Synthesis,characterization, and in vitro biological evaluation of highly stable diversely functionalized superparamagnetic iron oxide nanoparticles

In this article, we report the design and synthesis of a series of well-dispersed superparamagnetic iron oxide nanoparticles (SPIONs) using chitosan as a surface modifying agent to develop a potential T ₂ contrast probe for magnetic resonance imaging (MRI). The amine, carboxyl, hydroxyl, and thiol functionalities were introduced on chitosan-coated magnetic probe via simple reactions with small reactive organic molecules to afford a series of biofunctionalized nanoparticles. Physico-chemical characterizations of these functionalized nanoparticles were performed by TEM, XRD, DLS, FTIR, and VSM. The colloidal stability of these functionalized iron oxide nanoparticles was investigated in presence of phosphate buffer saline, high salt concentrations and different cell media for 1 week. MRI analysis of human cervical carcinoma (HeLa) cell lines treated with nanoparticles elucidated that the amine-functionalized nanoparticles exhibited higher amount of signal darkening and lower T ₂ relaxation in comparison to the others. The cellular internalization efficacy of these functionalized SPIONs was also investigated with HeLa cancer cell line by magnetically activated cell sorting (MACS) and fluorescence microscopy and results established selectively higher internalization efficacy of amine-functionalized nanoparticles to cancer cells. These positive attributes demonstrated that these nanoconjugates can be used as a promising platform for further in vitro and in vivo biological evaluations.     

In Situ Evaluation of Nanoparticle–Protein Interactions by Dynamic Magnetic Susceptibility Measurements

The use of dynamic magnetic susceptibility measurements is reported to study nanoparticle–protein interactions in situ. The technique consists of measuring the rotational diffusivity of thermally blocked magnetic nanoparticles (MNPs) in protein solutions. To illustrate the technique, the effect of nanoparticle zeta potential in carboxymethyl‐dextran‐coated MNPs and their interaction with model anionic and cationic proteins, such as bovine serum albumin (BSA), immunoglobulin G (IgG), fibrinogen (FIBR), apo‐transferrin (TRANS), lysozyme (LYZ), and histone (HIS), in a range of protein concentrations is studied. Experiments indicate that interactions between the negatively charged particles and the negatively charged proteins BSA, IgG, FIBR, and TRANS are negligible. However, positively charged proteins LYZ and HIS readily absorb onto the nanoparticles, as evidenced by an increase in size and eventual aggregation of the particles. Onset of this effect seems to happen at a lower concentration of HIS compared with LYZ. The technique could be applied to other particle surface coatings and to particles in complex protein mixtures, such as whole blood and serum, allowing systematic in situ studies of nanoparticle–protein interactions.     

Altering protein surface charge with chemical modification modulates protein–gold nanoparticle aggregation

Gold nanoparticles (AuNP) can interact with a wide range of molecules including proteins. Whereas significant attention has focused on modifying the nanoparticle surface to regulate protein–AuNP assembly or influence the formation of the protein “corona,” modification of the protein surface as a mechanism to modulate protein–AuNP interaction has been less explored. Here, we examine this possibility utilizing three small globular proteins—lysozyme with high isoelectric point (pI) and established interactions with AuNP; α-lactalbumin with similar tertiary fold to lysozyme but low pI; and myoglobin with a different globular fold and an intermediate pI. We first chemically modified these proteins to alter their charged surface functionalities, and thereby shift protein pI, and then applied multiple methods to assess protein–AuNP assembly. At pH values lower than the anticipated pI of the modified protein, AuNP exposure elicits changes in the optical absorbance of the protein–NP solutions and other properties due to aggregate formation. Above the expected pI, however, protein–AuNP interaction is minimal, and both components remain isolated, presumably because both species are negatively charged. These data demonstrate that protein modification provides a powerful tool for modulating whether nanoparticle–protein interactions result in material aggregation. The results also underscore that naturally occurring protein modifications found in vivo may be critical in defining nanoparticle–protein corona compositions.     

