Surface Functionalized Cationic Lipid-DNA Complexes for Gene Delivery: PEGylated Lamellar Complexes Exhibit Distinct DNA-DNA Interaction Regimes

Abstract

Cationic lipid-DNA (CL-DNA) complexes are abundantly used in nonviral gene therapy clinical applications. Surface functionality is the next step in developing these complexes as competent, target-specific gene carriers. Poly(ethylene glycol) (PEG) is the natural choice to serve as a protective coat or act as a tether for a specific ligand on the surface of these complexes due to its biocompatibility and ability to convey stealth-like properties. Understanding the effect of PEG on the internal structure and surface properties of CL-DNA complexes is essential in developing vectors with more complex derivatives of PEG, such as Arg-Gly-Asp (RGD)-based peptide-PEG-lipids. We report on x-ray diffraction studies to probe the internal structure of CL-DNA complexes consisting of a ternary mixture of cationic lipids, neutral lipids, and PEG-lipids. The PEG-coated complexes are found to exhibit a structure consistent with the lamellar phase. In addition, three distinct DNA interchain interaction regimes were found to exist, due to a), repulsive long-range electrostatic forces; b), short-range repulsive hydration forces; and c), novel polymer-induced depletion attraction forces in two dimensions. Optical microscopy and reporter gene assays further demonstrate the incorporation of the PEG-lipids into the lamellar CL-DNA complexes under biologically relevant conditions, revealing surface modification. Both techniques show that PEG-lipids with a polymer chain of molecular weight 400 do not provide adequate shielding of the PEGylated CL-DNA complexes, whereas PEG-lipids with a polymer chain of molecular weight 2000 confer stealth-like properties. This surface functionalization is a crucial initial step in the development of competent vectors for in vivo systemic gene delivery and suggests that a second type of surface functionality can be added specifically for targeting by the incorporation of peptide-PEG-lipids.

New multivalent cationic lipids reveal bell curve for transfection efficiency versus membrane charge density: lipid–DNA complexes for gene delivery

“…Another approach could involve introducing specific interactions via derivatives of PEG-lipids. By themselves, PEG-lipids of sufficient chain length are able to block the unspecific electrostatic interactions between cells and CL-DNA complexes [39].…”

Cationic liposome–nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing

“…The added PEG polymer coat induces repulsive interactions with a range on the scale of the size of the polymer chain [51][52][53][54]. Complexes which contain sufficient PEG-lipids (such that the PEG chains transition from the mushroom to the brush conformation) sterically stabilize CL-DNA complexes against flocculation caused by van der Waals attractions [55][56][57]. Experiments have shown that PEGylated complexes resist aggregation even after extensive centrifugation [40].…”

Transfection mediated by pH-sensitive sugar-based gemini surfactants; potential for in vivo gene therapy applications

“…Indeed, we and others have previously shown that PEG-lipids act via stabilizing the lamellar phase of the lipoplex lipid phase while this relatively hydrophilic coating to the lipoplex shields it from interactions with protein or other blood components [20][21][22][23]. Nevertheless, the presence of a lamellar-phase stabilizing PEG coating can impair intracellular gene delivery by frustrating endosomal release of the cargo from cationic lipoplexes [21,24].…”