A. S. Hoffman

pH-Responsive Polymeric Micelle Carriers for siRNA Drugs

The ability of small interfering RNA (siRNA) to efficiently silence the expression of specific genes provides the basis for exciting new therapies based on RNA interference (RNAi). The efficient intracellular delivery of siRNA from cell uptake through the endosomal trafficking pathways into the cytoplasm remains a significant challenge. Previously we described the synthesis of a new family of diblock copolymer siRNA carriers using controlled reversible addition-fragmentation chain transfer (RAFT) polymerization. The carriers were composed of a positively charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA binding and a second pH-responsive endosome releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios and butyl methacylate (BMA). Here we describe the development of a new generation of siRNA delivery polymers based on this design that exhibit enhanced transfection efficiency and low cytotoxicity. This design incorporates a longer endosomolytic block with increased hydrophobic content to induce micelle formation. These polymers spontaneously form spherical micelles in the size range of 40 nm with CMC (critical micelle concentration) values of approximately 2 μg/mL based on dynamic light scattering (DLS), (1)H NMR, electron microscopy, and selective partitioning of the small molecule pyrene into the hydrophobic micelle core. The siRNA binding to the cationic shell block did not perturb micelle stability or significantly increase particle size. The self-assembly of the diblock copolymers into particles was shown to provide a significant enhancement in mRNA knockdown at siRNA concentrations as low as 12.5 nM. Under these conditions, the micelle-based systems showed an 89% reduction in GAPDH mRNA levels as compared to only 23% (10 nM siRNA) for the nonmicelle system. The reduction in mRNA levels becomes nearly quantitative as the siRNA concentration is increased to 25 nM and higher. Flow cytometry analysis of fluorescent-labeled siRNA showed uptake in 90% of cells and a 3-fold increase in siRNA per cell compared to a standard lipid transfection agent. These results demonstrate the potential utility of this carrier design for siRNA drug delivery.

Bioinspired nanocarriers designed to enhance intracellular delivery of biotherapeutics

The biotechnology and pharmaceutical industries have developed a wide variety of potential therapeutics based on the molecules of biology: DNA, RNA and proteins. While these therapeutics have tremendous potential, effectively formulating and delivering them has also been a widely recognized challenge. A variety of viruses and toxins have evolved multi-functional biopolymer complexes to solve this problem by directing uptake and enhancing biomolecular transport to the cytoplasm from the low pH endosomal compartment. Here, we have designed and synthesized bio-inspired, pH-responsive polymeric carriers that mimic the multifunctional design of biology. These nanocarriers target and direct cellular uptake, as well as enhance cytosolic delivery by disrupting endosomal membranes in pH-dependent fashion. We show that the encrypted polymeric carriers significantly enhance the delivery oligonucleotides and peptides to the cytoplasm of cultured hepatocytes and macrophages, demonstrating the potential of this approach to therapeutic and vaccine development.