Chitosan-modified poly(D,L-lactide-co-glycolide) nanospheres for improving siRNA delivery and gene-silencing effects

Abstract

Chitosan (CS) surface-modified poly(D,L-lactide-co-glycolide) (PLGA) nanospheres (NS) for a siRNA delivery system were evaluated in vitro. siRNA-loaded PLGA NS were prepared by an emulsion solvent diffusion (ESD) method, and the physicochemical properties of NS were investigated. The level of targeted protein expression and siRNA uptake were examined in A549 cells. CS-modified PLGA NS exhibited much higher encapsulation efficiency than unmodified PLGA NS (plain-PLGA NS). CS-modified PLGA NS showed a positive zeta potential, while plain-PLGA NS were negatively charged. siRNA uptake studies by observation with confocal laser scanning microscopy (CLSM) indicated that siRNA-loaded CS-modified PLGA NS were more effectively taken up by the cells than plain-PLGA NS. The efficiencies of different siRNA preparations were compared at the level of targeted protein expression. The gene-silencing efficiency of CS-modified PLGA NS was higher and more prolonged than those of plain-PLGA NS and naked siRNA. This result correlated with the CLSM studies, which may have been due to higher cellular uptake of CS-modified PLGA NS due to electrostatic interactions. It was concluded that CS-modified PLGA NS containing siRNA could provide an effective siRNA delivery system.

Fig. 1

Experimental schedule for the evaluation of luciferase activity in A549-Luc cells.

Fig. 2

Scanning electron micrograph of siRNA-loaded plain-PLGA NS (magnification × 10,000, scale bar = 1 μm).

Fig. 3

Cumulative release (% of amount loaded) of siRNA from PLGA NS. Release was studied in PBS (pH 7.4) at 37 °C. Results are the means ± SD of three samples.

Fig. 4

Confocal laser microscopic images of A549 cells following 4-h uptake of Cy3-labeled siRNA (50 nM) preparations. (A) Naked siRNA and (B) siRNA/DOTAP complexes were used to treat A549 cells. F-actin in A549 cells was stained with Alexa Fluor® 488-conjugated phalloidin; green fluorescence. Scale bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Confocal laser microscopic images of A549 cells following 4-h uptake of the different PLGA NS preparations containing Cy3-labeled siRNA (50 nM): (A) Plain-PLGA NS, (B) CS-modified PLGA NS, (C) siRNA/NS complexes. PLGA NS were stained with 6-coumarin as a fluorescence marker (green fluorescence). Scale bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Inhibition of luciferase expression in A549-Luc cells by different siRNA preparations. Luciferase activity was measured at 48 and 120 h after siRNA addition. (A) A549-Luc cells were treated with different Luc-siRNA preparations. (B) A549-Luc cells were treated with different control-siRNA preparations and blank (empty) CS-modified PLGA NS. Results are the means ± SD (n = 3–6), ∗p < 0.01, significantly different compared with control.