Effects of a Delocalizable Cation on the Headgroup of Gemini Lipids on the Lipoplex-Type Nanoaggregates Directly Formed from Plasmid DNA

Abstract

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl imidazolium) alkanes], referred as (C16Im)2Cn (where Cn is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, α, and effective charge ratios, ρeff, of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low α value) and a fingerprint-type, that coexist with the cluster-type at moderate α composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low α value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.

Influence of Polyplex Formation on the Performance of Star-Shaped Polycationic Transfection Agents for Mammalian Cells

“…While this is most likely correct for linear DNA, it has been shown the effective charge density of plasmid DNA can be considerably lower. Aicart gives values between´0.19 and´0.46 for a plasmid of similar size as the one used by us [22]. A comparison with linear DNA would be required to determine the effective charge density of the pDNA in our case.…”

Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors

“…The net charge of the MVCV, the nucleic acid, and of the complex formed by both, together with the electrostatic interaction between the MVCV and the NA, are analyzed by means of electrophoretic mobility which can be directly related with zeta potential (ζ), the property usually reported in the literature [45,54,90]. Both properties inform about the electroneutrality ratio in terms of molar charge ratio ( eff = 1) or mass ratio ( / GV NA mm ) between the gene vector (m GV ) and nucleic acid (m NA ) masses.…”

“…The size and morphology of the complexes are studied by static (SLS) or dynamic (DLS) light scattering and electron microscopy (EM, TEM, and cryo-TEM) methods [60,93,95,97]. The well-organized structures of the complexes have been determined by diffraction (SAXS or SANS) and electron microscopy (TEM and cryo-TEM) [45,65]. The fluidity of the gene vector (preferently when they are lipids) has been studied by means of fluorescence anisotropy [93].…”

“…In most cases, as the effective charge of the multivalent cationic agent is lower than its nominal one, it must be experimentally determined, as reported elsewhere [45].…”

“…Additionally, the positive charge of the univalent cationic vectors match its nominal one (+1), but in many cases MVCVs show a lower effective charge, and thus, it must be initially known as well. Both, MVCV and pDNA effective charges, can be experimentally determined following a procedure recently reported based on a ζ-potential study [45]. The procedure permits, in a first step, to obtain the effective charge of any MVCV by measuring ζ-potential at different mass ratios ( / GV NA mm ) of the complex formed with a long linear DNA or short linear RNA (for which its effective charge match its nominal one, i.e., -2 per bp) [45].…”