Sensorineural hearing loss, a subset of all clinical hearing loss, may be correctable through the use of gene therapy. We are testing a delivery system of therapeutics through a 3 cell-layer round window membrane model (RWM model) that may provide an entry of drugs or genes to the inner ear. We designed an in vitro RWM model similar to the RWM (will be referred to throughout the paper as RWM model) to determine the feasibility of using superparamagnetic iron oxide (Fe3O4) nanoparticles (SPION) for targeted delivery of therapeutics to the inner ear.
The RWM model is a 3 cell-layer model with epithelial cells cultured on both sides of a small intestinal submucosal (SIS) matrix and fibroblasts seeded in between. Dextran encapsulated nanoparticle clusters 130 nm in diameter were pulled through the RWM model using permanent magnets with flux density 0.410 Tesla at the pole face. The SIS membranes were harvested at day 7 and then fixed in 4% paraformaldehyde. Transmission electron microscopy and fluorescence spectrophotometry were used to verify transepithelial transport of the SPION across the cell-culture model. Histological sections were examined for evidence of SPION toxicity, as well to generate a timeline of the position of the SPION at different times. SPION also were added to cells in culture to assess in vitro toxicity.
Transepithelial electrical resistance measurements confirmed epithelial confluence, as SPION crossed a membrane consisting of three co-cultured layers of cells, under the influence of a magnetic field. Micrographs showed SPION distributed throughout the membrane model, in between cell layers, and sometimes on the surface of cells. TEM verified that the SPION were pulled through the membrane into the culture well below. Fluorescence spectrophotometry quantified the number of SPION that went through the SIS membrane. SPION showed no toxicity to cells in culture.
A three-cell layer model of the human round window membrane has been constructed. SPION have been magnetically transported through this model, allowing quantitative evaluation of prospective targeted drug or gene delivery through the RWM. Putative in vivo carrier superparamagnetic nanoparticles may be evaluated using this model.
Purpose: This study was performed to compare the cytotoxicity and magnetic resonance (MR) contrast in diverse cultured cells and xenograft tumors models of two ultra-small superparamagnetic iron oxides (USPIOs), thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION) and monocrystalline iron oxide nanoparticles (MION-47).
Materials and methods: Transmission electron microscopy (TEM) images and R2 relaxivity values of the TCL-SPION and MION-47 were obtained and the cell viability and cell growth velocity of treated and untreated human fibroblasts and human umbilical vein endothelial cells (HUVEC) were evaluated. The effect of TCL-SPION and MION-47 on the secretion of interlukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), the production of nitric oxides and the mitochondrial membrane potentials in murine macrophage cells (RAW264.7) was compared. Human hepatocellular carcinoma cells (HepG2, 5x105) were subcutaneously injected into nude mice (BALB/c) and in vivo MR imaging of tumors before and after injection with TCL-SPION or MION-47 (12.5 mg Fe/kg) was performed on a 1.5 Tesla MRI scanner.
Results: On TEM images, the average core diameter of TCL-SPION was 9 nm whereas that of MION-47 was 5 nm. TCL- SPION (345.0 ± 6.2 mM-1sec-1) had higher relaxivity (R2) than MION-47 (130.7 ± 1.1 mM-1sec-1). Significant changes in cell viability and growth were not found in human fibroblasts and HUVEC exposed to TCL-SPION and MION-47. However, IL-6 and TNF-α secretions increased dose-dependently and significantly in the macrophages treated with MION-47 or TCL-SPION. TCL-SPION had a lower stimulatory effect on IL-6 secretions than did MION-47 (P <0.05) and nitric oxides were produced in the macrophages by MION-47 but not TCL-SPION. A change in the mitochondrial membrane potential of the macrophages was observed 24 hours after the exposure, and MION-47 induced more collapses of the mitochondrial membrane potential than did TCL-SPION. In the in vivo MR imaging, 33.0 ± 1.3% and 7.5 ± 0.4% signal intensity decrease on T2*-weighted images was observed in the tumors injected with TCL-SPION and MION-47, respectively.
Conclusion: Due to the modified surface properties and larger core size of its iron oxide nanoparticles, TCL-SPION achieves lower cytotoxicity and better tumor MR contrast than MION-47. Our study suggests that TCL-SPION may be used as a new platform for tumor imaging and therapy monitoring.
