Impact of PEG Chain Length on the Physical Properties and Bioactivity of PEGylated Chitosan/siRNA Nanoparticles in Vitro and in Vivo

Abstract

PEGylation of cationic polyplexes is a promising approach to enhance the stability and reduce unspecific interaction with biological components. Herein, we systematically investigate the impact of PEGylation on physical and biological properties of chitosan/siRNA polyplexes. A series of chitosan-PEG copolymers (CS-PEG2k, CS-PEG5k and CS-PEG10k) were synthesized with similar PEG mass content but with different molecular weight. PEGylation with higher molecular weight and less grafting degree resulted in smaller and more compacted nanoparticles with relatively higher surface charge. PEGylated polyplexes showed distinct mechanism of endocytosis, which was macropinocytosis and caveolae-dependent and clathrin-independent. In vitro silencing efficiency in HeLa and H1299 cells was significantly improved by PEGylation and CS-PEG5k/siRNA achieved the highest knockdown efficiency. Efficient silence of ribonucleotide reductase subunit M2 (RRM2) in HeLa cells by CS-PEG5k/siRRM2 significantly induced cell cycle arrest and inhibited cell proliferation. In addition, PEGylation significantly inhibited macrophage phagocytosis and unspecific interaction with red blood cells (RBCs). Significant extension of in vivo circulation was achieved only with high molecular weight PEG modification (CS-PEG10k), whereas all CS/siRNA and CS-PEG/siRNA nanoparticles showed similar pattern of biodistribution with major accumulation in liver and kidney. These results imply that PEGylation with higher molecular weight PEG and less grafting rate is a promising strategy to improve chitosan/siRNA nanocomplexes performance both in vitro and in vivo.

Diethylaminoethyl- chitosan as an efficient carrier for siRNA delivery: Improving the condensation process and the nanoparticles properties

“…It is well known that the folic acid receptor is often overexpressed in malignant tissues of epithelial origin, such as ovarian carcinomas [37]. The presence of PEG chains provides an improved stability to nanoparticles [38][39][40] and depending on the N/P ratio knockdown efficiencies from 80% to 95% can be achieved [39]. For HeLa and OV-3 cell lines, higher silencing efficiencies would be expected due to a more efficient targeted cell uptake.…”

Effects of PEG surface density and chain length on the pharmacokinetics and biodistribution of methotrexate-loaded chitosan nanoparticles

“…Different MWs of mPEG (2, 5, and 10 kDa) were conjugated to CS, and it was found that PEG with higher MW and less grafting density is a promising strategy to improve CS/siRNA NP performance both in vitro and in vivo. 20 It was also proved by Chen et al 21 that methotrexate (MTX)loaded mPEG (2000 Da)-conjugated CS NPs could enhance their targeting ability and prolong blood circulation even better than folic acid (FA), a targeting ligand for nanocarriers. 21 MTX {(2S)-2-[[4-[(2,4-diaminopteridin-6-yl)-methylethylamino]benzoyl]amino] pentanedioic acid)} is a stoichiometric inhibitor of dihydrofolate reductase.…”

Bioinspired Shielding Strategies for Nanoparticle Drug Delivery Applications

“…In practice nanoparticles have been optimized with various PEG formulations, for example 10 kDa molecular weight PEG best reduced clearance for chitosan/siRNA nanoparticles delivered intravenously, while 1 kDa molecular weight PEG was best for oral administration of prodrug-based micelles. 16–17 …”