Diagnostic Tumor Imaging Using Renally Excretable Nanoparticles: Focus on Active and Passive Targeting

Introduction
Depending on their size, shape and surface functionalities, nanoparticles can passively extravasate and accumulate in the tumor tissue through the enhanced permeability and retention (EPR) effect. Being an accumulative process, this effect favors nanoparticles with long blood retention time. Renally excretable, ultrasmall nanoparticles with short blood half-lives are therefore less prone to passive tumor targeting as they rapidly diffuse back to the vasculature and re-enter the systemic circulation, which results in only transient intratumoral presence without substantial retention. To prevent their rapid efflux from malignant tissues by increasing the interactions between nanoparticles and tumor cells as well as by improving cellular nanoparticle uptake, the strategy of active or ligand-mediated targeting is pursued. Here, we describe the development of renally excretable dendritic polyglycerols (dPGs) functionalized with different targeting units to differentiate between active and passive tumor targeting.

Methods
Fluorescent dye labels for optical imaging and small camelid single-domain antibodies (sdAbs) as targeting units - both equipped with maleimide functionalities - were simultaneously attached in a one-pot reaction to thiol groups of the dPGs. As the presented work focusses on the epidermal growth factor receptor (EGFR) acting as a model receptor, an EGFR-specific sdAb was attached to the dPGs to obtain an active targeting probe. In parallel, a probe with similar surface characteristics but a nonspecific sdAb (passive targeting) was synthesized. Both conjugates were purified using affinity chromatography, which selectively separates the sdAb-conjugated dPGs.

Results
In vitro binding studies on different human epithelial cancer cell lines using dye-labeled sdAb-conjugated dPGs showed a high specificity, co-localization and a receptor-mediated cellular uptake of the EGFR-specific probes. Optical imaging studies using murine xenografts revealed a substantial accumulation of the EGFR-specific probes in comparison to its nonspecific counterparts and a minimum off-target accumulation of both conjugates.

Discussion and Conclusion
The direct comparison of specific and nonspecific probes with similar surface characteristics allows the straight-forward preclinical discrimination between active and potential passive tumor targeting of renally excretable nanoparticles in small animal models. Furthermore, it provides important information on the extent to which ligand-mediated targeting contributes to total nanoparticle accumulation in malignant and normal tissues.