Stefaan Soenen

Cytotoxic effects of iron oxide nanoparticles and implications for safety in cell labelling

The in vitro labelling of cultured cells with iron oxide nanoparticles (NPs) is a frequent practice in biomedical research. To date, the potential cytotoxicity of these particles remains an issue of debate. In the present study, 4 different NP types (dextran-coated Endorem, carboxydextran-coated Resovist, lipid-coated magnetoliposomes (MLs) and citrate-coated very small iron oxide particles (VSOP)) are tested on a variety of cell types, being C17.2 neural progenitor cells, PC12 rat pheochromocytoma cells and human blood outgrowth endothelial cells. Using different NP concentrations, the effect of the NPs on cell morphology, cytoskeleton, proliferation, reactive oxygen species, functionality, viability and cellular homeostasis is investigated. Through a systematic study, the safe concentrations for every particle type are determined, showing that MLs can lead up to 67.37 ± 5.98 pg Fe/cell whereas VSOP are the most toxic particles and only reach 18.65 ± 2.07 pg Fe/cell. Using these concentrations, it is shown that for MRI up to 500 cells/μl labelled with VSOP are required to efficiently visualize in an agar phantom in contrast to only 50 cells/μl for MLs and 200 cells/μl for Endorem and Resovist. These results highlight the importance of in-depth cytotoxic evaluation of cell labelling studies as at non-toxic concentrations, some particles appear to be less suitable for the MR visualization of labelled cells.

High Intracellular Iron Oxide Nanoparticle Concentrations Affect Cellular Cytoskeleton and Focal Adhesion Kinase‐Mediated Signaling

Iron oxide nanoparticle internalization exerts detrimental effects on cell physiology for a variety of particles, but little is known about the mechanism involved. The effects of high intracellular levels of four types of iron oxide particles (Resovist, Endorem, very small organic particles, and magnetoliposomes (MLs)) on the viability and physiology of murine C17.2 neural progenitor cells and human blood outgrowth endothelial cells are reported. The particles diminish cellular proliferation and affect the actin cytoskeleton and microtubule network architectures as well as focal adhesion formation and maturation. The extent of the effects correlates with the intracellular concentration (= iron mass) of the particles, with the biggest effects for Resovist and MLs at the highest concentration (1000 microg Fe mL(-1)). Similarly, the expression of focal adhesion kinase (FAK) and the amount of activated kinase (pY397-FAK) are affected. The data suggest that high levels of perinuclear localized iron oxide nanoparticles diminish the efficiency of protein expression and sterically hinder the mature actin fibers, and could have detrimental effects on cell migration and differentiation.

Intracellular nanoparticle coating stability determines nanoparticle diagnostics efficacy and cell functionality

Iron oxide nanoparticles (NPs) are frequently employed in biomedical research as magnetic resonance (MR) contrast agents where high intracellular levels are required to clearly depict signal alterations. To date, the toxicity and applicability of these particles have not been completely unraveled. Here, we show that endosomal localization of different iron oxide particles results in their degradation and in reduced MR contrast, the rate of which is governed mainly by the stability of the coating. The release of ferric iron generates reactive species, which greatly affect cell functionality. Lipid-coated NPs display the highest stability and furthermore exhibit intracellular clustering, which significantly enhances their MR properties and intracellular persistence. These findings are of considerable importance because, depending on the nature of the coating, particles can be rapidly degraded, thus completely annihilating their MR contrast to levels not detectable when compared to controls and greatly impeding cell functionality, thereby hindering their application in functional in vivo studies.

Stimuli-responsive electrospun fibers and their applications

Stimuli-responsive electrospun nanofibers are gaining considerable attention as highly versatile tools which offer great potential in the biomedical field. In this critical review, an overview is given on recent advances made in the development and application of stimuli-responsive fibers. The specific features of these electrospun fibers are highlighted and discussed in view of the properties required for the diverse applications. Furthermore, several novel biomedical applications are discussed and the respective advantages and shortcomings inherent to stimuli-responsive electrospun fibers are addressed (136 references).

Addressing the problem of cationic lipid-mediated toxicity: The magnetoliposome model

The high biocompatibility and versatile nature of liposomes made these particles keystone components in many hot-topic research areas. For transfection and cell labelling purposes, synthetic cationic lipids are often added, but in most studies, little attention has been paid to their cytotoxic effects. In the present work, cationic magnetoliposomes (MLs), i.e. iron oxide cores enwrapped by a phospholipid bilayer (dimyristoylphosphatidylcholine or sphingomyelin) doped with cationic lipids (1,2-distearoyl-3-trimethylammonium propane), serve as a model to examine cationic lipid toxicity. Mechanisms of cytotoxic effects were found to be either dependent or independent of actual particle internalisation according to data obtained in the absence or presence of several endocytosis inhibitors. The former seem to be caused by the generation of reactive oxygen species (ROS) leading to a Ca2+ influx at high ROS levels. The latter are due to a destabilisation of the cell plasma membrane upon transfer of the cationic lipid from the ML bilayer into the plasma membrane. However, these adverse effects can be diminished by the use of a ROS scavenger, a Ca(2+)-channel blocker or by modulating the liposome size, lipid bilayer constitution or by stabilising the membrane by anchoring it on a solid core. Careful attention must be paid in terms of assessing cell viability as the effects are highly time dependent and the data suggest the incompatibility of using the well-known MTT assay when high levels of ROS species are generated.

