Endothelial delivery of antioxidant enzymes loaded into non-polymeric magnetic nanoparticles

https://doi.org/10.1016/j.jconrel.2010.05.003Get rights and content

Abstract

Antioxidant enzymes have shown promise as a therapy for pathological conditions involving increased production of reactive oxygen species (ROS). However the efficiency of their use for combating oxidative stress is dependent on the ability to achieve therapeutically adequate levels of active enzymes at the site of ROS-mediated injury. Thus, the implementation of antioxidant enzyme therapy requires a strategy enabling both guided delivery to the target site and effective protection of the protein in its active form. To address these requirements we developed magnetically responsive nanoparticles (MNP) formed by precipitation of calcium oleate in the presence of magnetite-based ferrofluid (controlled aggregation/precipitation) as a carrier for magnetically guided delivery of therapeutic proteins. We hypothesized that antioxidant enzymes, catalase and superoxide dismutase (SOD), can be protected from proteolytic inactivation by encapsulation in MNP. We also hypothesized that catalase-loaded MNP applied with a high-gradient magnetic field can rescue endothelial cells from hydrogen peroxide toxicity in culture. To test these hypotheses, a family of enzyme-loaded MNP formulations were prepared and characterized with respect to their magnetic properties, enzyme entrapment yields and protection capacity. SOD- and catalase-loaded MNP were formed with average sizes ranging from 300 to 400 nm, and a protein loading efficiency of 20–33%. MNP were strongly magnetically responsive (magnetic moment at saturation of 14.3 emu/g) in the absence of magnetic remanence, and exhibited a protracted release of their cargo protein in plasma. Catalase stably associated with MNP was protected from proteolysis and retained 20% of its initial enzymatic activity after 24 h of exposure to pronase. Under magnetic guidance catalase-loaded MNP were rapidly taken up by cultured endothelial cells providing increased resistance to oxidative stress (62 ± 12% cells rescued from hydrogen peroxide induced cell death vs. 10 ± 4% under non-magnetic conditions). We conclude that non-polymeric MNP formed using the controlled aggregation/precipitation strategy are a promising carrier for targeted antioxidant enzyme therapy, and in combination with magnetic guidance can be applied to protect endothelial cells from oxidative stress mediated damage. This protective effect of magnetically targeted MNP impregnated with antioxidant enzymes can be highly relevant for the treatment of cardiovascular disease and should be further investigated in animal models.

Graphical abstract

Introduction

Magnetically guided delivery strategies have the potential to enhance the therapeutic profile of a broad range of pharmaceuticals by increasing their distribution to the site of action, while minimizing off-target interactions [1]. Therefore, encapsulation in Magnetically-targeted NanoParticles (MNP) can increase efficiency and limit adverse effects of drug cargoes, facilitating the introduction of novel pharmacological interventions in clinical use. In support of this concept, the feasibility of using MNP for targeted delivery of small molecule pharmaceuticals, gene vectors, and cells has been addressed in a number of recent in vitro and animal model studies [2], [3], [4], [5], [6]. However, the utility and therapeutic potential of MNP for site-specific delivery of biologically active enzymes has remained largely unexplored due to considerable challenges in the design of such formulations.

Maintaining protein integrity and functionality in the process of nano- or microparticles formulation is not a trivial concern [7]. Stability of proteins incorporated in particulate systems based on biodegradable polymers, polylactide or poly(lactide-co-glycolide), has been investigated in a number of recent studies [8], [9], [10] using lysozyme, growth hormone, tetanus toxoid and growth factors as model cargoes. The primary mechanisms responsible for the compromised protein stability in these formulations have been shown to be related to the particle interior acidification due to autocatalytic degradation of the matrix polymer, as well as irreversible protein aggregation [7], both adversely affecting the structural integrity and biological activity of the encapsulated agents.

In this study a novel formulation method based on the use of a biocompatible fatty acid calcium salt as a non-polymeric particle matrix component was developed and applied to create a family of MNP further characterized in vitro as a platform for magnetically guided delivery of therapeutic enzymes. To combat the challenges of enzyme delivery, an ideal MNP formulation approach would not only enable loading of therapeutically adequate amounts of protein without compromising the biological activity, but would also create a sub-micron sized, magnetically responsive, biocompatible carrier structure that provides protection from potential proteolytic deactivation while allowing permeability for the enzyme substrate.

To design such MNP we chose antioxidant enzymes, catalase and superoxide dismutase (SOD), as model biotherapeutics. These enzymes consist of several subunits (two in SOD, Mw 36 kD, and four in catalase, Mw 240 kD) and contain active centers with coordinated metals, which decompose superoxide anion and hydrogen peroxide, respectively, i.e., reactive oxygen species (ROS) that are postulated to cause vascular oxidative stress involved in pathogenesis of many maladies, including hypertension, stroke, ischemia, inflammation, myocardial infarction and restenosis [11], [12], [13], [14]. ROS are small molecules that have been shown to diffuse almost as rapidly across synthetic materials that are used to form nanocarriers as they do across biological membranes [15]. Therefore, encapsulated enzymes retain the capacity to decompose their substrates while remaining protected from proteolytic degradation.

