Phospholipid-polyethylenimine conjugate-based micelle-like nanoparticles for siRNA delivery

Abstract

Gene silencing using small interfering RNA (siRNA) is a promising therapeutic strategy for the treatment of various diseases, in particular, cancer. Recently, our group reported on a novel gene carrier, the micelle-like nanoparticle (MNP), based on the combination of a covalent conjugate of phospholipid and polyethylenimine (PLPEI) with polyethylene glycol (PEG) and lipids. These long-circulating MNPs loaded with plasmid DNA-mediated gene expression in distal tumors after systemic administration in vivo. In the current study, we investigated the potential of MNPs for siRNA delivery. MNPs were prepared by condensing siRNA with PLPEI at a nitrogen/phosphate ratio of 10, where the binding of siRNA is complete. The addition of a PEG/lipid coating to the PLPEI complexes generated particles with sizes of ca. 200 nm and a neutral surface charge compared with positively charged PLPEI polyplexes without the additional coating. MNPs protected the loaded siRNA against enzymatic digestion and enhanced the cellular uptake of the siRNA payload. MNPs carrying green fluorescent protein (GFP)-targeted siRNA effectively downregulated the gene in cells that stably express GFP. Finally, MNPs were non-toxic at a wide range of concentrations and for different cell lines.

Fig. 1

Schematic representation of the self-assembly process of micelle-like nanoparticles (MNPs)

Fig. 2

PLPEI/siRNA complex formation. a. Gel retardation and b. relative binding affinity measured by ethidium bromide displacement assay of PLPEI and PEI complexes with siRNA at varying N/P ratios

Fig. 3

Protection of a. siRNA within PLPEI complexes and b. MNPs against serum and RNAse III degradation

Fig. 4

Cytotoxicity of MNPs, PLPEI complexes, PEI 1.8 kDa complexes, and PEI 25 kDa complexes towards a. NIH/3 T3 cells and b. B16F10 cells at different PEI concentrations. Relative cell viability was expressed as a percentage of control cells

Fig. 5

Cellular uptake of FAM-labeled siRNA in various complexes and MNPs. a. Changes in FACS histograms indicative of siRNA-positive cells after 24 h of incubation following 4 h of treatment with different formulations. b. Bars represent quantitative analysis of FACS histograms in (a) to obtain the percentage of cells positive for FAM-siRNA. Data are expressed as the mean±SD (n=3; *p<0.05 vs free siRNA and PEI 1.8 complexes)

Fig. 6

(Color online) Intracellular trafficking of a–c MNPs and d free FAM-siRNA after different incubation times with B16 cells. The nuclei (blue) were stained with Hoechst dye. The internalized FAM-siRNA appears green and the cytoplasmic boundaries (red) were marked with Lysotracker Red

Fig. 7

a. siRNA-mediated downregulation of the target gene and b. toxicity of MNPs. Experiments were performed in stably transfected c166-GFP cells using GFP-siRNA. Cells were treated with MNPs for 4 h, and the GFP fluorescence was analyzed by FACS after 48 h incubation. A non-targeting control duplex (Negative-siRNA) was used as a non-specific control siRNA. PEI 25 kDa was used as positive control. Data are expressed as the mean±SD (n=3; *p<0.05 vs non-treated control cells). Cytotoxicity studies were performed under the same conditions as FACS experiments. Relative cell viability was expressed as a percentage of untreated control cells