Increased Cellular Uptake of Biocompatible Superparamagnetic Iron Oxide Nanoparticles into Malignant Cells by an External Magnetic Field

Superparamagnetic iron oxide nanoparticles (SPIONs) are used as delivery systems for different therapeutics including nucleic acids for magnetofection-mediated gene therapy. The aim of our study was to evaluate physicochemical properties, biocompatibility, cellular uptake and trafficking pathways of the custom-synthesized SPIONs for their potential use in magnetofection. Custom-synthesized SPIONs were tested for size, shape, crystalline composition and magnetic behavior using a transmission electron microscope, X-ray diffractometer and magnetometer. SPIONs were dispersed in different aqueous media to obtain ferrofluids, which were tested for pH and stability using a pH meter and zetameter. Cytotoxicity was determined using the MTS and clonogenic assays. Cellular uptake and trafficking pathways were qualitatively evaluated by transmission electron microscopy and quantitatively by inductively coupled plasma atomic emission spectrometry. SPIONs were composed of an iron oxide core with a diameter of 8–9 nm, coated with a 2-nm-thick layer of silica. SPIONs, dispersed in 0.9% NaCl solution, resulted in a stable ferrofluid at physiological pH for several months. SPIONs were not cytotoxic in a broad range of concentrations and were readily internalized into different cells by endocytosis. Exposure to neodymium-iron-boron magnets significantly increased the cellular uptake of SPIONs, predominantly into malignant cells. The prepared SPIONs displayed adequate physicochemical and biomedical properties for potential use in magnetofection. Their cellular uptake was dependent on the cell type, and their accumulation within the cells was dependent on the duration of exposure to an external magnetic field.     

Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.     

Glutaric acid as a spacer facilitates improved intracellular uptake of LHRH-SPION into human breast cancer cells

Superparamagnetic iron oxide nanoparticles (SPIONs) bound directly to luteinizing hormone releasing hormone (LHRH) have shown high efficiency for intracellular uptake to breast cancer cells, MDA-MB-435S.luc. We demonstrate in this communication that inclusion of a small spacer molecule such as glutaric acid (Glu) in between SPION and LHRH increases further receptor mediated intracellular uptake. LHRH-bound SPIONs with and without the spacer molecule were nontoxic.     

Synthesis of chemically functionalized superparamagnetic nanoparticles as delivery vectors for chemotherapeutic drugs

Superparamagnetic iron oxide nanoparticles (SPIONs) are in clinical use for disease detection by MRI. A major advancement would be to link therapeutic drugs to SPIONs in order to achieve targeted drug delivery combined with detection. In the present work, we studied the possibility of developing a versatile synthesis protocol to hierarchically construct drug-functionalized-SPIONs as potential anti-cancer agents. Our model biocompatible SPIONs consisted of an iron oxide core (9-10 nm diameter) coated with polyvinylalcohols (PVA/aminoPVA), which can be internalized by cancer cells, depending on the positive charges at their surface. To develop drug-functionalized-aminoPVA-SPIONs as vectors for drug delivery, we first designed and synthesized bifunctional linkers of varied length and chemical composition to which the anti-cancer drugs 5-fluorouridine or doxorubicin were attached as biologically labile esters or peptides, respectively. These functionalized linkers were in turn coupled to aminoPVA by amide linkages before preparing the drug-functionalized-SPIONs that were characterized and evaluated as anti-cancer agents using human melanoma cells in culture. The 5-fluorouridine-SPIONs with an optimized ester linker were taken up by cells and proved to be efficient anti-tumor agents. While the doxorubicin-SPIONs linked with a Gly-Phe-Leu-Gly tetrapeptide were cleaved by lysosomal enzymes, they exhibited poor uptake by human melanoma cells in culture.     