Enzyme-loaded non-polymeric MNP were formulated in the present study by the controlled aggregation/precipitation method using mild aqueous conditions, and the key features of this new protein delivery platform were investigated, including the magnetic properties, enzymatic activity and capacity to protect cargo proteins from proteolysis. Additionally, we tested the hypothesis that under magnetic guidance MNP can efficiently deliver catalase to cultured vascular endothelial cells, and thereby provide therapeutically adequate protection against oxidative stress.

Section snippets

Reagents

Ferric chloride hexahydrate, ferrous chloride tetrahydrate, sodium oleate (99% pure), Pluronic F-127, xanthine, xanthine oxidase, 2-(N-morpholino) ethane sulfate (MES), and pronase were purchased from Sigma-Aldrich (St Louis, MO). Uranyl acetate was from Electron Microscopy Sciences. Catalase and Cu, Zn superoxide dismutase, both from bovine liver, were purchased from Calbiochem (La Jolla, CA). Iodogen and Dylight 488 NHS ester were purchased from Pierce Biotechnology (Rockford, IL). Other

MNP size and magnetic properties

Catalase-loaded MNP were formed with an average size of 303 ± 38 nm per digital analysis of multiple TEM images (example in Fig. 1A). In agreement with these results, dynamic light scattering analysis of equivalent formulations showed that MNP had a mean hydrodynamic size of 340 ± 29 nm (Fig. 1B and C). The zeta potential of blank and catalase-loaded MNP revealed a similar dependence on pH, equaling − 7.9 ± 1.3 mV and − 9.3 ± 1.1 mV at pH 7.5, and + 8.6 ± 0.8 mV and + 9.7 ± 0.6 mV at pH 5.5, respectively.

The

Discussion

In the present study a novel type of superparamagnetic nanoparticle formulation (MNP) applicable for targeted delivery of antioxidant enzymes, SOD and catalase, was developed and characterized. The underlying formulation approach is based on the entrapment of biologically active enzymes in non-polymeric MNP formed by controlled aggregation of oleate-stabilized nanocrystalline iron oxide in the presence of calcium, and enables high protein loading yields, controllable MNP size, and magnetic

Conclusions

This study describes a novel biocompatible magnetic nanocarrier system suitable for efficient encapsulation of antioxidant enzymes, SOD and catalase, with preserved biological activity. The protection of enzymatic activity from proteolysis by encapsulation in MNP was further demonstrated with catalase, a larger and more labile enzyme, which is therefore more challenging to protect. Furthermore, via magnetic targeting the carrier system demonstrated a therapeutic effect by combating a severe

Acknowledgements

The authors would like to thank Vladimir Shuvaev for assistance in determination of SOD activity, Ann-Marie Chacko for fluorescent labeling of catalase, and Eric Simone for particle characterization advice. This work was funded by the National Institute of Health (NIH RO1 HL073940, RO1 HL087036 and PO1 HL079063) (VRM, EH) and the National Center for Research Resources (Grant number UL1RR024134), the Transdisciplinary Program in Translational Medicine and Therapeutics of the Institute for

References (39)

  • J.H. Brasen et al.

    Extracellular superoxide dismutase accelerates endothelial recovery and inhibits in-stent restenosis in stented atherosclerotic Watanabe heritable hyperlipidemic rabbit aorta

    J. Am. Coll. Cardiol.

    (2007)
  • S. Laurent et al.

    Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications

    Chem. Rev.

    (2008)
  • M. Chorny et al.

    Magnetically driven plasmid DNA delivery with biodegradable polymeric nanoparticles

    FASEB J.

    (2007)
  • M. Namdeo et al.

    Magnetic nanoparticles for drug delivery applications

    J. Nanosci. Nanotechnol.

    (2008)
  • O. Veiseh et al.

    Inhibition of tumor-cell invasion with chlorotoxin-bound superparamagnetic nanoparticles

    Small

    (2009)
  • H.L. Ma et al.

    Magnetic targeting after femoral artery administration and biocompatibility assessment of superparamagnetic iron oxide nanoparticles

    J. Biomed. Mater. Res. A

    (2008)
  • M. Lewin et al.

    Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells

    Nat. Biotechnol.

    (2000)
  • S.P. Schwendeman

    Recent advances in the stabilization of proteins encapsulated in injectable PLGA delivery systems

    Crit. Rev. Ther. Drug Carrier Syst.

    (2002)
  • C.F. van der Walle et al.

    Current approaches to stabilising and analysing proteins during microencapsulation in PLGA

    Expert Opin. Drug Deliv.

    (2009)
  • Cited by (105)

    • Enzyme–polymeric/inorganic metal oxide/hybrid nanoparticle bio-conjugates in the development of therapeutic and biosensing platforms

      2021, Journal of Advanced Research
      Citation Excerpt :

      Enzymes, based on catalytic activities, are considered as a key part of the development of health systems [1].

    View all citing articles on Scopus
    1

    Contributed equally to this work.

    View full text