Cationic Nanoparticles Assembled from Natural-Based Steroid Lipid for Improved Intracellular Transport of siRNA and pDNA

Abstract

Developing new functional biomaterials from biocompatible natural-based resources for gene/drug delivery has attracted increasing attention in recent years. In this work, we prepared a series of cationic nanoparticles (Diosarg-DOPE NPs) by assembly of a natural steroid diosgenin-based cationic lipid (Diosarg) with commercially-available helper lipid 1,2-dioleoyl-sn-glycero-3-phosphorethanolamine (DOPE). These cationic Diosarg-DOPE NPs were able to efficiently bind siRNA and plasmid DNA (pDNA) via electrostatic interactions to form stable, nano-sized cationic lipid nanoparticles instead of lamellar vesicles in aqueous solution. The average particle size, zeta potentials and morphologies of the siRNA and pDNA complexes of the Diosarg-DOPE NPs were examined. The in vitro cytotoxicity of NPs depends on the dose and assembly ratio of the Diosarg and DOPE. Notably, the intracellular transportation efficacy of the exogenesis siRNA and pDNA could be greatly improved by using the Diosarg-DOPE NPs as the cargoes in H1299 cell line. The results demonstrated that the self-assembled Diosarg-DOPE NPs could achieve much higher intracellular transport efficiency for siRNA or pDNA than the cationic lipid Diosarg, indicating that the synergetic effect of different functional lipid components may benefit the development of high efficiency nano-scaled gene carriers. Moreover, it could be noted that the traditional "lysosome localization" involved in the intracellular trafficking of the Diosarg and Diosarg-DOPE NPs, indicating the co-assembly of helper lipid DOPE, might not significantly affect the intracellular localization features of the cationic lipids.

Keywords: diosgenin; intracellular uptake; pDNA; siRNA; steroid.

Scheme 1

Preparation of Diosarg-1,2-dioleoyl-$\mathrm{sn}$-glycero-3-phosphor-ethanolamine (DOPE) nanoparticles as gene (siRNA and DNA) carriers for intracellular gene transportation.

Figure 1

Transmission electron microscopy (TEM) photographs of the Diosarg-DOPE aggregates: (a) Diosarg-DOPE (2:1) nanoparticles (NPs); (b) Diosarg-DOPE (1:1) NPs; (c) Diosarg-DOPE (1:2) NPs; scale bar: 200 nm.

Figure 2

Storage stability of the Diosarg-DOPE (2:1, 1:1, 1:2) NPs in distilled water after 0 day, 7 days and 14 days; the average particle size was measured by the dynamic light scattering instrument.

Figure 3

Average particle sizes and zeta potentials of the Diosarg/siRNA and Diosarg-DOPE NPs/siRNA (a,b), as well as the Diosarg/pDNA (c) and Diosarg-DOPE NPs/pDNA (d) complexes under the +/− charge ratios from 0 to 30 determined by DLS.

Figure 4

Cytotoxicity of H1299 cells incubated with the Diosarg lipid and Diosarg-DOPE NPs at various +/− charge ratios (pDNA 0.5 μg/well), with the Diosarg lipid at the same +/− charge ratio as the control.

Figure 5

Intracellular uptake of the Diosarg lipid and Diosarg-DOPE (2:1, 1:1, 1:2) NPs/Cy3-siRNA complexes (+/− = 10) in H1299 cells measured by flow cytometry (10,000 cells for each sample) and observed by fluorescence microscopy (blue: DAPI (4’,6-diamidino-2-phenylindole) stained nuclei; red: Cy3-labeled siRNA).

Figure 6

Intracellular uptake of the Diosarg lipid and Diosarg-DOPE (2:1, 1:1, 1:2)/Cy3-pDNA complexes (+/− = 10) in H1299 cells measured by flow cytometry (10,000 cells for each sample) and observed by fluorescence microscopy (blue: DAPI stained nuclei; red: Cy3-labeled pDNA).

Figure 7

In vitro luciferase gene transfection efficiencies of the Diosarg lipid and Diosarg-DOPE (2:1, 1:1, 1:2) NPs/pDNA complexes at various +/− charge ratios in H1299 cells.

Figure 8

Intracellular localization of the Diosarg lipid and Diosarg-DOPE (1:1)/pDNA complexes (+/− = 10) in H1299 cells observed and recorded by fluorescence microscopy (blue: DAPI stained cell nuclei; green: Lysotracker-stained lysosome; red: Cy3-labeled pDNA).