Protein adsorption onto Fe3O4 nanoparticles with opposite surface charge and its impact on cell uptake

Nanoparticles (NPs) engineered for biomedical applications are meant to be in contact with protein-rich physiological fluids. These proteins are usually adsorbed onto the NP surface, forming a swaddling layer called protein corona that influences cell internalization. We present a study on protein adsorption onto different magnetic NPs (MNPs) when immersed in cell culture medium, and how these changes affect the cellular uptake. Two colloids with magnetite cores of 25 nm, same hydrodynamic size and opposite surface charge were in situ coated with (a) positive polyethyleneimine (PEI-MNPs) and (b) negative poly(acrylic acid) (PAA-MNPs). After few minutes of incubation in cell culture medium the wrapping of the MNPs by protein adsorption resulted in a 5-fold size increase. After 24 h of incubation large MNP-protein aggregates with hydrodynamic sizes 1500 to 3000 nm (PAA-MNPs and PEI-MNPs respectively) were observed. Each cluster contained an estimated number of magnetic cores between 450 and 1000, indicating the formation of large aggregates with a "plum pudding" structure of MNPs embedded into a protein network of negative surface charge irrespective of the MNP_core charge. We demonstrated that PEI-MNPs are incorporated in much larger amounts than the PAA-MNPs units. Quantitative analysis showed that SH-SY5Y cells can incorporate 100 per cent of the added PEI-MNPs up to about 100 pg per cell, whereas for PAA-MNPs the uptake was less than 50 percent. The final cellular distribution showed also notable differences regarding partial attachment to the cell membrane. These results highlight the need to characterize the final properties of MNPs after protein adsorption in biological media, and demonstrate the impact of these properties on the internalization mechanisms in neural cells.Comment: 32 pages, 10 figure

Iron oxide nanoparticles and derivatives for biomedical imaging and application in cancer diagnosis and siRNA therapy

This paper was presented at the 3rd Micro and Nano Flows Conference (MNF2011), which was held at the Makedonia Palace Hotel, Thessaloniki in Greece. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, Aristotle University of Thessaloniki, University of Thessaly, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute.Our studies have focused on the application of imaging-capable nanoparticulate agents for the delivery of small RNA-based tumor therapy. One example includes magnetic nanoparticles (MN), which have traditionally been utilized as contrast agents for magnetic resonance imaging. The probes typically consist of a dextran-coated superparamagnetic iron oxide core (for magnetic resonance imaging), labeled with Cy5.5 dye (for near-infrared in vivo optical imaging), and conjugated to synthetic small interfering RNA (siRNA) molecules targeting model or therapeutic genes. We have explored the potential of these nanoparticles as delivery modules for small interfering RNA to tumors. Furthermore, we have investigated the feasibility of combining the imaging and delivery capabilities of these nanoparticles for the tracking of siRNA bioavailability. The versatile functionalization potential of MN has allowed us to control properties of the agents, such as uptake mechanism and target organ distribution. The tumoral accumulation of MNsiRNA results in a remarkable level of target-gene down-regulation. Repeated treatment with MN-siBIRC5, targeting the tumor-specific anti-apoptotic gene, birc5, leads to the induction of apoptosis in the tumors and an overall reduction in tumor growth rate. More recently, we have synthesized a second generation of nanoparticles, which combine the capability for high-resolution magnetic resonance imaging with detection by ultrasensitive surface enhanced Raman scattering

Growth mechanism of nanostructured superparamagnetic rods obtained by electrostatic co-assembly

We report on the growth of nanostructured rods fabricated by electrostatic co-assembly between iron oxide nanoparticles and polymers. The nanoparticles put under scrutiny, {\gamma}-Fe2O3 or maghemite, have diameter of 6.7 nm and 8.3 nm and narrow polydispersity. The co-assembly is driven by i) the electrostatic interactions between the polymers and the particles, and by ii) the presence of an externally applied magnetic field. The rods are characterized by large anisotropy factors, with diameter 200 nm and length comprised between 1 and 100 {\mu}m. In the present work, we provide for the first time the morphology diagram for the rods as a function of ionic strength and concentration. We show the existence of a critical nanoparticle concentration and of a critical ionic strength beyond which the rods do not form. In the intermediate regimes, only tortuous and branched aggregates are detected. At higher concentrations and lower ionic strengths, linear and stiff rods with superparamagnetic properties are produced. Based on these data, a mechanism for the rod formation is proposed. The mechanism proceeds in two steps : the formation and growth of spherical clusters of particles, and the alignment of the clusters induced by the magnetic dipolar interactions. As far as the kinetics of these processes is concerned, the clusters growth and their alignment occur concomitantly, leading to a continuous accretion of particles or small clusters, and a welding of the rodlike structure.Comment: 15 pages, 10 figures, one tabl

The effects of aggregation and protein corona on the cellular internalization of iron oxide nanoparticles

