Anti-myeloma Effects of Conjugated Arginyl-Glycyl-Aspartic Acid Ultrafine Iron Oxide Nanoparticles Loaded with Xanthohumol

Multiple myeloma (MM) is a plasma-cell cancer characterized by accumulation of malignant cells preferentially in the bone marrow. This disease accounts for about 1% of all cancers and remains incurable with a short survival time of 3 to 6 years in patients. Our previous studies have investigated the potential anti-myeloma effect of Xanthohumol (XN). To improve its inhibitory effect on MM cell growth and bioavailability, the strategy of nanoparticles (NPs) and target delivery is applied. The purpose of this study is to improve the biocompatibility of XN through encapsulation onto ultrafine iron oxide nanoparticles (uIONPs) and evaluate the cytotoxic and apoptogenic effects of uIONP/XN with arginyl-glycylaspartic acid (RGD), a small peptide, conjugated as a targeting ligand for human MM cells. uIONPs were conjugated with Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1- carboxylate (Sulfo-SMCC) and cyclic RGD and then loaded with Xanthohumol. Characterization of the loaded and conjugated nanoparticles was confirmed by comparing the size and zeta potential. Viability effects of the RGD-uIONP/XN treatment (0 μM – 30 μM) were assessed in 3 human MM cell lines (RPMI8226, U266, and NCI-H929) using PrestoBlue assays after 24-, 48-, and 72- hour treatment. Apoptosis induction was analyzed in treated RPMI 8226 cells by western blotting for expression of proapoptotic proteins (cleaved caspase-3 and cleaved PARP1). This mechanism was also assessed in RPMI 8226 cells co-cultured with human bone marrow stromal (HS-5) cells in a Transwell model which mimics the bone marrow microenvironment in vivo. RGD-uIONP/XN showed dose- and time-dependent cytotoxic effects across the three MM cell lines tested, demonstrating the greatest cytotoxicity to the RPMI8226 cell line. RPMI8226 cells treated with 30 μM RGD-uIONP/XN for 24 hours did not show significant increases in propapoptotic protein expression. The prepared RGDuIONP/ XN shows potential as a MM treatment. Further research with alternative NPs and uIONPs with modified conjugated ligands could lead to improved efficacy of treatment for MM