Ultra-small superparamagnetic iron oxides; Magnetic resonance imaging; TCL-SPION; MION-47; Tumor targeting.
Magnetic separation in biomedical applications is based on differential magnetophoretic mobility (MM) of microparticulate matter in viscous media. Typically, the difference in MM is obtained by selectively labeling the target cells with superparamagnetic iron oxide nanoparticles(SPIONs). We have measured the MM of monodisperse, polystyrene microspheres (PSMs), with and without attached SPIONs as a model of cell motion induced by nanoparticle magnetization, using variable H field and Cell Tracking Velocimetry (CTV). As a model of paramagnetic microparticle motion, the MM measurements were performed on the same PSMs in paramagnetic gadolinium solutions, and on spores of a prokaryotic organism, Bacillus globigii (shown to contain paramagnetic manganese). The CTV analysis was sensitive to the type of the microparticle magnetization, producing a value of MM independent of the applied H field for the paramagnetic species, and a decreasing MM value with an increasing field for superparamagnetic species, as predicted from theory. The SPION-labeled PSMs exhibited a saturation magnetization above H ≅ 64,000 A m−1 (or 0.08 tesla). Based on those data, the average saturation magnetizations of the SPIONs was calculated and shown to vary between different commercial sources. The results demonstrate sensitivity of the CTV analysis to different magnetization mechanisms of the microparticles.
Our goal is to develop the functionalized superparamagnetic
iron oxide nanoparticles (SPIONs) demonstrating the capacities
to be delivered in liver specifically and to be dispersed in
physiological environment stably. For this purpose, SPIONs
were coated with polyvinylbenzyl-O-β-D-galactopyranosyl-D-gluconamide (PVLA) having
galactose moieties to be recognized by asialoglycoprotein
receptors (ASGP-R) on hepatocytes. For use as a control, we also
prepared SPIONs coordinated with 2-pyrrolidone. The sizes, size
distribution, structure, and coating of the nanoparticles were
characterized by transmission electron microscopy (TEM),
electrophoretic light scattering spectrophotometer (ELS), X-ray
diffractometer (XRD), and Fourier transform infrared (FT-IR),
respectively. Intracellular uptake of the PVLA-coated SPIONs was
visualized by confocal laser scanning microscopy, and their
hepatocyte-specific delivery was also investigated through
magnetic resonance (MR) images of rat liver. MRI experimental
results indicated that the PVLA-coated SPIONs possess the more
specific accumulation property in liver compared with control,
which suggests their potential utility as liver-targeting MRI
Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely utilized for the diagnosis and therapy of specific diseases, as magnetic resonance imaging (MRI) contrast agents and drug-delivery carriers, due to their easy transportation to targeted areas by an external magnetic field. For such biomedical applications, SPIONs must have multifunctional characteristics, including optimized size and modified surface. However, the biofunctionality and biocompatibility of SPIONs with various surface functional groups of different sizes have yet to be elucidated clearly. Therefore, it is important to carefully monitor the cytotoxicity and genotoxicity of SPIONs that are surfaced-modified with various functional groups of different sizes. In this study, we evaluated SPIONs with diameters of approximately 10 nm and 100~150 nm, containing different surface functional groups. SPIONs were covered with −O− groups, so-called bare SPIONs. Following this, they were modified with three different functional groups – hydroxyl (−OH), carboxylic (−COOH), and amine (−NH2) groups – by coating their surfaces with tetraethyl orthosilicate (TEOS), (3-aminopropyl)trimethoxysilane (APTMS), TEOS-APTMS, or citrate, which imparted different surface charges and sizes to the particles. The effects of SPIONs coated with these functional groups on mitochondrial activity, intracellular accumulation of reactive oxygen species, membrane integrity, and DNA stability in L-929 fibroblasts were determined by water-soluble tetrazolium, 2′,7′-dichlorodihydrofluorescein, lactate dehydrogenase, and comet assays, respectively. Our toxicological observations suggest that the functional groups and sizes of SPIONs are critical determinants of cellular responses, degrees of cytotoxicity and genotoxicity, and potential mechanisms of toxicity. Nanoparticles with various surface modifications and of different sizes induced slight, but possibly meaningful, changes in cell cytotoxicity and genotoxicity, which would be significantly valuable in further studies of bioconjugation and cell interaction for drug delivery, cell culture, and cancer-targeting applications.