Cell viability and MRI performance of highly efficient polyol-coated magnetic nanoparticles

This work aimed at determining conditions that would allow us to control the size of the NPs and create a system with characteristics apt for biomedical applications. We describe a comprehensive study on the synthesis and physical characterization of two highly sensitive sets of triethylene glycol (TREG) and polyethylene glycol (PEG)-coated superparamagnetic iron oxide nanoparticles (SPIONs) to be evaluated for use as magnetic resonance (MR) contrast agents. The ferrofluids demonstrated excellent colloidal stability in deionized water at pH 7.0 as indicated by dynamic light scattering (DLS) data. The magnetic relaxivities, r ₂, were measured on a 1.5 T clinical MRI instrument. Values in the range from 205 to 257 mM^?1 s^?1 were obtained, varying proportionally to the SPIONs’ sizes and coating nature. Further in vitro cell viability tests and in vivo biodistribution analyses of the intravenously administered nanoparticles showed that the prepared systems have good biocompatibility and migrate to several organs, mainly the meninges, spleen, and liver. Based on these results, our findings demonstrated the potential utility of these nanosystems as clinical contrast agents for MR imaging.     

Characterization of PEI-coated superparamagnetic iron oxide nanoparticles for transfection: Size distribution,colloidal properties and DNA interaction

Superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyethylenimine. Here, we briefly describe the synthesis as well as DNA:PEI:SPION complexes and the characterization of the compounds according to their particle size, ζ-potential, morphology, DNA complexing ability, magnetic sedimentation, and colloidal stability. PEI coating of SPIONs led to colloidally stable beads even in high salt concentrations over a wide pH range. DNA plasmids and PCR products encoding for green fluorescent protein were associated with the described beads. The complexes were added to cells and exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency.     

Effect of ground electrode on charge injection and surface potential of corona charged polyethylene film

The surface potential decay measurement is a simple and low cost tool to assess electrical properties of insulation materials; therefore, understanding the physical mechanisms of the surface potential decay becomes necessary. With our recent space charge measurement results on corona charged samples, bipolar charge injection on corona charged samples had been observed. Based on this new fact, it is anticipated that the ground electrode should have significant effect during corona charging and subsequently decay processes. In the paper, low density polyethylene (LDPE) film with gold ground electrode was compared with LDPE film with aluminium ground to study effect of ground electrode on charge injection and surface potential decay processes. Charging current during the corona charging, surface potential decay and space charge dynamics after corona charging in the samples with either gold coated or aluminium ground electrode were measured. Differences have been observed for gold ground electrode when compared with aluminium ground electrode. Higher work function of gold electrode is responsible for the observed differences. A preliminary simulation has also attempted to show that the bipolar injection may take place in corona charged LDPE films.     

Ex vivo assessment of polyol coated-iron oxide nanoparticles for MRI diagnosis applications: toxicological and MRI contrast enhancement effects

Polyol synthesis is a promising method to obtain directly pharmaceutical grade colloidal dispersion of superparamagnetic iron oxide nanoparticles (SPIONs). Here, we study the biocompatibility and performance as T2-MRI contrast agents (CAs) of high quality magnetic colloidal dispersions (average hydrodynamic aggregate diameter of 16-27 nm) consisting of polyol-synthesized SPIONs (5 nm in mean particle size) coated with triethylene glycol (TEG) chains (TEG-SPIONs), which were subsequently functionalized to carboxyl-terminated meso-2-3-dimercaptosuccinic acid (DMSA) coated-iron oxide nanoparticles (DMSA-SPIONs). Standard MTT assays on HeLa, U87MG, and HepG2 cells revealed that colloidal dispersions of TEG-coated iron oxide nanoparticles did not induce any loss of cell viability after 3 days incubation with dose concentrations below 50 μg Fe/ml. However, after these nanoparticles were functionalized with DMSA molecules, an increase on their cytotoxicity was observed, so that particles bearing free terminal carboxyl groups on their surface were not cytotoxic only at low concentrations (<10 μg Fe/ml). Moreover, cell uptake assays on HeLa and U87MG and hemolysis tests have demonstrated that TEG-SPIONs and DMSA-SPIONs were well internalized by the cells and did not induce any adverse effect on the red blood cells at the tested concentrations. Finally, in vitro relaxivity measurements and post mortem MRI studies in mice indicated that both types of coated-iron oxide nanoparticles produced higher negative T2-MRI contrast enhancement than that measured for a similar commercial T2-MRI CAs consisting in dextran-coated ultra-small iron oxide nanoparticles (Ferumoxtran-10). In conclusion, the above attributes make both types of as synthesized coated-iron oxide nanoparticles, but especially DMSA-SPIONs, promising candidates as T2-MRI CAs for nanoparticle-enhanced MRI diagnosis applications.     

