Balancing Cationic and Hydrophobic Content of PEGylated siRNA Polyplexes Enhances Endosome Escape, Stability, Blood Circulation Time, and Bioactivity <i>in Vivo</i>

A family of pH-responsive diblock polymers composed of poly[(ethylene glycol)-<i>b</i>-[(2-(dimethylamino)ethyl methacrylate)-<i>co</i>-(butyl methacrylate)], PEG-(DMAEMA-<i>co</i>-BMA), was reversible addition–fragmentation chain transfer (RAFT) synthesized with 0–75 mol % BMA in the second polymer block. The relative mole % of DMAEMA and BMA was varied in order to identify a polymer that can be used to formulate PEGylated, siRNA-loaded polyplex nanoparticles (NPs) with an optimized balance of cationic and hydrophobic content in the NP core based on siRNA packaging, cytocompatibility, blood circulation half-life, endosomal escape, and <i>in vivo</i> bioactivity. The polymer with 50:50 mol % of DMAEMA:BMA (polymer “50B”) in the RAFT-polymerized block efficiently condensed siRNA into 100 nm NPs that displayed pH-dependent membrane disruptive behavior finely tuned for endosomal escape. <i>In vitro</i> delivery of siRNA with polymer 50B produced up to 94% protein-level knockdown of the model gene luciferase. The PEG corona of the NPs blocked nonspecific interactions with constituents of human whole blood, and the relative hydrophobicity of polymer 50B increased NP stability in the presence of human serum or the polyanion heparin. When injected intravenously, 50B NPs enhanced blood circulation half-life 3-fold relative to more standard PEG-DMAEMA (0B) NPs (<i>p</i> < 0.05), due to improved stability and a reduced rate of renal clearance. The 50B NPs enhanced siRNA biodistribution to the liver and other organs and significantly increased gene silencing in the liver, kidneys, and spleen relative to the benchmark polymer 0B (<i>p</i> < 0.05). These collective findings validate the functional significance of tuning the balance of cationic and hydrophobic content of polyplex NPs utilized for systemic siRNA delivery <i>in vivo</i>

Dual MMP7-Proximity-Activated and Folate Receptor-Targeted Nanoparticles for siRNA Delivery

A dual-targeted siRNA nanocarrier has been synthesized and validated that is selectively activated in environments where there is colocalization of two breast cancer hallmarks, elevated matrix metalloproteinase (MMP) activity and folate receptor overexpression. This siRNA nanocarrier is self-assembled from two polymers containing the same pH-responsive, endosomolytic core-forming block but varying hydrophilic, corona-forming blocks. The corona block of one polymer consists of a 2 kDa PEG attached to a terminal folic acid (FA); the second polymer contains a larger (Y-shaped, 20 kDa) PEG attached to the core block by a proximity-activated targeting (PAT), MMP7-cleavable peptide. In mixed micelle smart polymer nanoparticles (SPNs) formed from the FA- and PAT-based polymers, the proteolytically removable PEG on the PAT polymers shields nonspecific SPN interactions with cells or proteins. When the PAT element is cleaved within an MMP-rich environment, the PEG shielding is removed, exposing the underlying FA and making it accessible for folate receptor-mediated SPN uptake. Characterization of mixed micelles prepared from these two polymers revealed that uptake and siRNA knockdown bioactivity of a 50% FA/50% PAT formulation was dependent on both proteolytic activation and FA receptor engagement. MMP activation and delivery of this formulation to breast cancer cells expressing the FA receptor achieved greater than 50% protein-level knockdown of a model gene with undetectable cytotoxicity. This modular nanoparticle design represents a new paradigm in cell-selective siRNA delivery and allows for stoichiometric tuning of dual-targeting components to achieve superior targeting specificity

Macrophage-Specific RNA Interference Targeting via “Click”, Mannosylated Polymeric Micelles

Macrophages represent an important therapeutic target, because their activity has been implicated in the progression of debilitating diseases such as cancer and atherosclerosis. In this work, we designed and characterized pH-responsive polymeric micelles that were mannosylated using “click” chemistry to achieve CD206 (mannose receptor)-targeted siRNA delivery. CD206 is primarily expressed on macrophages and dendritic cells and upregulated in tumor-associated macrophages, a potentially useful target for cancer therapy. The mannosylated nanoparticles improved the delivery of siRNA into primary macrophages by 4-fold relative to the delivery of a nontargeted version of the same carrier (<i>p</i> < 0.01). Further, treatment for 24 h with the mannose-targeted siRNA carriers achieved 87 ± 10% knockdown of a model gene in primary macrophages, a cell type that is typically difficult to transfect. Finally, these nanoparticles were also avidly recognized and internalized by human macrophages and facilitated the delivery of 13-fold more siRNA into these cells than into model breast cancer cell lines. We anticipate that these mannose receptor-targeted, endosomolytic siRNA delivery nanoparticles will become an enabling technology for targeting macrophage activity in various diseases, especially those in which CD206 is upregulated in macrophages present within the pathologic site. This work also establishes a generalizable platform that could be applied for “click” functionalization with other targeting ligands to direct siRNA delivery