Characterization, stability, cellular uptake and intracellular trafficking of peptide gold nanostructures in mammalian cells

Gold nanoparticles (AuNPs) have been widely investigated as potential nanocarriers for drug delivery. This potentially important role requires an understanding of cellular transport mechanisms that may be harnessed in facilitating highly efficacious nanocarrier-mediated drug delivery. In the present study, two different cysteineterminated peptides were conjugated with AuNPs (20 nm). Peptide A has a C-terminal Lys-Asp-Glu-Leu (KDEL) motif, named KDEL peptide, which is expected to interact with the KDEL receptor (KDELR) for retrograde trafficking, while peptide B is designed with a random sequence that has no known specific receptor or other cell membrane protein binding activity. Radio-ligand binding studies were conducted to characterize AuNP-peptide binding, suggesting both covalent and non-covalent interactions. The interactions of serum proteins with AuNP-peptide nanoconjugates were determined using gel electrophoresis and dynamic light scattering. Serum proteins rapidly bound the nanoconjugatcs (within 15 min). The cellular uptake of free peptides and nanoconjugates into mouse myogenie (So18) cells was investigated in the absence or presence of serum. In the absence of serum, peptides presented as nanoconjugates showed significantly higher intracellular fluorescence intensities compared to those in the presence of serum (P<0.05), suggesting that serum proteins inhibit AuNP-mediated peptide delivery. The cellular uptake of nanoconjugatcs was also confirmed using transmission electron microscopy. These data suggest that AuhiP-peptide nanoconjugates arc a potential platform for intracellular delivery of therapeutics. The mechanisms regulating their uptake and intracellular trafficking were further investigated using KDEL peptide gold (AuNP-KDEL) nanostructures. In a pulse-chase study, time-course sampling revealed that AuNP-delivered KDEL peptides were rapidly localized to the endoplasmic reticulum (ER) in 5 to 15 min, and after 1 h the majority of peptides were localized to the ER. Clathrin-coated vesicles and Golgi apparatus were also involved during the intracellular trafficking of AuNP-KDEL nanostructures. Furthermore, overexpression of KDELR significantly enhanced KDEL peptide uptake of both free and AuNP-conjugated forms (P<0.05). These data indicate that the ALINP-KDEI, nanostructures are internalized via a clathrin-mediated pathway and trafficked to the ER via a retrograde transport pathway, bypassing the lysosomal degradation pathway. Further studies are needed to evaluate the efficacy of drug delivery using these nanostructures.