superparamagnetic iron oxide nanoparticles; surface functional groups; cytotoxicity; genotoxicity
Nanotechnology has given scientists new tools for the development of advanced materials for the detection and diagnosis of disease. Iron oxide nanoparticles (SPIONs) in particular have been extensively investigated as novel magnetic resonance imaging (MRI) contrast agents due to a combination of favorable superparamagnetic properties, biodegradability, and surface properties of easy modification for improved in vivo kinetics and multifunctionality. This review discusses the basics of MR imaging, the origin of SPION’s unique magnetic properties, recent developments in MRI acquisition methods for detection of SPIONs, synthesis and post-synthesis processes that improve SPION’s imaging characteristics, and an outlook on the translational potential of SPIONs.
Chelation therapy involving organic chelators for treatment of heavy metal intoxication can cause cardiac arrest, kidney overload, mineral deficiency, and anemia.
In this study, superparamagnetic iron oxide nanoparticles (SPIONs) modified with an edible biopolymer poly(γ-glutamic acid) (PGA) were synthesized by coprecipitation method, characterized and evaluated for their removal efficiency of heavy metals from a metal solution, and simulated gastrointestinal fluid (SGIF).
Instrumental characterization of bare- and PGA-SPIONs revealed 7% coating of PGA on SPIONs with a spherical shape and an iron oxide spinel structure belonging to magnetite. The particle sizes as determined from transmission electron microscopy images were 8.5 and 11.7 nm for bare- and PGA-SPIONs, respectively, while the magnetization values were 70.3 and 61.5 emu/g. Upon coating with PGA, the zeta potentials were shifted from positive to negative at most of the environmental pH (3–8) and biological pH (1–8), implying good dispersion in aqueous suspension and favorable conditions for heavy metal removal. Batch studies showed rapid removal of lead and cadmium with the kinetic rates estimated by pseudo-second-order model being 0.212 and 0.424 g/mg·min, respectively. A maximum removal occurred in the pH range 4–8 in deionized water and 5–8 in SGIF corresponding to most gastrointestinal pH except for the stomach. Addition of different ionic strengths (0.001–1 M sodium acetate) and essential metals (Cu, Fe, Zn, Mg, Ca, and K) did not show any marked influence on lead removal by PGA-SPIONs, but significantly reduced the binding of cadmium. Compared to deionized water, the lead removal from SGIF was high at all pH with the Langmuir monolayer removal capacity being 98.70 mg/g for the former and 147.71 mg/g for the latter. However, a lower cadmium removal capacity was shown for SGIF (23.15 mg/g) than for deionized water (31.13 mg/g).
These results suggest that PGA-SPIONs could be used as a metal chelator for clinical treatment of metal poisoning.
superparamagnetic iron oxide nanoparticles; poly(γ-glutamic acid); heavy metals; chelation therapy; gastrointestinal pH; kinetics
Experimental tissue fusion benefits from the selective heating of superparamagnetic iron oxide nanoparticles (SPIONs) under high frequency irradiation. However, the metabolic pathways of SPIONs for tissue fusion remain unknown. Hence, the goal of this in vivo study was to analyze the distribution of SPIONs in different organs by means of magnetic resonance imaging (MRI) and histological analysis after a SPION-containing patch implantation.
SPION-containing patches were implanted in rats. Three animal groups were studied histologically over six months. Degradation assessment of the SPION-albumin patch was performed in vivo using MRI for iron content localization and biodistribution.
No SPION degradation or accumulation into the reticuloendothelial system was detected by MRI, MRI relaxometry, or histology, outside the area of the implantation patch. Concentrations from 0.01 μg/mL to 25 μg/mL were found to be hyperintense in T1-like gradient echo sequences. The best differentiation of concentrations was found in T2 relaxometry, susceptibility-sensitive gradient echo sequences, and in high repetition time T2 images. Qualitative and semiquantitative visualization of small concentrations and accumulation of SPIONs by MRI are feasible. In histological liver samples, Kupffer cells were significantly correlated with postimplantation time, but no differences were observed between sham-treated and induction/no induction groups. Transmission electron microscopy showed local uptake of SPIONs in macrophages and cells of the reticuloendothelial system. Apoptosis staining using caspase showed no increased toxicity compared with sham-treated tissue. Implanted SPION patches were relatively inert with slow, progressive local degradation over the six-month period. No distant structural alterations in the studied tissue could be observed.
