLL-CsA) or maintained (AOT/PLL/PGA-CsA) neuroprotective

Conflict of interest

The author reports no conflicts of interest in this work.

Acknowledgments

We kindly thank Ms. Barbara Korzeniak for her excellent technical assistance. The research was funded by the Polish-Norwegian Research Programme operated by the National Centre for Research and Development under the Norwegian Financial Mechanism 2009–2014 in the frame of Project Contract No Pol-Nor/199523/64/2013 NanoNeucar.

References (58)

  • M. Guzmán et al.

    Formation and characterization of cyclosporine-loaded nanoparticles

    J. Pharm. Sci.

    (1993)
  • J.L. Italia et al.

    PLGA nanoparticles for oral delivery of cyclosporine: nephrotoxicity and pharmacokinetic studies in comparison to Sandimmune Neoral®

    J. Control. Release

    (2007)
  • J. Jaiswal et al.

    Preparation of biodegradable cyclosporine nanoparticles by high-pressure emulsification-solvent evaporation process

    J. Control. Release

    (2004)
  • P. Aksungur et al.

    Development and characterization of cyclosporine A loaded nanoparticles for ocular drug delivery: cellular toxicity, uptake, and kinetic studies

    J. Control. Release

    (2011)
  • Y.T. Cheung et al.

    Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research

    Neurotoxicology

    (2009)
  • F.M. Lopes et al.

    Comparison between proliferative and neuron-like SH-SY5Y cells as an in vitro model for Parkinson disease studies

    Brain Res.

    (2010)
  • D. Jantas et al.

    The attenuating effect of memantine on staurosporine-, salsolinol- and doxorubicin-induced apoptosis in human neuroblastoma SH-SY5Y cells

    Neurochem. Int.

    (2008)
  • D. Jantas et al.

    Neuroprotective effects of metabotropic glutamate receptor group II and III activators against MPP(+)-induced cell death in human neuroblastoma SH-SY5Y cells: the impact of cell differentiation state

    Neuropharmacology

    (2014)
  • D. Jantas et al.

    Neuroprotective effects of mGluR II and III activators against staurosporine- and doxorubicin-induced cellular injury in SH-SY5Y cells: new evidence for a mechanism involving inhibition of AIF translocation

    Neurochem. Int.

    (2015)
  • M. Richter et al.

    SK channel activation modulates mitochondrial respiration and attenuates neuronal HT-22 cell damage induced by H2O2

    Neurochem. Int.

    (2015)
  • V. Schaeffer et al.

    Selective regulation of neurosteroid biosynthesis in human neuroblastoma cells under hydrogen peroxide-induced oxidative stress condition

    Neuroscience

    (2008)
  • G. Wendt et al.

    Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death

    Neuroscience

    (2014)
  • M. Piotrowski et al.

    Polyelectrolyte-coated nanocapsules containing undecylenic acid: synthesis, biocompatibility and neuroprotective properties

    Colloids Surf. B Biointerfaces

    (2015)
  • K. Domañska-Janik et al.

    Neuroprotection by cyclosporin A following transient brain ischemia correlates with the inhibition of the early efflux of cytochrome C to cytoplasm

    Brain Res. Mol. Brain Res.

    (2004)
  • L. Schultz et al.

    Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models

    Toxicol. In Vitro

    (2015)
  • C.P. Fall et al.

    Visualization of cyclosporin A and Ca2+-sensitive cyclical mitochondrial depolarizations in cell culture

    Biochim. Biophys. Acta

    (1999)
  • A. Xiao et al.

    The cyclophilin D/Drp1 axis regulates mitochondrial fission contributing to oxidative stress-induced mitochondrial dysfunctions in SH-SY5Y cells

    Biochem. Biophys. Res. Commun.

    (2017)
  • Y. Muramatsu et al.

    Neuroprotective efficacy of FR901459, a novel derivative of cyclosporin A, in in vitro mitochondrial damage and in vivo transient cerebral ischemia models

    Brain Res.

    (2007)
  • S. Gandhi et al.

    Mechanism of oxidative stress in neurodegeneration

    Oxid. Med. Cell. Longev.

    (2012)
  • Cited by (0)

    1

    These authors contributed equally to this work.

    2

    Current address: International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga, Braga 4715-330, Portugal.

    View full text