Radiosensitizing Effect of Dextran-Coated Iron Oxide Nanoparticles on Malignant Glioma Cells.

The potential of standard methods of radiation therapy is limited by the dose that can be safely delivered to the tumor, which could be too low for radical treatment. The dose efficiency can be increased by using radiosensitizers. In this study, we evaluated the sensitizing potential of biocompatible iron oxide nanoparticles coated with a dextran shell in A172 and Gl-Tr glioblastoma cells in vitro. The cells preincubated with nanoparticles for 24 h were exposed to ionizing radiation (X-ray, gamma, or proton) at doses of 0.5-6 Gy, and their viability was assessed by the Resazurin assay and by staining of the surviving cells with crystal violet. A statistically significant effect of radiosensitization by nanoparticles was observed in both cell lines when cells were exposed to 35 keV X-rays. A weak radiosensitizing effect was found only in the Gl-Tr line for the 1.2 MeV gamma irradiation and there was no radiosensitizing effect in both lines for the 200 MeV proton irradiation at the Bragg peak. A slight (ca. 10%) increase in the formation of additional reactive oxygen species after X-ray irradiation was found when nanoparticles were present. These results suggest that the nanoparticles absorbed by glioma cells can produce a significant radiosensitizing effect, probably due to the action of secondary electrons generated by the magnetite core, whereas the dextran shell of the nanoparticles used in these experiments appears to be rather stable under radiation exposure.

Potential of Magnetic Hyperthermia to Stimulate Localized Immune Activation.

Magnetic hyperthermia (MH) harnesses the heat-releasing properties of superparamagnetic iron oxide nanoparticles (SPIONs) and has potential to stimulate immune activation in the tumor microenvironment whilst sparing surrounding normal tissues. To assess feasibility of localized MH in vivo, SPIONs are injected intratumorally and their fate tracked by Zirconium-89-positron emission tomography, histological analysis, and electron microscopy. Experiments show that an average of 49% (21-87%, n = 9) of SPIONs are retained within the tumor or immediately surrounding tissue. In situ heating is subsequently generated by exposure to an externally applied alternating magnetic field and monitored by thermal imaging. Tissue response to hyperthermia, measured by immunohistochemical image analysis, reveals specific and localized heat-shock protein expression following treatment. Tumor growth inhibition is also observed. To evaluate the potential effects of MH on the immune landscape, flow cytometry is used to characterize immune cells from excised tumors and draining lymph nodes. Results show an influx of activated cytotoxic T cells, alongside an increase in proliferating regulatory T cells, following treatment. Complementary changes are found in draining lymph nodes. In conclusion, results indicate that biologically reactive MH is achievable in vivo and can generate localized changes consistent with an anti-tumor immune response.

Granzyme B Functionalized Nanoparticles Targeting Membrane Hsp70-Positive Tumors for Multimodal Cancer Theranostics.

Functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as potential clinical tools for cancer theranostics. Membrane-bound 70 kDa heat shock protein (mHsp70) is ubiquitously expressed on the cell membrane of various tumor types but not normal cells and therefore provides a tumor-specific target. The serine protease granzyme B (GrB) that is produced as an effector molecule by activated T and NK cells has been shown to specifically target mHsp70 on tumor cells. Following binding to Hsp70, GrB is rapidly internalized into tumor cells. Herein, it is demonstrated that GrB functionalized SPIONs act as a contrast enhancement agent for magnetic resonance imaging and induce specific tumor cell apoptosis. Combinatorial regimens employing stereotactic radiotherapy and/or magnetic targeting are found to further enhance the therapeutic efficacy of GrB-SPIONs in different tumor mouse models.

Detection of experimental myocardium infarction in rats by MRI using heat shock protein 70 conjugated superparamagnetic iron oxide nanoparticle.