Proteasomes regulate the duration of erythropoietin receptor activation by controlling down-regulation of cell surface receptors

The binding of erythropoietin (Epo) to its receptor leads to the transient phosphorylation of the Epo receptor (EpoR) and the activation of intracellular signaling pathways. Inactivation mechanisms are simultaneously turned on, and Epo-induced signaling pathways return to nearly basal levels after 30-60 min of stimulation. We show that proteasomes control these inactivation mechanisms. In cells treated with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal (LLnL) or lactacystin, EpoR tyrosine phosphorylation and activation of intracellular signaling pathways (Jak2, STAT5, phosphatidylinositol 3-kinase) were sustained for at least 2 h. We show that this effect was due to the continuous replenishment of the cell surface pool of EpoRs in cells treated with proteasome inhibitors. Proteasome inhibitors did not modify the internalization and degradation of Epo.EpoR complexes, but they allowed the continuous replacement of the internalized receptors by newly synthesized receptors. Proteasome inhibitors did not modify the synthesis of EpoRs, but they allowed their transport to the cell surface. N-Ac-Leu-Leu-norleucinal, but not lactacystin, also inhibited the degradation of internalized Epo.EpoR complexes, most probably through cathepsin inhibition. The internalized EpoRs were not tyrosine-phosphorylated, and they did not activate intracellular signaling pathways. Our results show that the proteasome controls the down-regulation of EpoRs in Epo-stimulated cells by inhibiting the cell surface replacement of internalized EpoRs.     

Comprehensive Evaluation of microRNA Expression Profiling Reveals the Neural Signaling Specific Cytotoxicity of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) through N-Methyl-D-Aspartate Receptor

Though nanomaterials are considered as drug carriers or imaging reagents targeting the central nervous system their cytotoxicity effect on neuronal cells has not been well studied. In this study, we treated PC12 cells, a model neuronal cell line, with a nanomaterial that is widely accepted for medical use, superparamagnetic iron oxide nanoparticles (SPIONs). Our results suggest that, after treated with SPIONs, the expression pattern of the cellular miRNAs changed widely in PC12 cells. As potential miRNA targets, NMDAR, one of the candidate mRNAs that were selected using GO and KEGG pathway enrichment, was significantly down regulated by SPIONs treatment. We further illustrated that SPIONs may induce cell death through NMDAR suppression. This study revealed a NMDAR neurotoxic effect of SPIONs and provides a reliable approach for assessing the neurocytotoxic effects of nanomaterials based on the comprehensive annotation of miRNA profiling.     

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe₃O₄) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.     

Effect of iron chelators on the transferrin receptor in K562 cells

Delivery of iron to K562 cells by diferric transferrin involves a cycle of binding to surface receptors, internalization into an acidic compartment, transfer of iron to ferritin, and release of apotransferrin from the cell. To evaluate potential feedback effects of iron on this system, we exposed cells to iron chelators and monitored the activity of the transferrin receptor. In the present study, we found that chelation of extracellular iron by the hydrophilic chelators desferrioxamine B, diethylenetriaminepentaacetic acid, or apolactoferrin enhanced the release from the cells of previously internalized 125I-transferrin. Presaturation of these compounds with iron blocked this effect. These chelators did not affect the uptake of iron from transferrin. In contrast, the hydrophobic chelator 2,2-bipyridine, which partitions into cell membranes, completely blocked iron uptake by chelating the iron during its transfer across the membrane. The 2,2-bipyridine did not, however, enhance the release of 125I-transferrin from the cells, indicating that extracellular iron chelation is the key to this effect. Desferrioxamine, unlike the other hydrophilic chelators, can enter the cell and chelate an intracellular pool of iron. This produced a parallel increase in surface and intracellular transferrin receptors, reaching 2-fold at 24 h and 3-fold at 48 h. This increase in receptor number required ongoing protein synthesis and could be blocked by cycloheximide. Diethylenetriaminepentaacetic acid or desferrioxamine presaturated with iron did not induce new transferrin receptors. The new receptors were functionally active and produced an increase in 59Fe uptake from 59Fe-transferrin. We conclude that the transferrin receptor in the K562 cell is regulated in part by chelatable iron: chelation of extracellular iron enhances the release of apotransferrin from the cell, while chelation of an intracellular iron pool results in the biosynthesis of new receptors.     

Intermediate steps in cellular iron uptake from transferrin. Detection of a cytoplasmic pool of iron, free of transferrin.

