No abstract

Cell surface glycosaminoglycans (GAGs) are responsible for the majority of respiratory syncytial virus (RSV) attachment to cultured cells leading to infection. The viral glycoprotein G binds to GAGs and was thought to be the viral attachment protein, but more recently virus lacking the G protein was shown to be infectious in cell culture. We have compared the GAG dependence of a recombinant, green fluorescent protein-expressing virus containing the F protein as its only viral glycoprotein (rgRSV-F) to isogenic complete virus containing all three viral glycoproteins (rgRSV-SGF). Attachment and infection by each virus was found to be largely dependent on cell surface heparan sulfate (HS) based on the finding that both activities were inhibited by preincubation of virus with soluble HS, by removal of HS from target cells by enzymatic treatment or mutation, or by pretreatment of the target cells with basic fibroblast growth factor (bFGF), which binds HS. These results, coupled with the previous finding that SH is not involved in virion binding (S. Techaarpornkul, N. Barretto, and M. Peeples, 2001, J. Virol. 75, 6825-6834), suggest that, in the context of the virion, both the G and F proteins bind to HS. Interestingly, both rgRSV-F and rgRSV-SGF retained significant binding activity and infectivity despite these treatments, suggesting an alternate productive attachment and infection pathway. This property of GAG independence was particularly apparent for rgRSV-F virions, which retained nearly half of its attachment and infection activities in most of these experiments. Comparison of the attachment and infection activities of rgRSV-SGF and rgRSV-F virions with a Chinese hamster ovary cell line and a derivative thereof that is defective in GAG synthesis indicated that approximately 50% of rgRSV-SGF attachment is due to G protein-GAG binding, 25% to F protein-GAG binding, and 25% to an independent pathway. This alternative pathway presumably is mediated by the sole remaining viral surface protein, F, although the formal possibility exists that some other virion-associated protein is involved.

This study suggests that CS-TPP is straightforward to prepare, safe and exhibits significantly improved siRNA delivery potential in vitro.

Abstract. The aim of this study was to investigate chitosan/siRNA complexes formulated with various chitosan salts (CS) including chitosan aspartate (CS-Asp), chitosan glutamate (CS-Glu), chitosan acetate (CS-Ac), and chitosan hydrochloride (CS-HCl) for in vitro siRNA delivery into stable and constitutive enhanced green fluorescent protein (EGFP)-expressing HeLa cells. The CS/siRNA complexes were characterized by 2% agarose gel electrophoresis and investigated for their transfection efficiency in stable and constitutive EGFP-expressing HeLa cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The formation of complexes CS/siRNA is mainly dependent on the weight ratio, whereas salt form and molecular weight has less effect. The particle sizes of the complete complexes were in the range of 270-373 nm except the complete complex of CS-Ac, with a slightly positive charge of less than 2 mV. The ability of CS to transfer functionally active siRNA into cell culture is mainly dependent on the weight ratio and molecular weight of CS whereas salt form of CS has less effect. The high gene-silencing efficiency was observed with low MW of CS (20 kDa) and high weight ratio of 32. Over 80% average cell viabilities were observed for CS/ siRNA complexes in all weight ratios comparison to untreated cells. This study suggests CS salts have the potential to be used as safe siRNA delivery vectors.

The objective of this study was to investigate the transfection efficiency of chitosan hydroxybenzotriazole (CS-HOBT) for in vitro nucleic acid delivery. The results revealed that CS-HOBT was able to condense with DNA/small interfering double-stranded RNA molecules (siRNA). Illustrated by agarose gel electrophoresis, complete complexes of CS-HOBT/DNA were formed at a weight ratio of above 3, whereas those of CS-HOBT/siRNA were formed at a weight ratio of above 4 (CS molecular weights [MWs] 20 and 45 kDa) and above 2 (CS MWs 200 and 460 kDa). Gel electrophoresis results indicated that binding of CS-HOBT and DNA or siRNA depended on the MW and weight ratio. The particle sizes of CS-HOBT/nucleic acid complexes were in nanosize range. The highest transfection efficiency of CS-HOBT/DNA complex was found at a weight ratio of 2, with the lowest CS MW of 20 kDa. The CS-HOBT-mediated siRNA silencing of the enhanced green fluorescent protein gene occurred maximally with 60% efficiency. The CS-HOBT/siRNA complex with the lowest CS MW of 20 kDa at a weight ratio of 80 showed the strongest inhibition of gene expression. For cytotoxicity studies, over 80% the average cell viabilities of the complexes were observed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. This study suggests that CS-HOBT is straightforward to prepare, is safe, and exhibits significantly improved nucleic acid delivery potential in vitro.

No abstract

Chitosan (CS) has a high potential for gene delivery into mammalian cells. However, its uptake mechanism is not well clarified. We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine), caveolae-mediated endocytosis (genistein), macropinocytosis (LY 29004 and wortmannin), microtubuli polymerization (nocodazole) and of membrane cholesterol recycle (methyl-β-cyclodextrin) on the transfection efficiency with CS/pEGFP complexes and on the internalization of CS/rhodamine-labeled pEGFP complexes by hepatoma cell line (Huh 7 cells). The transfection was blocked by nocodazole, genistein, and methyl-β-cyclodextrin, respectively. CS/DNA complexes internalization was clearly inhibited by genistein. We conclude that the complexes uptake predominantly by caveolin-mediated pathways. In addition, fluorescence colocalization studies with acidotropic probes, LysoSensor dye, illustrated that CS/DNA complexes are targeted to lysosomes for the degradation after internalization.