Molecular imaging of activated platelets via antibody-targeted ultra-small iron oxide nanoparticles displaying unique dual MRI contrast
Graphical abstract
Introduction
Despite significant advances in diagnostic and therapeutic technologies, cardiovascular disease (CVD) remains the global leading cause of death, accounting for 17.3 million deaths per year, and is expected to grow to more than 23.6 million by 2030 [1]. This represents 30% of all global deaths and 80% of this occurs in low- and middle-income countries. CVD claims more lives than all forms of cancers combined [1]. Of all CVDs, stroke and coronary artery disease account for more than 70% of all deaths [2].
The most common form of CVD and also the leading cause of sudden death is atherosclerosis, a chronic progressive inflammatory disease of the arterial vessels. The process of atherosclerosis involves a complex interplay between various cells, particular leukocytes and platelets [3]. Unstable, vulnerable atherosclerotic plaques can rupture and cause thrombosis, resulting in myocardial infarction (MI) and stroke. Recent studies have confirmed that (micro)thrombi containing activated platelets exist long before the presentation of sudden coronary death, indicating that vessel occlusion is often preceded by a variable period of localized coagulation and inflammation [4]. Currently, the prevention of MI and stroke is limited due to the lack of sensitive imaging methods. Those available usually involve invasive procedures such as coronary angiograms, which are potentially associated with complications, including death caused by MI or bleeding. Hence, there is a great need for new diagnostic strategies to determine whether the individual patient is at risk of MI or stroke, which then would allow for effective and early preventative treatment and improved clinical outcome.
Molecular imaging, the non-invasive visualization of fundamental (disease) biomarkers in living organisms, holds the promise to transform the diagnosis of CVD, profoundly impacting future clinical CVD care. Non-invasive detection of thrombosis employs various contrast agents that are equipped with moieties targeting the biological features associated with thrombosis formation such as fibrin, factor XIII, and activated platelets [5], [6]. Compared to other modalities, molecular MRI has great potential for thrombus detection. Unlike positron emission tomography (PET) or computed tomography (CT) methods, MRI does not employ ionizing radiation; unlike ultrasound and optical methods, MRI provides deep tissue penetration; and it is more advantageous than PET, because MRI has much higher spatial resolution (sub-millimeter). The principle drawback of molecular MRI is the lower sensitivity of contrast agent detection compared to nuclear techniques and therefore the choice of a highly abundant target and the design of sensitive and specific molecular probes are critical. For molecular imaging of thrombosis, platelets are an ideal choice for targeting since they are the major component of thrombi [7].
To achieve high accuracy of disease diagnosis, attempts to combine complementary information obtained from different imaging techniques, including MRI, PET, CT and optical microscopy [8], [9], [10], [11] have been made. However, differences in depth penetrations and spatial/time resolutions of various imaging devices can lead to difficulties and discrepancies when matching images, resulting in interpretation inaccuracies [8], [9]. The development of dual imaging strategies that employ a single imaging technique and a single instrumental system such as MRI would provide significant advantages.
There are two widely used T1 and T2/T2* - weighted MRI contrast agents. Gadolinium (Gd) based agents enhance signal due to T1 shortening at lower concentrations providing positive image contrast. At high concentrations, e.g. in the bolus following injection, Gd agents null the signal due to T2/T2* shortening. Iron oxide nanoparticles generally provide negative image contrast due to T2/T2* shortening. The T1 effect or bright spot imaging is preferred since the location of the imaging agent is more readily distinguishable from potential artefacts produced by tissue interfaces, hemorrhage or signal cancellations at water-fat interfaces, which all produce negative contrast effects [12]. T1-weighted imaging is particularly advantageous in imaging of vessel thrombosis, as a sufficient T1 shortening effect allows generation of positive contrast between a thrombus (appearing bright in the image) and surrounding tissues and blood (dark). T2-contrast agents, such as magnetic iron oxide nanoparticles, have shown limited toxicity and have been proven to be one of the most promising contrast agents for clinical use because iron is naturally found in the body [13]. These agents can be detected at a relative lower concentration and a sub-millimeter areas using T2-weighted or T2* (susceptibility) weighted imaging. However, the limitations of iron oxide agents include the lack of specificity in heterogeneous anatomy and potential confusion with effects from bleeding, calcification, metal deposits (such as endogenous iron) and other susceptibility artefacts [14].
