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

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.