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.     

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.     

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.     

Particle size dependence of relaxivity for silica-coated iron oxide nanoparticles

We investigate the particle size dependence of the relaxivity of hydrogen protons in an aqueous solution of iron oxide (Fe₃O₄) nanoparticles coated in silica for biocompatibility. The T₁ and T₂ relaxation times for various concentrations of silica-coated nanoparticles were determined by a magnetic resonance scanner. We find that the relaxivity increased linearly with increasing particle size. The T₂ relaxivity (R₂) is more than 50 times larger than the T₁ relaxivity (R₁) for the nanoparticle contrast agent, which reflects the fact that the T₂ relaxation is mainly influenced by outer sphere processes. The high R₂/R₁ ratio demonstrates that silica-coated iron oxide nanoparticles may serve as a T₂ contrast agents in magnetic resonance imaging with high efficacy.     

Exclusive T2 MRI contrast enhancement by mesoporous carbon framework encapsulated manganese oxide nanoparticles

Single mode (either T₁ or T₂) contrast agents employed during magnetic resonance imaging owe their advantage over their dual counterparts to the fact that they do not involve any quenching caused by interference between the two modes. The chemistry involving oxides of manganese is highly significant due to their applicability as MRI contrast agents. Manganese oxides are usually known to display a dominant T₁ relaxation enhancement. But, in this work, an engineered structure of manganese oxide (Mn₂O₃) nanoparticles encapsulated within mesoporous carbon frameworks was developed which exhibited dominant T₂ contrast enhancement, through regulation of contact between the magnetic ion and water. Microstructural characterization revealed that the mesoporous carbon frameworks were spherical in shape and the nanoparticles within them had an average size of 40–50 nm. Relaxivity measurement, MRI experiments and cell viability assay convincingly established the system as a new class of biocompatible T₂ based magnetic resonance imaging agent.     

Enhanced ferrite nanoparticles as MRI contrast agents

Enhanced ferrite nanoparticles are a new class of contrast agents for magnetic resonance imaging (MRI). The enhanced ferrites are synthesized by reverse micelles technique to form iron core and oxide or ferrite shell preventing further oxidation of the nanoparticles. The nanoparticles are further functionalized using dopamine and PEG-600 to increase the solubility of the high magnetic moment nanoparticles. ¹H relaxation measurements of aqueous solutions of the nanoparticles were conducted at 2.4 T. The relaxivities r₁ and r₂, representing the slopes of these curves, are 7.19 and 9.96 s⁻¹ mM⁻¹, respectively. These values should be compared with relaxivities of 4–5 s⁻¹ mM⁻¹ corresponding to commonly used commercial contrast agents in human MR examinations.     

Effect of pH, citrate treatment and silane-coupling agent concentration on the magnetic, structural and surface properties of functionalized silica-coated iron oxide nanocomposite particles

Superparamagnetic iron oxide nanoparticles were synthesized by coprecipitation of iron chloride salts at various pH values (9, 10, 11 and12) that were adjusted using an ammonia solution. Increasing the pH from 9 to 12 led to decreases in the size of iron oxide nanoparticles from 7.9±1.4 to 5±0.6 nm and the saturation magnetization (M_s) from 82.73 to 67.14 emu/g, respectively, when analyzed with transmission electron microscopy (TEM) and vibrating sample magnetometer (VSM). X-ray diffraction patterns as well as M_s values showed that magnetite is the dominantly synthesized phase in the examined pH values. Unmodified iron oxide nanoparticles were coated with silica via the hydrolysis and condensation of tetraethyl orthosilicate (TEOS), designated P1 particles. The size distribution diagram of P1 particles showed two regions with mean sizes of 143.3±15.4 and 216.9±13.7 nm corresponding to silica and iron oxide@silica particles, respectively. Stabilization of iron oxide nanoparticles using sodium citrate prior to coating with silica (P2 particles) resulted in nanocomposites with a mean size of 275±16.1 nm and an M_s value of 2.9 emu/g. Subsequently, the surface of P2 particles was functionalized by amine groups using N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (EDS). Results obtained from the measurement of zeta potential revealed that the highest value of isoelectric point (PI) change, indicating a more efficient surface functionalization, occurs when the EDS concentration of 90 mM is used, as compared to that for particles aminated using 25 and 180 mM EDS.     

