MMP-Responsive, Proximity-activated Targeting Polymeric Nanoparticles for siRNA Delivery

SUMMARY A smart polymeric nanoparticle (SPN) with matrix metalloproteinase-7 (MMP-7) dependent proximityactivated targeting (PAT) has been designed and tested for targeted intracellular siRNA delivery to MMP-7 overexpressing tissues (i.e., tumor metastases). With this novel PAT-SPN design, a removable PEG corona mitigates cytotoxicity and nonspecific uptake in normal tissues. Exposure to MMP (in proximity to a tumor) unmasks the underlying cationic layer that mediates cellular uptake. Finally, pH-responsiveness from the particle core assists escape from endo-lysosomal pathways and promotes access to cytoplasmic RNAi machinery. Here, it was verified that the PAT-SPN produced increased cell uptake and siRNA activity when it was “pre-activated” by MMP-7. This PAT-SPN design shows significant promise for targeted siRNA delivery to cancer metastases. INTRODUCTION Small interfering RNA (siRNA) has significant potential to evolve into a new class of pharmaceuticals, but delivery technologies that enable robust, tissuespecific intracellular delivery must be developed before effective clinical translation can be achieved. Cancerspecific targeting of nanoparticulate drug carriers is often approached by optimizing carrier biophysical properties for nonspecifically enhanced permeation and retention in tumors or through active biochemical targeting of cell surface receptors that are overexpressed on cancer cells. Here, we have investigated an alternative approach to achieving tissue specific siRNA delivery based on environmentally-dependent or “proximity-activated targeting” (PAT) of smart polymer micelle-based siRNA carriers. The PAT-SPN described here was designed to be activated in the presence of MMP-7. MMPs are a group of zinc-dependent endopeptidases that mediate matrix remodeling and cell migration (i.e., tissue invasion during metastasis). Overexpression of MMP-7 has been associated with the occurrence of metastasis in breast cancer 1,2 , and the current carrier is designed for targeted activation within MMP-7-rich metastatic sites. To achieve this functionality, an MMP-7-responsive peptide linker has been incorporated between the surface of a previously-tested SPN and a corona-forming PEG. The SPN has a cationic corona for siRNA condensation and a pH-responsive, endosomolytic core that mediates efficient siRNA cytosolic delivery. 3 The removable PEG corona blocks nonspecific uptake in normal tissues, and upon exposure to MMP (in the proximity to tumors), the underlying cationic layer is exposed, triggering cellular uptake (Figure 1). Figure 1. Overview of MMP-7 Proximity-Activated siRNA-Delivery Vehicle. The first frame shows the composition of the polymeric components that selfassemble into nanoparticle. The base SPN polymer enables siRNA electrostatic loading and pH-responsive endo-lysosomal escape activity. The complete PAT-SPN incorporates PEG shielding removable in MMP-7-rich environments (i.e., breast cancer metastases). MMP-7 activity exposes the cationic component of the SPN polymer, triggering cell uptake, pH-dependent endosomal escape, and cytosolic siRNA delivery. EXPERIMENTAL METHODS An MMP-7 cleavable peptide (H-VPLSLYSGCGOH) was selected, 4 made by solid phase peptide synthesis, and purified by HPLC. Human MMP-7 proteolytic degradability of this peptide was proved by SDS-PAGE prior to its incorporation into the PAT-SPN. Orthogonal, site-selective chemistry was used to attach a 5 kDa PEG and a reversible addition-fragmentation chain transfer (RAFT) polymerization chain transfer agent (CTA) to the opposite ends of the peptide. Subsequently, RAFT polymerizations were carried out to grow a diblock copolymer p(DMAEMA)-b-p(DMAEMA-PAA-BMA) (SPN) from this PEG-peptide macro-CTA. The resultant quad-block construct (PAT-SPN) was characterized by NMR and size exclusion chromatography (SEC). Nanoparticles were formed by adding concentrated polymer solutions from ethanol into an excess of PBS. Particle diameter was measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The MMP-7-dependent proximity activation was confirmed by SEC, DLS, and zeta potential. Enhanced intracellular uptake of MMP-7 treated PAT-SPNs was investigated in MDA-MB-231 