Cells sense invading pathogens using a network of molecular sensors, many of which recognize viral nucleic acids to induce an antiviral state characterized by production of type-I interferon and pro-inflammatory cytokines. The recent and ongoing discovery of these pattern recognition receptors (PRRs) has generated significant interest in developing vaccine adjuvants and cancer immunotherapeutics using synthetic nucleic acids targeting receptors that comprise the cytosolic immune surveillance network. However, the therapeutic potential of these nucleic acids has been limited by nuclease degradation, poor intracellular uptake, and inefficient delivery to cytosolic targets. To address these drug delivery challenges, we are developing ‘smart’ pH-responsive nanoparticles (NPs) that actively enhance delivery of nucleic acids to cytosolic PRRs.
Our initial investigations utilized a recently described amphiphilic diblock copolymer composed of a cationic dimethylamino ethyl methacrylate (DMAEMA) first block and a pH-responsive and endosome destabilizing second block. Polymers self-assemble into ~30 nm micellar NPs in aqueous media and facilitate facile electrostatic complexation of 5’ triphosphorylated RNA (5’ppp-RNA), a ligand for the cytosolic PRR retinoic acid-inducible gene 1 (RIG-I). NPs significantly enhanced RNA delivery to the RIG-I pathway in macrophages as well as several cancer cell lines, resulting in increased production of type-I interferon (IFN) in 5’ppp-dependent manner.
Having demonstrated that endosomolytic nanoparticles can promote 5’ppp-RNA delivery to the RIG-I pathway, we aimed to develop an optimized delivery platform with increased biocompatibility. Towards this end, we have used reversible addition-fragmentation chain transfer (RAFT) polymerization to synthesize a small library of amphiphilic diblock copolymers with a polyethylene glycol (PEG) first block and a second copolymer block comprising variable compositions of DMAEMA and a methacrylate monomer of variable alkyl chain length. The percentage of DMAEMA in the second block was varied between 60 and 100% and ethyl, butyl, hexyl, octyl, or lauryl methacrylate were used as co-monomers. The effect of second block composition and alkyl chain length on micelle assembly, RNA complexation, and pH-dependent membrane-destabilization were evaluated using dynamic light scattering, gel electrophoresis, and red blood cell hemolysis, respectively. The efficiency of nucleic acid complexation was dependent on both composition and alkyl chain length and resultant particles were <100 nm in diameter. Significantly, pH-dependent membrane destabilization demonstrated a non-linear dependence on both composition and alkyl chain length and could be tuned through control of polymer structure. Notably, these investigations revealed a novel polymer composition (70% DMAEMA and 30% hexyl methacrylate) that demonstrated the strongest membrane destabilizing activity at endosomal pH values. Ongoing investigations are focused on evaluating the ability of these carriers to enhance delivery of 5’ppp-RNA to the RIG-I pathway. Collectively, these studies establish that endosomolytic polymer nanoparticles can enhance delivery to the RIG-I pathway and establish new structure-property-activity relationships for endosomolytic polymers that may be exploited to achieve optimal delivery to cytosolic immune surveillance pathways.