Abstract 720: Dual MMP-7-proximity-activated and folate-targeted nanoparticles for siRNA delivery

We have developed a dual, folic acid (FA) and matrix metalloproteinase (MMP), proximity-activated targeting (PAT) smart polymeric nanoparticle (SPN) for tumor-specific siRNA delivery. The nanocarrier was designed to expose concealed FA ligands specifically in regions of elevated MMP activity, resulting in targeted uptake by folate receptor-expressing cells. Site-selective chemistry and reversible addition-fragmentation chain transfer (RAFT) polymerization were used to prepare diblock copolymers {p(DMAEMA)-b-p(DMAEMA-PAA-BMA) (SPN)} terminated with either MMP-7 peptide coupled to a Y-shaped 20 kDa PEG (PAT-SPN) or a 2 kDa linear PEG coupled to FA (FA-SPN). These amphiphilic polymers were co-assembled in aqueous media, forming mixed micelles consisting of 0, 25, 50, 75, 100 mol% FA and presenting zeta potentials of -0.21, -0.20, +1.75, +6.71 and +12.2 mV, respectively. Cleavage by MMP-7 was confirmed by decreased particle diameter (e.g., 25% FA micelle diameter decreased from 54 to 48 nm) and simultaneous increase in zeta potential (-0.20 to +8.11 mV). These data suggest that MMP-7 removal of the PEG corona from the nanocarriers revealed the underlying polycationic charge of the pDMAEMA core. Intracellular uptake of PAT/FA-SPNs was investigated in FA receptor-expressing breast cancer cells by confocal microscopy and flow cytometry. Pre-treatment with exogenous MMP-7 to mimic the proteolytically active microenvironment of human breast cancers, elevated cellular uptake 2.3-fold for single targeting PAT-SPN and 4.6-fold for dual-targeting PAT/FA-SPN compared to intact PAT-SPN. Cell uptake of uncleaved PAT/FA-SPN was statistically equivalent to untreated cells. These data suggest that the 20 kDa Y-shaped PEG effectively shields both the underlying cationic charge and the FA, and also blocks nonspecific cellular interactions of the SPN. 75-100% FA-SPN polymer reduced the dependency of cellular uptake on MMP-7 activity. Excess FA reduced the uptake of FA-SPN-containing micelles, consistent with FA receptor-dependent uptake following MMP activation. Functional protein knockdown delivered by PAT/FA-SPN was validated using luciferase-expressing MDA-MB-231 cells. 50% PAT/FA-SPN, pre-activated with MMP-7and loaded with luciferase siRNA, resulted in significant suppression of luciferase expression relative to controls (no MMP activation or competitive inhibition with free FA or scrambled siRNA), confirming the efficient dual PAT/FA targeting and endosomal escape of this designed nanoparticle. Unlike other similar technologies, this micelle design is optimized for use in vivo. Animal studies confirm an extended blood half-life, appropriate organ distribution, minimal toxicity and significant siRNA activity in mammary tumors. 1. Li, H. et al. Adv. Func. Mater. 2013. 10.1002/adfm.201202215 Support DoD CDMRP (W81XWH-10-1-0445/0446); Vanderbilt Institute of Nanoscale Science & Engineering Citation Format: Todd D. Giorgio, Hongmei Li, Martina Miteva, Kellye C. Kirkbride, Ming Cheng, Chris E. Nelson, Craig L. Duvall. Dual MMP-7-proximity-activated and folate-targeted nanoparticles for siRNA delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 720. doi:10.1158/1538-7445.AM2014-720