Hybrid inorganic-organic capsules for efficient intracellular delivery of novel siRNAs against influenza A (H1N1) virus infection

Abstract

The implementation of RNAi technology into the clinical practice has been significantly postponing due to the issues regarding to the delivery of naked siRNA predominantly to target cells. Here we report the approach to enhance the efficiency of siRNA delivery by encapsulating the siRNA into new carrier systems which are obtained via the combination of widely used layer-by-layer technique and in situ modification by sol-gel chemistry. We used three types of siRNAs (NP-717, NP-1155 and NP-1496) in encapsulated form as new therapeutic agents against H1N1 influenza virus infection. By employing the hybrid microcontainers for the siRNA encapsulation we demonstrate the reduction of viral nucleoprotein (NP) level and inhibition of influenza virus production in infected cell lines (MDCK and A549). The obtained hybrid carriers based on assembled biodegradable polyelectrolytes and sol-gel coating possess several advantages such as a high cell uptake efficiency, low toxicity, efficient intracellular delivery of siRNAs and the protection of siRNAs from premature degradation before reaching the target cells. These findings underpin a great potential of versatile microencapsulation technology for the development of anti-viral RNAi delivery systems against influenza virus infection.

Figure 1

The schematic illustration of the intracellular delivery of antiviral siRNA against influenza A virus using SiO₂-coated hybrid capsules.

Figure 2

(A) The schematic illustration of the fabrication of SiO₂-coated capsules and the encapsulation of antiviral siRNA. (B) SEM image of SiO₂-coated capsules. (C,D) TEM images of hollow SiO₂ -coated capsules.

Figure 3

Confocal microscopy analysis of distribution of PA-1630-FAM in used carriers: (A) CaCO₃ microparticles; (B) (PARG/DEXS)₃ capsules; (C) SiO₂-coated capsules. The plots below CLSM images are the representative fluorescence intensity profiles over the microcapsule. (D) Cumulative loss of PA-1630-FAM after dissolution of CaCO₃ in SiO₂-coated and (PARG/DEXS)₃ capsules, respectively.

Figure 4

The uptake of fluorescently labeled siRNA (PA-1630-FAM) in MDCK (A) and A549 (B) cells in vitro. Confocal microscopy was used to compare the uptake and cellular localization of non-encapsulated and encapsulated siRNAs in SiO₂-coated capsules after 8 h incubation with MDCK and A549 cells.

Figure 5

(A) CLSM images demonstrating the intracellular release of PA-1630-FAM from SiO₂-coated capsules and further PA-1630-FAM distribution in A549 cells at different incubation period. (B) Flow cytometry analysis of SiO₂-coated capsules uptake in A549 cells at different capsules-to-cell ratio and incubation time. (C) Cell viability of A549 cells incubated with SiO₂-coated capsules at different capsules-to-cell ratios for various time intervals (24, 48, or 72 hours).

Figure 6

(A) Schematic diagrams showing the location of each siRNA in the viral genome. (B) Results of electrophoresis of the NP-1496 siRNA PCR products extracted from MDCK cells. (C) ELISA analysis showing the reduction of viral NP level in infected A549 cells treated with different anti-viral siRNAs in encapsulated form (50 pmol–1 capsule/cell; 250 pmol–5 capsules/cell; 500 pmol–10 capsules/cell). (D) Comparison of antiviral effect of NP-1496 siRNA delivered by siRNA/PEI polyplexes and SiO₂-coated capsules (50 pmol–1 capsule/cell; 250 pmol–5 capsules/cell; 500 pmol–10 capsules/cell). (E) Inhibition of influenza virus production by different dose of siRNAs in encapsulated form. Viral titers was measured 72 h after infection in a hemagglutination assay. The data are presented as means ± standard deviation (SD) from three independent experiments. Note that some hemagglutination unit values were identical for the triplicate experiments, and so standard deviation values are 0.