90Y-CA/SPIONs for dual magnetic hyperthermia-radionuclide nanobrachytherapy of solid tumours.

Radiolabelled superparamagnetic iron oxide nanoparticles (SPIONs) are a promising nanomaterial for the development of dual radiation/hyperthermia cancer therapy. To that purpose, flower-shaped SPIONs with an exceptional heating capability were synthesised and coated with citrate, dextran or (3-aminopropyl)triethoxysilane. Both non-coated and coated SPIONs were nontoxic to CT-26 mouse colon cancer cells up to 1.0 mg ml-1in vitro. In an oscillating magnetic field, citrate-coated SPIONs (CA/SPIONs) displayed the highest heating rate (SAR âˆ¼ 253 W g-1) and the strongest hyperthermia effects against CT-26 cells. Labelling of the CA/SPIONs by the90Y radionuclide, emitting ß-radiation with an average/maximum energy of 0.94/2.23 MeV, and deep tissue penetration generated90Y-CA/SPIONs intended for the therapy of solid tumours. However, intravenous injection of90Y-CA/SPIONs in CT-26 xenograft-bearing mice resulted in low tumour accumulation. On the contrary, intratumoural injection resulted in long-term retention at the injection site. A single intratumoural injection of 0.25 mg CA/SPIONs followed by 30-min courses of magnetic hyperthermia for four consecutive days caused a moderate antitumour effect against CT-26 and 4T1 mouse tumour xenografts. Intratumoural application of 1.85 MBq/0.25 mg90Y-CA/SPIONs, alone or combined with hyperthermia, caused a significant (P ≤ 0.01) antitumour effect without signs of systemic toxicity. The results confirm the suitability of90Y-CA/SPIONs for monotherapy or dual magnetic hyperthermia-radionuclide nanobrachytherapy (NBT) of solid tumours.

Trastuzumab Conjugated Superparamagnetic Iron Oxide Nanoparticles Labeled with 225Ac as a Perspective Tool for Combined α-Radioimmunotherapy and Magnetic Hyperthermia of HER2-Positive Breast Cancer.

It has been proven and confirmed in numerous repeated tests, that the use of a combination of several therapeutic methods gives much better treatment results than in the case of separate therapies. Particularly promising is the combination of ionizing radiation and magnetic hyperthermia in one drug. To achieve this objective, magnetite nanoparticles have been modified in their core with α emitter 225Ac, in an amount affecting only slightly their magnetic properties. By 3-phosphonopropionic acid (CEPA) linker nanoparticles were conjugated covalently with trastuzumab (Herceptin®), a monoclonal antibody that recognizes ovarian and breast cancer cells overexpressing the HER2 receptors. The synthesized bioconjugates were characterized by transmission electron microscopy (TEM), Dynamic Light Scattering (DLS) measurement, thermogravimetric analysis (TGA) and application of 131I-labeled trastuzumab for quantification of the bound biomolecule. The obtained results show that one 225Ac@Fe3O4-CEPA-trastuzumab bioconjugate contains an average of 8-11 molecules of trastuzumab. The labeled nanoparticles almost quantitatively retain 225Ac (>98%) in phosphate-buffered saline (PBS) and physiological salt, and more than 90% of 221Fr and 213Bi over 10 days. In human serum after 10 days, the fraction of 225Ac released from 225Ac@Fe3O4 was still less than 2%, but the retention of 221Fr and 213Bi decreased to 70%. The synthesized 225Ac@Fe3O4-CEPA-trastuzumab bioconjugates have shown a high cytotoxic effect toward SKOV-3 ovarian cancer cells expressing HER2 receptor in-vitro. The in-vivo studies indicate that this bioconjugate exhibits properties suitable for the treatment of cancer cells by intratumoral or post-resection injection. The intravenous injection of the 225Ac@Fe3O4-CEPA-trastuzumab radiobioconjugate is excluded due to its high accumulation in the liver, lungs and spleen. Additionally, the high value of a specific absorption rate (SAR) allows its use in a new very perspective combination of α radionuclide therapy with magnetic hyperthermia.

Gamma-Camera Direct Imaging of the Plasma and On/Intra Cellular Distribution of the 99mTc-DPD-Fe3O4 Dual-Modality Contrast Agent in Peripheral Human Blood.

