Active versus passive targeting of renally excretable nanoparticles using multimodal diagnostic tumor imaging

Objectives
Integration of multiple imaging modalities onto a single scaffold obviates the need to administer several compounds with different pharmacokinetics, increases the sensitivity of the detection and gives a deeper insight into the pathophysiological processes. This requires a scaffold with multiple attachment sites, a high tumor binding affinity, and a rapid renal elimination profile. In this regard, dendritic polyglycerols (dPGs) are well-defined, globular, highly biocompatible macromolecules with a nano-size (2-20 nm), narrow size distribution (PDI <1.26) and numerous surface functionalities, which make them amenable to a wide range of chemical modifications [1]. Previous studies done on 3H and 64Cu radiolabeled dPGs show their great potential as platforms for diagnostic applications [2]. Here, the development of dPGs as dual-modal agents for epidermal growth factor receptor (EGFR) specific tumor imaging is described.

Methods
A one-pot strategy was employed for simultaneous attachment of fluorescent labels for optical imaging (cy3/cy7) and macrocyclic chelators based on a 1,4,7-triazacyclononane system for 64Cu (PET tracer) to thiol anchoring groups of the dPGs. A small camelid single-domain antibody (sdAb) representing a potential recognition agent for EGFR as targeting vector was attached (1). In parallel, a probe with similar surface characteristics but an EGFR unspecific sdAb (control) was synthesized (2). The conjugates were purified using affinity chromatography, which selectively separates the antibody-conjugated multimodal conjugates. In vitro and in vivo studies were conducted to assess its diagnostic potential.

Results
64Cu labeling was achieved under ambient temperature and physiological pH. Binding studies on A431 and FaDu cells using 64Cu and dye-labeled 1 and 2 showed a high specificity, colocalization and a receptor-mediated cellular uptake of 1. Intravenous injection of the 1 and 2 on mouse xenografted models studies using PET and optical imaging revealed an overwhelming tumor accumulation of the EGFR-specific 1 in comparison to the EGFR-unspecific 2 and a minimum off-target accumulation of both conjugates.

Conclusions
These results show the great potential of dendritic polyglycerols as multimodal platforms for various biomedical applications.