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In vivo magnetic resonance imaging of pancreatic tumors using iron oxide nanoworms targeted with PTR86 peptide

https://doi.org/10.1016/j.colsurfb.2017.06.051Get rights and content

Highlights

  • Magnetic nanoworms were developed for multimodal diagnosis of pancreatic tumors.

  • A novel cyclic peptide PTR86 was used to target somatostatin receptors.

  • In vitro experiments confirmed the cellular uptake efficacy of targeted nanoworms.

  • In vivo MR imaging showed accumulation of nanoworms in the tumor areas.

  • Ex vivo fluorescence imaging displayed tissue accumulation of targeted nanoworms.

Abstract

To cut the high mortality rate of malignant disease such as pancreatic cancer, development of newly diagnostic probes for early stage detection of tumor lesions is required. Multimodal imaging nanoprobes allowing targeted and real time functional/anatomical imaging of tumors meet the demands. For this purpose, a MRI/optical dual-modality probe based on biodegradable magnetic iron oxide nanoworms has been developed. The cross-linked surface of nanoworms were anchored to fluorescent dyes and to FITC.PTR86; a novel synthetic peptide with high affinity towards somatostatin receptors. Combination of various in vitro techniques including Prussian blue staining, fluorescent microscopy and fluorescence activated cell sorting (FACS) have been performed to explore the interaction of developed nanoprobe with pancreatic tumor cell lines. Together with in vivo studies in a xenograft mouse model of human pancreatic adenocarcinoma and ex vivo investigations, the results show the efficient imaging and targeting of pancreatic tumors by our newly developed nanosystem using both MRI and optical imaging modalities.

Introduction

As early detection of cancer substantially increases the chance of cure, different modalities such as PET (Positron Emission Tomography), SPECT (Single Photon Emission Computed Tomography), x-ray CT (Computed Tomography), MRI (Magnetic Resonance Imaging) and NIRF (Near Infrared Fluorescence), have been developed for personal cancer diagnosis. However, to shape the wish for clear-cut imaging, single-modality imaging methods tend to be insufficient, since each of these imaging methods has its own restrictions in terms of sensitivity, spatial resolution and depth of signal penetration that might be addressed by combining two or more such modalities [1].

The very recent emergence of hybrid scanner like PET-MRI brings new hopes for precise imaging of cancer. At the time of being, the PET tracer and the MRI probe are separately injected to the patient to obtain different information from each modality [2]. However, integration of the probes into a single platform avoids differential biodistribution that results from introducing a cocktail of imaging agents to the body [3].

Nanoscopic objects are suitable platforms for buildup of multimodal diagnostics due to their large surface area, which offers the possibility to functionalize them with various molecules such as tumor biomarkers and signaling agents [4]. In this regard, superparamagnetic iron oxide nanoparticles (SPIONs) with their inherent magnetic properties and biodegradability are one of the most attractive materials for development of diagnostics and theranostics [5]. Most studies till far have been focused on SPIONs with spherical morphology, which were mostly synthesized through co-precipitation of iron salts through in situ coating with biocompatible sugar, polymer or protein [6]. At a specific ratio of the iron salts and the coating materials, the spherical iron oxide core could join together along one dimension to produce 60–100 nm long structures named nanoworms [7]. These nanoparticles (NPs) revealed to produce stronger MRI signals than their component spherical ones [8], and also displayed longer bioavailability [7]. The elongated shape and large surface area of these particles let them to carry increased number of targeting moieties. Hence, the synchronized and cooperative binding of these biomarkers to the tumor surface can extensively improve the accumulation of these particles in the target region and can potentially help clinician to image small lesions [9].

Pancreatic cancer is a highly aggressive tumor type with very low survival rate [10]. Its high mortality rate demands the development of more sensitive diagnostic agents. Early stage imaging of pancreatic lesions might help for efficient treatment and the saving of lives. Many pancreatic tumors display overexpression of somatostatin receptors (SSTR), a protein with five different subtypes (SSTR 1–5). During the progression of the pancreatic cancer, the tumor changes its strategy by altering the upregulations of the receptor subtypes. Therefore, a biomarker capable of targeting all subtypes of SSTR is desired. Such a biomarker meeting these demands have been already developed by us [11]. PTR86 is a synthetic somatostatin analogue with backbone cyclic core peptide with affinity towards SSTR 1–5.

Herein, we report a multifunctional nanoworm platform that combines MRI/optical imaging capability and SSTR specificity for pancreatic cancer detection. The physicochemical properties of the synthesized nanostructures were extensively characterized via various techniques. In addition, in vitro, in vivo and ex vivo experiments were carried out to carefully evaluate cellular uptake efficacy and tumor MR imaging performance.

Section snippets

Materials

Ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), Dextran (Mr 15000–20000), epichlorohydrin, monochloroacetic acid (MCA), 1,10-phenanthroline, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC), 2-(N-morpholino)ethanesulfonic acid (MES), hydroxylamine hydrochloride, potassium ferrocyanide, sodium acetate trihydrate, ammonium hydroxide solution (28%) and DAPI dihydrochloride were purchased from Sigma-Aldrich. Nuclear Fast Red was acquired by Vector

Synthesis and characterization of nanoworms

The TEM image of CLNW is shown in Fig. 1a, in which the elongated and worm shape of the nanostructures is clearly visible. The morphology of nanostructures was examined at different conjugation steps and based on the TEM results, the worm shape of IONWs stayed intact over subsequent functionalization. In addition, the size of the iron-core was measured by TEM to be between 5–10 nm.

Table 1 shows the hydrodynamic size (size distribution by intensity), polydispersity index (PDI) and the zeta

Conclusion

In this study, preparation and preclinical evaluation of a multifunctional nanoprobe for dual MRI and optical imaging of pancreatic tumors have been demonstrated. The nanoprobe contains the magnetic ion cores for MRI, was conjugated to fluorescent dyes for fluorescence imaging and was anchored to FITC.PTR86; a novel backbone cyclic core peptide targeting SSTR 1–5. Combination of various in vitro techniques such as Prussian blue staining, fluorescent microscopy and FACS using PANC-1 cell lines

Conflict of interest

The authors declare no competing financial interest.

Acknowledgments

The authors would like to thank Mag. Daniel Gruber at the Core Facility Cell Imaging and Ultrastructure Research, Vienna for her help and support in TEM imaging. The authors greatly acknowledge Prof. Manfred Ogris and Dr. Haider Sami for discussion and advice for nanoparticle synthesis. The authors thank Prof. Franz Gabor for access to DLS facility and Ms. Julia Maier for assistance in FACS experiments.

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