Reductively Responsive siRNA-Conjugated Hydrogel Nanoparticles for Gene Silencing

Abstract

A critical need still remains for effective delivery of RNA interference (RNAi) therapeutics to target tissues and cells. Self-assembled lipid- and polymer-based systems have been most extensively explored for transfection with small interfering RNA (siRNA) in liver and cancer therapies. Safety and compatibility of materials implemented in delivery systems must be ensured to maximize therapeutic indices. Hydrogel nanoparticles of defined dimensions and compositions, prepared via a particle molding process that is a unique off-shoot of soft lithography known as PRINT (Particle Replication in Non-wetting Templates), were explored in these studies as delivery vectors. Initially, siRNA was encapsulated in particles through electrostatic association and physical entrapment. Dose-dependent gene silencing was elicited by PEGylated hydrogels at low siRNA doses without cytotoxicity. To prevent disassociation of cargo from particles after systemic administration or during post-fabrication processing for surface functionalization, a polymerizable siRNA pro-drug conjugate with a degradable, disulfide linkage was prepared. Triggered release of siRNA from the prodrug hydrogels was observed under a reducing environment while cargo retention and integrity were maintained under physiological conditions. Gene silencing efficiency and cytocompatibility were optimized by screening the amine content of the particles. When appropriate control siRNA cargos were loaded into hydrogels, gene knockdown was only encountered for hydrogels containing releasable, target-specific siRNAs, accompanied by minimal cell death. Further investigation into shape, size, and surface decoration of siRNA-conjugated hydrogels should enable efficacious targeted in vivo RNAi therapies.

Reductively Responsive Hydrogel Nanoparticles with Uniform Size, Shape, and Tunable Composition for Systemic siRNA Delivery in Vivo

“…37 Compared to the direct siRNA incorporation method previously reported, 37 the current formulation strategy made particles and loaded siRNA in two separate steps. We first prepared 80 × 320 nm hydrogel nanoparticles using the PRINT based continuous particle fabrication instrument with a high efficiency.…”

“…32–36 We previously reported the use of PRINT based hydrogel nanoparticles to deliver siRNA in vitro . 37 In that work, siRNA was loaded when nanoparticles were being fabricated, requiring water to be used as solvent (see further explanations in Supporting Information, Part 2), which does not favor continuous particle fabrication process. Therefore, batch fabrication method was used.…”

Designing hydrogels for controlled drug delivery

“…Microgels can be produced with microfluidics and micromolding methods, and nanogels can be produced with emulsion and nanomolding techniques 75 . As an example, a molding technique consisting of particle replication in nonwetting templates (PRINT) produced monodisperse hydrogel particles 76–78 ranging from below 200 nm to 10 μm.…”

Future of the Particle Replication in Nonwetting Templates (PRINT) Technology

“…[44] The PRINT process allows direct physical entrapment of siRNA in the PEG hydrogel particles. [22] We were able to achieve significant siRNA knockdown with monodisperse, cylindrical PEG particles ( d = 200 nm; h = 200 nm) fabricated with siRNA electrostatically entrapped. However, subsequent efforts to modulate in vivo behavior by conjugation of targeting ligands to the particle surface resulted in extensive premature release of siRNA.…”