Synthesis of amino-group functionalized superparamagnetic iron oxide nanoparticles and applications as biomedical labeling probes

Superparamagnetic iron oxide (SPIO) nanoparticles were synthesized by coprecipitation technique and further functionalized with amino-group to obtain amino-group functionalized (amino-SPIO) nanoparticles. The X-ray diffraction results reveal the structure of amino-SPIO nanoparticles, from which the average iron core diameter is approximately 10 nm by calculation; while Zetasizer reveals their hydrodynamic diameter are mainly distributed in the range of 40?C60 nm. These nanoparticles can be taken up by liver tissue, resulting in dramatically darkening of liver tissue under T2-magnetic resonance imaging (MRI). The spin?Cspin relaxivity coefficient of these nanoparticles is 179.20 mM^?1 s^?1 in a 1.5 T magnetic resonance system. In addition, amino-SPIO nanoparticles were conjugated to Tat (FITC) peptide and incubated with neural stem cells in vitro, the authors can detect the positive-labeling (labeled) neural stem cells showing green fluorescence, which indicates Tat (FITC) peptide-derivated amino-SPIO nanoparticles are able to enter cells. Furthermore, it was also find significant negative T2 contrast enhancement when compared with the non-nanoparticles-labeled neural stem cells in T2-weighted MRI. The amino-SPIO nanoparticles show promising potential as a new type of labeling probes, which can be used in magnetic resonance-enhanced imaging and fluorescence diagnosis.     

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.     

Applications of polymeric micelles with tumor targeted in chemotherapy

The chitosan-coated magnetic nanoparticles (CS MNPs) were in situ synthesized by cross-linking method. In this method; during the adsorption of cationic chitosan molecules onto the surface of anionic magnetic nanoparticles (MNPs) with electrostatic interactions, tripolyphosphate (TPP) is added for ionic cross-linking of the chitosan molecules with each other. The characterization of synthesized nanoparticles was performed by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS/ESCA), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), thermal gravimetric analysis (TGA), and vibrating sample magnetometry (VSM) analyses. The XRD and XPS analyses proved that the synthesized iron oxide was magnetite (Fe₃O₄). The layer of chitosan on the magnetite surface was confirmed by FTIR. TEM results demonstrated a spherical morphology. In the synthesis, at higher NH₄OH concentrations, smaller sized nanoparticles were obtained. The average diameters were generally between 2 and 8?nm for CS MNPs in TEM and between 58 and 103?nm in DLS. The average diameters of bare MNPs were found as around 18?nm both in TEM and DLS. TGA results indicated that the chitosan content of CS MNPs were between 15 and 23?% by weight. Bare and CS MNPs were superparamagnetic. These nanoparticles were found non-cytotoxic on cancer cell lines (SiHa, HeLa). The synthesized MNPs have many potential applications in biomedicine including targeted drug delivery, magnetic resonance imaging?(MRI), and magnetic hyperthermia.     

In situ preparation of high relaxivity iron oxide nanoparticles by coating with chitosan: A potential MRI contrast agent useful for cell tracking

