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. 2012 Jun 10;160(2):172-6.
doi: 10.1016/j.jconrel.2011.11.030. Epub 2011 Dec 2.

Alkane-modified short polyethyleneimine for siRNA delivery

Avi Schroeder  1 , James E Dahlman, Gaurav Sahay, Kevin T Love, Shan Jiang, Ahmed A Eltoukhy, Christopher G Levins, Yingxia Wang, Daniel G Anderson
Affiliations

Alkane-modified short polyethyleneimine for siRNA delivery

Avi Schroeder et al. J Control Release. .

Abstract

RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer-lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials.

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Figures

Fig. 1
Fig. 1
Conjugating lipid tails to PEI via an epoxide ring opening reaction. (A) A schematic representation of the chemical reaction. Epoxide terminated alkane was dissolved in ethanol and reacted with PEI at 90 °C for 48 h to form lipid–PEI hybrids. (B) The molecular weight of alkane-modified polyethyleneimine as a function of lipid:PEI mole ratio was determined using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-ToF MS).
Fig. 2
Fig. 2
The spontaneous self-assembled mean particle size and zeta potential of lipid–PEI compounds, as a function of the lipid-tail:PEI mole ratio. (A) As the number of conjugated lipid tails increases — the particle size increases. The polydispersity of the particles was <0.1. (B) The Zeta potential of the compounds was measured at pH 7.4 and 5.3.
Fig. 3
Fig. 3
Gene knockdown capacity of alkylated PEI compounds. RNAi and cell viability were tested in HeLa cells that express both Renilla and firefly luciferase. (A) Lipid–PEI compounds were complexed with anti-firefly siRNA and then incubated with the cells. Mean knockdown levels increased as the number of lipid tails conjugated to the PEI increased. (B) Cell viability was measured by recording the expression of an off-target gene — Renilla luciferase and is relative to non-treated cells. In both graphs error bars represent the standard deviation (N=8).
Fig. 4
Fig. 4
Cellular uptake of lipid–PEI–siRNA complexes. The uptake of lipid–PEI compounds complexed with Cy5-labled siRNA by HeLa cells was imaged using an Opera spinning disk confocal microscope. The uptake of the lipid:PEI mole ratio of 0.56 (A) is compared to that of lipid:PEI mole ratio of 2.91 (B). Five different representative images are presented for each compound (i–v) and image (vi) represents an enlarged image. Punctate structures can be noticed in panel A (vi), while cytoplasmic as well as punctuate structures can be seen in panel B (vi). Blue is Hoechst dye.
Fig. 5
Fig. 5
Binding affinity between alkane-modified PEI to siRNA. PEI with higher conjugation levels had weaker binding affinity to siRNA in comparison to lower conjugation levels. This was noticed when testing the affinity between modified PEI to siRNA at various N/P ratios (A), and after incubating siRNA-complexed particles with heparin sodium salt (Sigma) at various concentrations (B). Bands represent free (non-bound) siRNA as detected on an agarose gel.
Fig. 6
Fig. 6
EM images of alkane-modified PEI particles. Alkane-modified PEI particles were stained with phosphotungstate and imaged by EM. (A) particles at a lipid:PEI ratio of 0.56 were clearly noticed on the grid; on the other hand, particles with a lipid:PEI ratio of 2.91 (B) were unstable during the imaging preparation procedure, although silhouettes of the particles could be noticed on the grid, and excess stained material was removed from the figure by false coloring.
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