Systemic bioavailability may play a role in specific SPION implant toxicity and therefore the local degradation process is a further aspect to be assessed in future studies.
superparamagnetic iron oxide nanoparticles; metabolism; distribution
Breast cancer remains one of the most prevalent and lethal malignancies in women. The inability to diagnose small volume metastases early has limited effective treatment of stage 4 breast cancer. Here we report the rational development and use of a multifunctional superparamagnetic iron oxide nanoparticle (SPION) for targeting metastatic breast cancer in a transgenic mouse model and imaging with magnetic resonance (MR). SPIONs coated with a copolymer of chitosan and polyethylene glycol (PEG) were labeled with a fluorescent dye for optical detection and conjugated with a monoclonal antibody against the neu receptor (NP-neu). SPIONs labeled with mouse IgG were used as a non-targeting control (NP-IgG). These SPIONs had desirable physiochemical properties for in vivo applications such as near neutral zeta potential and hydrodynamic size around 40 nm, and were highly stable in serum containing medium. Only NP-neu showed high uptake in neu expressing mouse mammary carcinoma (MMC) cells which was reversed by competing free neu antibody, indicating their specificity to the neu antigen. In vivo, NP-neu was able to tag primary breast tumors and significantly, only NP-neu bound to spontaneous liver, lung, and bone marrow metastases in a transgenic mouse model of metastatic breast cancer, highlighting the necessity of targeting for delivery to metastatic disease. The SPIONs provided significant contrast enhancement in MR images of primary breast tumors; thus, they have the potential for MRI detection of micrometastases, and provide an excellent platform for further development of an efficient metastatic breast cancer therapy.
iron oxide; nanoparticle; genetically engineered mouse model; HER2/neu; theranostics; breast cancer; metastases
The purpose of the study was to develop tumor specific, water dispersible superparamagnetic iron oxide nanoparticles (SPIONs) and evaluate their efficacy as a contrast agent in magnetic resonance imaging (MRI). We have developed SPIONs capped with citric acid/2-bromo-2-methylpropionic acid which are compact, water dispersible, biocompatible having narrow range of size dispersity (8–10 nm), and relatively high T2 relaxivity (R2 = 222L · mmol−1 · sec−l). The targeting efficacy of unconjugated and folic acid-conjugated SPIONs (FA-SPIONS) was evaluated in a folic acid receptor overexpressing and negative tumor cell lines. Folic acid receptor-positive cells incubated with FA-SPIONs showed much higher intracellular iron content without any cytotoxicity. Ultrastructurally, SPIONs were seen as clustered inside the various stages of endocytic pathways without damaging cellular organelles and possible mechanism for their entry is via receptor mediated endocytosis. In vitro MRI studies on tumor cells showed better T2-weighted images in FA-SPIONs. These findings indicate that FA-SPIONs possess high colloidal stability with excellent sensitivity of imaging and can be a useful MRI contrast agent for the detection of cancer.
superparamagnetic iron oxide nanoparticles (SPION); cellular internalization; magnetic resonance imaging (MRI); cancer-cell targeting ligand
Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.
Conventional corticosteroid suspensions for the intra-articular treatment of arthritis suffer from limitations such as crystal formation or rapid clearance from the joint. The purpose of this study was to investigate an innovative alternative consisting of corticosteroid encapsulation into magnetically retainable microparticles.
Microparticles (1 or 10 μm) containing both superparamagnetic iron oxide nanoparticles (SPIONs) and dexamethasone 21-acetate (DXM) were prepared. In a preliminary study, we compared the persistence of microparticles of both sizes in the joint. A second study evaluated the influence of a subcutaneously implanted magnet near the knee on the retention of magnetic microparticles in the joint by in vivo imaging. Finally, the efficacy of 10-μm microparticles was investigated using a model of antigen-induced arthritis (AIA) in mice. Phosphate-buffered saline, DXM suspension, SPION suspension, blank microparticles and microparticles containing only SPIONs were used as controls. Arthritis severity was assessed using 99mTc accumulation and histological scoring.
