Abstract
1190
Objectives Hyperbranched polyglyercols (HPGs) are highly water-soluble polymers that can be readily synthesized with low polydispersity. Ranging from 0.5 kDa to 500 kDa in molecular weight, HPGs are characterized by the abundance of hydroxyl groups. These functional groups can be readily derivatized with different biochemical entities, providing multimodal utility. The excellent biocompatibility of HPGs has led them to be evaluated as synthetic protein substitutes in blood, as well as drug delivery vehicles. In this study, we aim to evaluate the use of HPGs as a potential scaffold for imaging carbonic anhydrase IX (CA-IX) expression with SPECT. CA-IX is a protein that is overexpressed in solid tumours in response to depleting oxygen levels in the microenvironment, and is a surrogate marker for hypoxia.
Methods 46-kDa HPG was obtained by ring-opening multibranching polymerization of glycidol using dioxane as the reaction medium. For derivatization, 1,2-diols were converted to aldehydes with periodic acid treatment. 4-(2-Aminoethyl)benzenesulfonamide (AEBSA) or tyramine and p-NH2-Bn-DOTA were coupled via reductive amination with NaBH3CN to yield the precursors DOTA-HPG46k-AEBSA and DOTA-HPG46k-Tyramine (as control). 111In labeling was performed in HEPES buffer (pH 5.3) at 95⁰C for 30 min, followed by PD-10 column and amicon purification. The stability of both tracers was assessed in mouse plasma with HPLC. SPECT imaging and biodistribution studies were performed in immunodeficient mice bearing CA-IX expressing HT-29 tumours.
Results On average 10 DOTA chelators and 25 AEBSA moieties were coupled to DOTA-HPG46k-AEBSA, and 8 DOTA chelators and 20 tyramine moieties were coupled to DOTA-HPG46k-Tyramine. 111In-DOTA-HPG46k-AEBSA was obtained in 53 ± 13% decay-corrected radiochemical yield with >97% radiochemical purity and 1.8 ± 0.5 Ci/μmol specific activity. 111In-DOTA-HPG46k-Tyramine was obtained in 54 ± 25% decay-corrected radiochemical yield with >99% radiochemical purity and 1.5 ± 0.6 Ci/μmol specific activity. Both tracers were stable in mouse plasma with negligible decomposition after 24 h incubation at 37 °C. Although uptake was mostly peripheral, administration of 111In-DOTA-HPG46k-AEBSA allowed for the visualization of tumour xenografts at 48 h p.i. with better contrast than 111In-DOTA-HPG46k-Tyramine. Both tracers were excreted by the hepatobiliary pathway. From biodistribution analysis at 48 h p.i., uptake in tumour xenografts were 10.60 ± 1.51 and 9.08 ± 1.79 for 111In-DOTA-HPG46k-AEBSA and 111In-DOTA-HPG46k-Tyramine, respectively. This corresponded to tumour-to-muscle ratios of 11.29 ± 1.14 and 7.44 ± 2.97.
Conclusions The development of molecular imaging scaffolds, particularly those that can be readily repurposed for therapeutic application, is of immense interest. 111In-DOTA-HPG46k-AEBSA was synthesized in good radiochemical yield, purity and specific activity; however tracer uptake and contrast was not significantly better than 111In-DOTA-HPG46k-Tyramine. While HPGs remain a flexible platform or vehicle, the derivatives evaluated in this study lack sufficient selectivity for CA-IX to be effective imaging agents.