Synthetic Antiferromagnetic Gold Nanoparticles as Bimodal Contrast Agents in MRI and CT-An Experimental In Vitro and In Vivo Study.

The use of multimodal contrast agents can potentially overcome the intrinsic limitations of individual imaging methods. We have validated synthetic antiferromagnetic nanoparticles (SAF-NPs) as bimodal contrast agents for in vitro cell labeling and in vivo cell tracking using magnetic resonance imaging (MRI) and computed tomography (CT). SAF-NP-labeled cells showed high contrast in MRI phantom studies (r2* = 712 s-1 mM-1), while pelleted cells showed clear contrast enhancement in CT. After intravenous SAF-NP injection, nanoparticles accumulated in the liver and spleen, as visualized in vivo by significant MRI contrast enhancement. Intravenous injection of SAF-NP-labeled cells resulted in cell accumulation in the lungs, which was clearly detectable by using CT but not by using MRI. SAF-NPs proved to be very efficient cell labeling agents for complementary MRI- and CT-based cell tracking. Bimodal monitoring of SAF-NP labeled cells is in particular of interest for applications where the applied imaging methods are not able to visualize the particles and/or cells in all organs.

A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency.

Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.

Intraarterial route increases the risk of cerebral lesions after mesenchymal cell administration in animal model of ischemia.

Mesenchymal stem cells (MSCs) are a promising clinical therapy for ischemic stroke. However, critical parameters, such as the most effective administration route, remain unclear. Intravenous (i.v.) and intraarterial (i.a.) delivery routes have yielded varied outcomes across studies, potentially due to the unknown MSCs distribution. We investigated whether MSCs reached the brain following i.a. or i.v. administration after transient cerebral ischemia in rats, and evaluated the therapeutic effects of both routes. MSCs were labeled with dextran-coated superparamagnetic nanoparticles for magnetic resonance imaging (MRI) cell tracking, transmission electron microscopy and immunohistological analysis. MSCs were found in the brain following i.a. but not i.v. administration. However, the i.a. route increased the risk of cerebral lesions and did not improve functional recovery. The i.v. delivery is safe but MCS do not reach the brain tissue, implying that treatment benefits observed for this route are not attributable to brain MCS engrafting after stroke.

Easy and Efficient Cell Tagging with Block Copolymer-Based Contrast Agents for Sensitive MRI Detection in Vivo.

Superparamagnetic iron oxide nanoparticles (MNPs) together with magnetic resonance imaging (MRI) are the preferred tools for monitoring the fate and biodistribution of administered cells in stem cell therapy studies. Commercial MNPs need transfection agents and long incubation times for sufficient cell labeling and further in vivo cell detection. In this work, we have synthesized MNPs coated with pluronic F127 and tetronic 908, and validated their applicability as contrast agents for MRI cell detection on two different cell types: rat mesenchymal stem cells (MSCs) and multipotent neural progenitor cell line from mice (C17.2). No transfection agent was needed for a complete MNP internalization, and the uptake was only dependent on MNP concentration in medium and limited on the incubation time. By combining in vivo MRI and ex vivo histology microscopy, we have demonstrated the MRI signal detected corresponded exclusively to labeled cells and not to free particles. Pluronic F127- and tetronic 908-coated MNPs represent promising contrast agents for stem cell tracking due to their ease of use in preparation, their efficiency for cell labeling, and their high sensitivity for in vivo cell detection.

Monitoring the Bystander Killing Effect of Human Multipotent Stem Cells for Treatment of Malignant Brain Tumors.

Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683) in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI) and magnetic resonance imaging (MRI). Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1) outliers can be detected earlier, (2) GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3) a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents.

Assessment of bystander killing-mediated therapy of malignant brain tumors using a multimodal imaging approach.

INTRODUCTION: In this study, we planned to assess if adult stem cell-based suicide gene therapy can efficiently eliminate glioblastoma cells in vivo. We investigated the therapeutic potential of mouse Oct4(-) bone marrow multipotent adult progenitor cells (mOct4(-) BM-MAPCs) in a mouse glioblastoma model, guided by multimodal in vivo imaging methods to identify therapeutic windows. METHODS: Magnetic resonance imaging (MRI) of animals, wherein 5 × 10(5) syngeneic enhanced green fluorescent protein-firefly luciferase-herpes simplex virus thymidine kinase (eGFP-fLuc-HSV-TK) expressing and superparamagnetic iron oxide nanoparticle labeled (1 % or 10 %) mOct4(-) BM-MAPCs were grafted in glioblastoma (GL261)-bearing animals, showed that labeled mOct4(-) BM-MAPCs were located in and in close proximity to the tumor. Subsequently, ganciclovir (GCV) treatment was commenced and the fate of both the MAPCs and the tumor were followed by multimodal imaging (MRI and bioluminescence imaging). RESULTS: In the majority of GCV-treated, but not phosphate-buffered saline-treated animals, a significant difference was found in mOct4(-) BM-MAPC viability and tumor size at the end of treatment. Noteworthy, in some phosphate-buffered saline-treated animals (33 %), a significant decrease in tumor size was seen compared to sham-operated animals, which could potentially also be caused by a synergistic effect of the immune-modulatory stem cells. CONCLUSIONS: Suicide gene therapy using mOct4(-) BM-MAPCs as cellular carriers was effective in reducing the tumor size in the majority of the GCV-treated animals leading to a longer progression-free survival compared to sham-operated animals. This treatment could be followed and guided noninvasively in vivo by MRI and bioluminescence imaging. Noninvasive imaging is of particular interest for a rapid and efficient validation of stem cell-based therapeutic approaches for glioblastoma and hereby contributes to a better understanding and optimization of a promising therapeutic approach for glioblastoma patients.

