Nanoscale DNA - Zwıtterionıc Vesicle Formulation Compacted For Gene Delivery: Adiabatic Differential Scanning Calorimetric Study
differential scanning microcalorimetry, divalent metal cation, dna-liposome self-assembly, non-viral gene delivery, thermodynamics
E Süleymano?lu. Nanoscale DNA - Zwıtterionıc Vesicle Formulation Compacted For Gene Delivery: Adiabatic Differential Scanning Calorimetric Study. The Internet Journal of Nanotechnology. 2003 Volume 1 Number 1.
Nucleic acids-membrane associations comprise the least functionally studied macromolecular assembly, yet attract the attention of researchers due to their potential in the field of gene therapy (
Materials and Methods
Synthetic dipalmitoylphosphatidylcholine (DPPC) and calf thymus DNA were a kind gift of Prof. P. Balgavy (J. A. Comenius University – Bratislava, Slovakia). EDTA was purchased from Sigma Chemical Co., St. Louis, MO, USA. MgCl2.6H2O, NaHPO4 and NaCl were obtained from Merck, Darmstadt, Germany. The presented nucleic acid concentrations and the molar ratios are based on on the average nucleotide molecular weight of 308 calculated from the known DNA composition employed (Uhrikova,
Preparation of vesicles
1.2 mM lipid in standard SSC buffer, pH=7.2 was used in all experiments and was stored at 4°C. The formation of a thin layer of lipids of a 15 ml round-bottomed flask was achieved by a hand-shaking and hydration of in SSC buffer at arround 70°C. Vortexing of the lipid with the desired aqueous solution above the gel-to-liquid crystalline phase transition of the lipid (T m ) for arround 30 min resulted in multilamellar vesicles. The DNA concentration used troughout all experiments was 1.8 mM based on the abovementioned assumption. Unilamellar vesicles were obtained by extrusion of multilamellar vesicle suspension through two stacked polycarbonate filters (Nucleopore, Inc.) of 100 nm pore size at arround 60°C. Repeated extrusion (10 times) through the extruder (Lipex Biomembranes, Inc., Vancouver, B.C., Canada) creates homogeneous vesicle suspension. This allows the preparation of vesicles with a mean diameter of 90 nm and a trap volume in the range of 1.5 – 2.0 l/mole.
Results And Discussion
Although double-stranded DNA (dsDNA) has been shown to bind to zwitterionic lipids (
In this type of complex formation, the measured value of Tm=41.9 and ΔHcal=31.9 kJ/mol were determined. The interaction with liposomes results in the significant decrease of excess apparent specific heat capacity. The next three curves to the bottom marked as DPPC
– DNA – Mg 2+ show the phase behaviour of ternary mixtures of DPPC:DNA:Mg 2+ in equimolar 1:1:1, 1:3:1 and 1:5:1 ratios with increased DNA amount, respectively. The equimolar peak possesses narrower signal, compared to DPPC vesicles' peak, with further DNA phase separation. The Tm value remained, however ΔHcal diminished to 9.7 kJ/mol. The specific heat capacity remains upon complexation with divalent cation. The main phase transitions shifts somehow to 41.7°C. Intere stingly the DNA phase peak moves further to 89°C. At the 1:1:1 molar ratio of the tripple complexation, the self - assemlies display two peaks. The first one is at main phase transition temperature corresponding to the melting of DNA-lipid aggregate. The second one is at 86 °C and corresponds for the DPPC-DNA complex. The former peak is attributed to the stabilization of the DNA secondary structure by a tight packing of DNA molecules several unilamellar vesicles, bridged by Mg 2+ -ions. This is a particular case of liposome surface induced nucleic acid condensation of the “spaghetti and meatballs” structure (
The tripple complex of DNA-metal ion-phosphatioline vesicle remains stable at different incubation times, which is in agreement with small- and wide-angle X-ray scattering measurements . Apparently, Mg 2+ decreases the DNA efective radii and creates groove narrowing, by ligand binding to six or eight water molecules, or alternatively to nucleic acid phosphate in the minor groove in a fully hydrated state (
It is well-established that such a positively charged particle deliver DNA into cultured cells by electrostatic mechanisms of binding to their negatively charged membranes. Liposomes enter cells by various routes, such as through endocytic pathway and direct membrane fusion. Gene delivery designs involving receptor mediated transfer face problems, since endosomes fuse rapidly with lysosomes, thus degrading the nucleic acids. The ternary complex between nucleic acid, divalent inorganic cation and extruded liposome formulated from zwitterionic lipid, described in the work herein, can deliver genes into cells via direct fusion with the cell membrane. This model is is accordance with recent proposal (
The close supervision of Prof. P. Balgavy (Bratislava) and Dr. J. Bagelova (Kosice) is highiy appreciated.
Erhan SÜLEYMANOGLU DEM ILAÇ PHARMACEUTICALS, Büklüm Sokak, No: 15/7, Kavaklidere, 06660 – Ankara, Tel: 00-90-312-417-02-51, Fax: 00-90-312-417-09-16, web – site: http://www.demilac.com; E-mail: email@example.com; firstname.lastname@example.org