Affibody modified and radiolabeled gold–Iron oxide hetero-nanostructures for tumor PET, optical and MR imaging
Introduction
Advancement on the research of different imaging modalities such as positron emission tomography (PET), single photon emission computed tomography (SPECT), optical imaging (OI), magnetic resonance imaging (MRI), computed tomography (CT), ultrasound, etc. has greatly improved their imaging sensitivity, specificity, and spatial and temporal resolution, which render them powerful tools for diseases detection. More recently, it has been widely recognized that combining certain functional imaging techniques (PET, OI, etc.) with anatomic imaging modalities (MRI, CT, etc.) together can provide complementary information and thus offer synergistic advantages over any single modality alone. For example, PET/CT combines functional PET and three-dimensional high resolution CT [1], [2]. It has shown many applications in cancer imaging and been fully integrated into routine clinical practice [3], [4]. Comparing to CT, MRI offers even higher soft tissue resolution while without any radiation exposure to patients. As encouraged by the success of PET/CT, combination of PET and MRI (PET/MRI) has been actively pursued in the imaging community for the last several years. Several research groups have reported development of PET/MRI systems for small-animal studies [5], [6]. More importantly, PET/MRI in a single device with simultaneous imaging acquisition has also been introduced into clinical applications recently [7], [8], [9]. Comparing with PET/CT, PET/MRI provides better soft-tissue contrast and more functional information with lower radiation exposure, thereby raising it to be an attractive choice of future imaging modality.
With the rapid development of imaging instrumentations, there are urgent demands in developing multimodal molecular probes such as dual-labeled probes for PET/MRI pre-clinically and clinically. This type of agents could be biomolecules labeled with both PET radionuclide and MRI metal or nanoparticle (NPs) that are capable of providing specific dual-modal imaging. Recently iron oxide nanoparticle (IONP) has been modified with the Arg-Gly-Asp (RGD) peptide and radiometal chelators and labeled with 64Cu for both PET and MRI of integrin αvβ3 expression in tumor [10], [11]. These pioneer studies demonstrate the feasibility of preparation of PET/MRI probes and further use of them for cancer imaging in vivo. However, until now the majority of studies on PET/MRI probe development have simply focused on the use of IONP which is composed by a single component and could suffer from certain limitations as listed below. First, coupling of different chemical functionalities on a mono-component NP surface could be a low yield synthetic process; Second, the presence of targeting biomolecules, functional labeling moieties and drugs on the same NP surface may compromise the targeting and imaging capability and the subsequent in vivo performance of the NP conjugates; Lastly, modification of a single component NP with multiple different ligands simultaneously or sequentially is a relative complex process that could have low reproducibility on the product preparation. It usually generates heterogeneous products which could be consisted by different ratios of ligands on the NPs. This problem could severely hamper their further translation into clinic.
One of the promising solutions is to use multicomponents NPs, which have at least two different NPs within one structure [12], [13]. Unlike regular single-component nanospheres, dumbbell-like NPs (DBNPs) have two distinctive NP surfaces (Fig. 1A), so that many biological targeting molecules, drug molecules, or labeling moieties can be specifically anchored on each of NP surface, eliminating their intimate contact and interference. The recent progress in synthesis and application of Au–Fe3O4 DBNPs (Au-IONPs) had attracted much attention for its potential as multifunctional probes in diagnosis and therapy [13], [14], [15], [16], [17]. Furthermore, Au-IONPs are constructed with both a magnetic (IO) and an optically active plasmonic (Au) unit, which is also suitable for simultaneous optical and magnetic detection [12]. But all the previous reported studies regarding Au-IONPs are solely focused on in vitro studies. Moreover, the average sizes of those whole hetero-nanostructures varied in the range from 15 to 20 nm with a broad size distribution. There was not much attention paid to the size and quality control of hetero-nanostructures when they were used as probes or delivery vehicles in vitro studies. To achieve highly sensitive and efficient tumor cell detection in vivo, the hetero-nanostructures need to be monodisperse so that each individual NP has nearly identical physical and chemical properties for controlled biodistribution, bioelimination and contrast effects. Moreover, it is important to systematically optimize various nanostructure parameters (such as size, surface chemistry) at a time to enhance the detection sensitivity and tumor accumulation of those engineered hetero-nanostructures. Considering the size, shape, and surface modification have profound impact to the in vivo performance of the NPs and smaller size NPs are usually preferred for in vivo targeted imaging, herein, we report the development and use of a highly monodispersed dumbbell-shaped Au–IONP (size: ∼4 nm in diameter for Au–NP and 8 nm in diameter for IONP, characterized by TEM) for the development of a PET/Optical/MRI probe for cancer imaging in small living animals.