Superparamagnetic iron-oxide based contrast agents can provide important diagnostic information regarding the assessment of cardiac inflammatory diseases. The aim of the study was to analyze whether nanoparticles conjugated to recombinant 70-kDa heat shock protein (Hsp70-SPION) can be applied for the detection of acute myocardium infarct by MRI. Cellular experiments demonstrated increased CD40-mediated uptake of Hsp70-SPIONs in comparison to non-conjugated SPIONs. Following induction of an acute infarct in rats by ligation of the left anterior descending artery SPIONs and Hsp70-SPION conjugates were injected intravenously on day 4. The animals underwent sequential MRI that showed the presence of the particles in the infarcted zone. Subsequent biodistribution analyses with the help of method on non-linear magnetic response indicated the preferential accumulation of the Hsp70-SPIONs in the heart tissue that was further confirmed with histological analyses. The study demonstrated that an acute infarct can be visualized by MRI using Hsp70-functionalized SPION conjugates. FROM THE CLINICAL EDITOR: Superparamagnetic iron oxides nanoparticles (SPIONs) have been studied extensively as a contrast agent for MRI. Their tissue specificity can be further enhanced by conjugation with various ligands. In this study, the authors conjugated superparamagnetic nanoparticles to 70-kDa heat shock protein (Hsp70-SPION) to investigate the feasibility for the detection of acute myocardium infarct. The positive findings would suggest that this approach might be used clinically in the future.

Magnetic Relaxation Switching Assay Using IFNα-2b-Conjugated Superparamagnetic Nanoparticles for Anti-Interferon Antibody Detection.

Type I interferons, particularly IFNα-2b, play essential roles in eliciting adaptive and innate immune responses, being implicated in the pathogenesis of various diseases, including cancer, and autoimmune and infectious diseases. Therefore, the development of a highly sensitive platform for analysis of either IFNα-2b or anti-IFNα-2b antibodies is of high importance to improve the diagnosis of various pathologies associated with the IFNα-2b disbalance. For evaluation of the anti-IFNα-2b antibody level, we have synthesized superparamagnetic iron oxide nanoparticles (SPIONs) coupled with the recombinant human IFNα-2b protein (SPIONs@IFNα-2b). Employing a magnetic relaxation switching assay (MRSw)-based nanosensor, we detected picomolar concentrations (0.36 pg/mL) of anti-INFα-2b antibodies. The high sensitivity of the real-time antibodies' detection was ensured by the specificity of immune responses and the maintenance of resonance conditions for water spins by choosing a high-frequency filling of short radio-frequency pulses of the generator. The formation of a complex of the SPIONs@IFNα-2b nanoparticles with the anti-INFα-2b antibodies led to a cascade process of the formation of nanoparticle clusters, which was further enhanced by exposure to a strong (7.1 T) homogenous magnetic field. Obtained magnetic conjugates exhibited high negative MR contrast-enhancing properties (as shown by NMR studies) that were also preserved when particles were administered in vivo. Thus, we observed a 1.2-fold decrease of the T2 relaxation time in the liver following administration of magnetic conjugates as compared to the control. In conclusion, the developed MRSw assay based on SPIONs@IFNα-2b nanoparticles represents an alternative immunological probe for the estimation of anti-IFNα-2b antibodies that could be further employed in clinical studies.

Targeting Brain Tumors with Mesenchymal Stem Cells in the Experimental Model of the Orthotopic Glioblastoma in Rats.

Despite multimodal approaches for the treatment of multiforme glioblastoma (GBM) advances in outcome have been very modest indicating the necessity of novel diagnostic and therapeutic strategies. Currently, mesenchymal stem cells (MSCs) represent a promising platform for cell-based cancer therapies because of their tumor-tropism, low immunogenicity, easy accessibility, isolation procedure, and culturing. In the present study, we assessed the tumor-tropism and biodistribution of the superparamagnetic iron oxide nanoparticle (SPION)-labeled MSCs in the orthotopic model of C6 glioblastoma in Wistar rats. As shown in in vitro studies employing confocal microscopy, high-content quantitative image cytometer, and xCelligence system MSCs exhibit a high migratory capacity towards C6 glioblastoma cells. Intravenous administration of SPION-labeled MSCs in vivo resulted in intratumoral accumulation of the tagged cells in the tumor tissues that in turn significantly enhanced the contrast of the tumor when high-field magnetic resonance imaging was performed. Subsequent biodistribution studies employing highly sensitive nonlinear magnetic response measurements (NLR-M2) supported by histological analysis confirm the retention of MSCs in the glioblastoma. In conclusion, MSCs due to their tumor-tropism could be employed as a drug-delivery platform for future theranostic approaches.

