Skeleton-Controlled pDNA Delivery of Renewable Steroid-Based Cationic Lipids, the Endocytosis Pathway Analysis and Intracellular Localization

Abstract

Using renewable and biocompatible natural-based resources to construct functional biomaterials has attracted great attention in recent years. In this work, we successfully prepared a series of steroid-based cationic lipids by integrating various steroid skeletons/hydrophobes with (l-)-arginine headgroups via facile and efficient synthetic approach. The plasmid DNA (pDNA) binding affinity of the steroid-based cationic lipids, average particle sizes, surface potentials, morphologies and stability of the steroid-based cationic lipids/pDNA lipoplexes were disclosed to depend largely on the steroid skeletons. Cellular evaluation results revealed that cytotoxicity and gene transfection efficiency of the steroid-based cationic lipids in H1299 and HeLa cells strongly relied on the steroid hydrophobes. Interestingly, the steroid lipids/pDNA lipoplexes inclined to enter H1299 cells mainly through caveolae and lipid-raft mediated endocytosis pathways, and an intracellular trafficking route of "lipid-raft-mediated endocytosis→lysosome→cell nucleic localization" was accordingly proposed. The study provided possible approach for developing high-performance steroid-based lipid gene carriers, in which the cytotoxicity, gene transfection capability, endocytosis pathways, and intracellular trafficking/localization manners could be tuned/controlled by introducing proper steroid skeletons/hydrophobes. Noteworthy, among the lipids, Cho-Arg showed remarkably high gene transfection efficacy, even under high serum concentration (50% fetal bovine serum), making it an efficient gene transfection agent for practical application.

Keywords: endocytosis pathway; gene delivery; serum-compatible; steroid; structure–function relationships.

Scheme 1

Molecular structures of the steroid-based cationic lipids.

Figure 1

(a) Agarose-gel retardant assay of pDNA binding affinity for the steroid-based cationic lipids under various +/− charge ratios; (b) Heparin competition assay of the stability of the steroid-based cationic lipids/pDNA lipoplexes (± = 15).

Figure 2

Cytotoxicity of the steroid-based cationic lipids with various concentrations (from 0 to 150 µg/mL) in: (a) H1299 cells; and (b) HeLa cells.

Figure 2

Cytotoxicity of the steroid-based cationic lipids with various concentrations (from 0 to 150 µg/mL) in: (a) H1299 cells; and (b) HeLa cells.

Figure 3

Luciferase gene transfection efficiency for the steroid-based cationic lipids/pDNA lipoplexes under various +/− charge ratios in: (a) H1299; and (b) HeLa cell lines. Commercially available bPEI-25K and Lipo2000 were used as controls.

Figure 4

Luciferase gene transfection efficiency for the steroid-based cationic lipids/pDNA lipoplexes under various +/− charge ratios in the absence/presence of serum in: (a) H1299; and (b) HeLa cell lines. bPEI-25K and Lipo2000 were used as controls.

Figure 5

Luciferase gene transfection efficiency for the steroid-based cationic lipids/pDNA lipoplexes under +/− charge ratios of 5 and 10 in the presence of various concentrations of serum in H1299 cell lines.

Figure 6

Intracellular uptake of the steroid-based cationic lipids/pDNA lipoplexes in HeLa cells analyzed by FACS. Lipo2000 and bPEI-25k were utilized as the controls for gene transfection in the optimized dose.

Figure 7

Endocytosis pathway analysis of the steroid-based cationic lipids/pDNA lipoplexes (± = 10) in H1299 cells by measuring the relative luciferase gene expression in the presence of different endocytosis-specific inhibitors: Chloropromazine (CPZ, clathrin-mediated endocytosis inhibitor); Methyl-β-cyclodextrin (MBCD, lipid-raft mediated endocytosis inhibitor); Genistein (GENI, caveolae mediated endocytosis inhibitor); Amiloride (AMIL, macropinocytosis mediated endocytosis inhibitor); and Nocodazole (NOCO, microtubule and actin inhibitor).

Figure 8

Fluorescence images (400×) for intracellular localization of the steroid-based cationic lipids/Cy3-pDNA lipoplexes after 4 h incubation in H1299 cells. Lipofectamine2000 (Lipo2000) was used as a reference. Red: Cy3-labeled pDNA; green: Lysotracker stained endosome/lysosome; blue: DAPI stained cell nuclei.