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.     

Static Magnetic Field Dictates Protein Corona Formation on the Surface of Glutamine‐Modified Superparamagnetic Iron Oxide Nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) have become important tools for the imaging and detecting of prevalent diseases for many years. Scientists usually harness their attraction to a static magnetic field (SMF) to increase targeting efficiency and minimize side effects. To prolong blood circulation time and minimize reticuloendothelial system clearance, SPIONs are increasingly designed with a negatively charged surface. Understanding how a SMF affects the SPIONs with a negative surface charge is fundamental to any potential downstream applications of SPIONs as drug delivery carriers and bio‐separation nanoparticles. The goal of our study is to investigate the effect of SMF treatment (204 mT) on the in vitro and in vivo protein corona formed on negatively charged SPIONs. The results reveal that the amount of protein and the composition of protein corona is directly related to the SMF treatment. Compared with the in vivo protein corona, SMF treatment exercises considerable influence on the composition of the in vitro protein corona. The in vitro protein corona formed on SPIONs modulates the secretion of inflammatory cytokines from cells. To the best of our knowledge, this report describes the first demonstration of a SMF as an influencing factor on protein corona formation in vivo. Our results help to elucidate the biological mechanisms of SPIONs with SMF treatment and suggest that the protein corona effect should be considered during the development of a magnetic target.     

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.     

Cellular uptake of folate-conjugated lipophilic superparamagnetic iron oxide nanoparticles

We prepared five folate-conjugated lipophilic superparamagnetic iron oxide nanoparticles (F₅-Liposuperparamagnetic iron oxide nanoparticles(SPIONs), 5.5 and 11 nm) and investigated their cellular uptake with KB cells, which is one of the representative folate-receptor over-expressing human epidermoid carcinoma cells, using MRI. The cellular uptake tests with the respective 5.5 and 11 nm F₅-LipoSPIONs at a fixed particle concentration showed appreciable amount of receptor-mediated uptakes and the specificity was higher in 5.5 nm SPIONs, due to its higher folic acid (FA) density, without inhibition. However, the numbers of the particles taken up under FA inhibition were similar, irrespective of their sizes.     

In situ thermolysis of magnetic nanoparticles using non-hydrated iron oleate complex

A novel strategy for the fabrication of nanostructured materials based on preparation of metallic surfactants is presented and some examples are demonstrated in this article. The suggested synthetic procedure of metal oleate is universal, potentially able to produce bulk quantities, and can be applicable to the synthesis of other metal oxide and metal nanoparticles. In general, organometallic compounds are quite expensive and are mostly classified as a highly toxic substance. In this study, we used simple, inexpensive, and eco-friendly approaches to prepare the metallic surfactants. As an example, non-hydrated iron oleate (FeOl) complexes are prepared as precursors for the in situ-fabricated superparamagnetic iron oxide nanoparticles (SPIONs) by thermolysis. The different coordination of the non-hydrated FeOl complexes are directly relating to the competition between nucleation and crystal growth. The in situ preparation of SPIONs involves the reaction of metal nitrate and carboxylic acid at 120 °C to synthesize the non-hydrated FeOl complexes and following the thermolysis of FeOl at 300 °C in non-coordination solvent. The coordination modes and distinct thermal behaviors of intermediates non-hydrated FeOl complexes are comparatively investigated by means of thermo-analytic techniques complimented by differential scanning calorimetry, thermal gravimetric analysis (TGA) and infrared spectroscopy (FTIR). The potential chemical structures of non-hydrated FeOl and their reaction mechanism by thermolysis were elucidated. The resulting lipid-coated SPIONs were characterized by transmission electron microscope, FTIR, differential temperature analysis, and TGA. These data suggested a bimodal interaction of organic shell and nanoparticle surface, with chemically absorbed inner layer and physically absorbed outer layer of carboxylic acid.     

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.     

Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy

Intravenously injected microbubbles (MBs) can be utilized as ultrasound contrast agent (CA) resulting in enhanced image quality. A novel CA, consisting of air filled MBs stabilized with a shell of polyvinyl alcohol (PVA) has been developed. These spherical MBs have been decorated with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as both ultrasound and magnetic resonance imaging (MRI) CA. In this study, a mathematical model was introduced that determined the shell thickness of two types of SPIONs decorated MBs (Type A and Type B). The shell thickness of MBs is important to determine, as it affects the acoustical properties. In order to investigate the shell thickness, thin sections of plastic embedded MBs were prepared and imaged using transmission electron microscopy (TEM). However, the sections were cut at random distances from the MB center, which affected the observed shell thickness. Hence, the model determined the average shell thickness of the MBs from corrected mean values of the outer and inner radii observed in the TEM sections. The model was validated using simulated slices of MBs with known shell thickness and radius. The average shell thickness of Type A and Type B MBs were 651 nm and 637 nm, respectively.     

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.     

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.     

Magnetic and in vitro heating properties of implants formed in situ from injectable formulations and containing superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microparticles for magnetically induced local hyperthermia

The biological and therapeutic responses to hyperthermia, when it is envisaged as an anti-tumor treatment modality, are complex and variable. Heat delivery plays a critical role and is counteracted by more or less efficient body cooling, which is largely mediated by blood flow. In the case of magnetically mediated modality, the delivery of the magnetic particles, most often superparamagnetic iron oxide nanoparticles (SPIONs), is also critically involved. We focus here on the magnetic characterization of two injectable formulations able to gel in situ and entrap silica microparticles embedding SPIONs. These formulations have previously shown suitable syringeability and intratumoral distribution in vivo. The first formulation is based on alginate, and the second on a poly(ethylene-co-vinyl alcohol) (EVAL). Here we investigated the magnetic properties and heating capacities in an alternating magnetic field (141 kHz, 12 mT) for implants with increasing concentrations of magnetic microparticles. We found that the magnetic properties of the magnetic microparticles were preserved using the formulation and in the wet implant at 37 °C, as in vivo. Using two orthogonal methods, a common SLP (20 W g⁻¹) was found after weighting by magnetic microparticle fraction, suggesting that both formulations are able to properly carry the magnetic microparticles in situ while preserving their magnetic properties and heating capacities.     

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.     

Multifunctional iron platinum stealth immunomicelles: targeted detection of human prostate cancer cells using both fluorescence and magnetic resonance imaging

Superparamagnetic iron oxide nanoparticles (SPIONs) are the most common type of contrast agents used in contrast agent-enhanced magnetic resonance imaging (MRI). Still, there is a great deal of room for improvement, and nanoparticles with increased MRI relaxivities are needed to increase the contrast enhancement in MRI applied to various medical conditions including cancer. We report the synthesis of superparamagnetic iron platinum nanoparticles (SIPPs) and subsequent encapsulation using PEGylated phospholipids to create stealth immunomicelles (DSPE-SIPPs) that can be specifically targeted to human prostate cancer cell lines and detected using both MRI and fluorescence imaging. SIPP cores and DSPE-SIPPs were 8.5 ± 1.6 nm and 42.9 ± 8.2 nm in diameter, respectively, and the SIPPs had a magnetic moment of 120 A m²/kg iron. J591, a monoclonal antibody against prostate specific membrane antigen (PSMA), was conjugated to the DSPE-SIPPs (J591-DSPE-SIPPs), and specific targeting of J591-DSPE-SIPPs to PSMA-expressing human prostate cancer cell lines was demonstrated using fluorescence confocal microscopy. The transverse relaxivity of the DSPE-SIPPs, measured at 4.7 Tesla, was 300.6 ± 8.5 s^?1 mM^?1, which is 13-fold better than commercially available SPIONs (23.8 ± 6.9 s^?1 mM^?1) and ~3-fold better than reported relaxivities for Feridex^® and Resovist^®. Our data suggest that J591-DSPE-SIPPs specifically target human prostate cancer cells in vitro, are superior contrast agents in T ₂-weighted MRI, and can be detected using fluorescence imaging. To our knowledge, this is the first report on the synthesis of multifunctional SIPP micelles and using SIPPs for the specific detection of prostate cancer.     

