Plasmon-resonant gold nanorods (GNRs) have attracted much recent attention for their potential as multifunctional agents in theragnostics, an integrated approach to diagnostic imaging and therapy.1,2,3
GNRs are well known for their very high extinction coefficients at near-infrared (NIR) wavelengths; when excited at plasmon resonance, they can serve simultaneously as optical contrast agents and as photothermal transducers capable of mediating local heating effects.4,5,6,7,8,9
These resonances are tunable as a function of size and shape: the absorption and scattering cross sections both increase rapidly with particle volume, whereas the plasmon frequencies are sensitive to particle anisotropy and aspect ratio. For in vivo
applications, nanoparticles with NIR resonances are particularly favored because of the relatively high transmittivity of biological tissues in the spectral range between 750 and 1300 nm. Other examples of NIR-absorbing Au nanoparticles used in theragnostic applications include nanoshells,10,11
and aggregates of spherical nanoparticles.13,14
In order to be considered for translation to clinical studies, nanoparticles and their functionalized derivatives must pass a preclinical evaluation commonly referred to as adsorption, distribution, metabolism, excretion and toxicity (ADMET) profiling. These are performed in vivo using standard animal models, but are usually preceded by in vitro cell-based assays for preliminary evaluation of selective targeting and cytotoxicity. Cell-based assays provide a rapid and cost-effective method for evaluating three practical issues that affect the viability of nanoparticle agents for in vivo use: (i) surface functionalization to enable targeted delivery while avoiding nonspecific adsorption and uptake, (ii) long-term dispersion stability in fluids of high ionic strength, as it relates to targeting efficacy, and (iii) minimal cytotoxicity at high dosages. While each issue can be addressed independently in relatively straightforward fashion, addressing all three criteria at once is more challenging because biocompatibility may be compromised by the coatings and surfactants responsible for nanoparticle targeting and dispersion stability, and vice versa.
The criteria above present a particularly vexing problem for anisotropic nanoparticles such as GNRs, whose synthesis involves high concentrations of cetyltrimethylammonium bromide (CTAB), a cationic surfactant with membrane-compromising properties. CTAB has a poor biocompatibility profile, with in vitro
toxicological studies yielding IC50
values in the low micromolar range.15,16,17
Furthermore, CTAB-coated nanoparticles are susceptible to nonspecific cell uptake, even at very low surfactant levels.18,19
While submicromolar CTAB concentrations may have little adverse effect on cell viability, the effort to target nanoparticles to diseased cells is nonetheless compromised by residual CTAB, as it heightens the possibility of collateral photothermal damage caused by subsequent NIR irradiation. These studies indicate that GNRs and other formulations containing CTAB will require a rigorous purification procedure prior to clinical testing.4
The unresolved issue of biocompatibility belies the numerous methods of surface coating methodologies developed for GNRs and other nanoparticles in recent years.5-13,19-24
Most of these appear to be useful for exploratory investigations in a laboratory setting, although few if any have been subject to the rigors of thorough preclinical evaluation. For example, a widely used method of nanoparticle coating involves the electrostatic physisorption of polyelectrolytes, which can provide dispersion stability as well as a foundation for immobilizing antibodies or protein biomarkers.5,8,20 , 21 ,22 ,23 ,24
However, the stability and biocompatibility of nanoparticles functionalized in this manner cannot be assumed, as the surface binding energies are often variable or attenuated under physiological conditions, with possible leaching of the physisorbed species.
In this paper we evaluate polystyrenesulfonate sodium salt (PSS, 70 kDa) as a peptizing agent and detergent for the efficient removal of CTAB from GNR suspensions. Our interests are driven by a need to identify a reliable and ultimately scalable process for producing batch quantities of GNRs with good dispersion control and low cytotoxicity, to support their application as imaging and theragnostic agents in a clinical setting. PSS is commonly used as a nontoxic peptizing agent in numerous commercial products, and thus generally regarded as a safe additive.25
However, we find that PSS-coated GNRs can retain surprisingly high levels of in vitro
cytotoxicity even after exhaustive membrane dialysis or ultrafiltration. We ascribe this to the presence of a persistent PSS—CTAB complex that gradually desorbs from the GNR surface. CTAB also appears to play a cooperative role in PSS adsorption, but the PSS—CTAB complex can be removed from GNRs by centrifugation and replaced with fresh polyelectrolyte or other surfactants, to the point that cytotoxic effects are no longer observed.