Automated Fluorine-18 Radiolabelling via Alkyne-azide Cycloaddition Reaction on Dual Peptide-functionalized Liposomes Surface for in vivo PET Imaging

Marco Iannone ^1* Marcelo Kravicz ² Paolo Rainone ^3,4 Antonia Antoniou ⁵ Stefano Stucchi ¹ Silvia Valtorta ^3,4,6 Arianna Amenta ⁵ Elia Anna Turolla ^1,2 Sara Pellegrino ⁷ Daniele Passarella ⁵ Elisa Vino ¹ Sergio Todde ^1,2,4 Francesca Re ² Pierfausto Seneci ⁵ Rosa Maria Moresco ^2,3,4

• ¹ Tecnomed, Fondazione dell'Università degli Studi di Milano-Bicocca, Monza, Italy
• ² Department of Medicine and Surgery, University of Milano Bicocca, Monza, Italy
• ³ Department of Nuclear Medicine, San Raffaele Scientific Institute (IRCCS), Milan, Lombardy, Italy
• ⁴ Istituto di Bioimmagini e Sistemi Biologici Complessi (IBSBC-CNR), Milan, Lombardy, Italy
• ⁵ Department of Chemistry, Faculty of Science and Technology, University of Milan, Milan, Lombardy, Italy
• ⁶ National Biodiversity Future Center, Palermo, Sicily, Italy
• ⁷ Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Milan, Milan, Lombardy, Italy

Labeled nanoparticles can be monitored within the body using PET imaging, offering real-time insight into their pharmacokinetics and biodistribution. In the present work, liposomes are labeled with the radionuclide fluorine-18, exploiting the "surface radiolabeling" approach. Two alkyne-DOPE (dioleoylphosphatidylethanolamine) constructs are embedded in the bulk of the liposome bilayer composed of cholesterol and sphingomyelin, and radiolabeling is performed either via copper(I)-catalyzed cycloaddition "click" reaction (CuAAC), or cyclooctyne-driven copper-free "click" reaction (CyOctC) modality, using a suitable fluorine-18 labeled azide, obtaining good results in terms of yields, purity, stability and automation of entire radiosynthesis process. In addition, radiolabeling is also performed on liposome formulations functionalized with (i) a peptide deriving from the receptor-binding domain of apolipoprotein E (mApoE), (ii) and with a metalloproteinase (MMP)-sensitive lipopeptide (MSLP). The in vivo uptake in an orthotopic mouse model of glioma (Gli36ΔEGFR cell line) by PET/CT is performed. The results show a higher tumor/background ratio, a faster clearance rate and a lower uptake in healthy brain tissue and peripheral districts for mApoE and MSLP-functionalized liposomes, in comparison with non-functionalized ones, that prompt for further characterization. On the contrary, radiolabeled liposome uptake is higher in most peripheral organs for non-functionalized liposomes. Hence, fluorine-18 labeled liposomes can be reliably used for in vivo PET tracking of multifunctionalized nanoparticles, enabling effective investigation of their potential as drug delivery systems.