The effect of static magnetic fields and tat peptides on cellular and nuclear uptake of magnetic nanoparticles

Abstract

Magnetic nanoparticles are widely used in bioapplications such as imaging (MRI), targeted delivery (drugs/genes) and cell transfection (magnetofection). Historically, the impermeable nature of both the plasma and nuclear membranes hinder potential. Researchers combat this by developing techniques to enhance cellular and nuclear uptake. Two current popular methods are using external magnetic fields to remotely control particle direction or functionalising the nanoparticles with a cell penetrating peptide (e.g. tat); both of which facilitate cell entry. This paper compares the success of both methods in terms of nanoparticle uptake, analysing the type of magnetic forces the particles experience, and determines gross cell response in terms of morphology and structure and changes at the gene level via microarray analysis. Results indicated that both methods enhanced uptake via a caveolin dependent manner, with tat peptide being the more efficient and achieving nuclear uptake. On comparison to control cells, many groups of gene changes were observed in response to the particles. Importantly, the magnetic field also caused many change in gene expression, regardless of the nanoparticles, and appeared to cause F-actin alignment in the cells. Results suggest that static fields should be modelled and analysed prior to application in culture as cells clearly respond appropriately. Furthermore, the use of cell penetrating peptides may prove more beneficial in terms of enhancing uptake and maintaining cell homeostasis than a magnetic field.

Introduction

In recent years nanotechnology has reached a stage that allows nanoparticles (NPs) with magnetic properties to be functionalised with specific ligands for various clinical applications, such as site-specific drug/gene delivery, magnetic resonance imaging, hyperthermic treatment for cancer cells and tumour targeting [1], [2]. The term NP refers to particles less than 1μm in size, typically less than 200 nm. Thus they are of a similar length scale to biomolecules, making NPs ideal for combining with biomolecules for medical applications [3]. Due to their size, they can penetrate practically any tissue, including the blood brain barrier, and tissues protected by tight junctions. In addition, the resultant increase in surface area to volume ratio compared to other therapeutic molecules creates more scope for surface modifications and conjugation to functionally specific ligands [4].

However the cell and nuclear membranes present a substantial hurdle to the use of magnetic NPs that are not actively translocated into cells [5]. Currently several methods are adopted to overcome this, each with its own limitations. For example microinjection, electroporation and liposomes have all been used to deliver a variety of agents into cell cultures, but these techniques are limited by their respective large scale unfeasibility, cytotoxicity and low efficacy [6], [7], [8], [9]. Viral vectors have also been shown to be highly efficient at delivering nucleic acids into cells, and have been considered for use in siRNA and gene therapies, however safety concerns have delayed their clinical application [1], [10]. When considering NP delivery, the two common cell uptake routes capitalised on are via receptor-mediated endocytosis, which is a specific uptake mechanism naturally employed by cells (e.g. using transferring derivatised particles) or via non-specific uptake, typically pinocytosis or fluid phase uptake (e.g. using dextran derivatised particles). However both of these routes give varying degrees of success with regards uptake, neither achieve nuclear uptake, and any attached cargo (such as drugs or genes) get degraded in the endosome [11].

In order to improve cellular and nuclear uptake, one of the more promising approaches is the use of NPs conjugated to cell penetrating peptides (CPPs). CPPs are a family of proteins defined by the presence of a domain conferring the ability to cross the plasma membrane, termed the protein transduction domain (PTD). By using this PTD, CPPs are taken into cells via what appears to partly be a receptor-independent pathway. The 1988 discovery that purified HIV transactivator of transcription (tat) peptide could induce transcription in cell culture had led to its classification as the first CPP, with many other naturally derived peptides to follow [12], [13], [14], [15], [16]. It has been known for several years that various types of molecules have been successfully internalised following conjugation to CPPs [17]. Indeed, the efficiency of tat peptide derivatised NPs to increase cellular uptake has been widely demonstrated by comparison with the non-conjugated form [18].

In addition to the use of CPPs, cell uptake of magnetic NPs can be further assisted by use of an external magnetic field [19]. External magnetic fields have been shown to assist uptake of various biomolecules, for example, using a magnetic field can enhance the efficiency of virus internalisation by up to 100 fold [20]. The presence of a magnetic field can also help capture and remove target cells [21]. The use of external magnetic fields to enhance nuclear uptake of magnetic NPs has also been capitalised on via a process, termed magnetofection, which can achieve high transfection rates with extremely low concentrations of particles. For example, gene expression can be increased several thousand fold compared to standard transfection methods upon short-term incubation [Chemicell.com]. Magnetofection is now a commercially available and viable alternative to other common transfection techniques.

In this study, magnetic NPs, coated with a thin layer of gold, have been synthesised and functionalised with the HIV1-tat peptide PTD (approximately 6 nm). Fibroblasts were subsequently challenged in culture with the particles, in the presence and absence of a 350 mT static magnetic field. The results strongly suggest that magnetofection techniques should be critically analysed as to the exact forces the cells are perceiving prior to use as cells clearly respond to even weak field strengths, which may have a long standing influence on cell behaviour. Furthermore, results strongly support the use of CPPs such as tat peptide as a prospective nuclear targeting vehicle.