Phospholipid-Nucleic Acid Complexation: Biomolecular Energetics of DNA-Mg(2+)-Phosphatidylcholine Ternary Complex Formation, Compaction and Relevance as Lipoplex Formulation

Abstract

Thermodynamic features related to preparation and use of self-assemblies formed between multilamellar and unilamellar zwitterionic liposomes and polynucleotides with various conformation and sizes are presented. The divalent metal cation induced adsorption, aggregation and adhesion between single- and double-stranded polyribonucleotides and phosphatidylcholine vesicles was followed by differential adiabatic scanning microcalorimetry. Nucleic acid condensation and compaction mediated by Mg(2+) was followed, with regard to interfacial interaction with unilamellar vesicles. Microcalorimetric measurements of synthetic phospholipid vesicles and poly(ribo) nucleotides and their ternary complexes with inorganic cations were used to build the thermodynamic model of their structural transitions. The increased thermal stability of the phospholipid bilayers is achieved by affecting their melting transition temperature by nucleic acid induced electrostatic charge screening. Measurements give evidence for the stabilization of polynucleotide helices upon their association with liposomes in presence of divalent metal cations. Such an induced aggregation vesicles either leads to heterogeneous multilamellar DNA-lipid arrangements, or to DNA-induced bilayer destabilization and lipid fusion. The further employment of these polyelectrolyte nanostructures as an improved formulations in therapeutic gene delivery trials, as well as in DNA chromatography is discussed.

Keywords: differential scanning microcalorimetry; non-viral gene delivery; polyelectrolyte phospholipid-polynucleotide nanostructures; thermotropic phase behaviour.

Figure 1

Microcalorimetric phase behaviour of DPPC multilayers in the presence of poly(ribo) nucleotides and Mg²⁺. Thermograms depict the thermal properties of ternary DNA-metal cation-lipid complexes and their components as ΔCp vs temperature plot. Heat flow is represented by the downward arrow. Concentration of synthetic biopolymers were: 1 mM of (A):(U) bp of poly(A:U) duplex, and 1 mM of DPPC (assuming average molecular mass of 770), and DNA (assuming average molecular mass of base pair of 643) in 10 mM HEPES/10 mM NaCl; pH=7.2, cell volume 1.5 ml. Differential scanning calorimetric measurements were performed using SETARAM® DSC microcalorimeter, equipped with Hewlett-Packard PC and with company supplied computer programme. (a) DPPC-MLV-Mg²⁺; (b) poly(A:U)n-Mg²⁺; (c) DPPC-MLV- Mg²⁺-poly(A:U)_n; (d) DPPC-MLV-calf thymus DNA.

Figure 2

Thermotropic phase behaviour of calf thymus DNA in complex with DPPC in the presence of Mg²⁺. (a) DPPC-MLV; (b) equimolar mixture of DPPC-ULV- Mg²⁺-DNA; (c) DNA- Mg²⁺ binary complex; (d) unbound calf thymus DNA.

Figure 3

UV-melting curves of DPPC-MLV-Mg²⁺dispersions. The main phase transition tmperature of the lipids is shifted towards higher values. Thermotropic phase behaviour of lipids is presented as measurements of the change in optical density as a function of temperature. Values were recorded by stepwise increasing the temperature using 3 ml cuvettes thermostatized within ±0.3°C by circulating water bath connected to the cuvette holder, as described in Materials and Methods. Recordings were performed after maintenance of the samples in the cuvette holder for 5 min. at each teperature value. Data is presented as a mean ± S.D. of 6 measurements, and plotted using MATLAB® software. Inlet shows the aggregation and kinetics of adsorption of DPPC-MLV and ULV with poly(ribo) nucleotides or DNA in the presence of Mg²⁺ at constant temperature at 550 nm. Their initial absorbance vary with respect to each other due to their different lamelarity, curvature and homogeneity. The stock solutions of poly(ribo) nucleotides and calf thymus DNA were prepared freshly and used immediately before measurements. After zeroing the initial apparent absorption, their complexation with lipid vesicles was followed during the first 30 sec. Mg²⁺ were added to the binary mixture of ploy-nucleotides and liposomes at the final concentration of 0.5 mM and the mixture was mixed in the cuvette. The folded ternary complex is destroyed by EDTA treatment. The evaluation of their complex formation was performed, as outlined in Materials and Methods.

Figure 4

Schematic representation of formation of DNA-surfactant (a) and DNA-liposome aggregates (b-c). Plamid DNA behave differently from larger nucleic acids. (a) DNA first cover the neutral liposome with adsorbed Mg²⁺ on its surface. DNA itself leads to aggregation between two neighbouring ULVs (c). Charge neutralization brings about a local destabilization and membrane rupture and resealing of both lipid vesicle bilayers into larger structure, in which nucleic acid is sandwiched between the phospholipid bilayers. This can result in additional adhesions of other ULVs to form a multilamellar arrangements. DNA-phospholipid vesicle complexes probably fuse to a condensed and heterogeneous multilamellar structures depending on nucleic acid type and concentration. The inlets depict fluorescent images of relaxed and condensed DNA in the presence of surfactant, as described (21). Drawing is not to scale.

Figure 5

Proposed mechanism of Mg²⁺-induced internalization of neutral liposome-DNA formulations. Drawing is not to scale.