Higher stabilities of positive and negative charge on tetrafluoroethylene–hexafluoropropylene copolymer (FEP) electrets treated with titanium-tetrachloride vapor

Tetrafluoroethylene–hexafluoropropylene copolymer (FEP) films were treated with titanium-tetrachloride vapor in a molecular-layer deposition process. As a result of the surface treatment, significant improvements of the thermal and temporal charge stability were observed. Charge-decay measurements revealed enhancements of the half-value temperatures and the relaxation times of positively charged FEP electrets by at least 120 °C and two orders of magnitude, respectively. Beyond previous publications on fluoropolymer electrets with surface modification, we here report enhanced charge stabilities of the FEP films charged in negative as well as in positive corona discharges. Even though the improvement for negatively charged FEP films is moderate (half-value temperature about 20 °C higher), our experiments show that the asymmetry in positive and negative charge stability that is typical for FEP electrets can be overcome by means of chemical surface treatments. The results are discussed in the context of the formation of modified surface layers with enhanced charge-trapping properties.     

Electrokinetic transport of nanoparticles to opening of nanopores on cell membrane during electroporation

Nanoparticle transport to the opening of the single nanopore created on the cell membrane during the electroporation is studied. First, the permeabilization of a single cell located in a microchannel is investigated. When the nanopores are created, the transport of the nanoparticles from the surrounding liquid to the opening of one of the created nanopores is examined. It was found that the negatively charged nanoparticles preferably move into the nanopores from the side of the cell membrane that faces the negative electrode. Opposite to the electro-osmotic flow effect, the electrophoretic force tends to draw the negatively charged nanoparticles into the opening of the nanopores. The effect of the Brownian force is negligible in comparison with the electro-osmosis and the electrophoresis. Smaller nanoparticles with stronger surface charge transport more easily to the opening of the nanopores. Positively charged nanoparticles preferably enter the nanopores from the side of the cell membrane that faces the positive electrode. On this side, both the electrophoretic and the electro-osmotic forces are in the same directions and contribute to bring the positively charged particles into the nanopores.     

Size-controlled synthesis of superparamagnetic iron oxide nanoparticles and their surface coating by gold for biomedical applications

The size mono-dispersity, saturation magnetization, and surface chemistry of magnetic nanoparticles (NPs) are recognized as critical factors for efficient biomedical applications. Here, we performed modified water-in-oil inverse nano-emulsion procedure for preparation of stable colloidal superparamagnetic iron oxide NPs (SPIONs) with high saturation magnetization. To achieve mono-dispersed SPIONs, optimization process was probed on several important factors including molar ratio of iron salts [Fe³⁺ and Fe²⁺], the concentration of ammonium hydroxide as reducing agent, and molar ratio of water to surfactant. The biocompatibility of the obtained NPs, at various concentrations, was evaluated via MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay and the results showed that the NPs were non-toxic at concentrations <0.1 mg/mL. Surface functionalization was performed by conformal coating of the NPs with a thin shell of gold (∼4 nm) through chemical reduction of attached gold salts at the surface of the SPIONs. The Fe₃O₄ core/Au shell particles demonstrate strong plasmon resonance absorption and can be separated from solution using an external magnetic field. Experimental data from both physical and chemical determinations of the changes in particle size, surface plasmon resonance optical band, phase components, core–shell surface composition, and magnetic properties have confirmed the formation of the mono-dispersed core–shell nanostructure.     

Protein Corona Modulates Distribution and Toxicological Effects of Silver Nanoparticles In Vivo

Exploitation of silver nanoparticles (AgNPs) in biomedicine represents more than one third of their overall applications. Despite their wide use, detailed toxicokinetic data and information on their action mechanisms in vivo are still scarce. One important obstacle is their fate and transformation patterns in biological environments where AgNPs get a “new face” after interaction with biomolecules, particularly proteins. The impact of protein corona on AgNP effects in vivo is eludicated. The in vivo effects of AgNPs prepared with two different protein coronas, albumin, and metallothionein, with polymer‐coated AgNPs are compared in male and female Wistar rats after intravenous administration. The results demonstrate that the character of the protein coronas on the AgNP surface affects not only distribution, but also oxidative stress response and genotoxicity in tissues of rat exposed to AgNPs. Additionally, sex‐related effects are observed. By emphasizing the importance of protein corona formation and sex‐related response, the obtained data support a reliable evidence base needed for assessing the health risks of the steadily increasing human exposure to AgNPs.     

