Targeted diagnostic magnetic nanoparticles for medical imaging of pancreatic cancer☆
Introduction
During the last decades the development of early diagnostic methods for various tumours enabled an improvement in the treatment of cancer patients. This achievement, however, has not been achieved for pancreatic ductal adenocarcinoma (PDAC). PDAC is currently the fourth leading cause of cancer death in the United States of America [1]. Furthermore it is anticipated to become the second leading cause of cancer-related deaths in the year 2030 [1], [2]. PDAC possesses a mortality rate of up to 80% within the first 6 months after diagnosis [3], and the 5-year survival rate is only 6.7% [4], [5].
The paramount problem with pancreatic adenocarcinoma is that in most cases this cancer is diagnosed only in late stages, after possible metastasis spread, especially into the liver. Only 10%–15% of patients are diagnosed in the early stages of the disease [6].
Magnetic resonance imaging (MRI), computed tomography (CT), and single photon emission computed tomography–computer tomography (SPECT–CT) are state of the art in the diagnosis of pancreatic tumours. However, these medical imaging methods often don't have the ability to distinguish pancreatitis from pancreatic cancer [7], which is even worse as pancreatitis is a risk factor for carcinogenesis. Even the addition of more sophisticated imaging techniques such as diffusion weighted imaging is of no help in this effort [8].
In preclinical MRI studies, such as s of tumour cell migration and regional tumour growth, contrast enhanced MRI is used for both, diagnostic purposes and therapeutic monitoring. (Super-)paramagnetic nanoparticles offer the advantage, that they can be visualized in the morphologic standard T1-weighted (T1w) and T2-weighted (T2w) sequences.
NPs represent a novel class of therapeutics and diagnostics for cancer therapy. Recent studies demonstrated that therapeutics bound to or encapsulated into NPs provide an enhanced efficacy as well as reduced side effects compared to the respective unbound therapeutic entities [9], [10], [11]. Due to the leaky vasculature within a variety of tumours combined with their poor lymphatic drainage, nanoparticles can selectively accumulate in the tumour tissue following intravenous injection [12]. In addition, the attachment of targeting ligands that can bind to receptors or other biochemical structures which are presented and overexpressed on the surface of cancer cells, enhances the interaction with these cells resulting in further increase of accumulation in the tumour. Moreover, the targeting ligands also promote internalization by receptor-mediated endocytosis [13], [14], which is a prerequisite for advanced intracellular therapeutic approaches such as interference with the cells' metabolism via siRNA. Tumour-associated antigens that are already at earlier stages highly expressed on the cell surface of cancer cells but not in neighbouring tissue or only in negligible amounts are ideal for tumour targeting. Attachment or incorporation of diagnostic markers thus could allow the detection of the tumour by the targeted NPs using medical imaging. Since pancreatic adenocarcinomas are tumours with a very low perfusion (hypovascularized tumours) and hence radiologically low contrast agent uptake, a nanoparticle accumulation in these tumours, therefore, would be of great help for diagnosis as well as therapy.
Previous studies showed that these magnetic NPs (MNPs) consisting of recombinant human serum albumin (rHSA) and a magnetic core are non-toxic in vitro and in vivo and exhibited promising in vitro MRI-behaviour [15]. The main advantage of MNPs is their ability to covalently bind different molecules to their surface for presentation to cellular molecular units such as receptors. Loaded NPs were able to release the drug or diagnostic agent after internalization and procession of the particles in the targeted tissue, by using different binding techniques as described by Wartlick et al. and Weber et al. [11], [16]. This fact underlines the versatility of these particles in the scope of modular nanosystem platforms.
Recently, it was shown that the galectin-family (Gal), especially galectin-1 (Gal-1), acts as functional receptors for tissue plasminogen activator (t-PA) [17]. The binding is specific with a strong affinity and, hence, may provide a promising strategy for pancreatic cancer targeting. The expression of Gal-1 was reported to be upregulated in pancreatic cancer cells but is not expressed in adjacent normal tissues or adjacent inflammatory pancreas [18], [19].
The prime objective of the present study is to investigate different in vivo imaging procedures for their ability to improve the pancreatic cancer diagnosis using non-toxic human serum albumin nanoparticles that offer the possibility for attachment of a variety of targeting ligands and, as a further step, allow the binding of drugs (theranostics). As targeting moiety glycosylated peptides derived from t-PA, Gal-1's natural ligand, were bound covalently to previously developed [15] magnetic maghemite-containing nanoparticles (MNPs). The targeted MNPs were physico-chemically characterized, and their potential in vivo toxicity was determined by histology. The in vivo imaging methods included MRI as well as SPECT-CT and handheld gamma camera after radiolabelling the MNPs with 67Ga.
Section snippets
Nanoparticle preparation
MNPs were prepared following a method previously described by Rosenberger et al. [15]. For the incorporation of the magnetic γ-CAN maghemite NPs (CAN, (NH4)2Ce(IV)(NO3)6-γ-Fe2O3 NPs) into the rHSA nanoparticles, rHSA (Sigma-Aldrich, Steinheim, Germany) was dissolved to 100 mg/mL in 10 mM NaCl solution. 2000 μg or 4000 μg iron, respectively, were added to the protein solution and incubated for 1 h at 20 °C (Eppendorf thermomixer, 300 rpm, Hamburg, Germany). Afterwards desolvation took place by addition
Physico-chemical characterization of the nanoparticles
In a first step, magnetic nanoparticles (MNPs) were prepared using rHSA as shell protein and CAN-maghemite NPs as contrast agent for MRI. To ensure that the iron is encapsulated into the rHSA NP matrix, transmission electron microscopy pictures were taken previously. In these pictures, CAN-maghemite is visible in form of black spots surrounded by the grey rHSA nanoparticle matrix in transmission electron microscopy images [15]. The MNPs were characterized by PCS to obtain particle diameter, PDI
Discussion
In the present preclinical study, MNPs with and without an attached targeting moiety for pancreatic cancer were tested with respect to their capabilities for in vivo medical imaging using MRI, SPECT–CT, and a handheld gamma camera. Increased relaxation rates in MRI or increased radiation ratios for tumour/muscle in SPECT–CT and handheld gamma camera imaging were found for targeted as well as for non-targeted MNPs evidencing their uptake by the tumours. This uptake can be explained by passive
Conclusions
The present study demonstrated that all imaging modalities – MRI, SPECT-CT, and handheld gamma camera – were able to detect an in vivo accumulation of human serum albumin nanoparticles over time in PANC1-tumours, a tumour-model for adenocarcinoma of the pancreas. Although nanoparticles – like other colloids including liposomes or many macromolecules in general – were taken up by the reticuloendothelial system (RES), especially the liver to a high percentage (up to 90% of the administered dose),
Acknowledgements
This project has received funding from the European Union's Seventh Program for research, technological development and demonstration under grant agreement no 263307 (SaveMe large-scale collaborative project).
We thank Prof. Hans-Joachim Gabius (Ludwig-Maximilians University, Munich, Germany) for the generosity in providing recombinant galectin-1, -3, -4 proteins and their truncated forms which made possible studying the interactions of the t-PA and t-PA peptides by MST.
L. Gil-Iceta acknowledges
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Conflicts of interests: The authors declare no competing financial interest.
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These authors have equally contributed to the study.