⁶⁴Cu-labelled dendritic polyglycerol derivatives for biodistribution studies

Objectives
Dendritic polyglycerols (dPG) and dendritic polyglycerol sulfates (dPGS) are easily synthesized macromolecules. These polymers are highly water-soluble, biocompatible, practically non-toxic and non-immunogenic. Hence, these properties make them highly interesting for application in biomedicine and offers excellent prospects for the development of new non-invasive strategies for the diagnosis and treatment of diseases. dPGS have already been developed as fully synthetic heparin analogues that show anti-inflammatory properties and accumulation in tumor tissue, which makes them promising agents for therapeutic applications. Since information about the biodistribution and the metabolism of dPGS in living systems is quite scarce, one approach is to radiolabel them so as to be able to track them with, for example, positron emission tomography (PET). ⁶⁴Cu has suitable decay characteristics that allow for PET imaging and a variety of chelator systems are available for attachment to these macromolecules using appropriate anchor groups. Herein, we report the conjugation of bifunctional chelating agents based on bis(2-pyridylmethyl)triaza-cyclononane [DMPTACN] onto the dPG/dPGS scaffolds. The structure of DMPTACN allows for the introduction of various functional groups, such as carboxylic, maleimide or isothiocyanate groups, for coupling to various functional groups
(amino and mercapto surface groups) on the dPG derivatives. ⁶⁴Cu-labeling experiments and stability studies of the resulting radiocopper complexes are presented and discussed.
Methods & materials
DMPTACN was synthesized by a 10-step process starting from diethylenetriamine to form a TACN ring containing tosyl protecting groups. After deprotection and subsequent introduction of two pendant 2-picolyl arms, coupling
groups, such as a carboxylic acid, maleimide or isothiocyanate, have been attached. The dPG and dPGS samples can be synthesized on a kilogram scale by utilizing ring-opening and copolymerization reactions. Surface modification with
amino and mercapto groups provides the target molecules needed for attachment of copper-binding agents. After conjugation of BFCAs to the dPG/dPGS, the compounds were purified by size exclusion chromatography. ⁶⁴Cu-labeling of DMPTACN-dPG(dPGS) conjugates were performed using [⁶⁴Cu]CuCl₂ in 0.1 M MES/NaOH buffer at a pH of 5.5 at room temperature, resulting in a radiochemical purity of higher than 95% within a few minutes. Animal experiments were carried out in male Wistar rats.
Results & conclusion
dPG and dPGS have been synthesized with various percentages of amine and mercapto functionalities capable of coupling to BFCAs based on a DMPTACN backbone with maleimide, carboxylic and isothiocyanate groups. The
conjugates can be purified by size exclusion chromatography, and have been obtained in good yields. ⁶⁴Cu-labeling experiments confirmed rapid copper(II) complex formation under mild conditions. The ⁶⁴Cu-labelled conjugates exhibit high in vitro stability in human serum. These probes can thus be utilized to derive quantitative distribution data in vivo. Biodistribution and pharmacokinetic properties of ⁶⁴Cu-labelled dendritic polyglycerol derivatives can be conveniently studied by PET.
Financial disclosure
This work is part of a research initiative within the Helmholtz-Portfoliothema “Technologie und Medizin - Multimodale Bildgebung zur Aufklaerung des In vivo-Verhaltens von polymeren Biomaterialien”.