The diagnosis and treatment of cancers at the molecular level would be greatly enhanced by the ability to deliver contact agents and/or potentially therapeutic agents into specific cancer cells and cellular compartments in living systems. Such monodisperse Au–IONP provides a robust nanoplatform for surface-specific modification with both targeting molecules and PET reporters in a highly efficient and reliable manner. Specifically, the Fe3O4 component in the Au–IONP can serve as a T2 reporter for MRI, and the IO surface was first coated with the polyethylene glycolated (PEGylated) dopamine linkers, followed by specifically conjugating anti-epidermal growth factor receptor (EGFR) Affibody proteins (for example: Ac-Cys-ZEGFR: 1907, amino acid sequence, Ac-C-VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPS -QSANLLAEAKKLNDAQAPK) (Fig. 1B). Affibody proteins are a new class of engineered scaffold proteins with a three-helix bundle structure. Comparing to antibodies, they have certain advantages for tumor-targeted imaging, including their much smaller size and lower molecular weight (58-amino acid residues, 7 kDa), faster tumor targeting ability, more well-defined structure which could potentially be site-specifically modified and can be chemically synthesized. Affibodies have shown high promise for development of tumor targeting agents [18], [19], [20], [21], [22], [23], [24]. Several anti-EGFR Affibody proteins including ZEGFR:1907 with high affinities in nM ranges have been reported and used for tumor imaging and potential radionuclide therapy [25], [26], [27], [28], [29], [30]. Since EGFR is a well established tumor biomarker and overexpressed in a wide range of human tumors [31]. modification of the Au–IONP with anti-EGFR Affibody molecules, like Ac-Cys-ZEGFR:1907 [30], makes the resulting nanoprobe capable for early diagnosis of various EGFR positive tumors. To further prepare the multimodal nanoprobe, the Au component of the Au-IONP was surface-specifically modified with thiolated PEG linkers via Au-S bonds, followed by coupling with the radiometal chelators, 2-S-(4-isothiocyanatobenzyl)-1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (p–SCN–Bn-NOTA). Radiolabeling the NOTA and Ac-Cys-ZEGFR:1907 modified Au–IONP (NOTA-Au-IONP-Affibody) with PET radionuclide, 64Cu, results in an EGFR targeted PET/Optical/MR Imaging probe, 64Cu-NOTA-Au-IONP-Affibody (Fig. 1B). We therefore consider that Affibody molecules are promising affinity ligands for making Au-IONPs targetable without adverse effect on their in vivo performance and giving those NPs targeting ability toward EGFR-positive tumors.
Section snippets
Instrumentation and materials
Hydrogen tetrachloroaurate (III) hydrate was ordered from Strem Chemicals, Inc.. N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (sulfo-NHS), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Pierce Biotechnology. PEG bisamine [Molecular weight (MW) = 2000 and 3000] and methoxyPEG amine 2000 (m-PEG-2000-NH2) were ordered from Sigma/Aldrich. The metal chelator, p–SCN–Bn-NOTA, was obtained from Macrocyclics. Unless otherwise mentioned, all other
NOTA-Au-IONP-Affibody preparation and characterization
First, the highly monodisperse Au-IONPs were synthesized according to the modified procedures described in the previous publications [12], [14], in which the injection condition of iron precursor (iron pentacarbonyl) and subsequent heating rate played important roles in determining the quality of obtained NPs (including shape and size distribution). The size of iron oxide component within Au-IONPs was dependant on the ratio of gold seeds to iron precursor. A constant heating rate at 8 °C/min
Discussion
Nanoparticles based molecular probes for imaging have attracted enormous attention because of their unique properties and multifunctionalities as nanoscale platforms. With the rapid development of imaging instrumentations, PET/MRI in a single device with simultaneous imaging acquisition has been introduced into clinical applications recently [1], [3], [10], [35]. There are urgent demands in developing multimodal molecular probes such as probes for PET/MRI. Among the current well-developed
Conclusions
In summary, the Au-IONPs can not only offer two functional nanocompositions for the site-specific attachment of various targeting biomolecules, reporting moieties and drugs after surface modification, they also contain both IO and Au NPs, which could be served for simultaneous magnetic and optical theronostic purpose. We developed a simple surface modification method, which is feasible for bioconjugation with various types of biomolecules, allowing versatile conjugations between Au-IONPs and
Acknowledgments
This work was partially supported by DOE Stanford Molecular Imaging Research and Training Program (DE-SC0008397), NCI of Cancer Nanotechnology Excellence Grant CCNE-TR U54 CA119367, CA151459, and National High Technology Research and Development Program of China (863 Plan, 2006AA027Z4B3). We thank Dr. Laura Jean Pisani for assistance in obtaining MR images.
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Meng Yang and Kai Cheng contributed equally to this work.