1H NMR Detection of superparamagnetic nanoparticles at 1T using a microcoil and novel tuning circuit

Magnetic beads containing superparamagnetic iron oxide nanoparticles (SPIONs) have been shown to measurably change the nuclear magnetic resonance (NMR) relaxation properties of nearby protons in aqueous solution at distances up to approximately 50 microm. Therefore, the NMR sensitivity for the in vitro detection of single cells or biomolecules labeled with magnetic beads will be maximized with microcoils of this dimension. We have constructed a prototype 550 microm diameter solenoidal microcoil using focused gallium ion milling of a gold/chromium layer. The NMR coil was brought to resonance by means of a novel auxiliary tuning circuit, and used to detect water with a spectral resolution of 2.5 Hz in a 1.04 T (44.2MHz) permanent magnet. The single-scan SNR for water was 137, for a 200 micros pi/2 pulse produced with an RF power of 0.25 mW. The nutation performance of the microcoil was sufficiently good so that the effects of magnetic beads on the relaxation characteristics of the surrounding water could be accurately measured. A solution of magnetic beads (Dynabeads MyOne Streptavidin) in deionized water at a concentration of 1000 beads per nL lowered the T(1) from 1.0 to 0.64 s and the T2 * from 110 to 0.91 ms. Lower concentrations (100 and 10 beads/nL) also resulted in measurable reductions in T2 *, suggesting that low-field, microcoil NMR detection using permanent magnets can serve as a high-sensitivity, miniaturizable detection mechanism for very low concentrations of magnetic beads in biological fluids.     

Ultrasound assisted chitosan coated iron oxide nanoparticles: Influence of ultrasonic irradiation on the crystallinity,stability, toxicity and magnetization of the functionalized nanoparticles

Due to unique reaction conditions of the acoustic cavitation process, ultrasound-assisted synthesis of nanoparticles has attracted increased research attention. In this study, we demonstrate the effect of ultrasonic irradiation on the crystallinity, stability, biocompatibility, and magnetic properties of chitosan-coated superparamagnetic iron oxide nanoparticles (CS-SPIONs). CS solution and colloidal suspension of SPIONs were mixed and sonicated using an ultrasonic probe of 1.3 cm tip size horn, frequency (20 kHz), and power (750 W). Different samples were sonicated for 1.5, 5, and 10 min with corresponding acoustic powers of 67, 40 and 36 W, and the samples were denoted S_1.5, S_5, and S₁₀, respectively. The samples were characterized using X-ray diffractometer (XRD), Energy dispersive X-ray (EDX), Transmission electronic microscope (TEM), Fourier transform infrared spectroscopy (FTIR), Zeta sizer, and vibrating sample magnetometer (VSM). Cell cytotoxicity and cell uptake were investigated with human embryonic kidney 293 (HEK-293) cells through MTT assay and Prussian blue staining, respectively. The sharp peaks of the XRD pattern were disappearing with an increase in the sonication period but a decrease in acoustic power. EDX analysis also demonstrates that atomic and weight percentages of the various elements in the samples were decreasing with an increase in the sonication period. However, the Zeta potential (ζ) values increase with an increase in the sonication period.The saturation magnetization (M_s) of the S_1.5 before and after the coating is 62.95 and 86.93 emu/g, respectively. Cell cytotoxicity and uptake of the S_1.5 show that above 70% of cells were viable at the highest concentration and the longest incubation duration. Importantly, the CS-SPIONs synthesized by the sonochemical method are non-toxic and biocompatible.     

Complex polarization-phase and spatial-frequency selections of laser images of blood-plasma films in diagnostics of changes in their polycrystalline structure

We present a theoretical formalism of correlation phase analysis of laser images of human blood plasma with spatial-frequency selection of manifestations of mechanisms of linear and circular birefringence of albumin and globulin polycrystalline networks. Comparative results of the measurement of coordinate distributions of the correlation parameter—the modulus of the degree of local correlation of amplitudes—of laser images of blood plasma taken from patients of three groups—healthy patients (donors), rheumatoid-arthritis patients, and breast-cancer patients—are presented. We investigate values and ranges of change of statistical (the first to fourth statistical moments), correlation (excess of autocorrelation functions), and fractal (slopes of approximating curves and dispersion of extrema of logarithmic dependences of power spectra) parameters of coordinate distributions of the degree of local correlation of amplitudes. Objective criteria for diagnostics of occurrence and differentiation of inflammatory and oncological states are determined.     

