Phospholipid-modified polyethylenimine-based nanopreparations for siRNA-mediated gene silencing: implications for transfection and the role of lipid components

Abstract

The clinical application of gene silencing mediated by small interfering RNA (siRNA) has been limited by the lack of efficient and safe carriers. Phospholipid modification of low molecular weight polyethylenimine (PEI 1.8 kDa) dramatically increased its gene down-regulation capacity while keeping cytotoxicity levels low. The silencing efficacy was highly dependent on the nature of the lipid grafted to PEI and the polymer/siRNA ratio employed. Phosphoethanolamine (DOPE and DPPE) and phosphocholine (PC) conjugation did not change the physicochemical properties and siRNA binding capacity of PEI complexes but had a large impact on their transfection and ability to down-regulate Green Fluorescent Protein (GFP) expression (60%, 30% and 5% decrease of GFP expression respectively). We found that the micelle-forming structure of DOPE and DPPE-PEI dramatically changed PEI's interaction with cell membranes and played a key role in promoting PEI 1.8 kDa transfection, completely ineffective in the absence of the lipid modification.

From the clinical editor: While siRNA-based gene silencing methods could have numerous clinical applications, efficient delivery remains a major challenge. This team reports that DOPE-PEI and DPPE-PEI based micelle-forming nanostructures may be able to provide an efficient vector for siRNA transfection.

Keywords: Lipid conjugation; Micelle; Phospholipid; Polyethylenimine; RNA interference; siRNA delivery.

Figure 1

Self-association of phospholipid-PEI conjugates. The critical micelle concentration (CMC) of conjugates was determined from fluorescence of the pyrene incorporated into the hydrophobic core of the micelles. The absence of a sharp increase in the fluorescence indicated the minimal solubilization capacity of the PC-PEI conjugate and non-modified PEI.

Figure 2

Analysis of complex formation. (A) Gel retardation of complexes at varying N/P ratios. No migration of siRNA into the gel indicates the complex formation at N/P ≥ 3. (B) Particle size and zeta potential of complexes as at N/P ratio of 16. (C) Protection of siRNA within complexes against RNAse III degradation.

Figure 3

GFP silencing efficacy of phospholipid-PEI/siRNA complexes (PC-PEI, DPPE-PEI and DOPE-PEI) prepared at various N/P ratios. Data are expressed as the mean ± SD (n=3). (ANOVA, *# P< 0.05 vs scramble siRNA formulations).

Figure 4

Cytotoxicity of free phospholipid-PEI conjugates (A) and free phospholipids (B) towards GFP-c166 cells. Data are expressed as the mean ±SD (n=3).

Figure 5

Intracellular delivery mediated by phospholipid –PEIs conjugates in c166 cells. (A) Cellular uptake of phospholipid-PEIs prepared at N/P 16 ratio after 4 h of incubation. The nuclei (blue) were stained with Hoechst dye. The internalized DY-547-siRNA appears in red. (B) Intracellular trafficking of DOPE-PEI/siRNA complexes after 4h of incubation. Selected images from sequentially numbered z-stacks are shown. C) Cellular uptake of the complexes prepared with fluorescence-labeled siRNA after 1h and 4 h of treatment with the complexes. The mean fluorescence intensity of cells after treatment with the complexes is shown. Data are expressed as the mean ± SD (n=3).

Figure 6

Analysis of endosomal escape of DOPE-PEI and DPPE-PEI complexes (N/P 16). The silencing efficacy of complexes was measured after pre-incubation of cells with the endosomal acidification inhibitors chloroquine and bafilomycin A1 and compared with PEI 25 kDa (N/P 4). (one-way ANOVA, Turkey's test, * P< 0.05, n.s. no significant differences)