Due to their capacity of encapsulating more corticosteroid and their increased joint retention, the 10-μm microparticles were more suitable vectors than the 1-μm microparticles for corticosteroid delivery to the joint. The presence of a magnet resulted in higher magnetic retention in the joint, as demonstrated by a higher fluorescence signal. The therapeutic efficacy in AIA of 10-μm microparticles containing DXM and SPIONs was similar to that of the DXM suspension, proving that the bioactive agent is released. Moreover, the anti-inflammatory effect of DXM-containing microparticles was more important than that of blank microparticles or microparticles containing only SPIONs. The presence of a magnet did not induce a greater inflammatory reaction.
This study confirms the effectiveness of an innovative approach of using magnetically retainable microparticles as intra-articular drug delivery systems. A major advantage comes from a versatile polymer matrix, which allows the encapsulation of many classes of therapeutic agents (for example, p38 mitogen-activated protein kinase inhibitors), which may reduce systemic side effects.
Targeted delivery of contrast agents is a highly desirable strategy for enhancing diagnostic efficiency and reducing side effects and toxicity. Water-soluble and tumor-targeting superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by loading hydrophobic SPIONs into micelles assembled from an amphiphilic block copolymer poly(ethylene glycol)- poly(ɛ-caprolactone) (PEG-PCL) bearing folate in the distal ends of PEG chains. Compared to the water-soluble SPIONs obtained by small molecular surfactant coating, ultrasmall SPION encapsulation with PEG-PCL micelles (PEG-PCL-SPIONs) simultaneously increases transverse (r2) and decreases longitudinal (r1) magnetic resonance (MR) relaxivities of water proton in micelle solution, leading to a notably high r2/r1 ratio up to 78, which makes the PEG-PCL-SPIONs a highly sensitive MR imaging (MRI) T2 contrast agent. The mean size of folate-attached SPION micelles (Fa-PEG-PCL-SPIONs) is 44 ± 3 nm on average, ideal for in vivo MRI applications in which long circulation is greatly determined by small particle size and is highly desirable. Prussian blue staining of BEL-7402 cells over-expressing folate receptors, after incubation with micelle-containing medium, demonstrated that folate functionalization of the magnetic particles significantly enhanced their cell uptake. The potential of Fa-PEG-PCL-SPIONs as a potent MRI probe for in vivo tumor detection was assessed. At 3 hours after intravenous injection of the Fa-PEG-PCL-SPION solution into mice bearing subcutaneous xenografts of human BEL-7402 hepatoma, a 41.2% signal intensity decrease was detected in the T2-weighted MR images of the tumor, indicating the efficient accumulation of Fa-PEG-PCL-SPIONs in the tumor tissue.
tumor targeting; magnetic resonance imaging; polymeric micelles; superparamagnetic iron oxide
The potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various biomedical applications, including magnetic resonance imaging (MRI), sensing, and drug delivery, requires that their surface be derivatized to be hydrophilic and biocompatible. We report here the design and synthesis of a compact and water-soluble zwitterionic dopamine sulfonate (ZDS) ligand with strong binding affinity to SPIONs. After ligand exchange, the ZDS coated SPIONs exhibit small hydrodynamic diameters (HD), and stability with respect to time, pH, and salinity. Furthermore, small ZDS coated SPIONs were found to have a reduced non-specific affinity (compared to negatively charged SPIONs) towards serum proteins; streptavidin/dye functionalized SPIONs were bioactive and thus specifically targeted biotin receptors.
superparamagnetic iron oxide nanoparticles; zwitterionic dopamine sulfonate; hydrodynamic diameter; stability; serum binging test; biotin labeling
An antibody-directed nonviral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles and a gastric cancer-associated CD44v6 single-chain variable fragment (scFvCD44v6,-PEG-g-PEI-SPION), was constructed as a gastric cancer-targeting and magnetic resonance imaging (MRI)-visible nanocarrier for small interfering RNA (siRNA) delivery. Biophysical characterization of PEG-g-PEI-SPION and scFvCD44v6-PEG-g-PEI-SPION was carried out, including siRNA condensation capacity, cell viability, and transfection efficiency. Both the targeting and nontargeting nanocarriers were effective for transferring siRNA in vitro. The cellular uptake and distribution of nanoparticles complexed with siRNA was analyzed by fluorescence imaging and immunofluorescent staining. Moreover, the gastric cancer-targeting effect was verified in vivo by MRI and histology analysis. These results indicate that scFvCD44v6-PEG-g-PEI-SPION is a promising nonviral vector for gastric cancer gene therapy and diagnosis.