Synthetic antiferromagnetic nanoparticles as potential contrast agents in MRI.

We present the top-down synthesis of a novel type of MRI T2 contrast agent with great control over size and shape using a colloidal lithography technique. The resulting synthetic antiferromagnetic nanoparticles (SAF-NPs) yield improved relaxivities compared to superparamagnetic iron oxide alternatives (SPIONs). For T2 weighted imaging, the outer sphere relaxation theory has shown that the sensitivity of a T2 contrast agent is dependent on the particle size with an optimal size that exceeds the superparamagnetic limit of SPIONs. With the use of the interlayer exchange coupling effect, the SAF-NPs presented here do not suffer from this limit. Adjusting the outer sphere relaxation theory for spherical particles to SAF-NPs, we show both theoretically and experimentally that the SAF-NP size can be optimized to reach the r2 maximum. With measured r2 values up to 355 s(-1) mM(-1), our SAF-NPs show better performance than commercial alternatives and are competitive with the state-of-the-art. This performance is confirmed in an in vitro MRI study on SKOV3 cells.

Sensitive in vivo cell detection using size-optimized superparamagnetic nanoparticles.

Magnetic nanoparticle (MNP) enabled cell visualization with magnetic resonance imaging (MRI) is currently an intensively studied area of research. In the present study, we have synthesized polyethylene glycolated (PEG) MNPs and validated their suitability as MR cell labeling agents in in vitro and in vivo experiments. The labeling of therapeutic potent mesenchymal stem cells (MSCs) with small core and large core MNPs was evaluated. Both MNPs were, in combination with a transfection agent, stably internalized into the MSCs and didn't show an effect on cell metabolism. The labeled cells showed high contrast in MRI phantom studies. For quantification purposes, the MRI contrast generating properties of cells labeled with small core MNPs were compared with large core MNPs and with the commercial contrast agent Endorem. MSCs labeled with the large core MNPs showed the highest contrast generating properties in in vitro phantom studies and in in vivo intracranial stereotactic injection experiments, confirming the size-relaxivity relationship in biological systems. Finally, the distribution of MSCs pre-labeled with large core PEGylated MNPs was visualized non-invasively with MRI in a glioma model.

Temperature determination of resonantly excited plasmonic branched gold nanoparticles by X-ray absorption spectroscopy.

The fields of bioscience and nanomedicine demand precise thermometry for nanoparticle heat characterization down to the nanoscale regime. Since current methods often use indirect and less accurate techniques to determine the nanoparticle temperature, there is a pressing need for a direct and reliable element-specific method. In-situ extended X-ray absorption fine structure (EXAFS) spectroscopy is used to determine the thermo-optical properties of plasmonic branched gold nanoparticles upon resonant laser illumination. With EXAFS, the direct determination of the nanoparticle temperature increase upon laser illumination is possible via the thermal influence on the gold lattice parameters. More specifically, using the change of the Debye-Waller term representing the lattice disorder, the temperature increase is selectively measured within the plasmonic branched nanoparticles upon resonant laser illumination. In addition, the signal intensity shows that the nanoparticle concentration in the beam more than doubles during laser illumination, thereby demonstrating that photothermal heating is a dynamic process. A comparable temperature increase is measured in the nanoparticle suspension using a thermocouple. This good correspondence between the temperature at the level of the nanoparticle and at the level of the suspension points to an efficient heat transfer between the nanoparticle and the surrounding medium, thus confirming the potential of branched gold nanoparticles for hyperthermia applications. This work demonstrates that X-ray absorption spectroscopy-based nanothermometry could be a valuable tool in the fast-growing number of applications of plasmonic nanoparticles, particularly in life sciences and medicine.

Surface modification of gamma-Fe2O3@SiO2 magnetic nanoparticles for the controlled interaction with biomolecules.

Modifying the surface of magnetic nanoparticles (MNPs) to allow for controlled interaction with biomolecules enables their implementation in biomedical applications such as contrast agents for magnetic resonance imaging, labels in magnetic biosensing or media for magnetically assisted bioseparation. In this paper, self-assembly of trialkoxysilanes is used to chemically functionalize the surface of gamma-Fe2O3@SiO2 core-shell particles. First, the silane deposition procedure was optimized using infrared analysis in order to obtain maximum packing density of the silanes on the particles. The surface coverage was determined to be approximately 8 x 10(14) molecules/cm2. It was shown that the magnetic, crystalline, and morphological properties of the MNPs were not altered by deposition of a thin silane coating. The optimized procedure was transferred for the deposition of aldehyde and poly(ethylene glycol) (PEG) presenting silanes. The presence of both silanes on the particle surface was confirmed using XPS and FTIR. The interaction of proteins with silane-modified MNPs was monitored using a Bradford protein assay. Our results demonstrate that, by introducing aldehyde functions, the MNPs are capable of covalently binding human IgG while retaining their specific binding capacity. Maximum surface coverage occurs at 46 microg antibodies per mg particle, which corresponds to 35 antibodies bound to an average sized MNP (54 nm in diameter). The human IgG functionalized MNPs exhibit a high degree of specificity (approximately 90%) and retained a binding capacity of 32%. Using the same approach, streptavidin was coupled onto the MNPs and the biotin binding capacity was determined using biotinylated fluorescein. At maximum surface coverage, a biotin binding capacity of 1500 pmol/mg was obtained, corresponding to a streptavidin activity of 76%. On the other hand, by introducing PEG functions the non-specific adsorption of serum proteins could be significantly suppressed down to approximately 3 microg/mg. We conclude that self-assembly of silane films creates a generic platform for the controlled interactions of MNPs with biomolecules.