Quantification via Inductively-Coupled Plasma Optical Emission Spectroscopy (ICP-OES) of the Cellular Internalization and Nuclear Localization of Gold Nanoparticles Passivated with BSA-SV40 Large T NLS Conjugates after Incubation with Human Cervical Cancer (HeLa) Cells

Abstract

Rhodamine-labeled, cysteine-modified SV40 large T NLS peptide sequences were conjugated in varying amounts (~ 3 to 15 molar ratio) with bovine serum albuin (BSA) via the heterobifunctional linker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). These conjugates were then used to passivate nanomolar aliquots of citrate-coated gold nanoparticles of varying diameter (5, 10, 15, and 20 nm), and the stability of these nanoparticle complexes were evaluated with respect to: 1) amount of large T-BSA per nanoparticle (found to be stable under the following BSA:nanoparticle ratios: 5 nm diameter = 125:1, 10 nm diameter = 250:1, 15 nm diameter = 250:1, and 20 nm diameter = 500:1), 2) ionic strength of solution (critical coagulation concentration values above 0.85 M), and 3) temperature (found to be stable at 4, 25, and 37 degrees Celsius). A robust method was developed using inductively-coupled plasma optical emission spectroscopy (ICP-OES) as a means to quantify the internalization of nanoparticle complexes after incubation with human cervical cancer cells (HeLa) under many differing conditions. It was determined cellular internalization increased as a function of: 1) increasing amount of large T per nanoparticle complex, 2) longer incubation times, 3) temperature (which is to say incubations at 4 degrees Celsius afforded nearly no internalization), and 4) increasing nanoparticle diameter. Additionally, data from a pulse-chase experiment demonstrated that these nanoparticle complexes tend to remain associated with HeLa cells in similar concentration up to twelve hours after initial exposure. Lastly, a sub-cellular fractionation kit was used to extract nuclei from HeLa cells post-incubation with 5 nm diameter gold nanoparticle complexes. It was observed that nuclear localization of these nanoparticle complexes increased as a function of large T:nanoparticle ratio, but that resulting cell viability decreased dramatically at the highest large T:nanoparticle ratio.