Targeting experimental orthotopic glioblastoma with chitosan-based superparamagnetic iron oxide nanoparticles (CS-DX-SPIONs).

BACKGROUND: Glioblastoma is the most devastating primary brain tumor of the central nervous system in adults. Magnetic nanocarriers may help not only for a targeted delivery of chemotherapeutic agents into the tumor site but also provide contrast enhancing properties for diagnostics using magnetic resonance imaging (MRI). METHODS: Synthesized hybrid chitosan-dextran superparamagnetic nanoparticles (CS-DX-SPIONs) were characterized using transmission electron microscopy (TEM) and relaxometry studies. Nonlinear magnetic response measurements were employed for confirming the superparamagnetic state of particles. Following in vitro analysis of nanoparticles cellular uptake tumor targeting was assessed in the model of the orthotopic glioma in rodents. RESULTS: CS-DX-SPIONs nanoparticles showed a uniform diameter of 55 nm under TEM and superparamagentic characteristics as determined by T1 (spin-lattice relaxation time) and T2 (spin-spin relaxation time) proton relaxation times. Application of the chitosan increased the charge from +8.9 to +19.3 mV of the dextran-based SPIONs. The nonlinear magnetic response at second harmonic of CS-DX-SPIONs following the slow change of stationary magnetic fields with very low hysteresis evidenced superparamagnetic state of particles at ambient temperatures. Confocal microscopy and flow cytometry studies showed an enhanced internalization of the chitosan-based nanoparticles in U87, C6 glioma and HeLa cells as compared to dextran-coated particles. Cytotoxicity assay demonstrated acceptable toxicity profile of the synthesized nanoparticles up to a concentration of 10 µg/ml. Intravenously administered CS-DX-SPIONs in orthotopic C6 gliomas in rats accumulated in the tumor site as shown by high-resolution MRI (11.0 T). Retention of nanoparticles resulted in a significant contrast enhancement of the tumor image that was accompanied with a dramatic drop in T2 values (P<0.001). Subsequent histological studies proved the accumulation of the nanoparticles inside glioblastoma cells. CONCLUSION: Hybrid chitosan-dextran magnetic particles demonstrated high MR contrast enhancing properties for the delineation of the brain tumor. Due to a significant retention of the particles in the tumor an application of the CS-DX-SPIONs could not only improve the tumor imaging but also could allow a targeted delivery of chemotherapeutic agents.

Ionizing radiation improves glioma-specific targeting of superparamagnetic iron oxide nanoparticles conjugated with cmHsp70.1 monoclonal antibodies (SPION-cmHsp70.1).

The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy for theranostics. Superparamagnetic iron oxide nanoparticles (SPIONs) are contrast negative agents that are used for the detection of tumors with MRI. Herein, we conjugated the Hsp70-specific antibody (cmHsp70.1) which is known to recognize mHsp70 to superparamagnetic iron nanoparticles to assess tumor-specific targeting before and after ionizing irradiation. In vitro experiments demonstrated the selectivity of SPION-cmHsp70.1 conjugates to free and mHsp70 in different tumor cell types (C6 glioblastoma, K562 leukemia, HeLa cervix carcinoma) in a dose-dependent manner. High-resolution MRI (11 T) on T(2)-weighted images showed the retention of the conjugates in the C6 glioma model. Accumulation of SPION-cmHsp70.1 nanoparticles in the glioma resulted in a nearly 2-fold drop of T*(2) values in comparison to non-conjugated SPIONs. Biodistribution analysis using NLR-M(2) measurements showed a 7-fold increase in the tumor-to-background (normal brain) uptake ratio of SPION-cmHsp70.1 conjugates in glioma-bearing rats in comparison to SPIONs. This accumulation within Hsp70-positive glioma was further enhanced after a single dose (10 Gy) of ionizing radiation. Elevated accumulation of the magnetic conjugates in the tumor due to radiosensitization proves the combination of radiotherapy and application of Hsp70-targeted agents in brain tumors.