tumor targeting; CD44 variant 6; nonviral vector; small interfering RNA; magnetic resonance imaging
Superparamagnetic iron oxide nanoparticles (SPION) are being widely used for various biomedical applications, for example, magnetic resonance imaging, targeted delivery of drugs or genes, and in hyperthermia. Although, the potential benefits of SPION are considerable, there is a distinct need to identify any potential cellular damage associated with these nanoparticles. Besides focussing on cytotoxicity, the most commonly used determinant of toxicity as a result of exposure to SPION, this review also mentions the importance of studying the subtle cellular alterations in the form of DNA damage and oxidative stress. We review current studies and discuss how SPION, with or without different surface coating, may cause cellular perturbations including modulation of actin cytoskeleton, alteration in gene expression profiles, disturbance in iron homeostasis and altered cellular responses such as activation of signalling pathways and impairment of cell cycle regulation. The importance of protein-SPION interaction and various safety considerations relating to SPION exposure are also addressed.
SPION; cellular stress; cytotoxicity; DNA damage
In this study, we report formation of weblike fibrous nanostructure and nanoparticles of magnetic neodymium-iron-boron (NdFeB) via femtosecond laser radiation at MHz pulse repetition frequency in air at atmospheric pressure. Scanning electron microscopy (SEM) analysis revealed that the nanostructure is formed due to aggregation of polycrystalline nanoparticles of the respective constituent materials. The nanofibers diameter varies between 30 and 70 nm and they are mixed with nanoparticles. The effect of pulse to pulse separation rate on the size of the magnetic fibrous structure and the magnetic strength was reported. X-ray diffraction (XRD) analysis revealed metallic and oxide phases in the nanostructure. The growth of magnetic nanostructure is highly recommended for the applications of magnetic devices like biosensors and the results suggest that the pulsed-laser method is a promising technique for growing nanocrystalline magnetic nanofibers and nanoparticles for biomedical applications.
We have developed a compact and inexpensive microfluidic chip, the Self Assembled Magnetic filter, to efficiently remove magnetically tagged cells from suspension. The self-assembled magnetic filter consists of a microfluidic channel built directly above a self-assembled NdFeB magnet. Micrometer-sized grains of NdFeB assemble to form alternating magnetic dipoles, creating a magnetic field with a very strong magnitude B (from the material) and field gradient ∇B (from the configuration) in the microfluidic channel. The magnetic force imparted on magnetic beads is measured to be comparable to state-of-the-art microfabricated magnets, allowing for efficient separations to be performed in a compact, simple device. The efficiency of the magnetic filter is characterized by sorting non-magnetic (polystyrene) beads from magnetic beads (iron oxide). The filter enriches the population of non-magnetic beads to magnetic beads by a factor of >105 with a recovery rate of 90% at 1 mL/hr. The utility of the magnetic filter is demonstrated with a microfluidic device that sorts tumor cells from leukocytes using negative immunomagnetic selection, and concentrates the tumor cells on an integrated membrane filter for optical detection.
A targeted drug delivery system is the need of the hour. Guiding magnetic iron oxide nanoparticles with the help of an external magnetic field to its target is the principle behind the development of superparamagnetic iron oxide nanoparticles (SPIONs) as novel drug delivery vehicles. SPIONs are small synthetic γ-Fe2O3 (maghemite) or Fe3O4 (magnetite) particles with a core ranging between 10 nm and 100 nm in diameter. These magnetic particles are coated with certain biocompatible polymers, such as dextran or polyethylene glycol, which provide chemical handles for the conjugation of therapeutic agents and also improve their blood distribution profile. The current research on SPIONs is opening up wide horizons for their use as diagnostic agents in magnetic resonance imaging as well as for drug delivery vehicles. Delivery of anticancer drugs by coupling with functionalized SPIONs to their targeted site is one of the most pursued areas of research in the development of cancer treatment strategies. SPIONs have also demonstrated their efficiency as nonviral gene vectors that facilitate the introduction of plasmids into the nucleus at rates multifold those of routinely available standard technologies. SPION-induced hyperthermia has also been utilized for localized killing of cancerous cells. Despite their potential biomedical application, alteration in gene expression profiles, disturbance in iron homeostasis, oxidative stress, and altered cellular responses are some SPION-related toxicological aspects which require due consideration. This review provides a comprehensive understanding of SPIONs with regard to their method of preparation, their utility as drug delivery vehicles, and some concerns which need to be resolved before they can be moved from bench top to bedside.