Iron oxide nanocrystals are of considerable interest in nanoscience and nanotechnology because of their nanoscale dimensions, nontoxic nature, and superior magnetic properties. Colloidal solutions of magnetic nanoparticles (ferrofluids) with a high magnetite content are highly desirable for most molecular imaging applications. In this paper, we present a method for in situ coating of superparamagnetic iron oxide (SPIO) with chitosan in order to increase the content of magnetite. Iron chloride salts (Fe³⁺ and Fe²⁺) were directly coprecipitated inside a porous matrix of chitosan by Co-60 γ-ray irradiation in an aqueous solution of acetic acid. Following sonication, iron oxide nanoparticles were formed inside the chitosan matrix at a pH value of 9.5 and a temperature of 50 °C. The [Fe³⁺]:[Fe²⁺]:[NH₄OH] molar ratio was 1.6:1:15.8. The final ferrofluid was formed with a pH adjustment to approximately 2.0/3.0, alongside with the addition of mannitol and lactic acid. We subsequently characterized the particle size, the zeta potential, the iron concentration, the magnetic contrast, and the cellular uptake of our ferrofluid. Results showed a z-average diameter of 87.2 nm, a polydispersity index (PDI) of 0.251, a zeta potential of 47.9 mV, and an iron concentration of 10.4 mg Fe/mL. The MRI parameters included an R1 value of 22.0 mM⁻¹ s⁻¹, an R2 value of 202.6 mM⁻¹ s⁻¹, and a R2/R1 ratio of 9.2. An uptake of the ferrofluid by mouse macrophages was observed. Altogether, our data show that Co-60 γ-ray radiation on solid chitosan may improve chitosan coating of iron oxide nanoparticles and tackle its aqueous solubility at pH 7. Additionally, our methodology allowed to obtain a ferrofluid with a higher content of magnetite and a fairly unimodal distribution of monodisperse clusters. Finally, MRI and cell experiments demonstrated the potential usefulness of this product as a potential MRI contrast agent that might be used for cell tracking.     

Superparamagnetic FePt nanoparticles as excellent MRI contrast agents

Chemically disordered face-centered cubic (fcc) FePt nanoparticles (NPs) with a mean diameter of 9 nm were synthesized via pyrolysis of iron(III) ethoxide and platinum(II) acetylacetonate. The surface ligands of these NPs were then exchanged from oleic acid to tetramethylammonium hydroxide (TMAOH) to measure the longitudinal (T₁) and transverse (T₂) proton relaxation times of aqueous dispersion of FePt NPs. Magnetic resonance relaxometry reveals that TMAOH-capped FePt NPs have a higher T₂-shortening effect than conventional superparamagnetic iron oxide NPs, indicating that fcc-phase FePt NPs might be superior negative contrast agents for magnetic resonance imaging.     

Paramagnetic contribution of serum iron to the spin-spin relaxation rate (1/T 2) measured by MRI

Spin-spin relaxation timeT ₂ values of serum with and without iron were measured by magnetic resonance imaging (MRI) to find the proton relaxivity of Fe(III) in serum.T ₂ values in serum containing definite amounts of added iron were also measured before and after addition of ascorbic acid. The difference in the 1/T ₂ of serum with and without ascorbic acid was used for recalculation of the added iron values. Recalculated iron values confirm that the difference in healthy serum is caused by iron only. In addition, in order to find the paramagnetic contribution of serum iron,T ₂ values of iron-deficient, healthy and iron-overloaded serum were measured before and after addition of ascorbic acid. The difference in the 1/T ₂ values was then applied to the calculation of the serum iron values. The consistency of iron values determined from the difference to those by autoanalyzer suggests that the differences in diseased serum also represent the paramagnetic contribution of serum iron. The data imply that serum iron content in healthy, iron-deficient and iron-overloaded serum may be assessed by MRI.     

Study of carbon encapsulated iron oxide/iron carbide nanocomposite for hyperthermia

Magnetic nanocomposite has been synthesized successfully using biopolymer route which acts as a source of carbon for carbide formation. The present approach based on thermal decomposition represents a considerable advance over previous reports that often use high-energy procedures or costly and hazardous precursors. X-ray diffraction, high-resolution transmission electron microscopy and vibrating sample magnetometer have been used to characterize the composites. Multi phase formation is evident from X-ray diffraction in the as-prepared samples. Phase confirmation was further done from M (magnetization) versus T (temperature) curve indicating presence of different phases of carbide along with iron oxide. TEM study suggests formation of cuboidal shape nanocomposite using two different quenching conditions. Transmission electron microscopy also confirmed the formation of carbon layer in the vicinity of the Fe₃O₄/Fe₃C nanoparticles. The magnetic measurement shows that the composite nanoparticles exhibit a maximum magnetization of 60 emu g⁻¹ at room temperature. Biocompatibility study with three different cell lines (HeLa, MCF-7 and L929) confirms that these nanocomposites are biocompatible. Temperature versus time measurement in an AC field suggests good heating ability of the samples. These investigations indicate that these nanocomposites may be useful for bioapplications, in particular for hyperthermia.     

