The effect of N/P ratio on the in vitro and in vivo interaction properties of PEGylated poly[2-(dimethylamino)ethyl methacrylate]-based siRNA complexes.

PEG-PnBA-PDMAEMA triblock and PEG-PDMAEMA diblock copolymers are used as model systems for studying the role of N/P ratio on the in vivo behavior of PEGylated siRNA carriers in mice. The presence of a free/uncomplexed polymer population coexisting with siRNA complexes is established. A change in the N/P ratio exerts no significant influence on the in vivo biodistribution and ex vivo blood chemistry properties of the respective systems. Histological analysis of major organs indicates that the presence of uncomplexed polymer elicits toxicity to the organ that is associated with the clearance of the siRNA complexes from the circulation system. This effect can be eliminated by working at N/P ratios near the charge-neutralization point of the complexes.

Influence of nano-carrier architecture on in vitro siRNA delivery performance and in vivo biodistribution: polyplexes vs micelleplexes.

Micelle-based siRNA carriers ("micelleplexes") were prepared from the A-B-C triblock copolymer poly(ethylene glycol)-poly(n-butyl acrylate)-poly(2-(dimethylamino)ethyl methacrylate) (PEG-PnBA-PDMAEMA), and their in vitro performance and in vivo biodistribution properties were compared with the benchmark PEGylated and basic polycation systems PEG-PDMAEMA and PDMAEMA, respectively. The micelle architecture, incorporating increased PEG shielding and a larger particle size (∼50 nm) than polycation-based complexes (polyplexes; ∼10 nm), enhances siRNA delivery performance in two important aspects: in vitro gene silencing efficiency and in vivo tumor accumulation. The in vitro gene silencing efficiency of the micelleplexes (24% in HeLa cells) was significantly better than the statistically insignificant levels observed for PDMAEMA and PEG-PDMAEMA polyplexes under identical conditions. This enhancement is linked to the different mechanisms by which micelleplexes are internalized (i.e., caveolar, etc.) compared to PDMAEMA and PEG-PDMAEMA polyplexes. Folate-functionalization significantly improved micelleplex uptake but had negligible influence on gene-silencing efficiency, suggesting that this parameter is not limited by cellular internalization. In vivo biodistribution analysis revealed that siRNA delivered by micelleplexes was more effectively accumulated and retained in tumor tissues than that delivered by PEGylated polyplexes. Overall, the micelle particle size and architecture appear to improve in vitro and in vivo delivery characteristics without significantly changing other properties, such as cytotoxicity and resistance to enzymes and dissociation. The self-assembled nature of micelleplexes is expected to enable incorporation of imaging modalities inside the hydrophobic micelle core, thus combining therapeutic and diagnostic capabilities. The findings from the present study suggest that the micelleplex-type carrier architecture is a useful platform for potential theranostic and tumor-targeting applications.

Polymer-based siRNA delivery: perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery.

Gene therapy holds tremendous promise in the treatment of many genetic and acquired diseases. The future of gene therapy in humans, however, is contingent upon the discovery of safe and effective carriers of genetic material. Polymers represent a class of materials that can be extensively modified to meet the needs of a particular gene delivery system. A variety of polymer formulations have been proposed in the literature as potential carriers, most of which facilitate gene delivery by encapsulating, and in some cases, condensing nucleic acids into nano-sized particles which can then be taken up by cells. Crucial to successful delivery of the gene to a cell is the polymer's ability to protect its contents from degradation in the extracellular environment. A well-designed carrier will also promote cellular uptake and intracellular release of the nucleic acid. In the past, a common approach to gene therapy has been to transfect cells with a polymer-encapsulated DNA plasmid designed to replace a defective gene in the target-cell genome. Within the last few years, however, RNA interference (RNAi) has emerged as a novel therapeutic pathway by which harmful genes can be "silenced" by delivering complementary short interfering RNA (siRNA) to target cells. siRNA delivery facilitated by polymers, although very promising, suffers from many of the same limitations as DNA delivery. This review will (1) highlight the similarities and differences between these two methods of gene therapy and (2) discuss how some of the remaining challenges in siRNA delivery facilitated by polymers can be addressed by applying knowledge from the longer-studied problem of DNA delivery.