Assessing iron oxide nanoparticle toxicity in vitro: current status and future prospects

The in vitro labeling of stem or therapeutic cells with engineered nanoparticles with the aim of transplanting these cells into live animals and, for example, noninvasively monitoring their migration, is a hot topic in nanomedicine research. It is of crucial importance that cell-nanoparticle interactions are studied in depth in order to exclude any negative effects of the cell labeling procedure. To date, many disparate results can be found in the literature regarding nanoparticle toxicity due to the great versatility of different parameters investigated. In the present work, an overview is presented of different types of nanomaterials, focusing mostly on iron oxide nanoparticles, developed for biomedical research. The difficulties in assessing nanoparticle-mediated toxicity are discussed, an overview of some of the problems encountered using commercial (dextran-coated) iron oxide nanoparticles is presented, several key parameters are highlighted and novel methods suggested--emphasizing the importance of intracellular nanoparticle degradation and linking toxicity data to functional (i.e., cell-associated) nanoparticle levels, which could help to advance any progress in this highly important research topic.

Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap

The number of newly engineered nanomaterials is vastly increasing along with their applications. Despite the fact that there is a lot of interest and effort is being put into the development of nano-based biomedical applications, the level of translational clinical output remains limited due to uncertainty in the toxicological profiles of the nanoparticles (NPs). As NPs used in biomedicines are likely to directly interact with cells and biomolecules, it is imperative to rule out any adverse effect before they can be safely applied. The initial screening for nanotoxicity is preferably performed in vitro, but extrapolation to the in vivo outcome remains very challenging. In addition, generated in vitro and in vivo data are often conflicting, which consolidates the in vitro-in vivo gap and impedes the formulation of unambiguous conclusions on NP toxicity. Consequently, more consistent and relevant in vitro and in vivo data need to be acquired in order to bridge this gap. This is in turn in conflict with the efforts to reduce the number of animals used for in vivo toxicity testing. Therefore the need for more reliable in vitro models with a higher predictive power, mimicking the in vivo environment more closely, becomes more prominent. In this review we will discuss the current paradigm and routine methods for nanotoxicity evaluation, and give an overview of adjustments that can be made to the cultivation systems in order to optimise current in vitro models. We will also describe various novel model systems and highlight future prospects.

Electrospun cellulose acetate phthalate fibers for semen induced anti-HIV vaginal drug delivery

Despite many advances in modern medicine, human immunodeficiency virus (HIV) still affects the health of millions of people world-wide and much effort is put in developing methods to either prevent infection or to eradicate the virus after infection has occurred. Here, we describe the potential use of electrospun cellulose acetate phthalate (CAP) fibers as a tool to prevent HIV transmission. During the electrospinning process, anti-viral drugs can easily be incorporated in CAP fibers. Interestingly, as a result of the pH-dependent solubility of CAP, the fibers are stable in vaginal fluid (the healthy vaginal flora has a pH of below 4.5), whereas the addition of small amounts of human semen (pH between 7.4 and 8.4) immediately dissolves the fibers which results in the release of the encapsulated drugs. The pH-dependent release properties have been carefully studied and we show that the released anti-viral drugs, together with the CAP which has been reported to have intrinsic antimicrobial activity, efficiently neutralize HIV in vitro.

Assessing cytotoxicity of (iron-oxide based) nanoparticles: An overview of different methods exemplified with cationic magnetoliposomes

Iron oxide nanoparticles are the most widely used T(2)/T(2)* contrast agents and for biomedical research purposes, one of the main applications is the in vitro labeling of stem or therapeutic cells, allowing them to be subsequently tracked in vivo upon transplantation. To allow this, the nanoparticles used should not show any sign of cytotoxicity and not affect cellular physiology as this could impede normal cell functionality in vivo or lead to undesired side-effects. Assessing the biocompatibility of the nanoparticles has proven to be quite a difficult task. In the present work, a small overview of commonly used assays is presented in order to assess several aspects, such as cell viability, induction of reactive oxygen species, nanoparticle uptake, cellular morphology, cellular proliferation, actin cytoskeleton architecture and differentiation of stem cells. The main focus is on comparing the advantages and disadvantages of the different assays, highlighting several common problems and presenting possible solutions to these problems as well as pointing out the high importance of the relationship between intracellular nanoparticle concentration and cytotoxicity.

Magnetoliposomes: versatile innovative nanocolloids for use in biotechnology and biomedicine

The high biocompatibility and versatile nature of liposomes have made these particles keystone components in many hot-topic biomedical research areas. Liposomes can be combined with a large variety of nanomaterials, such as superparamagnetic iron oxide nanocores. Because the unique features of both the magnetizable colloid and the versatile lipid bilayer can be joined, the resulting so-called magnetoliposomes can be exploited in a great array of biotechnological and biomedical applications. In this article, we highlight the use of magnetoliposomes in immobilizing enzymes, both water-soluble and hydrophobic ones, as well as their potential in several biomedical applications, including MRI, hyperthermia cancer treatment and drug delivery. The goal of this article is not to list all known uses of magnetoliposomes but rather to present some conspicuous applications in comparison to other currently used nanoparticles.