^99mTc-labeled aminosilane-coated iron oxide nanoparticles for molecular imaging of α_νβ₃-mediated tumor expression and feasibility for hyperthermia treatment

Highlights

• Synthesis and characterization of RGD modified IO-NPs.

• In vitro % cell viability evaluation by MTT assay.

• In vivo biodistribution study about targeting ability.

• Scintigraphic imaging and hyperthermia treatment.

Abstract

Hypothesis

Dual-modality imaging agents, such as radiolabeled iron oxide nanoparticles (IO-NPs), are promising candidates for cancer diagnosis and therapy. We developed and evaluated aminosilane coated Fe₃O₄ (10 ± 2 nm) as a tumor imaging agent in nuclear medicine through 3-aminopropyltriethoxysilane (APTES) functionalization. We evaluated this multimeric system of targeted ^99mTc-labeled nanoparticles (NPs) conjugated with a new RGD derivate (cRGDfK-Orn₃-CGG), characterized as NPs-RGD as a potential thermal therapy delivery vehicle.

Experiments

Transmission Electron Microscopy (TEM) and spectroscopy techniques were used to characterize the IO-NPs indicating their functionalization with peptides. Radiolabeled IO-NPs (targeted, non-targeted) were evaluated with regard to their radiochemical, radiobiological and imaging characteristics. In vivo studies were performed in normal and α_νβ₃-positive tumor (U87MG glioblastoma) bearing mice. We also demonstrated that this system could reach ablative temperatures in vivo.

Findings

Both radiolabeled IO-NPs were obtained in high radiochemical yield (>98%) and proved stable in vitro. The in vivo studies for both IO-NPs have shown significant liver and spleen uptake at all examined time points in normal and U87MG glioblastoma tumor-bearing mice, due to their colloidal nature. We have confirmed through in vivo biodistribution studies that the non-targeted ^99mTc-NPs poorly internalized in the tumor, while the targeted ^99mTc-NPs-RGD, present 9-fold higher tumor accumulation at 1 h p.i. Accumulation of both IO-NPs in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells. The preliminary in vivo study of applied alternating magnetic field showed that the induced hyperthermia is feasible due to the aid of IO-NPs.

Introduction

Molecular imaging of cancer using nano-materials is potentially an important tool in diagnosis, therapy, and drug delivery [1], [2], [3], [4], [5]. Combined diagnosis and therapy through nanotechnology is attracting increased attention for cancer diagnosis and therapy. This integration of diagnostic imaging capability with therapeutic interventions, termed theranostics, is critical for addressing the challenges of cancer heterogeneity and adaptation. Dual-modality imaging agents, such as radiolabeled nanoparticles (NPs), are prominent candidates for a number of diagnostic applications, since they can combine the advantages of two different imaging modalities; e.g.: functional imaging with Single Photon Emission Computerized Tomography (SPECT) as well as Positron Emission Tomography (PET) and anatomical imaging, with Magnetic Resonance Imaging (MRI) [6], [7]. The combination of SPECT/PET with MRI has raised great expectations for highly sensitive and high-resolution imaging leading to the development of the first simultaneous imaging systems which is now available on both clinical and preclinical levels [8], [9]. NPs with dual modality SPECT/MRI and PET/MRI imaging properties must be designed in such a way to provide stable radiolabeling and high specific activity [10]. Although both SPECT and PET have their own advantages and limitations, SPECT tends to be a more robust tool because SPECT radionuclides are characterized by longer half-life and can be produced by small generators whilst most PET radionuclides have a short half-life and usually need a cyclotron, which limits their availability and exponentially increases the cost and complexity of the application. The most commonly used radionuclide in SPECT is ^99mTc because of its excellent physical properties (t_1/2 = 6 h, γ-radiation 140 keV) and extended availability through ⁹⁹Mo/^99mTc generators. It has been shown by many scientific groups, including ours, that in vivo administration of ^99mTc-radiolabeled NPs can provide accurate biodistribution profiles as well as visualization of excretion route of radioactivity in vivo for a period of up to 24 h [10], [11], [12].

