Biophysical properties of chitosan/siRNA polyplexes: profiling the polymer/siRNA interactions and bioactivity.

Chitosans are naturally occurring polymers widely used in life science to mediate intracellular uptake of nucleic acids such as siRNA. Four chitosans of fungal origin (Agaricus bisporus; molecular weights MW=44, 63, 93 and 143 kDa) were used in this study and profiled for size, viscosity and hydrodynamic radius using gel permeation chromatography (GPC). Polyplexes made of these chitosans and siRNA were developed and optimized for transfection efficacy in vitro. The characteristics of these polyplexes were low chitosan:siRNA ratios (4-8; N:P) similar positive zeta potential (20-30 mV) and comparable particle sizes (about 150 nm). Endogenous luciferase reporter gene down-regulation in human epithelial H1299 cells at nanomolar concentrations (37.5-150 nM) was significantly stronger for the lower molecular weight chitosans. The impact of these low N:P polyplexes on the cellular viability was minimal also at 150 nM. To help develop an understanding of these differences, an energetic profile of the molecular interactions and polyplex formation was established by isothermal titration calorimetry (ITC). The four polyplexes exhibited strong binding enthalpies delta H(bind)(-84 to -102 kcal/mol) resulting in nanomolar dissociation constants. Intracellular trafficking studies using rhodamine labeled siRNA revealed that polyplexes made from smaller MW chitosans exhibited faster cellular uptake kinetics than their higher MW counterpart. Transmission electron microscopy and small angle X-ray scattering studies (SAXS) revealed that the 44 kDa derived polyplexes exhibited regular spherical structure, whereas the 143 kDa chitosan polyplex was rather irregularly shaped. With regards to adverse effects these low N:P chitosan/siRNA formulations represent an interesting alternative to so far reported chitosan polyplexes that used vast N:P excess to achieve similar bioactivity.

Holzerny P ，Ajdini B ，Heusermann W ，Bruno K ，Schuleit M ，Meinel L ，Keller M ... -  《-》

The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing.

Liu X ，Howard KA ，Dong M ，Andersen MØ ，Rahbek UL ，Johnsen MG ，Hansen OC ，Besenbacher F ，Kjems J ... -  《BIOMATERIALS》

Phosphorylatable short peptide conjugated low molecular weight chitosan for efficient siRNA delivery and target gene silencing.

Kong F ，Liu G ，Sun B ，Zhou S ，Zuo A ，Zhao R ，Liang D ... -  《-》

Chitosan-graft-(PEI-β-cyclodextrin) copolymers and their supramolecular PEGylation for DNA and siRNA delivery.

Two water-soluble chitosan-graft-(polyethylenimine-β-cyclodextrin) (CPC) cationic copolymers were synthesized via reductive amination between oxidized chitosan (CTS) and low molecular weight polyethylenimine-modified β-cyclodextrin (β-CD-PEI). The two polycations, termed as CPC1 and CPC2, were characterized by proton nuclear magnetic resonance spectroscopy, gel permeation chromatography, and elemental analysis. These polycations exhibited good ability to condense both plasmid DNA (pDNA) and small interfering RNA (siRNA) into compact and spherical nanoparticles. Gene transfection activity of both polymers showed improved performance in comparison with native CTS in HEK293, L929, and COS7 cell lines. Further investigation of the gene transfection mediated by CPC2/DNA complexes showed both time-dependent and dose-dependent in the tested cell lines, where the polymer showed higher level luciferase expression than commercially available branched PEI (25 kDa) under the condition of high dose or extended time. Gene silencing activity mediated by CPC2/siRNA against luciferase expression showed superior knockdown effect in HEK293 and L929 cell lines. In addition, both polymers exhibited much lower cytotoxicity than PEI (25 kDa) in HEK293, L929, and COS7 cell lines. More interestingly, the pendent β-CD moieties of CPC copolymers allowed the supramolecular PEGylation though self-assembly of adamantyl-modified poly(ethylene glycol) with the β-CD moieties. The supramolecular PEGylation of the polyplexes significantly improved their stability under physiological conditions. The supramolecular PEGylated polyplexes of CPC with pDNA showed decreased transfection efficiency in all tested cell lines. However, remarkably, the supramolecular PEGylated polyplexes with siRNA exhibited even higher silencing efficiency in HEK293 and L929 cells (up to 84%), comparable to commercial DharmaFECT. The interesting mechanism for the enhanced silencing efficiency was discussed. With the pendent β-CD moieties on CTS chains, the system is expected to be further modified via inclusion complexation between β-CD unit and guest molecules to serve as a multifunctional delivery system.

Ping Y ，Liu C ，Zhang Z ，Liu KL ，Chen J ，Li J ... -  《-》

Gene silencing activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan.

Varkouhi AK ，Verheul RJ ，Schiffelers RM ，Lammers T ，Storm G ，Hennink WE ... -  《-》