The radiolabeled iron oxide nanoparticles constitute an attractive choice to be used as dual-modality contrast agents (DMCAs) in nuclear medical diagnosis, due to their ability to combine the benefits of two imaging modalities, for instance single photon emission computed tomography (SPECT) with magnetic resonance imaging (MRI). Before the use of any DMCA, the investigation of its plasma extra- and on/intra cellular distribution in peripheral human blood is of paramount importance. Here, we focus on the in vitro investigation of the distribution of 99mTc-DPD-Fe3O4 DMCA in donated peripheral human blood (the ligand 2-3-dicarboxypropane-1-1-diphosphonic-acid is denoted as DPD). Initially, we described the experimental methods we performed for the radiosynthesis of the 99mTc-DPD-Fe3O4, the preparation of whole blood and blood plasma samples, and their incubation conditions with 99mTc-DPD-Fe3O4. More importantly, we employed a gamma-camera apparatus for the direct imaging of the 99mTc-DPD-Fe3O4-loaded whole blood and blood plasma samples when subjected to specialized centrifugation protocols. The direct comparison of the gamma-camera data obtained at the exact same samples before and after their centrifugation enabled us to clearly identify the distribution of the 99mTc-DPD-Fe3O4 in the two components, plasma and cells, of peripheral human blood.

Radiolabeled Iron Oxide Nanoparticles as Dual Modality Contrast Agents in SPECT/MRI and PET/MRI.

During the last decades, the utilization of imaging modalities such as single photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI) in every day clinical practice has enabled clinicians to diagnose diseases accurately at early stages. Radiolabeled iron oxide nanoparticles (RIONs) combine their intrinsic magnetic behavior with the extrinsic character of the radionuclide additive, so that they constitute a platform of multifaceted physical properties. Thus, at a practical level, RIONs serve as the physical parent of the so-called dual-modality contrast agents (DMCAs) utilized in SPECT/MRI and PET/MRI applications due to their ability to combine, at real time, the high sensitivity of SPECT or PET together with the high spatial resolution of MRI. This review focuses on the synthesis and in vivo investigation of both biodistribution and imaging efficacy of RIONs as potential SPECT/MRI or PET/MRI DMCAs.

ZnO NPs immobilized by Alizarin as in vitro predictive and imaging biomarkers for protein amyloidosis.

Protein amyloidosis represents the main pathological hallmark of many incurable neurodegenerative disorders and protein misfolding diseases. Nanomaterials-based approaches give rise to diagnosis and/or prediction of these proteinopathies, with regards to the multifactorial nature of their pathogenesis. Herein, crystalline truncated hexagonal shaped naked ZnO nanoparticles (mean value 47.4 nm) have been solvothermally prepared and immobilized further with alizarin (Alzn) molecules (54%) to stand up to amyloidosis acting both as inhibitors and imaging agents, as well as antioxidants. Thioflavin-T (ThT) assay revealed that the resulted zinc oxide nanoparticles immobilized with alizarin (ZnO@Alzn NPs) inhibited in vitro insulin amyloids formation in a dose-dependent manner, while the kinetic mechanism of the phenomenon was recorded. In parallel, amyloid oligomers and plaques have been visualized by conventional optical microscopy upon protein co-incubation with ZnO@Alzn NPs, highlighting the imaging ability of the immobilized NPs. The antioxidant activity was monitored by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, through which it was shown that alizarin incorporation onto the inorganic core leads to the reduction of IC50 values from 221 µg/mL to 167 µg/mL. The enhanced free radical scavenging effects of ZnO@Alzn compared to the naked-ZnO NPs, features their prospect to serve additional functions.

99mTc-Labeled Iron Oxide Nanoparticles as Dual-Modality Contrast Agent: A Preliminary Study from Synthesis to Magnetic Resonance and Gamma-Camera Imaging in Mice Models.

The combination of two imaging modalities in a single agent has received increasing attention during the last few years, since its synergistic action guarantees both accurate and timely diagnosis. For this reason, dual-modality contrast agents (DMCAs), such as radiolabeled iron oxide (namely Fe3O4) nanoparticles, constitute a powerful tool in diagnostic applications. In this respect, here we focus on the synthesis of a potential single photon emission computed tomography/magnetic resonance imaging (SPECT/MRI) DMCA, which consists of Fe3O4 nanoparticles, surface functionalized with 2,3-dicarboxypropane-1,1-diphosphonic acid (DPD) and radiolabeled with 99mTc, [99mTc]Tc-DPD-Fe3O4. The in vitro stability results showed that this DMCA is highly stable after 24 h of incubation in phosphate buffer saline (~92.3% intact), while it is adequately stable after 24 h of incubation with human serum (~67.3% intact). Subsequently, [99mTc]Tc-DPD-Fe3O4 DMCA was evaluated in vivo in mice models through standard biodistribution studies, MR imaging and gamma-camera imaging. All techniques provided consistent results, clearly evidencing noticeable liver uptake. Our work documents that [99mTc]Tc-DPD-Fe3O4 has all the necessary characteristics to be a potential DMCA.