Previously, several attempts have been reported on the development of MRI imaging agents for molecular imaging of atherosclerosis and thrombosis. Among these, Gd-based agents were widely employed to image endothelial dysfunction and activation [15]; extracellular matrix (ECM - the major component of atherosclerostic lesions) via targeting ECM proteins such as collagen [16], neovascularization by targeting the αvβ3 integrin [17]: proteolytic enzymes during plaque development [18]; thrombus and plaque by targeting fibrin [19], [20], [21], [22], [23], [24]; and lipids – the major components of atherosclerotic plaques [25], [26]. Iron oxide nanoparticles were also used extensively to image endothelial dysfunction by targeting the expressed adhesion molecules on the endothelial surface [27]; macrophages resident in the plaques [28], [29]; and thrombus via targeting GPIIb/IIIa receptors expressed on activated platelets [30], [31], [32]. Although research on molecular imaging of CVD has been reported extensively, these MR imaging agents used only single imaging modes such as T1- or T2/T2*-weighted imaging.
Since both T1-positive and T2-negative contrast agents have their respective advantages and disadvantages, it is highly desirable to prepare a robust dual contrast agent for overcoming the limitations of single modality contrast agents. The simultaneous use of a contrast agent that can provide both T1 and T2 effects will significantly improve detection accuracy. Previously, we have reported the synthesis of ultra-small, water-soluble and biocompatible magnetic iron oxide nanoparticles as positive and negative dual contrast agents [13]. Here we report the development and functionalization of these nanoparticles for targeted imaging of thrombosis. The nanoparticles were also labelled with near infrared dyes to enable optical detection, and functionalized with single-chain antibodies (scFv) for targeting to activated platelets, which are critical players in atherosclerosis, thrombosis, and inflammation [33], [34]. Our strategic approach employing a dual mode, where two different T1 and T2-weighted imaging are performed simultaneously, can potentially provide highly sensitive and accurate diagnostic information.
Section snippets
Materials and methods
All reagents and solvents were obtained from standard commercial sources and were used as received.
Results and discussion
The present work is the first report on imaging of cardiovascular disease or thrombosis using MRI dual positive and negative 3.3 nm iron oxide contrast agents (DCIONs). The scFv-tagged DCIONs were prepared using a unique chemo-enzymatic approach involving copper-free click chemistry and Staphylococcus aureus sortase A enzyme conjugation. The antigen binding activity of the scFv-DCIONs was evaluated in a range of in vitro and in vivo experiments, including binding to in vitro human thrombi and
Conclusion
In conclusion, the presented data demonstrate a successful and unique approach for MR molecular imaging of thrombosis via a dual mode strategy employing positive and negative contrast iron oxide nanoparticles. DCIONs were functionalized and tagged with scFv using a combination of chemical and biological techniques. The conjugation was achieved with a high yield (94%) and a preserved scFv bioactivity was confirmed. The targeting of scFvantiGPIIb/IIIa-tagged DCIONs to activated GPIIb/IIIa
Acknowledgements
This work is funded by National Health and Medical Research Council (H.T.T: APP1037310), University of Queensland Early Career Grant (H.T.T.: UQECR1606713), the Australian Research Council (A.K.W.: CE140100036, K.P.: FT0992210), the National Heart Foundation (C.E.H: CR 11M 6066, X.W: 100517), and the National Natural Science Foundation of China (Z.L.: 81471657 and 81527901). The authors would like to acknowledge the Australian National Fabrication Facility (Queensland Node); National Imaging
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