Roger G. Harrison1, Luis F. F. Neves1, Yahya A. Lazrak1, David E. Martyn1, Peter S. McFetridge1, Daniel E. Resasco2, and Kenneth E. Bartels3. (1) Chemical, Biological and Materials Engineering, University of Oklahoma, 100 E. Boyd St., T-335, Norman, OK 73019, (2) Cbme, University of Oklahoma, 100 East Boyd, Room T-335, Norman, OK 73019, (3) Veterinary Clinical Sciences, Oklahoma State University, Veterinary Medical Teaching Hospital, Stillwater, OK 74078-2041
Single-walled carbon nanotubes (SWNTs) are unique in that they strongly absorb near-infrared (NIR) light, while biological systems have very low levels of absorption of NIR light. This project focuses on developing a means for targeting the SWNTs to the tumor vasculature. It is proposed to target the SWNTs by conjugation with the human annexin V protein. Human annexin V (molecular weight of 36,000) is a monomeric protein that binds with high affinity to phophatidylserine (PS) in phospholipid bilayers. PS is the most abundant anionic phospholipid of the plasma membrane and is tightly segregated to the internal side of the plasma membrane in most cell types. Anionic phospholipids are largely absent from the external surfaces of resting mammalian cells under normal conditions. Recently, it has been found that PS is expressed on the external surface of endothelial cells that line the blood vessels in tumors but is not expressed on the outside surface of the vascular endothelium in normal organs. Thus, annexin V can be used to specifically target the endothelial cells of the tumor vasculature. The underlying hypothesis is that a SWNT-annexin V conjugate injected into the bloodstream will localize in the tumor vasculature and that exposure of the tumor to NIR radiation will cause the tumor to be selectively killed by a combination of heating and cutting off its blood supply.
In this study, we have covalently coupled the human protein annexin V to carboxymethylcellulose adsorbed on single-walled carbon nanotubes (SWNTs) by using a zero-length crosslinking agent that couples carboxyl groups to primary amines (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, or EDC). The binding of annexin V (biotinylated) to phosphatidylserine allowed for the determination of the dissociation constant (Kd). The dissociation constant indicates a relatively high affinity of the receptor (phosphatidylserine) for the ligand (annexin V). The UV-vis-NIR absorption spectra of the SWNT-CMC-biotinylated annexin V complex was determined.
The SWNT-CMC-annexin V complex bound to human endothelial cells in vitro was subjected to laser irradiation at 980 nm on a 24-well plate in order to determine the effect of the energy adsorption by the SWNTs on cell viability. The cell viability was evaluated by performing the Alamar Blue assay, cell counting and cell observation with a microscope.