Elsevier

Biomaterials

Volume 26, Issue 13, May 2005, Pages 1565-1573
Biomaterials

Cytotoxicity suppression and cellular uptake enhancement of surface modified magnetic nanoparticles

https://doi.org/10.1016/j.biomaterials.2004.05.022Get rights and content

Abstract

The aim of this study was to modify the surfaces of superparamagnetic iron oxide nanoparticles (SPION) with pullulan in order to reduce the cytotoxicity and enhance the cellular uptake of the nanoparticles. In this study, we have prepared and characterised the pullulan coated superparamagnetic iron oxide nanoparticles (Pn-SPION) of size around 40–45 nm with magnetite inner core and hydrophilic outer shell of pullulan. We have investigated the effect of cellular uptake of uncoated and Pn-SPION on cell adhesion/viability, cytotoxicity, morphology and cytoskeleton organisation of human fibroblasts. Cell cytotoxicity/adhesion studies of SPIONs on human dermal fibroblasts showed that the particles are toxic and their internalisation resulted in disruption of cytoskeleton organisation of cells. On the other hand, Pn-SPIONs were found to be non-toxic and induced changes in cytoskeleton organisation different from that observed with SPION. Transmission electron microscopy results indicated that the SPION and Pn-SPION were internalised into cells via different mechanisms, thereby suggesting that the particle endocytosis behaviour is dependent on the surface characteristics of the nanoparticles.

Introduction

Superparamagnetic iron oxide nanoparticles have been recognised as a promising tool for the site-specific delivery of drugs and diagnostics agents [1], [2]. Magnetic properties and internalisation of particles in cells depend strongly on the size of the magnetic particles [3]. Particles below 100 nm are small enough both to evade reticuloendothelial system (RES) of the body as well as penetrate the very small capillaries within the body tissues [4]. Because of their hydrophobic surfaces and large surface area to volume ratio, in vivo use of nanoparticles is hampered by very rapid clearance of nanoparticles from the circulation by the RES. Avoidance of this obstacle is possible if the surfaces of these nanoparticles are made sufficiently hydrophilic, as these modifications prolong considerably the nanoparticle half-life in the circulation [5].

Surface characteristic of nanoparticles is a crucial factor that not only determines the biocompatibility of these magnetic materials but also plays an important role in cell adhesion on biomaterials [6], [7]. Adhesive interactions between cells and the extracellular matrix (ECM) or particles are governed primarily by integrins, a large family of cell surface adhesion receptors [8], [9]. Integrin-mediated cell adhesion is central to cell survival, differentiation and motility and is known to activate focal adhesion kinase (FAK), which plays a role in cytoskeleton reorganisation [10]. In addition, earlier we have shown that the event of endocytosis occurs by the reorganisation of cell cytoskeleton [11], [12]. This is because of the fact that the elastic properties of a cell are mainly due to the internal cytoskeleton consisting of three types of filamentous proteins: actin, microtubules and intermediate filaments, etc. [13]. The cytoskeleton plays a major role in many important fields such as cell shape, motility, division, adhesion and the connections the cell can realise with its environment [14]. Also there is a link between cytoskeleton components and endocytosis. Relation between the two processes is highly dynamic and may involve interactions between distinct protein complexes, depending on the nature of the cargo being internalised [15]. As a result, there could be different kind of changes in proteins responsible for maintaining cytoskeleton organisation, which may ultimately result from different pathways of particle internalisation. The nature and adhesion capacity of cells in the presence of nanoparticles as well as the subsequent cellular events such as endocytosis and changes in cytoskeleton organisation have not been fully elucidated yet.

Therefore, the objectives of this study were to prepare and characterise the surface modified superparamagnetic iron oxide nanoparticles (both SPION and Pn-SPION) and determine (i) the adhesion capacity, (ii) endocytosis behaviour and (iii) effect on cytoskeleton organisation of human fibroblasts as a result of nanoparticle internalisation. Pullulan, a nonionic polysaccharide, was chosen as a coating material for SPION due to the following properties: (i) high water solubility, (ii) no toxicity, (iii) usefulness as a plasma expander, (iv) non-immunogenic and (v) non-antigenic properties [16]. Also, pullulan is widely used as a food additive because of its safety in human use (http://www2.minlnv.nl/lnv/algemeen/vvm/codex/documenten/2003/CCFAC/fa36_36e.pdf). In addition, there are evidences for receptor-mediated hepatic uptake of pullulan in rats [17]. The influence of Pn-SPION on human fibroblasts in vitro has been determined as compared to SPION, in terms of cell adhesion/cytotoxicity, TEM and observation of F-actin and β-tubulin cytoskeleton by fluorescence microscopy.

Section snippets

Materials

Ferric chloride hexahydrate (FeCl3·6H2O>99%), ferrous chloride tetrahydrate (FeCl2·4H2O), pullulan, potassium thiocyanate (ACS reagent ⩾99.0%), ammonium persulphate (ACS reagent, ⩾99.0%) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma, England, UK, while sodium hydroxide (NaOH>99%) and hydrochloric acid (HCl>37% v/v) were obtained from Fluka, England, UK. Double distilled water was used for all the experiments.

Synthesis of Pn-SPION

Magnetic nanoparticles, SPION, were

FTIR studies

Fig. 1 displays the FTIR spectra of solid samples of SPION and SPION modified by the pullulan (Pn-SPION). The FTIR spectra of iron oxide exhibit strong bands in the low frequency region below 800 cm−1 due to the iron oxide skeleton. In other regions, the spectra of iron oxide have weak bands. The spectrum is consistent with magnetite (Fe3O4) and the signals associated to the magnetite appear as broad features at 408.9, 571.5 and 584.5 cm−1 [18]. The main characteristic vibrations of the pullulan

Discussion

The colloidal solution of Pn-SPION showed very high stability at neutral pH with no sedimentation observed even after 2 months of storage at room temperature. The strong anchoring of the pullulan molecules on the surface of iron oxide results in the steric stabilisation of the particles. Size distribution studies using TEM and AFM measurements showed that the particles have size less than 50 nm with inner magnetic core and outer polymeric shell (core-shell structure).

Cell adhesion/viability

Conclusions

In this paper, Pn-SPION of size about 40–50 nm having core-shell structure with magnetic core and polymeric shell have been prepared and characterised in vitro by various physicochemical means. The colloidal solution of nanoparticles showed high stability. The results of cell culture experiments have indicated that the Pn-SPIONs were non-toxic as comparing to SPIONs and because of the surface modification the cellular uptake of Pn-SPION could be enhanced. Pn-SPIONs were internalised by cells via

Acknowledgements

The authors would like to thank Professor Adam S.G. Curtis, Centre for Cell Engineering, IBLS, University of Glasgow, Glasgow, UK for encouraging us to work in his laboratory. Thanks are also due to Mr. Eoin Robertson and Mrs. M. Mullin, EM Unit, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK for their technical assistance.

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