Iron oxide (maghemite γ-Fe₂O₃ or magnetite Fe₃O₄) nanoparticles (IO-NPs) are one of the most popular nanostructures in medicine both for therapy and diagnosis [13]. IO-NPs possess unique characteristics that make them well-suited as probes for molecular imaging [10], [14]. Due to their important potential for hyperthermia-induced therapy, in vivo stem cell tracking and targeted drug delivery are established as theranostics [15], [16], [17], [18], [19]. Magnetic nanoparticles have played an important role in the development of hyperthermia for in vivo treatment of tumors, the first experimental investigations about the hyperthermia magnetic materials’ application dates back to 1957 [20]. Hyperthermia is usually applied as an adjunctive approach to chemotherapy and radiotherapy for cancer treatment [21], [22], [23]. Cancer cells are more susceptible to thermal sensitization than healthy ones due to their increased rate of cell cycling and to even decreased blood flow impairing oxygen and nutrient supply and inducing acidity [24], [25], [26], [27]. Thermotherapy based on IO-NPs, also known as magnetic fluid hyperthermia (MFH), is an attractive technique for thermotherapy of deep-seated tumors [15], [28], [29], [30]. Localized hyperthermia by directly injecting IO-NPs to the tumor site and applying alternating magnetic field (AMF) has been extensively investigated by several groups but few clinical trials have been tested so far [22], [31], [32], [33].

IO-NPs when modified with appropriate coatings, either organic (dextran, dendrimer, chitosan, etc.) or inorganicones (silica or gold particles), can provide an attractive approach for: (i) colloidal suspendability and subsequent protection against the formation of aggregates, and/or (ii) conjugation with fluorescent dyes for optical imaging, chelators for MRI/nuclear imaging, targeting moieties, or even drugs [34], [35], [36]. In the context of cancer, non-targeted NPs can accumulate in tumors through the enhanced permeability and EPR (Enhanced permeability and retention) effect, since the tumor vasculature is usually leaky and there is no lymphatic drainage in the tumor [7], [37]. Imaging agents based on passive targeting take advantage of the EPR effect [37]. However, the need for a more efficient, controllable and specific in vivo tumor targeting NP, although not necessary, is quite advantageous. Active targeting is achieved by functionalizing the NP surface with suitable vectors including peptides, mAbs and other biomolecules such as folic acid, which recognize characteristic epitopes at the surface of the diseased cells, thus increasing specific cell uptake [36].

Angiogenesis is essential for the development of malignant tumors and provides important targets for tumor diagnosis and therapy [38], [39]. The cell adhesion molecule α_νβ₃ integrin is a specific marker of angiogenesis, being over-expressed in activated and proliferating endothelial cells [38], [40]. The α_νβ₃ integrin is considered as an attractive target for addressing NPs to tumors [41]. In a great number of studies different type of NPs have been used to target integrins imaging the corresponding tissues, particularly in the endothelial cells of angiogenic blood vessels [41], [42]. In general peptides have gained a lot of attention as potent targeting ligands, Peptides based on the Arg-Gly-Asp (RGD) sequence present high affinity and selectivity for the α_νβ₃ integrin and are, therefore, useful in the noninvasive monitoring of tumor angiogenesis by molecular imaging techniques [43], [44], [45]. In particular, it has been shown that the cyclic Arg-Gly-Asp-D-Phe-Lys (cRGDfK) presents high affinity to the α_νβ₃ receptor and can specifically accumulate in several tumors including osteosarcomas, glioblastomas, melanomas, lung carcinomas, and breast cancer [40], [44], [46], [47]. Moreover, increased interest for RGD conjugated NPs [18], [45], [46], [48], [49], [50], [51], [52], [53] has prompted researchers to design RGD derivatives as successful receptor integrin α_νβ₃ targeting moieties in SPECT/MRI dual modality tumor imaging [50].

In the present study, we synthesized, characterized and evaluated aminosilane coated IO-NPs as a tumor imaging agent in nuclear medicine through 3-aminopropyltriethoxysilane (APTES). Following the preliminary evaluation of an ornithine-modified peptide cRGDfK-Orn₃-CGG, that considered to be appropriately designed for conjugation with NPs [54], we investigated its conjugation with IO-NPs (NPs-cRGDfK-Orn₃-CGG), named as NPs-RGD. Both targeted and non-targeted IO-NPs were radiolabeled with ^99mTc, based on an already published procedure [34]. The in vitro stability of both radiolabeled compounds, and their in vivo fate in normal mice and in mice bearing α_νβ₃-positive tumors, were investigated. The aim was to study the potential use of the multifunctional system of ^99mTc-labeled IO-NPs conjugated to the RGD (cRGDfK-Orn₃-CGG) peptide and evaluate its biological behavior, as a potential specific radiopharmaceutical product for early tumor-angiogenesis detection. Finally, we performed a proof of concept hyperthermia experiment for the in vivo evaluation of the system in hyperthermia therapy to assess the effect of magnetic resonance hyperthermia treatment. A hyperthermia session was performed in a U87MG glioblastoma tumor bearing animal model and tumor temperature increase was monitored with a near infrared camera.