Gummic acid stabilized γ-Fe2O3 aqueous suspensions for biomedical applications

Biomedical applications of magnetic nanoparticles depend critically on their preparation as aqueous colloidal suspensions, or ferrofluids, with long term stability under physiological conditions. Dispersion of the magnetic nanoparticles is generally achieved by the use of protein cages, polysaccharide, polypeptide and charged macromolecular coatings, which minimize interparticle magnetic interactions, particle agglomeration and precipitation. The synthesis and characterization of gummic-acid stabilized maghemite ferrofluids is reported. X-ray diffraction, transmission electron microscope and dynamic light scattering measurements give a γ-Fe₂O₃ magnetic core diameter of 8 nm and a nanocomposite particle hydrodynamic diameter of 50 nm. Mössbauer and magnetization measurements indicate the presence of isolated, sterically stabilized superparamagnetic nanoparticles resistant to aging, and thus, promising agents for the production of novel magneto-pharmaceuticals.     

Rhodamine B-Coated Gold Nanoparticles as Effective “Turn-on” Fluorescent Sensors for Detection of Zinc II Ions in Water

We designed a fluorescence resonance energy transfer system consisting of fluorophore Rhodamine B and gold nanoparticles for sensing of zinc ions in aqueous solution. The electrostatic attraction between positively charged N-atoms in Rhodamine molecules and negatively charged citrate corona of gold nanoparticles led to substantial fluorescence quenching. However, the quenching is switched off in the presence of zinc ions and therefore the system can be used as an effective “turn-on” fluorescence sensor. UV-Vis absorption, fluorescence spectroscopy, and transmission electron microscopy were used for sensor evaluation. The approach of “turn-on” fluorescence has real potential for sensing metallic ions in water.     

Irreversible Adsorption of Serum Proteins onto Nanoparticles

When a material comes in contact with serum or plasma, proteins will immediately adsorb to its surface. The extent of serum protein adsorption as well as the composition of the protein corona is thought to be decisive for the biological fate. The understanding of the mechanism underlying the concurrent adsorption of multiple proteins and the exact ways by which the adsorbed proteins interact with the biological setting, is still rudimentary. For both cases, a correct estimate of the composition of the protein corona is the key for an improved understanding. The protein corona composition is typically analyzed indirectly through analysis of the supernatant after protein desorption. However, in most cases the particles are not analyzed afterward in order to ensure that all proteins indeed have desorbed. Here, the results related to the analysis of the amounts of proteins in the corona are reported, focusing on the desorbed as well as the fraction of proteins that do not desorb. Irreversible protein adsorption can be observed in some cases. The results show that, in addition of the analysis of the supernatant, analysis of the particles is of critical importance to fully characterize the protein corona formed on nanoparticles.     

Nanoparticle Formation Mechanism in CVD Reactor with Ionization of Source Vapor

A new chemical vapor deposition (CVD) method, called ionization CVD, was developed, to produce non-agglomerated nanoparticles in which reactant gases are charged. A sonic-jet corona discharger was used as an ionizer in the developed nanoparticle generator. For a tetraethylorthosilicate (TEOS)/O₂ chemical system, SiO₂ nanoparticles were prepared. All particles formed by the ionization CVD were charged unipolarly. SEM micrographs of particles showed that the repulsive Coulombic force between charged particles reduces their coagulation rate and produces non-agglomerated nanoparticles that have a relatively high number concentration and small size. An external field was used to collect the charged particles onto Si wafers. These collected samples indicated that the deposition of charged particles could be controlled by the external electric field. Particle concentration measurement with a condensation nucleus counter at various TEOS concentrations suggested the particle formation mechanism in the ionization CVD was an ion-induced nucleation.     

Toxicity of superparamagnetic iron oxide nanoparticles: Research strategies and implications for nanomedicine

Superparamagnetic iron oxide nanoparticles （SPIONs） are one of the most versatile and safe nanoparticles in a wide variety of biomedical applications. In the past decades, considerable efforts have been made to investigate the potential adverse biological effects and safety issues associated with SPIONs, which is essential for the development of next-generation SPIONs and for continued progress in translational research. In this mini review, we summarize recent developments in toxicity studies on SPIONs, focusing on the relationship between the physicochemical properties of SPIONs and their induced toxic biological responses for a better toxicological understanding of SPIONs.     