Immunocompatibility of a new dual modality contrast agent based on radiolabeled iron-oxide nanoparticles.

Radiolabeled magnetic nanoparticles are promising candidates as dual-modality-contrast-agents (DMCA) for diagnostic applications. The immunocompatibility of a new DMCA is a prerequisite for subsequent in vivo applications. Here, a new DMCA, namely Fe3O4 nanoparticles radiolabeled with 68Ga, is subjected to immunocompatibility tests both in vitro and in vivo. The in vitro immunocompatibility of the DMCA relied on incubation with donated human WBCs and PLTs (five healthy individuals). Optical microscopy (OM) and atomic force microscopy (AFM) were employed for the investigation of the morphological characteristics of WBCs and PLTs. A standard hematology analyzer (HA) provided information on complete blood count. The in vivo immunocompatibility of the DMCA was assessed through its biodistribution among the basic organs of the mononuclear phagocyte system in normal and immunodeficient mice (nine in each group). In addition, Magnetic Resonance Imaging (MRI) data were acquired in normal mice (three). The combined OM, AFM and HA in vitro data showed that although the DMCA promoted noticeable activation of WBCs and PLTs, neither degradation nor clustering were observed. The in vivo data showed no difference of the DMCA biodistribution between the normal and immunodeficient mice, while the MRI data prove the efficacy of the particular DMCA when compared to the non-radiolabeled, parent CA. The combined in vitro and in vivo data prove that the particular DMCA is a promising candidate for future in vivo applications.

Hemocompatibility of gallium-68 labeled iron oxide nanoparticles coated with 2,3-dicarboxypropane-1,1-diphosphonic acid.

Dual-modality contrast agents (DMCA), such as radiolabeled magnetic nanoparticles, have attracted significant attention in diagnostic applications due to their potency for the timely and accurate diagnosis of diseases. The hemocompatibility of a candidate DMCA with human blood is essential for the investigation of its application in vivo. In this respect, here we focused on the evaluation of the hemocompatibility of a new DMCA, that is based on iron oxide nanoparticles (i.e. Fe3O4 magnetite), with human red blood cells (RBCs). The specific iron oxide nanoparticles are surface functionalized with 2,3-dicarboxypropane-1,1-diphosphonic acid (-DPD) and radiolabeled with gallium-68 (68Ga), resulting in 68Ga-DPD-Fe3O4. RBCs of five healthy individuals are incubated at room temperature for 120 min without and with 68Ga-DPD-Fe3O4 at concentrations 0.1 and 1.0 mg/ml. Optical microscopy (OM) and atomic force microscopy (AFM) are employed to assess detailed information on the overall morphological and geometrical characteristics of the entire cell at the microscopic (10-6 m) level and on the membrane morphology at the nanoscopic (10-9 m) level. In addition, a standard hematology analyzer (HA) is used to obtain complete blood count information. At the microscopic level, the combined OM, AFM and HA data revealed that the overall shape/size characteristics of RBCs were preserved upon incubation with 68Ga-DPD-Fe3O4. However, at the nanoscopic level, the AFM results revealed two different kinds of local deconstructions of the RBCs membrane, termed holes and ulcer-like abnormalities, that were observed in both the DMCA-free and DMCA-incubated samples. Holes did not exhibit any statistically significant difference upon incubation with the 68Ga-DPD-Fe3O4 DMCA. On the contrary, ulcer-like abnormalities exhibited two statistically significant differences upon incubation with the 68Ga-DPD-Fe3O4 DMCA. First, increased percentage of RBCs having at least one ulcer-like abnormality; in DMCA-incubated samples 78.6 ± 11.6% for CDMCA = 0.1 mg/ml and 80.4 ± 11.1% for CDMCA = 1.0 mg/ml, while in DMCA-free samples 61.2 ± 8.4% prior to and 63.6 ± 13.5% after incubation. Second, increased number of ulcer-like abnormalities per RBC; in DMCA-incubated samples 4.26 ± 0.62 for CDMCA = 0.1 mg/ml and 3.99 ± 0.97 for CDMCA = 1.0 mg/ml, while in DMCA-free samples 2.84 ± 0.54 prior to and 2.98 ± 0.50 after incubation. The combined OM, AFM and HA results prove fair hemocompatibility of the 68Ga-DPD-Fe3O4 DMCA with human RBCs, thus documenting its potential use in imaging applications.

Detection of Superoxide Alterations Induced by 5-Fluorouracil on HeLa Cells with a Cell-Based Biosensor.

