Slavko Kralj

Effect of surface charge on the cellular uptake of fluorescent magnetic nanoparticles

We report on the nanoparticle uptake into MCF10A neoT and PC-3 cells using flow cytometry, confocal microscopy, SQUID magnetometry, and transmission electron microscopy. The aim was to evaluate the influence of the nanoparticles’ surface charge on the uptake efficiency. The surface of the superparamagnetic, silica-coated, maghemite nanoparticles was modified using amino functionalization for the positive surface charge (CNPs), and carboxyl functionalization for the negative surface charge (ANPs). The CNPs and ANPs exhibited no significant cytotoxicity in concentrations up to 500xa0μg/cm3 in 24xa0h. The CNPs, bound to a plasma membrane, were intensely phagocytosed, while the ANPs entered cells through fluid-phase endocytosis in a lower internalization degree. The ANPs and CNPs were shown to be co-localized with a specific lysosomal marker, thus confirming their presence in lysosomes. We showed that tailoring the surface charge of the nanoparticles has a great impact on their internalization.

Magnetic Assembly of Superparamagnetic Iron Oxide Nanoparticle Clusters into Nanochains and Nanobundles

We report on the syntheses of magnetoresponsive, superparamagnetic nanostructures with highly anisotropic shapes, i.e., nanochains of controlled length and their bundles (nanobundles). These nanochains and nanobundles were obtained by the simultaneous magnetic assembly of superparamagnetic nanoparticle clusters (SNCs) and the fixation of the assembled SNCs with an additional layer of deposited silica, produced by a sol-gel process. This low-cost approach provides excellent length control of the short nanochains (approximately 6 or 14 SNCs per nanochain) and fine-tuning of the spacing between the neighboring SNCs inside an individual nanochain. Our magnetically responsive superparamagnetic nanostructures have a controlled aspect ratio, a uniform size, and a well-defined shape, and they express good colloidal stability. This general approach should lead to new, advanced applications of the nanochains and nanobundles in the treatment of cancer and in the ability to magnetically manipulate liquid and photonic crystals.

Binding of plasma proteins to titanium dioxide nanotubes with different diameters

Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices.

Protein interactions with layers of TiO2 nanotube and nanopore arrays: morphology and surface charge influence.

In the present work we investigate the key factors involved in the interaction of small-sized charged proteins with TiO2 nanostructures, i.e. albumin (negatively charged), histone (positively charged). We examine anodic nanotubes with specific morphology (simultaneous control over diameter and length, e.g. diameter - 15, 50 or 100nm, length - 250nm up to 10μm) and nanopores. The nanostructures surface area has a direct influence on the amount of bound protein, nonetheless the protein physical properties as electric charge and size (in relation to nanotopography and biomaterials electric charge) are crucial too. The highest quantity of adsorbed protein is registered for histone, for 100nm diameter nanotubes (10μm length) while higher values are registered for 15nm diameter nanotubes when normalizing protein adsorption to nanostructures surface unit area (evaluated from dye desorption measurements) - consistent with theoretical considerations. The proteins presence on the nanostructures is evaluated by XPS and ToF-SIMS; additionally, we qualitatively assess their presence along the nanostructures length by ToF-SIMS depth profiles, with decreasing concentration towards the bottom.nnnSTATEMENT OF SIGNIFICANCEnSurface nanostructuring of titanium biomedical devices with TiO2 nanotubes was shown to significantly influence the adhesion, proliferation and differentiation of mesenchymal stem cells (and other cells too). A high level of control over the nanoscale topography and over the surface area of such 1D nanostructures enables a direct influence on protein adhesion. Herein, we investigate and show how the nanostructure morphology (nanotube diameter and length) influences the interactions with small-sized charged proteins, using as model proteins bovine serum albumin (negatively charged) and histone (positively charged). We show that the protein charge strongly influences their adhesion to the TiO2 nanostructures. Protein adhesion is quantified by ELISA measurements and determination of the nanostructures total surface area. We use a quantitative surface charge model to describe charge interactions and obtain an increased magnitude of the surface charge density at the top edges of the nanotubes. In addition, we track the proteins presence on and inside the nanostructures. We believe that these aspects are crucial for applications where the incorporation of active molecules such as proteins, drugs, growth factors, etc., into nanotubes is desired.

Controlled heteroaggregation of two types of nanoparticles in an aqueous suspension.

Composite particles combining nanoparticles of different functional materials, as well as nanoclusters of nanoparticles of controlled size, can be synthesized by the assembly of nanoparticles in an aqueous suspension. Different interactions between the nanoparticles in the suspension can be applied for their heteroaggregation and controlled by engineering the surface properties of the nanoparticles. The heteroaggregation of nanoparticles in a suspension was studied on a model system composed of superparamagnetic carboxyl-functionalized silica-coated maghemite nanoparticles (cMNPs) (24 nm in size) and larger, amino-functionalized, silica nanoparticles (aSNPs) (92 nm). The heteroaggregates formed with electrostatic attractions between the nanoparticles displaying an opposite electrical surface charge, or with chemical interactions originating from covalent bonding between the molecules at their surfaces. The suspensions were characterized with measurements of the zeta-potential and dynamic light scattering (DLS). The heteroaggregates were analyzed by transmission (TEM) and scanning (SEM) electron microscopy. The kinetics of the heteroaggregation was followed by continuous monitoring of the changes in the average hydrodynamic size by DLS. The results show that covalent bonding is much more effective than attractive electrostatic interactions in terms of a much greater and more homogeneous coverage of the larger central aSNP by the smaller cMNPs in the outer layer.

