Molecularly targeted nanoparticles: an emerging tool for evaluation of expression of the receptor for advanced glycation end products in a murine model of peripheral artery disease

Marcin Woźniak, Christian J. Konopka, Agata Płoska, Jamila Hedhli, Anna Siekierzycka, Maciej Banach, Rafal Bartoszewski, Lawrence W. Dobrucki, Leszek Kalinowski, Iwona T. Dobrucki

Molecular imaging with molecularly targeted probes is a powerful tool for studying the spatio-temporal interactions between complex biological processes. The pivotal role of the receptor for advanced glycation end products (RAGE), and its involvement in numerous pathological processes, aroused the demand for RAGE-targeted imaging in various diseases. In the present study, we evaluated the use of a diagnostic imaging agent for RAGE quantification in an animal model of peripheral artery disease, a multimodal dual-labeled probe targeted at RAGE (MMIA-CML). PAMAM dendrimer was conjugated with Nε-carboxymethyl-lysine (CML) modified albumin to synthesize the RAGE-targeted probe. A control untargeted agent carried native non-modified human albumin (HSA). Bifunctional p-SCN-Bn-NOTA was used to conjugate the 64Cu radioisotope. Surgical right femoral artery ligation was performed on C57BL/6 male mice. One week after femoral artery ligation, mice were injected with MMIA-CML or MMIA-HSA labeled with 64Cu radioisotope and 60 min later in vivo microPET-CT imaging was performed. Immediately after PET imaging studies, the murine hindlimb muscle tissues were excised and prepared for gene and protein expression analysis. RAGE gene and protein expression was assessed using real-time qPCR and Western blot technique respectively. To visualize RAGE expression in excised tissues, microscopic fluorescence imaging was performed using RAGE-specific antibodies and RAGE-targeted and -control MMIA. Animals subjected to PET imaging exhibited greater MMIA-CML uptake in ischemic hindlimbs than non-ischemic hindlimbs. We observed a high correlation between fluorescent signal detection and radioactivity measurement. Significant RAGE gene and protein overexpression were observed in ischemic hindlimbs compared to non-ischemic hindlimbs at one week after surgical ligation. Fluorescence microscopic staining revealed significantly increased uptake of RAGE-targeted nanoparticles in both ischemic and non-ischemic muscle tissues compared to the control probe but at a higher level in ischemic hindlimbs. Ischemic tissue exhibited explicit RAGE dyeing following anti-RAGE antibody and high colocalization with the MMIA-CML targeted at RAGE. The present results indicate increased expression of RAGE in the ischemic hindlimb and enable the use of multimodal nanoparticles in both in vitro and in vivo experimental models, creating the possibility for imaging structural and functional changes with a RAGE-targeted tracer.