BACKGROUND: In vitro cell culture monitoring can be used as an indicator of cellular oxidative stress for the assessment of different chemotherapy agents. METHODS: A cell-based bioelectric biosensor was used to detect alterations in superoxide levels in the culture medium of HeLa cervical cancer cells after treatment with the chemotherapeutic agent 5-fluorouracil (5-FU). The cytotoxic effects of 5-fluorouracil on HeLa cells were assessed by the MTT proliferation assay, whereas oxidative damage and induction of apoptosis were measured fluorometrically by the mitochondria-targeted MitoSOX™ Red and caspase-3 activation assays, respectively. RESULTS: The results of this study indicate that 5-FU differentially affects superoxide production and caspase-3 activation when applied in cytotoxic concentrations against HeLa cells, while superoxide accumulation is in accordance with mitochondrial superoxide levels. Our findings suggest that changes in superoxide concentration could be detected with the biosensor in a non-invasive and rapid manner, thus allowing a reliable estimation of oxidative damage due to cell apoptosis. CONCLUSIONS: These findings may be useful for facilitating future high throughput screening of different chemotherapeutic drugs with a cytotoxic principle based on free radical production.

A Novel Metal-Based Imaging Probe for Targeted Dual-Modality SPECT/MR Imaging of Angiogenesis.

Superparamagnetic iron oxide nanoparticles with well-integrated multimodality imaging properties have generated increasing research interest in the past decade, especially when it comes to the targeted imaging of tumors. Bevacizumab (BCZM) on the other hand is a well-known and widely applied monoclonal antibody recognizing VEGF-A, which is overexpressed in angiogenesis. The aim of this proof-of-concept study was to develop a dual-modality nanoplatform for in vivo targeted single photon computed emission tomography (SPECT) and magnetic resonance imaging (MRI) of tumor vascularization. Iron oxide nanoparticles (IONPs) have been coated with dimercaptosuccinic acid (DMSA), for consequent functionalization with the monoclonal antibody BCZM radiolabeled with 99mTc, via well-developed surface engineering. The IONPs were characterized based on their size distribution, hydrodynamic diameter and magnetic properties. In vitro cytotoxicity studies showed that our nanoconstruct does not cause toxic effects in normal and cancer cells. Fe3O4-DMSA-SMCC-BCZM-99mTc were successfully prepared at high radiochemical purity (>92%) and their stability in human serum and in PBS were demonstrated. In vitro cell binding studies showed the ability of the Fe3O4-DMSA-SMCC-BCZM-99mTc to bind to the VEGF-165 isoform overexpressed on M-165 tumor cells. The ex vivo biodistribution studies in M165 tumor-bearing SCID mice showed high uptake in liver, spleen, kidney and lungs. The Fe3O4-DMSA-SMCC-BCZM-99mTc demonstrated quick tumor accumulation starting at 8.9 ± 1.88%ID/g at 2 h p.i., slightly increasing at 4 h p.i. (16.21 ± 2.56%ID/g) and then decreasing at 24 h p.i. (6.01 ± 1.69%ID/g). The tumor-to-blood ratio reached a maximum at 24 h p.i. (~7), which is also the case for the tumor-to-muscle ratio (~18). Initial pilot imaging studies on an experimental gamma-camera and a clinical MR camera prove our hypothesis and demonstrate the potential of Fe3O4-DMSA-SMCC-BCZM-99mTc for targeted dual-modality imaging. Our findings indicate that Fe3O4-DMSA-SMCC-BCZM-99mTc IONPs could serve as an important diagnostic tool for biomedical imaging as well as a promising candidate for future theranostic applications in cancer.

Gallium-68 Labeled Iron Oxide Nanoparticles Coated with 2,3-Dicarboxypropane-1,1-diphosphonic Acid as a Potential PET/MR Imaging Agent: A Proof-of-Concept Study.

The aim of this study was to develop a dual-modality PET/MR imaging probe by radiolabeling iron oxide magnetic nanoparticles (IONPs), surface functionalized with water soluble stabilizer 2,3-dicarboxypropane-1,1-diphosphonic acid (DPD), with the positron emitter Gallium-68. Magnetite nanoparticles (Fe3O4 MNPs) were synthesized via coprecipitation method and were stabilized with DPD. The Fe3O4-DPD MNPs were characterized based on their structure, morphology, size, surface charge, and magnetic properties. In vitro cytotoxicity studies showed reduced toxicity in normal cells, compared to cancer cells. Fe3O4-DPD MNPs were successfully labeled with Gallium-68 at high radiochemical purity (>91%) and their stability in human serum and in PBS was demonstrated, along with their further characterization on size and magnetic properties. The ex vivo biodistribution studies in normal Swiss mice showed high uptake in the liver followed by spleen. The acquired PET images were in accordance with the ex vivo biodistribution results. Our findings indicate that 68Ga-Fe3O4-DPD MNPs could serve as an important diagnostic tool for biomedical imaging.