Synthesis of Poly(meth)acrylates with Thioether and Tertiary Sulfonium Groups by ARGET ATRP and Their Use as siRNA Delivery Agents

The field of RNA interference depends on the development of safe and efficient carriers for short interfering ribonucleic acid (siRNA) delivery. Conventional cationic monomers for siRNA delivery have utilized the nitrogen heteroatom to produce cationic charges. Here, we polymerized cationic sulfonium (meth)­acrylate by activators regenerated by electron transfer (ARGET) atom transfer radical polymerization (ATRP) to form polymers with narrow molecular weight distributions for siRNA delivery. The tertiary sulfonium species was stable toward dealkylation in water but less stable in the polar aprotic solvent dimethyl sulfoxide. Block copolymers poly­(ethylene oxide) with poly­(meth)­acrylate containing sulfonium moieties were prepared as an siRNA delivery platform. Results suggested block copolymers were biocompatible up to 50 μg/mL <i>in vitro</i> and formed polyplexes with siRNA. Additionally, block copolymers protected siRNAs against endonuclease digestion and facilitated knockdown of glyceraldehyde 3-phosphate dehydrogenase (<i>Gapdh</i>) mRNA expression in murine calvarial preosteoblasts. The versatility, biocompatibility, and cationic nature of these tertiary sulfonium groups are expected to find widespread biological applications

Nanogel-Mediated RNAi Against Runx2 and Osx Inhibits Osteogenic Differentiation in Constitutively Active BMPR1A Osteoblasts

Trauma-induced heterotopic ossification (HO) and fibrodysplasia ossificans progressiva (FOP) are acquired and genetic variants of pathological bone formation occurring in soft tissues. Conventional treatment modalities target the inflammatory processes preceding bone formation. We investigated the development of a prophylaxis for heterotopic bone formation by addressing the biological basis for HO – dysregulation in the bone morphogenetic protein (BMP) signaling pathway. We previously reported the synthesis of cationic nanogel nanostructured polymers (NSPs) for efficient delivery of short interfering ribonucleic acids (siRNAs) and targeted gene silencing. Results suggested that nanogel:siRNA weight ratios of 1:1 and 5:1 silenced <i>Runx2</i> and <i>Osx</i> gene expression in primary mouse osteoblasts with a constitutively active (ca) BMP Receptor 1A (BMPR1A) by the Q233D mutation. Repeated RNAi treatments over 14 days significantly inhibited alkaline phosphatase activity in caBMPR1A osteoblasts. Hydroxyapatite (HA) deposition was diminished over 28 days in culture, though complete suppression of HA deposition was not achieved. Outcome data suggested minimal cytotoxicity of nanogel-based RNAi therapeutics, and the multistage disruption of BMP-induced bone formation processes. This RNAi based approach to impeding osteoblastic differentiation and subsequent bone formation may form the basis of a clinical therapy for heterotopic bone formation