The uptake of transferrin-bound iron by receptor-mediated endocytosis has been the subject of extensive experimental investigation. However, the path followed by iron (Fe) after release from transferrin (Tf) remains obscure. Once Fe is released from Tf within the endosome, it must be transported across the endosomal membrane into the cell. The present investigation describes the presence of a cytoplasmic Tf-free Fe pool which is detectable only when cells are detached from their culture dishes at low temperature, after initial incorporation of diferric transferrin at 37 degrees C. This cellular iron pool was greatly reduced if incubation temperatures were maintained at 37 degrees C or if cells were treated with pronase. Human melanoma cells (SK-MEL-28) in culture were prelabeled by incubation with human 125I-59Fe-transferrin for 2 h, washed, and reincubated at 4 degrees C or 37 degrees C in balanced salt solution in the presence or absence of pronase. The cells were then mechanically detached from the plates and separated into "internalized" and supernatant fractions by centrifugation. Approximately 90% of cellular 59Fe and 20% of 125I-Tf remained internalized when this reincubation procedure was carried out in balanced salt solution at 37 degrees C. However, at 4 degrees C, cellular internalized iron was reduced to approximately 50% of the initial value. The release of this component of cellular 59Fe (approximately 40% of total cell 59Fe) at 4 degrees C was completely inhibited in the presence of pronase and other general proteinases at 4 degrees C and at 37 degrees C, without affecting internalized transferrin levels. Similar results were obtained in fibroblasts and hepatoma cells, indicating that this phenomenon is not unique to melanoma cells. The characterization of this Tf-free cellular Fe pool which is detectable at low temperature may yield valuable insights into the metabolic fate of iron following its transport across the membrane of the endocytotic vesicle.     

Optimized labeling of bone marrow mesenchymal cells with superparamagnetic iron oxide nanoparticles and <Emphasis Type="Italic">in vivo</Emphasis> visualization by magnetic resonance imaging

Background  

Stem cell therapy has emerged as a promising addition to traditional treatments for a number of diseases. However, harnessing the therapeutic potential of stem cells requires an understanding of their fate in vivo. Non-invasive cell tracking can provide knowledge about mechanisms responsible for functional improvement of host tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) have been used to label and visualize various cell types with magnetic resonance imaging (MRI). In this study we performed experiments designed to investigate the biological properties, including proliferation, viability and differentiation capacity of mesenchymal cells (MSCs) labeled with clinically approved SPIONs.     

Magnetically labeled insulin-secreting cells

Iron oxide nanoparticles have been shown to magnetically label cells in order to visualize them in vivo via MR imaging. This technology has yet to be implemented in insulin secreting cells, thus it is not known whether the presence of these nanoparticles in the cytoplasm of the cells affects insulin secretion. This study investigates the effectiveness and consequence of labeling mouse insulinoma betaTC3 and betaTC-tet cells with monocrystalline iron oxide nanoparticles (MION). Our data show that MION can be internalized in both betaTC3 and betaTC-tet cells following a 24h exposure to 0.02mg/ml MION solution. The metabolic and secretory activities of both MION-labeled cell lines were statistically indistinguishable from sham treatment. Furthermore, cell viability and apoptosis remained constant throughout the cell's exposure to MION. Finally, MR images demonstrated significant contrast between labeled and sham-treated cells. Thus, labeling murine insulinoma cell lines with magnetic iron oxide nanoparticles does not hinder their insulin secretion, while it provides MR imaging contrast.     

Metabolic pathways of heparan sulfate proteoglycans in a rat parathyroid cell line.

The distribution of heparan sulfate (HS) proteoglycans in clonal rat parathyroid cells is regulated by the extracellular Ca2+ concentration, which is a principal factor for parathyroid cell function (Takeuchi, Y., Sakaguchi, K., Yanagishita, M., Aurbach, G. D., and Hascall, V. C. (1990) J. Biol. Chem. 265, 13661-13668). Increasing the concentration of extracellular Ca2+ in the physiological range redistributes HS proteoglycans from the cell surface to an intracellular compartment. We have now examined effects of the extracellular Ca2+ concentration on the metabolism of the HS proteoglycans in detail using [35S]sulfate metabolic labeling-chase experiments. Two distinct metabolic pathways were demonstrated: (i) the intracellular generation of HS chains from HS proteoglycans in prelysosomal compartments followed by their release into the medium (pathway 1), and (ii) intracellular generation of HS oligosaccharides from HS chains in prelysosomal compartments, which are eventually degraded into free sulfate in lysosomes (pathway 2). The HS oligosaccharides were exclusively present within the cells, whereas HS chains were found primarily in the medium. The cells do not internalize either HS proteoglycans or HS chains from the medium. These observations indicate that these two degradation pathways are independent. In addition to these pathways, approximately 15% of the HS proteoglycans were released into the medium as a proteoglycan form. Treatment of cells with chloroquine, a lysosomotropic agent, did not affect generation of HS chains but inhibited conversion of HS chains to HS oligosaccharides or to free sulfate and resulted in the release of HS chains from the cells. The drug did not affect metabolic pathway 1. The extracellular Ca2+ concentration did not alter these intracellular degradation pathways for HS proteoglycans in the parathyroid cells. Thus, extracellular Ca2+ appears to regulate only the distribution of HS proteoglycans between the cell surface and intracellular compartments, and the process of cycling between these compartments when extracellular Ca2+ is low.     

