P-glycoprotein-dependent trafficking of nanoparticle-drug conjugates.

P-gp plays a role in the cellular traffi cking of nanoscale drug carriers. In prior work, [ 8 ] we developed a gold nanoparticle delivery platform that preferentially targeted tumor stromal cells through surface presentation of macrolide small molecules, polarizing tumor associated macrophages towards an anti-tumor phenotype. Here, we use these novel nanoscale constructs to investigate the effects P-gp substrate presentation on the cellular traffi cking of PEGylated gold-nanorods. To investigate P-gp ligand-dependent cellular traffi cking of nanoparticles, a series of colloidal gold nanorods were synthesized and conjugated with substrates of P-gp that exhibit varying degrees of susceptibility to P-gp-mediated effl ux, as reported previously. [ 9 ] Figure 1 a illustrates the composition of these model nanoscale drug carriers, each comprised of 50 ± 8 × 13 ± 2 nm gold nanorods (Figure 1 b) surface functionalized with mixed (9:1) self-assembled monolayers of thiolated poly(ethylene glycol) (PEG) and one of the three thiol PEGylated macrolide antibiotics: azithromycin (Zithromax®), clarithromycin (Biaxin®), or tricyclic ketolide (TE-802). These gold nanorod (AuNR) conjugates are abbreviated hereafter as Azith-AuNRs, Clarith-AuNRs, and TriKeto-AuNRs, respectively. The macrolide ligands were synthesized by ‘ N -alkynylation of the corresponding desmethyl desosamine analogs, followed by Cu-catalyzed Huisgen cycloaddition (click) using an azide-modifi ed PEG-thiol (Supporting Data, Schemes S1–4). The gold nanorods were synthesized [ 8,10 ] and conjugated [ 11 ] as described previously (see Supporting Information for detailed methods). Photon correlation spectroscopy, laser Doppler electrophoresis measurements, and surface plasmon extinction spectra from the purifi ed nanoparticle conjugates indicate stable surface ligation that was maintained in 10% serum-containing cell growth media over the time course of the experiments (Supporting Data, Figures S1,2). Cellular uptake of the nanoparticle conjugates was assessed using a lung macrophage cell line previously shown to exhibit P-gp-dependent accumulation of macrolide compounds, [ 9b ]