Chimeric peptide-based radiopharmaceuticals for glioblastoma imaging and therapy by targeting mHsp70 and enhancing BBB penetration.

Glioblastoma (GBM) is the most aggressive form of malignant brain cancer. Here, we synthesized two chimeric peptide-based radiopharmaceuticals (Comb-1 and Comb-2) targeted to the membrane-bound form of heat shock protein 70 (mHsp70), which is overexpressed in GBM tissues, for future theranostic applications. The design concept features a DOTA chelator for coordination to different radiometals tethered directly or via a PEG linker to a chimeric peptide. The latter combines the Hsp70-targeting ability of the TPP sequence with the blood-brain barrier (BBB) penetration of another 7-amino acid sequence (d-PepH3) derived from the dengue virus capsid protein (DEN2C). The two compounds were successfully radiolabelled with gallium-67, suitable for single photon emission computed tomography (SPECT) imaging, or with lutetium-177 for β- therapy. Furthermore, the in vitro properties of the ligand, including lipophilicity (log D 7.4), human serum albumin (HSA) binding, and stability in human serum were evaluated. The presence of the TPP sequence affected the half-life of the combinatorial peptides. Cytometry assays performed with a fluorescent analogue of Comb-2 confirmed the binding to mHsp70-expressing U87-MG cells. In an in vitro model, all tracers demonstrated the ability to cross the BBB, indicating that conjugation of the mHsp70-targeting peptide to the PepH3 sequence did not impair its translocating properties. Biodistribution experiments with 67Ga-labeled compounds were performed in naive female CD1 mice and showed brain uptake at 2 min p.i., as well as renal excretion. For the best performing compound [67Ga]Ga-Comb-2 (0.60 ± 0.17% IA g-1 (injected activity per gram)), the biodistribution experiment was also performed with perfusion of the organs after sacrifice, and the results showed retention of radioactivity in the brain (0.14 ± 0.05% IA g-1). Further metabolic studies in murine urine and blood were performed after biodistribution, confirming the stability of the chimeric tracers.