Synthesis, characterization, and biological evaluation of novel multimodal dendrimer-based probe for targeted imaging of receptor for advanced glycation end-products

Objectives In this study we evaluated a novel Cu64-labeled RAGE-targeted probe as a multimodal PET-optical imaging agent to noninvasively assess changes in RAGE expression.

Methods The probe was synthesized by functionalization of G4 PAMAM dendrimer with the specific RAGE ligand Ne-carboxymethyl-lysine (CML) modified human serum albumin (HSA). Non-targeted probe contained unmodified HSA. Probe was rendered multimodal by conjugation of NOTA (for Cu64 PET imaging) and AlexaFluor 647 (for optical imaging). Targeting efficiency was optimized by tuning both the number of CML-HSA molecules per nanoparticle and the percentage of HSA modification with CML. Binding to RAGE-expressing cells was studied using flow cytometry. In vivo applicability was assessed in C57BL6 mice (n=12) subjected to surgical femoral artery ligation to induce hindlimb ischemia. At 1 wk after surgery Cu64-G4-CML (n=6) and Cu64-G4-HSA (n=6) were injected for PET-CT imaging. Mice were euthanized and organs excised for gamma well counting (GWC). Raw counts were corrected for background, decay, weight, and expressed as %ID/g.

Results Cell culture studies with flow cytometry and histology confirmed that Cu64-G4-CML which contained eight (n=8) CML groups per nanoparticle and 20% CML modification provided the highest affinity towards RAGE. Analysis of PET-CT images revealed favorable kinetics, rapid blood clearance, and a 3.4-fold higher uptake in ischemic hindlimb than that for the non-targeted probe. GWC demonstrated increased liver and lung retention paralleled by relatively low uptake in other critical organs.

Conclusions In our study we successfully synthesized, evaluated and demonstrated feasibility for non-invasive monitoring of RAGE expression with a newly synthesized RAGE-targeted multimodal nanoparticle.

Research Support The Foundation for Polish Science (LK, LWD), the National Science Centre (LK), American Heart Association (LWD)