Lipopolyplex Ternary Delivery Systems Incorporating C14 Glycerol-Based Lipids

Abstract

The structure, biophysical properties and biological behavior of lipopolyplex ternary gene delivery vectors incorporating novel C14 glycerol based lipids of varying alkyl chain geometry (containing cis, trans or alkyne double bonds) have been studied in the presence and absence of a bifunctional targeting peptide designed to both condense DNA and confer integrin-specific targeting. In vitro transfection studies in breast cancer MDA-MB-231 cells revealed that ternary formulations of lipid:peptide:DNA (LPD) complexes prepared using the aforementioned lipids possessed highly synergistic transfection activity up to 2500-fold higher than their respective lipid:DNA (LD) or peptide:DNA (PD) counterparts. Furthermore, the small structural differences in the lipid alkyl chain geometries also resulted in pronounced differences in transfection within each type of formulation, whereby the trans lipids showed best activity when formulated as LD complexes, whereas the cis lipids were superior in LPD formulations. Confocal fluorescence internalization studies using labeled components of the formulations showed both the lipid and the DNA of LD complexes to be trapped in endocytic compartments, whereas in the case of LPD complexes, the DNA was clearly released from the endosomal compartments and, together with the peptide, internalized within the cell nucleus. Physicochemical characterization of the formulations carried out by light and neutron scattering, zeta potential measurement, and negative staining electron microscopy detected major structural differences between LD and LPD complexes. Gel electrophoresis assays additionally showed differences between the individual lipids tested in each type of formulation. In conclusion, the superior transfection of the trans lipids in the LD complexes was thought to be attributed to superior DNA binding caused by a more closely matched charge distribution of the more rigid, trans lipids with the DNA. In the case of the LPD complexes, the DNA was thought to be predominantly condensed by the cationic portion of the peptide forming a central core surrounded by a lipid bilayer from which the targeting sequence partially protrudes. The more fluid, cis lipids were thought to confer better activity in this formulation due to allowing more of the targeting peptide sequence to protrude.

Delivery of siRNA using ternary complexes containing branched cationic peptides: the role of peptide sequence, branching and targeting

“…vesicle size measured by dynamic light scattering, which was indicative of the presence of very few multilamellar vesicles. The thickness and repeat distance (of D spacing) of 41.5 Å and 65 Å reported in this study agree well with values of 44.7 Å and 65 Å, previously reported for DOTMA : DOPE vesicles (1 : 1 molar ratio) 27 and with 39 Å and 65 Å reported for related DOTAP : DOPE vesicles (1 : 1 mola ratio). 34,35 Furthermore, the use of a Rs of 250 Å to fit the data agrees well with that used in previous analysis of similar systems.…”

“…We have previously shown for LPD complexes that endosomal release of the components is most likely mediated by the helper lipid DOPE, with the ''proton sponge'' effect of the cationic peptides in the formulations having only a negligible effect. 27 However, the most effective LPR formulations, using either (H 3 K) 4 B1-L1-[Y] or H 6 B1-L1-[Y], showed larger amounts of siRNA within the cells and that this higher level of siRNA persisted, at least up to 24 h after incubation ( Fig. 9h and Fig.…”

“…34,35 Furthermore, the use of a Rs of 250 Å to fit the data agrees well with that used in previous analysis of similar systems. [27][28][29] Significantly, the DOTMA:DOPE vesicles examined using SANS were those used to prepare the LPR complexes. SANS data for LPR complexes are shown in Fig.…”

Synthetic Nucleic Acid Delivery Systems: Present and Perspectives

“…The ternary lipid-gWIZ-Luc-DNA-peptide (LPD) complexes were found more efficient that the parent lipoplexes revealing a potentiation effect of the targeting peptide on lipoplex activity. 119 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 30 Moreover, in order to stabilize the lipoplexes against plasma proteins and uptake by the reticuloendothelial (RES) system, the same team introduced lipids 57a-d bearing oligoethylene glycol (oligoEG) moieties in the structure of the polar head and other structurally related congeners. 120 The best transfection efficiencies against 1HaEo-and SCFTE29o-cell lines were observed for lipoplexes generated from lipids 57a and 57b coformulated with DOPE at a weight ratio of 1/1 and at total lipid/pClLux DNA ratio of either 2/1 or 4/1.…”

Development of lipopolyplexes for gene delivery: A comparison of the effects of differing modes of targeting peptide display on the structure and transfection activities of lipopolyplexes

“…The addition of cell‐specific targeting ligands on the surface of liposome‐based nanoparticles enhances the cellular uptake and transfection efficiency. For instance, in a comparative study of the transfection efficiencies of lipopolyplexes formulated from bimodal peptides with and without targeting, lipopolyplexes with a scrambled targeting sequence showed 25% transfection, and lipopolyplexes with the targeting sequence removed showed 12% transfection, compared with lipopolyplexes with targeting sequence, indicating that the high transfection efficiency is at least partly a consequence of the targeting sequence. In another study, surface targeted liposomes bearing the GE sequence were compared with surface targeted liposomes with the sequence scrambled; the scrambled sequences had negligible binding to cells in vitro.…”