Doxorubicin‐Anchored Curcumin Nanoparticles for Multimode Cancer Treatment against Human Liver Carcinoma Cells

Curcumin (Curcuma longa L), a yellow‐colored Indian spice, receives immense attention for the prevention and treatment of various cancers. Despite the superlative therapeutic efficacy, its poor solubility and instability in the aqueous medium hinder the effectiveness of cancer treatment. The novel preparation of curcumin nanoparticles by mechanical grinding of curcumin crystals without any toxic organic solvents is described here for the first time. The surface of curcumin nanoparticles is modified with the negatively charged polyelectrolyte poly(sodium 4‐strynesulfonate) through hydrogen bonding, which is the key to increasing the solubility and stability in the aqueous medium. The negative surface charge is exploited to conjugate doxorubicin drug molecule on the surface of curcumin nanoparticles as evidenced by fluorescence quenching experiments. Doxorubicin‐conjugated curcumin nanoparticles have a higher solubility with an enhanced cytotoxic effect toward the human hepatocellular carcinoma cell line by a reactive‐oxygen‐species‐mediated p53‐dependent apoptotic pathway. The combination of chemotherapy and photodynamic therapy significantly enhances antitumor activity of doxorubicin‐conjugated curcumin nanoparticles, and is expected to be a promising anticancer agent with special reference to human liver carcinoma cells.     

Improving blood-compatibility of titanium by coating collagen-heparin multilayers

This work deals with improving the blood-compatibility of titanium by coating it with heparin (Hep) and collagen (Col) using a layer-by-layer (LBL) self-assembly technique. In the work described here, LBL-produced Hep-Col film growth is initialized by deposition of a layer of positively charged polyl-Lysine (PLL) on a titanium surface, which is negatively charged after treatment with NaOH, followed by formation of a multilayer thin film formed by alternating deposition of negatively charged heparin and positively charged collagen utilizing electrostatic interaction. The chemical composition, wettability, surface topography, mass and thickness of the film were investigated by Fourier transform infrared spectroscopy, water contact angle measurement, scanning electron microscopy, atomic force microscopy, electronic analytical semi-microbalances, and XP stylus profilometry. The in vitro platelet adhesion and activation were investigated by a static platelet adhesion test probing the lactate dehydrogenase (LDH) release of adherent platelets after lysis and by a P-selectin assay. The clotting time was examined by activated partial thromboplastin time (APTT) and prothrombin time (PT) assays. All obtained data showed that the LBL film can significantly decrease platelet adhesion and activation, and prolong clotting time of APTT and PT compared to untreated titanium. LBL-produced Hep-Col films on titanium display more excellent anticoagulation performance than on the surface of titanium.     

Development of citrate-stabilized Fe3O4 nanoparticles: Conjugation and release of doxorubicin for therapeutic applications

We demonstrate a single-step facile approach for the fabrication of citric acid functionalized (citrate-stabilized) Fe₃O₄ aqueous colloidal magnetic nanoparticles (CA-MNP) of size 8-10 nm using soft chemical route. The surface functionalization of Fe₃O₄ nanoparticles with citric acid was evident from infrared spectroscopy, thermal and elemental analyses, and zeta-potential measurements. The drug-loading efficiency of CA-MNP was investigated using doxorubicin hydrochloride (DOX) as a model drug to evaluate their potential as a carrier system. The quenching of fluorescence intensity and decrease in surface charge of drug loaded CA-MNP strongly suggest the interaction/attachment of drug molecules with CA-MNP. More specifically, the present investigation discusses a method for entrapping positively charged drugs onto the surface of negatively charged CA-MNP through electrostatic interactions and suggests that bound drug molecules will be released in appreciable amounts in the mild acidic environments of the tumors. Furthermore, the aqueous colloidal stability, optimal magnetization, good specific absorption rate (under external AC magnetic field) and cytocompatibility with cells suggested that CA-MNP is appropriate candidate for biomedical applications.     

Instability of equilibrium of the liquid dielectric surface and formation of regular cellular structures in the field of a corona discharge

It has been established experimentally that the equilibrium of the plane surface of transformer oil under a corona electrode (needle) becomes unstable when the critical voltage, which increases with the oil layer thickness, is attained at the corona point. When the voltage at the needle exceeds the critical value, regular static cells are formed on the oil surface with characteristic sizes decreasing upon an increase in voltage. The theoretically estimated parameters of the experiment are found to be close to the experimental data. Comparison of the parameters corresponding to the occurrence of instability in the equilibrium of the oil surface in the field of the corona discharge with the parameters of instability in the equilibrium of the charged surface of liquid helium in a uniform electric field [7] demonstrates the similarity of the effects.