Lipid raft-independent B cell receptor-mediated antigen internalization and intracellular trafficking

The Ag-specific B cell receptor (BCR) expressed by B lymphocytes has two distinct functions upon interaction with cognate Ag: signal transduction (generation of intracellular second messenger molecules) and Ag internalization for subsequent processing and presentation. While it is known that plasma membrane domains, termed lipid rafts, are involved in BCR-mediated signal transduction, the precise role of plasma membrane lipid rafts in BCR-mediated Ag internalization and intracellular trafficking is presently unclear. Using a highly characterized model system, it was determined that while plasma membrane lipid rafts can be internalized by B lymphocytes, lipid rafts do not represent a major pathway for the rapid and efficient internalization of cell surface Ag-BCR complexes. Moreover, internalized plasma membrane lipid rafts are delivered to intracellular compartments distinct from those to which the bulk of internalized Ag-BCR complexes are delivered. These results demonstrate that B lymphocytes, like other cell types, possess at least two distinct endocytic pathways (i.e., clathrin-coated pits and plasma membrane lipid rafts) that deliver internalized ligands to distinct intracellular compartments. Furthermore, Ag-BCR complexes differentially access these two distinct internalization pathways.     

Wheat germ agglutinin is selectively transported to multivesicular bodies.

Colloidal iron dextran particles bearing wheat germ agglutinin (WGA/FeDex) were bound by glycoconjugates expressed at the surface of HepG2 cells. Bound WGA/FeDex was internalized when cells were incubated at 37 degrees C and accumulated in intracellular structures which have the same buoyant density as the plasma membrane when examined on Percoll density gradients. The intracellular structures containing WGA/FeDex were identified as multivesicular bodies (MVB) by transmission electron microscopy. WGA/FeDex was not transported to lysosomes nor did it interfere with uptake and transport of GalBSA to lysosomes by the asialoglycoprotein receptor. WGA/FeDex was seen predominantly in non-coated invaginations at the cell surface, suggesting it may enter cells at a different site than GalBSA/FeDex. Highly enriched plasma membranes and MVBs containing superparamagnetic [125I]WGA/FeDex particles were prepared by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes prepared by HIMAC were enriched 30-fold for [125I]WGA/FeDex, 15-fold for alkaline phosphodiesterase I, and 9-fold for galactosyltransferase relative to the crude post-nuclear homogenate and consisted entirely of plasmalemmal sheets. Intracellular structures containing WGA/FeDex were enriched 35-fold for [125I]WGA/FeDex, 10-fold for alkaline phosphodiesterase I, and 10-fold for galactosyltransferase but did not contain lysosomal beta-galactosidase. WGA/FeDex has a different ultimate destination in HepG2 cells than ligands internalized by the asialoglycoprotein receptor and can be used to obtain highly enriched plasma membranes and MVBs from cultured cells.     

External magnetic field promotes homing of magnetized stem cells following subcutaneous injection

Background

Mesenchymal stem cells (MSCs) are multipotent stromal cells that have the ability to self-renew and migrate to sites of pathology. In vivo tracking of MSCs provides insights into both, the underlying mechanisms of MSC transformation and their potential as gene delivery vehicles. The aim of our study was to assess the ability of superparamagnetic iron oxide nanoparticles (SPIONs)-labeled Wharton’s Jelly of the human umbilical cord-derived MSCs (WJ-MSCs) to carry the green fluorescent protein (GFP) gene to cutaneous injury sites in a murine model.

