Targeted Delivery of Xanthohumol via RGD-conjugated µIONPs Induces Apoptosis and Inhibits Growth in Multiple Myeloma Cells

Introduction

Multiple Myeloma (MM) is a hematological malignancy characterized by the uncontrolled proliferation of monoclonal plasma cells within the bone marrow. This growth leads to severe complications, including anemia, hypercalcemia, bone lesions, and renal insufficiency. While proteasome inhibitors like Bortezomib (BTZ) are standard treatments, MM remains largely incurable due to the protective bone marrow microenvironment, which upregulates survival factors such as vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6). Furthermore, the systemic toxicity of traditional chemotherapies, like Bortezomib, necessitates safer alternatives. Xanthohumol (XN), a prenylated flavonoid from Humulus lupulus, offers potent anti-cancer properties, but suffers from rapid in vivo degradation. To enhance stability and delivery of XN in cells, we prepared ultrafine iron oxide nanoparticles (µIONP). RGD-conjugated µIONP were prepared and loaded with XN for targeted MM therapy. The RGD tripeptide is used to help specific binding to overexpressed integrins on the MM cell surface. The nanoparticle delivery vehicle was characterized based on its hydrodynamic dimensions and zeta potential. We also observed the characterization of µIONP loaded with XN (non-targeted drug control) and RGD-µIONP without loaded XN (vehicle control). Having established that XN was successfully loaded into the µIONPs, we will investigate its ability to inhibit MM cell growth and ability to upregulate proapoptotic protein expression relative to the effects observed with XN alone. Our hypothesis is that XN released from µIONP exerts inhibitory effects on multiple myeloma cell growth and induces apoptosis.

Methods

To address this hypothesis, we assessed cell viability using Presto Blue assays to examine the effects of XN alone on three MM cell lines (RPMI 8226, U266, and MM1.S). In addition, we measured cell viability using the Presto Blue assay with µIONP loaded with XN, RGD-µIONP without XN, and RGD-µIONP at a 40 µM XN concentration over time. Western blots were also performed on human RPMI 8226 and MM1.S cell lysates after 24 hours of XN treatment to assess proapoptotic protein expression, cleaved caspase 3, and cleaved PARP1.

Results

XN showed a time and dose-dependent decrease in cell viability over a period of time (24,48, and 72 hours) on the RPMI 8226 and MM1.S cells. RGD-µIONP showed a time-dependent decrease in cell viability on the RPMI 8226 and MM1.S cells as well. The Western blot results indicate that the potential mechanism of cell death at higher XN concentrations is apoptosis.

Conclusion

In conclusion, the results indicate that XN and µIONPs induce cell death via apoptosis in MM cell lines. Further experiments will be performed to evaluate the cytotoxic effects of RGD-µIONP, XN-µIONP, and RGD-µIONP/XN on U226 cells. As well as in comparison to the cytotoxic effects of XN alone, RGD-µIONP, XN-µIONP, and RGD-µIONP/XN with short-wave RF stimulation on the MM cell lines. Lastly, the mechanism of programmed cell death with the µIONPs in the U266 and MM.1S cell lines.