superparamagnetic iron oxide nanoparticles; SPIONs; targeted delivery; coating; functionalization; targeting ligands; toxicity
Superparamagnetic iron-oxide nanoparticles (SPIONs) are magnetic nanoparticles may be useful for early detection and treatment of cancers. SPIONs provide therapeutic drug-loading capabilities and targeting specificity through the use of antibodies or receptor specific tags. These versatile particles are prime candidates for improving current means to the treatment of cancer and potentially other diseases. This multi-disciplinary project includes various milestones and multiple aims: i) identify and optimize SPION design parameters that optimize aqueous stability and maximize amphiphilic character, ii) evaluate SPION drug storage and release characteristics, and iii) demonstrate binding and entry into targeted cells. The specific aim of this project is the coupling of cell-specific targeting agents to SPIONs. An antisense oligonucleotide against Survivin mRNA was synthesized by solid-phase DNA synthesis with an amino-terminated linker on the 3' end (5'CCCAGCCTTCCAGCTCCTTG-(CH2)6-3'NH2). SPIONs were prepared and then coated with a copolymer containing surface carboxylic acid groups (-COOH). The carboxylic acid groups were activated by treatment with N-((3-dimethylamino)-propyl)-Nethyl carbodiimide and coupled to oligonucleotides via the –NH2 terminus to the –COOH groups resulting in the formation of an amide linkage between the –COOH groups of the SPION and the –NH2 of the antisense oligonucleotide. Circular dichroism (CD) studies were performed to quantify/optimize coupling and to demonstrate antisense Survivin duplex formation was not inhibited by the presence of the SPION. CD results were correlated with agarose gel electrophoresis data and demonstrated oligonucleotides coupling to the SPION and that the SPION did not significantly alter duplex formation. Future studies will target cellular absorption and antisense binding to Survivin mRNA using confocal microscopy (Supported, in part, from NIH GM081348 grant and the WVU Research Corporation).
The aim of this research was to validate transcription magnetic resonance (MR) imaging (MRI) for gene transcript targeting in acute neurological disorders in live subjects. We delivered three MR probe variants with superparamagnetic iron oxide nanoparticles (SPION, a T2 susceptibility agent) linked to a phosphorothioate-modified oligodeoxynucleotide (sODN) complementary to c-fos mRNA (SPION-cfos) or β-actin mRNA (SPION-β-actin) and to sODN with random sequence (SPION-Ran). Each probe (1 μg Fe in 2 μl) was delivered via intracerebroventricular infusion to the left cerebral ventricle of male C57Black6 mice. We demonstrated SPION retention, measured as decreased T2* signal or increased R2* value (R2*=1/T2*). Animals that received the SPION-β-actin probe exhibited the highest R2* values, followed (in descending order) by SPION-cfos and SPION-Ran. SPION-cfos retention was localized in brain regions where SPION-cfos was present and where hybrids of SPION-cfos and its target c-fos mRNA were detected by in situ reverse transcription PCR. In animals that experienced cerebral ischemia, SPION-cfos retention was significantly increased in locations where c-fos mRNA increased in response to the ischemic insult; these elevations were not observed for SPION-β-actin and SPION-Ran. This study should enable MR detection of mRNA alteration in disease models of the central nervous system.
cardiac arrest; immediate early genes; oxidative stress; nanotechnology; signal transduction
To circumvent the limitations of using postmortem brain in molecular assays, we used avidin– biotin binding to couple superparamagnetic iron oxide nanoparticles (SPIONs) (15–20 nm) to phosphorothioate-modified oligodeoxynucleotides (sODNs) with sequence complementary to c-fos and β-actin mRNA (SPION-cfos and SPION-βactin, respectively) (14 –22 nm). The Stern–Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 × 106/m. We studied the feasibility of using the conjugates for in vivo magnetic resonance imaging (MRI) to monitor gene transcription, and demonstrated that these complexes at 40 μg of Fe per kilogram of body weight were retained at least 1 d after intracerebroventricular infusion into the left ventricle of C57Black6 mice. SPION retention measured by MRI as T2* or R2* maps (R2* = 1/T2*) was compared with histology of iron oxide (Prussian blue) and FITC-labeled sODN. We observed significant reduction in magnetic resonance (MR) T2* signal in the right cortex and striatum; retention of SPION-cfos and SPION-βactin positively correlated with c-fos and β-actin mRNA maps obtained from in situ hybridization. Histological examination showed that intracellular iron oxide and FITC-sODN correlated positively with in vivo MR signal reduction. Furthermore, in animals that were administered SPION-cfos and amphetamine (4 mg/kg, i.p.), retention was significantly elevated in the nucleus accumbens, striatum, and medial prefrontal cortex of the forebrain. Control groups that received SPION-cfos and saline or that received a SPION conjugate with a random-sequence probe and amphetamine showed no retention. These results demonstrated that SPION-sODN conjugates can detect active transcriptions of specific mRNA species in living animals with MRI.
