Synthesis of temozolomide loaded copper nanoclusters for glioblastoma multiforme theranostics

1122 Objectives: The goal of this study was to develop a 64Cu-incorporated copper nanocluster (64Cu-CuNCs) functionalized with temozolomide (TMZ), a front-line chemotherapeutic for the treatment of glioblastoma multiforme, with targeting of the overexpressed CXCR4 chemokine receptor using FC131 ligand. The designed synthesis protocol aimed to protect temozolomide, a prodrug, from premature activation to 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC), and further degradation to the inactive metabolite, 5-aminoimidazole-4-carboxamide (AIC). Methods: The 64Cu-CuNCs were synthesized in a sodium acetate/acetic acid buffer at pH 5.6 and at 4 °C by the reduction of 64CuCl2 + CuCl2 with sodium borohydride. The radiolabeling yield and radiochemical purity of the as-prepared 64Cu-CuNCs was measured using instant thin layer chromatography and the size of non-radioactive nanoclusters were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The surface of the nanocluster was modified with the thioctic acid (TA) terminated ligands TA-PEG-OMe, TA-PEG-FC131, and TA-TMZ in equal amounts. The surface modification of the 64Cu-CuNCs and the integrity of the TMZ conjugated to the 64Cu-CuNCs were monitored using high performance liquid chromatography (HPLC). Biodistribution of 64Cu-CuNCs-PEG-OMe in wild-type CD1 mice was conducted to evaluate the in vivo pharmacokinetic behavior. Results: The 64Cu-CuNCs were successfully synthesized with over 95% in both radiolabeling yield and radiochemical purity. The hydrodynamic diameter of the 64Cu-CuNCs was measured to be 5.5 ± 1.1 nm as determined by DLS while the core diameter of the 64Cu-CuNCs was approximately 2.6 ± 0.4 nm as determined by TEM. HPLC analyses showed that 98.2% of TA-TMZ was stable with less than 2% degradation post synthesis and its half-life was determined as 144.4 hours when stored at 4 C. Furthermore, each 64Cu-CuNC was conjugated with an average of 17 FC131 tumor-targeting agents and 28 TMZ on its surface. Initial invivo pharmacokinetic evaluation of 64Cu-CuNCs-PEG-OMe showed high blood (22.1 ± 3.5 %ID/g) retention and liver (24.5 ± 3.2 %ID/g) uptake at 1 hour post injection, followed by fast clearance at 24 hour post injection (blood: 1.0 ± 0.08 %ID/g, liver: 8.7 ± 1.1 %ID/g). Conclusions: We have prepared and optimized the synthesis of 64Cu-CuNCs loaded with TMZ and CXCR4 chemokine receptor targeting ligand, FC131, with desirable physicochemical properties. The rapid clearance and optimal organ distribution of 64Cu-CuNCs-PEG-OMe indicated the potential of theranostic 64Cu-CuNCs for glioblastoma multiforme imaging and therapy. Research Support: Funding for this project (MC-II-2017-661) was provided by the Children’s Discovery Institute of Washington University.