Calorimetric study of the interaction of binary DMTAP/DOTAP cationic liposomes with plasmid DNA

Abstract

Cationic liposomes have been suggested as possible agents for nonviral gene transfer. The interaction of plasmid DNA (pDNA) with dispersions of stable unilamellar cationic liposomes based on the binary lipid system 1,2-dimyristoyl-3-trimethyl-ammonium-propane (DMTAP):1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP) has been studied by using isothermal titration calorimetry (ITC), high-precision differential scanning calorimetry (DSC), dynamic light scattering (DLS), and circular dichroism (CD). Systematic calorimetric and DLS exploration of the DMTAP:DOTAP binary system reveals that single-bilayer liposomes are stable at the 4:1 molar ratio, exhibiting the main lipid-phase transition temperature at approximately 25.3 degrees C, and a total enthalpy change deltaH = 8.5 +/- 0.4 kcal/mol. The interaction of pDNA with unilamellar DMTAP:DOTAP vesicles was investigated by ITC experiments, which clearly distinguished endothermic binding between the phosphate and the ammonium groups from exothermic processes, driven by slow kinetics, corresponding to interliposomal, DNA-triggered aggregation that leads to the formation of large multilamellar liposome/pDNA assemblies. Lipid-added-to-pDNA and pDNA-added-to-lipid experiments have been carried out in order to systematically explore the interaction mechanisms. Complex ITC profiles are revealed, which may be linked to packing rearrangements of the pDNA molecules bound at the outer liposomal surface, possibly due to binding to more than one liposome or due to p-DNA-enhanced heterogeneity in the local lipid concentration. DNA-mediated aggregation effects are detected at high [ammonium]/[phosphate] molar ratios in the case of lipid-added-to-pDNA interactions and at relatively low [phosphate]/[ammonium] molar ratios in the case of pDNA-added-to-lipid.

DSC applications: nucleic acids and membrane interactions

“…For very weak or slow-kinetics interactions, the establishment of a stable state can be monitored and demonstrated by successive heating and cooling scans and the repeatability of the DSC thermograms. Characteristic results from Giatrellis et al 14 are displayed in Figure 8.2 . The DSC thermograms are considerably altered depending on the thermal history and the sequence of the DSC scans.…”

Calorimetric Investigations of Non-Viral DNA Transfection Systems

“…It is important to note that this is only a generalization, and factors such as the stoichiometry of the cationic lipid to DNA, the ionic strength of the formulation, the temperature of the formulation (both during and after formulation), and the incubation time can all have a significant effect on the resulting structure of the lipoplex (Giatrellis et al, 2009). Association of DNA with cationic lipids in a micellar or liposomal form leads to lamellar organization with DNA molecules sandwiched between lipid bilayers.…”

“…A key problem in the advancement of lipoplex transfection vectors, yet to be efficiently overcome, is the above mentioned inadequacy of transfection efficiency of current cationic lipid systems, attributed to the incomplete understanding of interaction mechanisms involved. While this is driving research efforts toward the design and synthesis of novel amphiphilic cationic compounds, as well as approaches for formulation of liposomal based vectors (Giatrellis et al, 2009), less effort is being directed towards better understanding the fundamental mechanism(s) by which liposome-mediated transfection occurs. Scheme 1.…”

“…The most commonly complex assemblies include DNA molecules sandwiched within liposomal bilayers, DNA electrostatically adsorbed onto the vesicles outer surface, DNA encapsulated in the aqueous phase of the liposomes, and DNA coated by a monolayer of cationic lipid envelop (Felgner and Ringold, 1989;Giatrellis et al, 2009). Therefore, lipoplexes have a broad range of morphologies from a hypothesized "beads on a string" arrangement proposed initially by Felgner (Felgner and Ringold, 1989) in their seminal paper, to more complicated such as spaghetti and meatballs structure (Sternberg et al, 1994), map-pin structures (characterized by spheroidal heads and tapering pins) (Sternberg et al, 1998)(See Figure 2), multilamellar or inverted hexagonal phase structures confirmed by highresolution synchrotron small-angle X-ray scattering (SAXS) experiments (Safinya, 2001) and sliding columnar phase (O'Hern, 1998).…”

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy

“…The application of cationic liposomes to gene therapy was first described in 1987 by Felgner (Felgner et al 1987). The lipoplexes are generally composed of a positively charged lipidic component (see Figure 2), such as dioleylpropyltrimethylammonium chloride, dioleoyl triethylammonium propane (DOTAP), or dimethylaminoethane carbamoyl cholesterol (DCChol), that is capable of complexing and condensing the DNA (Giatrellis et al 2009). Most lipoplex formulations include a ''helper'' lipid, such as dioleoylphosphatidyl-ethanolamine (DOPE, as seen in Figure 2), or cholesterol that provides added stability to the lipoplexes and enhances DNA release from endosomal compartments.…”