amphetamine; antisense delivery; aptamer; drug addiction; gene transcription; immediate-early genes; nanotechnology; signal transduction
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.
Superparamagnetic nanoparticles; Endocytosis; Magnetic field; Malignant cells; Internalization
Chitosan is the deacetylated form of chitin and used in numerous applications. Because it is a good dispersant for metal and/or oxide nanoparticle synthesis, chitosan and its derivatives have been utilized as coating agents for magnetic nanoparticles synthesis, including superparamagnetic iron oxide nanoparticles (SPIONs). Herein, we demonstrate the water-soluble SPIONs encapsulated with a hybrid polymer composed of polyelectrolyte complexes (PECs) from chitosan, the positively charged polymer, and dextran sulfate, the negatively charged polymer. The as-prepared hybrid ferrofluid, in which iron chloride salts (Fe3+ and Fe2+) were directly coprecipitated inside the hybrid polymeric matrices, was physic-chemically characterized. Its features include the z-average diameter of 114.3 nm, polydispersity index of 0.174, zeta potential of −41.5 mV and iron concentration of 8.44 mg Fe/mL. Moreover, based on the polymer chain persistence lengths, the anionic surface of the nanoparticles as well as the high R2/R1 ratio of 13.5, we depict the morphology of SPIONs as a cluster because chitosan chains are chemisorbed onto the anionic magnetite surfaces by tangling of the dextran sulfate. Finally, the cellular uptake and biocompatibility assays indicate that the hybrid polymer encapsulating the SPIONs exhibited great potential as a magnetic resonance imaging T2 contrast agent for cell tracking.
biocompatible polymer; chitosan; superparamagnetic iron oxide nanoparticle; nanomaterials
Determination of the fate of nanoparticles (NPs) in a biological system, or NP biodistribution, is critical in evaluating an NP formulation for nanomedicine. Current methods to determine NP biodistribution are greatly inadequate, due to their limited detection thresholds. Herein, proof of concept of a novel method for improved NP detection based on in situ polymerase chain reaction (ISPCR), coined “nanobarcoding,” is demonstrated.
Nanobarcoded superparamagnetic iron oxide nanoparticles (NB-SPIONs) were characterized by dynamic light scattering, zeta potential, and hyperspectral imaging measurements. Cellular uptake of Cy5-labeled NB-SPIONs (Cy5-NB-SPIONs) was imaged by confocal microscopy. The feasibility of the nanobarcoding method was first validated by solution-phase PCR and “pseudo”-ISPCR before implementation in the model in vitro system of HeLa human cervical adenocarcinoma cells, a cell line commonly used for ISPCR-mediated detection of human papilloma virus (HPV).
Dynamic light-scattering measurements showed that NB conjugation stabilized SPION size in different dispersion media compared to that of its precursor, carboxylated SPIONs (COOH-SPIONs), while the zeta potential became more positive after NB conjugation. Hyperspectral imaging confirmed NB conjugation and showed that the NB completely covered the SPION surface. Solution-phase PCR and pseudo-ISPCR showed that the expected amplicons were exclusively generated from the NB-SPIONs in a dose-dependent manner. Although confocal microscopy revealed minimal cellular uptake of Cy5-NB-SPIONs at 50 nM over 24 hours in individual cells, ISPCR detected definitive NB-SPION signals inside HeLa cells over large sample areas.
Proof of concept of the nanobarcoding method has been demonstrated in in vitro systems, but the technique needs further development before its widespread use as a standardized assay.
nanomedicine; in situ PCR; single cell; DNA-functionalized nanoparticles