Ultrasmall silica nanoparticle platforms for improved small molecular inhibitor delivery and efficacy

278 Objectives: To demonstrate that ultrasmall (sub 8-nm), multimodal (PET/optical), renal-clearable silica nanoparticles (Cornell prime dots or C’ dots) are an ideal drug delivery vehicle for enhanced delivery, efficacy, and improved therapeutic index of gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, in murine xenograft models of non-small cell lung cancer. Methods: Ultrasmall C’ dot nanoparticles (<8 nm diameter) functionalized with a gefitinib analog (APdMG) bound to an enzymatically-cleavable dipeptide (Phe-Lys) linker were synthesizedin aqueous environments using previously established methods and resulted in the generation of a gefitinib-nanoparticle drug conjugate (i.e., Gef-NDC). Varied drug-per-particle ratios (DPR = 11-56) were compared in vivovia PET imaging (89Zr-Gef-NDC) to ensure primary renal clearance. Uptake and potency of released gefitinib analog was validated in vitrousing flow cytometry (optical; Cy5) and target inhibition studies (Western blot), respectively. Efficacy of the optimized Gef-NDC construct was compared to free gefitinib in both in vitroand in vivomodels to evaluate LD50values, tumor growth inhibition, and overall survival. Results: Highly stable Gef-NDCs with varying DPRs (DPR = 11-56), demonstrated predominant renal clearance (DPR ≤40) in naive mice and a dose-dependent decrease in the levels of phosphorylated EGFR in ECLC26 non-small cell lung cancer cells. Gef-NDC constructs, optimized to demonstrate primary renal clearance (DPR = 40), were then further evaluated both in vitroand in vivo. Dose-dependent cellular uptake was observed for Gef-NDCs when incubated with ECLC26 cells. Evaluation of cytotoxicity demonstrated significantly enhanced killing of the Gef-NDC over free gefitinib, resulting in LD50vales of 9.6 nM and 3 uM, respectively. ECLC26 tumor-bearing mice were treated with either intravenous saline control (Days 0, 3, 6), oral gavage gefitinib (150 mg/kg/day), or intravenous Gef-NDC (3 nmoles; Days 0, 3, 6) while tumor volumes were monitored daily. Although tumor growth inhibition and overall survival were found to be fairly equivalent between treated groups, the amount of total gefitinib dose administered to mice in the NDC group was orders of magnitude less (360 nmoles) than the cohort receiving the native drug (78 umoles), equating to a 216-fold dose reduction. Additionally, evaluation of off-target (ear) tissue phosphorylated EFGR levels revealed reduced inhibition for Gef-NDC treated mice. Conclusions: To improve the delivery and efficacy of the small molecular inhibitor gefitinib, we engineered C’ dots to display varying densities (n= 11-56) of an enzymatically-cleavable gefitinib linker construct. It was demonstrated that particle DPRs can be accurately tuned over a wide range, allowing for the selection of optimal drug loading while maintaining dominant renal clearance. Optimized Gef-NDCs demonstrated enhanced delivery and therapeutic efficacy over that of the free drug for both in vitroand first-time in vivostudies. Overall, these results demonstrate that ultrasmall C’ dots function as an ideal drug delivery vehicle exhibiting favorable pharmacokinetics, high drug loading, low off-target toxicity, and antitumor efficacy at a fraction of the administered free drug dose.