breast cancer cells by flow cytometry and confocal microscopy. Luciferase gene knockdown by siRNA delivered by both the MMP-7activated and intact PAT-SPNs was evaluated in luciferase-expressing R221A (R221A-Luc) cancer cells. RESULTS AND DISCUSSION The quad-block PEG-peptide-p(DMAEMA)-bp(DMAEMA-BMA-PAA) (PAT-SPN) was found to have Mn = 37 kDa and a low polydispersity (PDI = 1.3) using SEC. These amphiphilic polymers self-assembled in aqueous media into 46 nm micelles, bearing a zeta potential of +5.8 mV. MMP-7-dependent cleavage of the PEG from the base SPN polymer was first confirmed by observing the polymer Mn change on SEC. In response to MMP-7, the polymer peak shifted toward a later elution time and a new peak appeared that correlated to the PEG reference. The responsiveness was further confirmed on DLS by a decrease in particle diameter (46 to 42 nm) and an increase in zeta potential from +5.8 to +14.4 mV, suggesting that MMP-7 cleavage of the distal PEG from the nanocarriers revealed the underlying polycationic charge. Lack of nonspecific cytotoxicity was investigated by the viability-associated luciferase expression level after treating R221A-Luc cells with varied amount of nanoparticles. Flow cytometry and confocal microscopy experiments confirmed that the uptake of PATSPN/nucleic acid therapeutics was elevated 2.5 fold following treatment of the complexes with MMP-7, mimicking the proteolytically active environment of MMP-7 rich human breast cancers (Figure 2). Figure 2. Flow cytometry analysis (left) and Confocal microscopy imaging (right) of MDA-MB-231 breast cancer cells treated with intact PAT-SPNs (PATSPN/FAM-dsDNA), PAT-SPNs pretreated with MMP-7 (MMP pretreated PAT-SPN/FAM-dsDNA) to remove the outer PEG layer, or no treatment (NT). (A-B) Mean fluorescence comparisons between experimental groups indicated that activation by MMP-7-dependent PEG cleavage noticeably increased cellular internalization of 21mer dsDNA-loaded PAT-SPN by 2.5-fold (data presented as mean +/standard error with n=3). p<0.05 compared with NT and # p<0.05 compared with PATSPN/FAM-dsDNA. (C) Fluorescent images of FAMdsDNA delivery to MDA-MB-231 cells visually confirmed that enhanced intracellular delivery is evident for PAT-SPN pretreated by MMP-7. Gene knockdown studies confirmed that siRNAmediated gene suppression using the PAT-SPNs was also enhanced when the carrier was pre-treated with MMP-7 (Figure 3), suggesting effective PAT. Figure 3. Luciferase activity suppression in R221A-Luc cancer cells treated with PAT-SPN loaded with luciferase siRNA with and without MMP-7 pretreatment. Charge ratios of 2, 4, and 6 were tested. Lipofectamine ® (Lipofec) was used as a positive control. Luciferase activity was reported relative to untreated cells (NT) (Data presented as mean +/standard error with n=3). p<0.05 compared with NT and # p<0.05 with Lipofec. CONCLUSION Herein, a multifunctional, „smart‟ siRNA carrier (PAT-SPN) designed to be activated in breast cancer metastases was successfully constructed. These amphiphilic polymers form micelles that are both MMP-7 and pH-responsive. Results of cellular uptake experimentsconfirmed the increased delivery of nucleic acidtherapeutics following the treatment of complexes withMMP-7, mimicking the proteolytically activeenvironment of human breast cancers. In addition, geneknockdown studies provided strong evidence supportingenhanced suppression of target protein expression inpresence of MMP-7. Evidence to date has indicated thatthis novel nanoscale vehicle has important advantages intargeted and effective therapeutic delivery to breastcancer metastases for gene knockdown. New versions ofPAT-SPNs suggest that variation of the PEG size in thePAT element may enable better charge-shielding andreduction of non-PAT uptake, and ongoing efforts arefocused in this area. REFERENCES1. Garcia, M. F. et al. Int. J. Clin. Pract. 2010, 91, 324.2. Beeghly-Fadiel, A. et al. Cancer Res. 2008, 68, 6453.3. Convertine, A. J. el al. J. Control. Release 2009, 133,221.4. Turk, B. E. et al. Nat. Biotechnol. 2001, 19, 661. ACKNOWLEDGMENTSThis research was supported by Department of DefenseCongressionally Directed Medical Research Programs(W81XWH-10-1-0445/0446) and the Vanderbilt Instituteof Nanoscale Science and Engineering.*