A Novel <scp>d</scp> -Amino Acid-Composed GRPR-Targeted Peptide in Glioma

Gastrin-releasing peptide receptor (GRPR) is overexpressed in glioma cells and can be specifically bound by Bombesin (BBN). Many molecular imaging agents based on the BBN structure have been tested in clinical trials and have shown promising results, but their <i>in vivo</i> stability needs to be improved. The aim of this study is to develop a novel d-amino acid-composed peptide BBN [as BBN(D)] and test its imaging performance by synthesizing two probes, i.e., BBN(D)-Cy5.5 and [⁶⁸Ga]Ga-DOTA-BBN(D), respectively, for near-infrared (NIR) fluorescence and PET/CT imaging. The HPLC purity of BBN(D)-Cy5.5 and DOTA-BBN(D) was >95%. And the mass spectrometry was used to confirm the identity of the product, LC-MS (ESI+) 528.5 was found [M+2H]/2. GRPR expression in U87-MG and glioma primary cells was confirmed by immunofluorescence. The <i>in vitro</i>. incubation of cells and BBN(D)-Cy5.5 exhibited a strong fluorescent signal and can be blocked by BBN. The <i>in vivo</i> subcutaneous and intracranial murine xenograft glioma models with two cell lines also demonstrated high specificity and tumor accumulation of BBN(D)-Cy5.5 and [⁶⁸Ga]Ga-DOTA-BBN(D) imaging tracers. PET/CT imaging with [⁶⁸Ga]Ga-DOTA-BBN(D) revealed optimal imaging at 40 min postinjection. Fluorescence imaging with BBN(D)-Cy5.5 was optimal at 4 h for subcutaneous models with tumor-to-background ratios peaked at 2.02 ± 0.136 and 2.00 ± 0.129 at 4 h. Biodistribution experiments confirmed that BBN(D) enhanced the accumulation of the probe in tumor tissue and endowed the probe with the ability to cross the blood-brain barrier; at 60 min postinjection of the probe through the teil vein, the uptake of [⁶⁸Ga]Ga-DOTA-BBN was less than 0.05%ID/g, while the uptake of [⁶⁸Ga]Ga-DOTA-BBN reached 0.70 ± 0.28%ID/g. The pathology and GRPR staining of <i>ex vivo</i> tissue from murine xenograft glioma models demonstrated strong consistency with fluorescence and PET/CT imaging. In conclusion, this study demonstrated that the d-amino acid-based BBN [BBN(D)] may provide specific imaging for glioma with high stability preoperatively and intraoperatively, warranting further exploration for clinical applications.