Engineered inorganic nanoparticles are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient : the particles become either coated by a corona of serum proteins or precipitate out of the solvent. In the present paper, we show that by changing the coating of iron oxide nanoparticles from a low-molecular weight ligand (citrate ions) to small carboxylated polymers (poly(acrylic acid)), the colloidal stability of the dispersion is improved and the adsorption/internalization of iron towards living mammalian cells is profoundly affected. Citrate-coated particles are shown to destabilize in all fetal-calf-serum based physiological conditions tested, whereas the polymer coated particles exhibit an outstanding dispersibility as well as a structure devoid of protein corona. The interactions between nanoparticles and human lymphoblastoid cells are investigated by transmission electron microscopy and flow cytometry. Two types of nanoparticle/cell interactions are underlined. Iron oxides are found either adsorbed on the cellular membranes, or internalized into membrane-bound endocytosis compartments. For the precipitating citrate-coated particles, the kinetics of interactions reveal a massive and rapid adsorption of iron oxide on the cell surfaces. The quantification of the partition between adsorbed and internalized iron was performed from the cytometry data. The results highlight the importance of resilient adsorbed nanomaterials at the cytoplasmic membrane.Comment: 21 pages, 11 figures, accepted at Biomaterials (2011

Interactions between sub-10 nm iron and cerium oxide nanoparticles and 3T3 fibroblasts : the role of the coating and aggregation state

Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of the cerium and iron oxide sub-10 nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), MW = 2000 g mol-1). Electrostatically adsorbed on the surfaces, the organic moieties provide a negatively charged coating in physiological conditions. We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. Only the bare and the citrate-coated nanoceria exhibit a slight decrease of the mitochondrial activity for cerium concentrations above 5 mM (equivalent to 0.8 g L-1). We also observe that the citrate-coated particles are internalized by the cells in large amounts, typically 250 pg per cell after a 24 h incubation for iron oxide. In contrast, the polymer-coated particles are taken up at much lower rates (< 30 pg per cell). The strong uptake shown by the citrate-coated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes. In conclusion, we show that the uptake of nanomaterials by living cells depends on the coating of the particles and on its ability to preserve the colloidal nature of the dispersions.Comment: 9 figures, 2 table

Encapsulation of human serum albumin in submicrometer magnetic poly(lactide-co-glycolide) particles as a model system for targeted drug delivery

Two types of iron oxide nanoparticles were synthesized by coprecipitation of Fe(II) and Fe(III) chlorides: water-dispersible γ-Fe2O3 and organic solvent-dispersible oleic acid-coated Fe3O4 particles. The nanoparticles, together with human serum albumin (HSA) serving as a model for a protein-type drug, were then incorporated in poly(lactide-co-glycolide) (PLGA) particles using double emulsion solvent evaporation technique. Morphology, size and particle size distribution of the resulting particles was analyzed by electron microscopy and dynamic light scattering. Iron oxide and HSA encapsulating efficiency was determined by Prussian Blue staining and micro-BCA assay, respectively

Smart systems related to polypeptide sequences

Increasing interest for the application of polypeptide-based smart systems in the biomedical field has developed due to the advantages given by the peptidic sequence. This is due to characteristics of these systems, which include: biocompatibility, potential control of degradation, capability to provide a rich repertoire of biologically specific interactions, feasibility to self-assemble, possibility to combine different functionalities, and capability to give an environmentally responsive behavior. Recently, applications concerning the development of these systems are receiving greater attention since a targeted and programmable release of drugs (e.g. anti-cancer agents) can be achieved. Block copolymers are discussed due to their capability to render differently assembled architectures. Hybrid systems based on silica nanoparticles are also discussed. In both cases, the selected systems must be able to undergo fast changes in properties like solubility, shape, and dissociation or swelling capabilities. This review is structured in different chapters which explain the most recent advances on smart systems depending on the stimuli to which they are sensitive. Amphiphilic block copolymers based on polyanionic or polycationic peptides are, for example, typically employed for obtaining pH-responsive systems. Elastin-like polypeptides are usually used as thermoresponsive polymers, but performance can be increased by using techniques which utilize layer-by-layer electrostatic self-assembly. This approach offers a great potential to create multilayered systems, including nanocapsules, with different functionality. Recent strategies developed to get redox-, magnetic-, ultrasound-, enzyme-, light-and electric-responsive systems are extensively discussed. Finally, some indications concerning the possibilities of multi-responsive systems are discussed.Postprint (published version

Bare and Polymer Coated Iron Oxide Superparamagnetic Nanoparticles for Effective Removal of U (VI) from Acidic and Neutral Aqueous Medium

Superparamagnetic {\gamma}-Fe2O3 nanoparticles (5 nm diameter) were synthesized in water. The bare particles exhibit good colloidal stability at ~ pH 2 because of the strong electrostatic repulsion with a surface charge of +25 mV. The polyacrylic acid (PAA)-coated particles exhibit remarkable colloidal stability at ~ pH 7 with abundant free carboxyl groups as reactive sites for subsequent functionalization. In this work, we used zeta potential analysis, transmission electron microscopy, small angle X-ray scattering, and Inductively coupled plasma mass spectrometry to investigate the adsorption behavior of U (VI) on bare and coated colloidal superparamagnetic nanoparticles at pH 2 and pH 7. At pH 2, uranyl ion (UO22+) absorbed on the surface of the bare particles with decreasing particle surface charge. This induced particle agglomeration. At pH 7, uranyl ion (UO22+) hydrolyzed and formed plate-like particles of uranium hydroxide that were ~ 50 nm in diameter. The PAA-coated iron oxide nanoparticles absorbed on the surface of these U (VI) hydroxide plates to form large aggregates that precipitate to the bottom of the dispersion. At both pH 2 and pH 7, the resulting U (VI)/nanoparticle complex can be easily collected and extracted from the aqueous environment via an external magnetic field. The results show that both bare and polymer-coated superparamagnetic {\gamma}-Fe2O3 nanoparticles are potential absorbents for removing U (VI) from water

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