Uptake of magnetic nanoparticles into cells for cell tracking

A challenge for future applications in nanotechnology is the functional integration of nano-sized materials into cellular structures. Here we investigated superparamagnetic Fe₃O₄ iron oxide nanoparticles coated with a lipid bilayer for uptake into cells and for targeting subcellular compartments. It was found that magnetic nanoparticles (MNPs) are effectively taken up into cells and make cells acquire magnetic activity. Biotin-conjugated MNPs were further functionalized by binding of the fluorescent tag streptavidin–fluorescein isothiocyanate (FITC) and, following uptake into cells, shown to confer magnetic activity and fluorescence labeling. Such FITC-MNPs were localized in the lysosomal compartment of cells which suggests a receptor-mediated uptake mechanism.     

The concentration dependence of relaxation times of hydrogen proton in the aqueous solution of iron ferrite magnetic nanoparticles

By the direct coprecipitation of the aqueous solution of iron salt and tetramethylammonium hydroxide solution the stable iron ferrite nanoparticles were formulated. These nanoparticles were found to have uniform sizes of about 7 nm, and also showed no coalescence in the aqueous solution for a few months. The superparamagnetic behavior of these nanoparticles was checked by a vibrating sample magnetometer. Also, the temperature dependence of saturation magnetization of nanoparticles was observed using a superconducting quantum interface device magnetometer. The relaxation times of T₁ and T₂ of hydrogen proton in the colloidal aqueous solution of magnetic nanoparticles were measured using a nuclear magnetic resonance spectrometer for the wide range of concentration of nanoparticles in the aqueous solution. The inverse of relaxation times was observed to be directly dependent on the concentration of nanoparticles.     

Magnetic,optical and relaxometric properties of organically coated gold–magnetite (Au–Fe3O4) hybrid nanoparticles for potential use in biomedical applications

We present the magnetic, optical and relaxometric properties of multifunctional Au–Fe₃O₄ hybrid nanoparticles (HNPs), as possible novel contrast agents (CAs) for magnetic resonance imaging (MRI). The HNPs have been synthesized by wet chemical methods in heterodimer and core–shell geometries and capped with oleylamine. Structural characterization of the samples have been made by X-ray diffraction and transmission electron microscopy, while magnetic properties have been investigated by means of Superconducting Quantum Interference Device-SQUID magnetometry experiments. As required for MRI applications using negative CAs, the samples resulted superparamagnetic at room temperature and well above their blocking temperatures. Optical properties have been investigated by analyzing the optical absorbtion spectra collected in UV–visible region. Relaxometric measurements have been performed on organic suspensions of HNPs and Nuclear Magnetic Resonance (NMR) dispersion curves have been obtained by measuring the longitudinal 1/T₁ and transverse 1/T₂ relaxation rates of solvent protons in the range 10 kHz/300 MHz at room temperature. NMR relaxivities r₁ and r₂ have been compared with ENDOREM^®, one of the commercial superparamagnetic iron oxide based MRI contrast agents. MRI contrast enhancement efficiencies have been investigated also by examining T₂-weighted MR images of suspensions. The experimental results suggest that the nanoparticles' suspensions are good candidates as negative CAs.     

Size distribution of magnetic iron oxide nanoparticles using Warren–Averbach XRD analysis

We use the Fourier transform based Warren–Averbach (WA) analysis to separate the contributions of X-ray diffraction (XRD) profile broadening due to crystallite size and microstrain for magnetic iron oxide nanoparticles. The profile shape of the column length distribution, obtained from WA analysis, is used to analyze the shape of the magnetic iron oxide nanoparticles. From the column length distribution, the crystallite size and its distribution are estimated for these nanoparticles which are compared with size distribution obtained from dynamic light scattering measurements. The crystallite size and size distribution of crystallites obtained from WA analysis are explained based on the experimental parameters employed in preparation of these magnetic iron oxide nanoparticles. The variation of volume weighted diameter (D_v, from WA analysis) with saturation magnetization (M_s) fits well to a core shell model wherein it is known that M_s=M_bulk(1?6g/D_v) with M_bulk as bulk magnetization of iron oxide and g as magnetic shell disorder thickness.