Uptake Inhibition to Improve Bioavailability of Nanoparticle-Mediated Growth Factor Delivery to the Corneal Epithelium

Nanoparticle (NP)-based drug delivery approaches may enhance the pharmacokinetics of protein therapies like epidermal growth factor (EGF). However, the small size that allows NPs to bypass barriers also confers rapid cellular internalization, which may lead to NP-encapsulated EGF bypassing its extracellular targets. In this study, we investigated the potential of sodium azidea nonspecific energy-dependent inhibitor of NP endocytosisto improve the functional efficacy of NP-mediated growth factor delivery. We generated NPs composed of FITC-labeled poly-(lactic-co-glycolic acid) (PLGA) using a double emulsion solvent evaporation method, encapsulating recombinant human EGF (rhEGF). The rhEGF-loaded NPs (EGF-NPs) were 170.7 ± 53.0 nm in diameter. EGF-NPs released most of their cargo over the course of 24 h. EGF-NPs were rapidly internalized by human corneal limbal epithelial (HCLE) cells, which included active processes as well as passive diffusion. Uptake was reduced by pretreatment with sodium azide in a dose-dependent manner. Treatment with a low concentration of sodium azide (0.01 mg/mL) prior to EGF-NPs resulted in approximately 25% reduction in cell uptake of EGF-NP after 24 h, without significantly affecting cell viability. Phosphorylated EGF receptor (P-EGFR) levels were higher after 24 h in cells pretreated with sodium azide prior to EGF-NP, suggesting persistent or continuous EGFR activation at extended time points. Reduced cell uptake corresponded with enhanced cellular response to EGF-NPs, with 0.01 mg/mL sodium azide pretreatment producing a significant increase in HCLE cell viability (36.8 ± 10.9%, P < 0.0001) after 24 h and a significantly greater wound closure (22.8 ± 17.9%, P = 0.04) after 12 h in a scratch assay. The inhibition of NP uptake may serve as an approach to enable growth factor-loaded NP-based therapies.