Influence of Biological Media on the Structure and Behavior of Ferrocene-Containing Cationic Lipid/DNA Complexes Used for DNA Delivery

Abstract

Biological media affect the physicochemical properties of cationic lipid-DNA complexes (lipoplexes) and can influence their ability to transfect cells. To develop new lipids for efficient DNA delivery, the influence of serum-containing media on the structures and properties of the resulting lipoplexes must be understood. To date, however, a clear and general picture of how serum-containing media influences the structures of lipoplexes has not been established. Some studies suggest that serum can disintegrate lipoplexes formed using certain types of cationic lipids, resulting in inhibition of transfection. Other studies have demonstrated that lipoplexes formulated from other lipids are stable in the presence of serum and able to transfect cells efficiently. In this paper, we describe the influence of serum-containing media on lipoplexes formed using the redox-active cationic lipid bis(n-ferrocenylundecyl)dimethylammonium bromide (BFDMA). This lipoplex system promotes markedly decreased levels of transgene expression in COS-7 cells as serum concentrations are increased from 0% to 2%, 5%, 10%, and 50% (v/v). To understand the cause of this decrease in transfection efficiency, we used cryogenic transmission electron microscopy (cryo-TEM) and measurements of zeta potential to characterize lipoplexes in cell culture media supplemented with 0%, 2%, 5%, 10%, and 50% serum. Cryo-TEM revealed that in serum-free media BFDMA lipoplexes form onion-like, multilamellar nanostructures. However, the presence of serum in the media caused disassociation of the intact multilamellar lipoplexes. At low serum concentrations (2% and 5%), DNA threads appeared to separate from the complex, leaving the nanostructure of the lipoplexes disrupted. At higher serum concentration (10%) disassociation increased, and bundles of multilamellae were discharged from the main multilamellar complex. In contrast, lipoplexes characterized in serum-free aqueous salt (Li2SO4) medium and in OptiMEM cell culture medium (no serum) did not exhibit significant structural changes. The zeta potentials of lipoplexes in serum-free media (salt medium and cell culture medium) were similar (e.g., approximately −35mV). Interestingly, the presence of serum caused zeta potentials to become less negative (about −20mV in OptiMEM and −10mV in Li2SO4), even though serum contains negatively charged entities that have been demonstrated to lead to more negative zeta potentials in other lipoplex systems. The combined measurements of zeta potential and cryo-TEM are consistent with the proposition that DNA threads separate from the lipoplex in the presence of serum, resulting in a decrease in the net negative charge of the surface of the lipoplex.

Incorporation of DOPE into lipoplexes formed from a ferrocenyl lipid leads to inverse hexagonal nanostructures that allow redox-based control of transfection in high serum

“…2). These results confirm those of our past studies, 55–57, 60 indicating that serum proteins have a deleterious effect on transfection of cells with BFDMA.…”

“…S9B†) suggests that addition of serum does not cause complete loss of the L α c nanostructure of the BFDMA RED lipoplex. This result appears consistent with these prior cryo-TEM results, 60 which indicated that some intact multilamellar, L α c nanostructures also appeared to survive addition of serum under certain conditions. The results shown in Fig.…”

“…In particular, in a study reported recently, cryo-TEM was used to characterize the nanostructures of BFDMA-containing lipoplexes (no DOPE) in the presence of serum and revealed that serum can promote dissociation of lipoplexes containing BFDMA. 60 Our SAXS-based characterization of the nanostructures of lipoplexes of BFDMA RED performed in the presence of serum (Fig. S9B†) suggests that addition of serum does not cause complete loss of the L α c nanostructure of the BFDMA RED lipoplex.…”

“…60, 66, 90 These studies have also suggested that the destruction of the lipoplexes by serum proteins underlies the low levels of cell transfection measured in the presence of serum. In particular, in a study reported recently, cryo-TEM was used to characterize the nanostructures of BFDMA-containing lipoplexes (no DOPE) in the presence of serum and revealed that serum can promote dissociation of lipoplexes containing BFDMA.…”

Physicochemical and biological characterization of 1,2-dialkoylamidopropane-based lipoplexes for gene delivery

“…Several processing and formulation factors are expected to affect the efficiency with which these events can take place and subsequently, their biological activity and the colloidal characteristics of the resultant particles. Examples of such factors include (1) lipid composition [15,24] (2) charge ratio [31,32,44] (3) type and ratio of helper lipid [8,13,34] (4) type of the hydrophobic anchor of PEG-lipids in PEGylated lipoplexes [17] and (5) complexing medium [12,22].…”