Gold Nanorods Mediate Tumor Cell Death by Compromising Membrane Integrity.

Light-activated therapies can be used to eradicate diseased cells and tissues in a non-invasive manner. Much attention has been focused on the emerging potential of photothermolysis (also referred to as optical hyperthermia), which involves the conversion of absorbed light into heat via nonradiative mechanisms. Photoactivated effects can be localized and intensified by employing exogenous agents with large absorption crosssections, confining damage to areas of interest with minimal collateral effects. In particular, targeted photothermolysis may be most effective when mediated by photothermal agents that absorb strongly at near-infrared (NIR) frequencies, to enable deeper penetration into biological tissues. Among the many materials investigated for NIR photoactivated imaging and therapy, plasmon-resonant gold nanorods (GNRs) and nanoshells appear to be some of the most effective agents to date. GNRs can be prepared with lengths on the order of 50 nm, a size compatible with long blood residency and permeation into tumors via their leaky vasculatures. GNRs support longitudinal plasmon resonances at NIR frequencies with higher quality factors than those of spherical gold nanoparticles at comparable resonance frequencies and are highly efficient at converting light energy into heat, particularly if embedded in media of low thermal conductivity. Recently, GNRs have been shown to be capable of generating two-photon luminescence (TPL) at sufficient intensities for single-particle detection and in vivo imaging. This latter property permits the real-time imaging of GNRs during their simultaneous application as photothermal agents in biological systems. While the therapeutic potential of nanoparticle-mediated photothermolysis is widely recognized, many causal relationships between local photothermal effects and cell injury remain to be defined. Heat-induced cell injury has traditionally been viewed as a systemic effect, characterized by phenotypic responses such as membrane blebbing, depolymerization of cytoskeletal filaments, thermal inactivation of membrane proteins and mitochondria, or increased production of heat shock proteins. These individual outcomes may be resolved at the subcellular level by using targeted nanoparticle delivery to administer localized photothermal effects. For example, nanosecond laser pulses have been used to induce cavitation in cells containing gold nanoparticles, resulting in transient increases in membrane permeability and inactivation of adsorbed proteins. These processes are quite distinct from those based on systemic changes in temperature. In this work we investigate the mechanisms and extent of photothermal injury inflicted by GNRs targeted to cell-surface receptors. Folate-conjugated GNRs were monitored in real time by TPL microscopy, and were observed to be particularly effective at inducing tumor cell necrosis when localized on the cell membrane. The mechanistic insights in this study reveal that the photothermal activity of GNRs and other nanoparticles extends beyond simple hyperthermia, and can be directed for maximum damage to cells using an appropriate targeting mechanism. GNRs (kmax = 765 nm) were prepared as previously described and functionalized with a folic acid conjugate by in situ dithiocarbamate formation, a recently developed method for the robust functionalization of gold surfaces (Fig. 1). The folate-conjugated nanorods (F-NRs) were targeted toward the plasma membrane of malignant KB cells, a tumor cell line known to overexpress the high-affinity folate receptor. Cells were observed to be densely coated with F-NRs C O M M U N IC A TI O N