2D-Mueller-matrix tomography of optically anisotropic polycrystalline networks of biological tissues histological sections

A new technique of Mueller-matrix mapping of the birefringent structure of biological preparations of human organs tissues is suggested. The algorithms of reconstruction of average values and magnitude of fluctuations of the phase (birefringence) and amplitude (dichroism) of optically anisotropic structure of myocardium and connective tissue component of the vaginal wall histological section are proposed. The magnitudes and ranges of changes in the statistical moments of the 1st-4th order that characterize the distribution of average values and magnitude of fluctuations of birefringence and dichroism of the myocardium and connective tissue of the vaginal tissues histological sections were determined. Joint studies of distributions of the characteristics of phase and amplitude of the anisotropy of myocardium and connective tissue component of the vaginal wall tissues of different states were performed. The cases of various necrotic changes in the myocardium and pathological conditions of the vagina wall (prolapse of the genitals) are examined. Balanced accuracy of the method of Mueller-matrix polarization-phase and diffuse tomography of optically anisotropic polycrystalline networks in the differentiation of necrotic and pathological changes in human organs is determined.     

Bifurcations accompanying monotonic instability of an equilibrium of a cosymmetric dynamical system

It is well known that equilibrium in a cosymmetric system in the general position is a member of a one-parameter family. In the present paper the Lyapunov-Schmidt method and the method of the central manifold are used to analyze bifurcations of such a family of equilibria as well as internal bifurcations: transitions of the type focus-node, node-saddle, and so on during motion along the family. A series of scenarios of branching of families of equilibria and the change in the structure of their arcs, consisting of equilibria of the same type, is described. Bifurcations of stable and unstable arcs, coalescence and decomposition of families of equilibria, bifurcation of the loss of smoothness by the family of equilibria, and branching of a small equilibrium cycle from a corner point of the family of equilibria are investigated in detail. The variability of the spectrum along a family gives rise to a variety of new phenomena that are not encountered in the classical case of an isolated equilibrium or in bifurcations of families of equilibria of a system with symmetry. They include protraction with respect to the branching parameter of the family of equilibria, Lyapunov instability of a family of equilibria with the attraction properties being preserved, and the appearance and disappearance of new stable and unstable arcs on the family of equilibria. (c) 2000 American Institute of Physics.     

Complex degree of mutual anisotropy of linear birefringence and optical activity of biological tissues in diagnostics of prostate cancer

We present theoretical fundamentals of polarization and correlation analysis of the optical anisotropy of biological tissues. Results of measurements of coordinate distributions of the complex degree of mutual anisotropy (CDMA) that are formed by birefringent structures of the prostate tissue with benign and malignant changes are compared. Magnitudes and ranges of variation of statistical (the firstto fourth-order distribution moments) and correlation (excess of autocorrelation functions) parameters of the coordinate CDMA distributions of histological sections of the prostate postoperative bioptic material are studied. Objective criteria of the diagnostics of the appearance of pathology and of the differentiation of the degree of its severity are determined.     

Reconstruction of synthesized holograms-Fresnel projectors at angles of incidence of reconstructing wave that exceed the angle of incidence of the reference wave in hologram synthesis

The possibility of the aberration-free reconstruction of a synthesized hologram-Fresnel projector in the case when the angles of incidence of the reconstructing plane wave exceed the angle of incidence of the plane reference wave during hologram synthesis is shown. The character of the dependence of admissible angles of incidence of the reconstructing wave on the parameters of the synthesis of hologram projectors is revealed and described mathematically. The results of an experimental study that confirms the possibility of the aberration-free reconstruction of hologram projectors at angles of incidence of the reconstructing plane wave that exceed the angle of incidence of the reference wave of the plane are presented. The results of the successful implementation of the projection photolithography process using a synthesized relief-phase reflection hologram-Fresnel projector are demonstrated.     

Theory of measurement of entangled states

Problem on reconstruction of state of finite-dimension quantum information transfer channel, pure or mixed, by results of measurements of needed number of observables, is considered. It is shown that in general case it is needed to measure incompatible observables in number exceeding by one dimension of space of vectors of state. Each of incompatible observables is measured in its statistically valuable series of measurements. In special case, when one of observables is a non-demolition observable, measurement of the other observables is needed for realization of control of property of non-demolition. In case of paired channel it is shown that results of measurements of observables that do not demolish states of sub-channels are characterized by mutual distribution of probabilities while results of measurement of over-classical observables are characterized by mutual correlation only. This correlation vanishes completely in case of pure unentangled states.