Targeted Delivery of a Novel Conjugate Nef-M1 Peptide and Single-Walled Carbon Nanotubes promotes Growth Inhibition and Mortality for Breast and Colon Tumorspheres

Abstract

The Nef-M1 peptide (Nef-M1) has been shown to be an inhibitor of growth and metastasis of breast cancer (BC) and colorectal cancer (CRC) cells. Since a nano-construct for Nef-M1 could enhance its efficacy, we developed three-dimensional (3-D) cultures of tumorspheres of BC and CRC cells and treated them with functionalized, single-walled carbon nanotubes (SWNTs) conjugated with Nef-M1 (SWNTs-Nef-M1) to evaluate inhibition of cell growth. We hypothesized that cancer cells cultured as tumorspheres would be more sensitive to SWNTs-Nef-M1 than to Nef-M1 alone. 3-D cultures of human BC cells (MDA-MB-231 and MDA-MB-468) and CRC cells (SW480) were developed with 1% Sea Prep Hydrogel in supplemented RPMI-1640 medium. SWNTs-Nef-M1 was prepared by use of thionine, and its structure was confirmed by ultraviolet (UV) spectral analysis and gel electrophoresis. The tumorspheres were treated with Nef-M1 or SWNTs-Nef-M1 to compare their relative effects. Internalization of SWNTs-Nef-M1 was evaluated by transmission electron microscopy. The viability/apoptosis status of the tumorspheres was established by use of ethidium bromide and acridine orange staining using fluorescent microscopy. Gel electrophoresis and UV spectral analysis confirmed formation of the SWNTs-Nef-M1 conjugate. After 3 weeks, 3-D cultures of BC and CRC cells developed as tumorspheres, which, in the presence of Nef-M1, showed reduced growth and increased apoptosis. This effect was greater in the presence of SWNTs-Nef-M1, consistent with enhanced delivery of Nef-M1 to the cells via SWNTs. This sets the stage for more detailed evaluation to quantify apoptosis and elaboration of the mechanism of increased apoptotic activity. Delivery of an apoptotic peptide, Nef-M1, into 3-D cultures of BC and CRC cells was achieved via a nanoparticle construct with SWNTs. Furthermore, compared to Nef-M1, SWNTs-Nef-M1 suggests more extensive apoptosis.