Targeting EGFR-overexpressed A431 cells with EGF-labeled silica-coated magnetic nanoparticles

Human epidermal growth-factor receptor (EGFR) has emerged as an attractive target for cancer therapy. In this study, amino- or carboxyl-functionalized silica-coated maghemite nanoparticles were conjugated with epidermal growth-factor (EGF) using five different binding modes: carbodiimide chemistry, two types of homo-bifunctional cross-linking reagents, and electrostatic interactions between the nanoparticles and the EGF. The nanoparticles and their aqueous suspensions were characterized by transmission electron microscopy, zeta-potential measurements and dynamic light scattering. The binding efficiency of the EGF to the nanoparticles was measured by flow cytometry using a specific anti-EGF antibody. The ability of EGF bioconjugates to target the EGF receptors was tested using EGFR over-expressing A431 cells in comparison to EGFR negative HeLa cells. Our results showed that the bioconjugates where the EGF was bonded by carbodiimide chemistry are the most effective for the specific targeting of EGFR-expressing cells in vitro.

The role of PVP in the bioavailability of Ag from the PVP-stabilized Ag nanoparticle suspension

We assessed the bioavailability of Ag from Ag nanoparticles (NPs), stabilized with polyvinylpyrrolidone (PVP), to terrestrial isopods which were exposed to 10, 100 and 1000xa0μg Ag NPs/g of dry food. Different Ag species were determined in the NP suspension that was fed to isopods: (i) total Ag by atomic absorption spectroscopy, (ii) the sum of Ag-PVP complexes and free Ag+ by anodic stripping voltammetry at the bismuth-film electrode, and (iii) free Ag+ by ion-selective potentiometry. The amounts of Ag species in the consumed food were compared to the masses of Ag accumulated in the isopod digestive glands. Our results show that all three Ag species (Ag NPs, Ag-PVP complexes and free Ag+) could be the source of bioaccumulated Ag, but to various degrees depending on the exposure concentration and transformations in the digestive system. We provide a proof that (i) Ag NPs dissolve and Ag-PVP complexes dissociate in the isopod digestive tract; (ii) the concentration of free Ag+ in the suspension offered to the test organisms is not the only measure of bioavailable Ag. The type of NP stabilizer along with the NP transformations in the digestive system needs to be considered in the creation of new computational models of the nanomaterial fate.

Ferromagnetic behavior and exchange bias effect in akaganeite nanorods

We report ferromagnetic-like properties and exchange bias effect in akaganeite (β-FeOOH) nanorods. They exhibit a Neel temperature TNu2009=u2009259u2009K and ferromagnetic-like hysteresis behavior both below and above TN. An exchange bias effect is observed below TN and represents an interesting behavior for akaganeite nanorods. These results are explained on the basis of a core-shell structure in which the core has bulk akaganeite magnetic properties (i.e., antiferromagnetic ordering) while the shell exhibits a disordered spin state. Thus, the nanorods show ferromagnetic properties and an exchange bias effect at the same time, increasing their potential for use in practical applications.

The chemically directed assembly of nanoparticle clusters from superparamagnetic iron-oxide nanoparticles

We describe the controlled synthesis of nanoparticle clusters (NCs) based on the chemically directed assembly of superparamagnetic iron-oxide nanoparticles. The NCs, with their “raspberry-like” shape, have a large effective surface area, uniform size, and contain a large fraction of the magnetic maghemite.

Harmful at non-cytotoxic concentrations: SiO2-SPIONs affect surfactant metabolism and lamellar body biogenesis in A549 human alveolar epithelial cells

Abstract The pulmonary delivery of nanoparticles (NPs) is a promising approach in nanomedicine. For the efficient and safe use of inhalable NPs, understanding of NP interference with lung surfactant metabolism is needed. Lung surfactant is predominantly a phospholipid substance, synthesized in alveolar type II cells (ATII), where it is packed in special organelles, lamellar bodies (LBs). In vitro and in vivo studies have reported NPs impact on surfactant homeostasis, but this phenomenon has not yet been sufficiently examined. We showed that in ATII-like A549 human lung cancer cells, silica-coated superparamagnetic iron oxide NPs (SiO2-SPIONs), which have a high potential in medicine, caused an increased cellular amount of acid organelles and phospholipids. In SiO2-SPION treated cells, we observed elevated cellular quantity of multivesicular bodies (MVBs), organelles involved in LB biogenesis. In spite of the results indicating increased surfactant production, the cellular quantity of LBs was surprisingly diminished and the majority of the remaining LBs were filled with SiO2-SPIONs. Additionally, LBs were detected inside abundant autophagic vacuoles (AVs) and obviously destined for degradation. We also observed time- and dose-dependent changes in mRNA expression for proteins involved in lipid metabolism. Our results demonstrate that non-cytotoxic concentrations of SiO2-SPIONs interfere with surfactant metabolism and LB biogenesis, leading to disturbed ability to reduce hypophase surface tension. To ensure the safe use of NPs for pulmonary delivery, we propose that potential NP interference with LB biogenesis is obligatorily taken into account.