Methods

WJ-MSCs were isolated from a fresh umbilical cord and were genetically transformed to carry the GFP gene using lentiviral vectors with magnetically labeled SPIONs. The SPIONs/GFP-positive WJ-MSCs expressed multipotent cell markers and demonstrated the potential for osteogenic and adipogenic differentiation. Fifteen skin-injured mice were divided into three groups. Group I was treated with WJ-MSCs, group II with SPIONs/GFP-positive WJ-MSCs, and group III with SPIONs/GFP-positive WJ-MSCs exposed to an external magnetic field (EMF). Magnetic resonance imaging and optical molecular imaging were performed, and images were acquired 1, 2, and 7 days after cell injection.

Results

The results showed that GFP could be intensively detected around the wound in vivo 24 h after the cells were injected. Furthermore, we observed an accumulation of WJ-MSCs at the wound site, and EMF exposure increased the speed of cell transport. In conclusion, our study demonstrated that SPIONs/GFP function as cellular probes for monitoring in vivo migration and homing of WJ-MSCs. Moreover, exposure to an EMF can increase the transportation efficiency of SPIONs-labeled WJ-MSCs in vivo.

Conclusions

Our findings could lead to the development of a gene carrier system for the treatment of diseases.

     

Carboxymethyl dextran-trimethyl chitosan coated superparamagnetic iron oxide nanoparticles: An effective siRNA delivery system for HIV-1 Nef

Gene therapy, including small interfering RNA (siRNA) technology, is one of the leading strategies that help to improve the outcomes of the current therapeutic systems against HIV-1 infection. The successful therapeutic application of siRNAs requires their safe and efficient delivery to specific cells. Here, we introduce a superparamagnetic iron oxide nanoparticle (SPION) for delivering siRNA against HIV-1 nef (anti-nef siRNA) into two cell lines, HEK293 and macrophage RAW 264.7. SPIONs were coated with trimethyl chitosan (TMC), and thereafter, different concentrations of SPION–TMC were coated with different ratios of a carboxymethyl dextran (CMD) to modify the physicochemical properties and improve the biological properties of the nanocarriers. The nanoparticles exhibited a spherical shape with an average size of 112 nm. The obtained results showed that the designed delivery route enhanced the uptake of siRNA into both HEK293 and RAW 264.7 cells compared with control groups. Moreover, CMD–TMC–SPIONs containing anti-nef siRNA significantly reduced the expression of HIV-1 nef in HEK293 stable cells. The modified siRNA-loaded SPIONs also displayed no toxicity or apoptosis-inducing effects on the cells. The CMD–TMC–SPIONs are suggested as potential nanocarriers for siRNA delivery in gene therapy of HIV-1 infection.     

Hydrogenase Activity of Mineral-Associated and Suspended Populations of Desulfovibrio desulfuricans Essex 6

The interactions between sulfate-reducing microorganisms and iron oxides influence a number of important redox-sensitive biogeochemical processes including the formation of iron sulfides. Enzymes, such as hydrogenase which catalyze the reversible oxidation of molecular hydrogen, are known to mediate electron transfer to metals and may contribute to the formation and speciation of ferrous sulfides formed at the cell–mineral interface. In the present study, we compared the whole cell hydrogenase activity of Desulfovibrio desulfuricans strain Essex 6 growing as biofilms on hematite (hematite-associated) or as suspended populations using different metabolic pathways. Hematite-associated cells exhibited significantly greater hydrogenase activity than suspended populations during sulfate respiration but not during pyruvate fermentation. The enhanced activity of the hematite-associated, sulfate-grown cells appears to be dependent on iron availability rather than a general response to surface attachment since the activity of glass-associated cells did not differ from that of suspended populations. Hydrogenase activity of pyruvate-fermenting cells was stimulated by addition of iron as soluble Fe(II)Cl₂ and, in the absence of added iron, both sulfate-reducing and pyruvate-fermenting cells displayed similar rates of hydrogenase activity. These data suggest that iron exerts a stronger influence on whole cell hydrogenase activity than either metabolic pathway or mode of growth. The location of hydrogenase to the cell envelope and the enhanced activity at the hematite surface in sulfate-reducing cells may influence the redox conditions that control the species of iron sulfides on the mineral surface.     

Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages

Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated versus dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs.