1. DNA-MEMBRANE RECOGNITIONS: FROM PROTOCELL EVOLUTION TO BIONANOTHERAPY
Erhan Süleymanoğlu
Department of Pharmaceutical Chemistry,
Gazi University, Faculty of Pharmacy,
06330 – Ankara, Turkey,
OBJECTIVES
To build thermodynamic model of the structural transitions of lipoplexes as virus resembling self-assemblies (virosomes) with further potential to be employed as efficient lipid-based gene delivery tools.
METHODS
Thermodynamic features of ternary lipid-DNA complexes were followed by biophysical measurements. Their secondary structures were determined by fluorescence spectroscopy. Fluorescent microscopy was used to visualize phospholipid-DNA arrangements.
HUMAN GENE THERAPY
LABORATORY SET-UPS FOR STUDYING DNA-LIPID INTERACTIONS
STRUCTURES OF DNA-LIPID COMPLEXES
Confocal Fluorescence Microscopy of Cellular Internalization of Lipoplexes A B D F E C
Fig. 6. Formation of lipoplexes. Anionic nucleic acid molecules are attracted (A) to the surface cationic charge of the positively charged liposomes (made from a cationic lipid and a neutral lipid mixture) forming complexes that eventually form compact nucleolipidic particles of various morphologies and sizes, some maintaining the roughly spherical vesicle structure (B) while others form non-liposomal structures (C) (N. Dass, 2005).
Cytotoxicity Profiles of Lipoplexes
X-Ray Diffraction of DNA/lipid complexes (München data)
Fig.1. A current view of entry routes of lipoplexes in target cell. Three mechanisms referred to as receptor-mediated endocytosis, lipid-mediated poration and lipid fusion are depicted to show the hurdles for efficient transfection.
Cellular barriers for efficient gene transfection
DNA/ surfactant/ liposome and DNA/ Me2+/ liposome structures
RESULTS
Fig. 2. Phase behaviour of DPPC multilayers in the presence of poly (ribo) nucleotides and Mg2+, as determined by fluorescence spectroscopy.
Proposed mechanism for cellular internalization of lipoplexes
Fig. 3. Fluorescence spectroscopic measurements of DPPC multibilayers upon their associations with various amounts of calf thymus DNA, surfactant and 0.5 mM Mg2+
CONCLUSIONS
Measurements give evidence for the stabilization of polynucleotide helices upon their association with liposomes in the presence of cations with various valency. Such an induced aggregation of vesicles either leads to heterogeneous multilamellar DNA-lipid arrangements, or to DNA-induced bilayer destabilization and lipid fusion. The described structures could be further employed as transfection competent polyelectrolyte nanostructures as an improved formulations in therapeutic gene delivery.
Acknowledgements
The financial support by TÜBİTAK within the frame of COST Project 111S495 is greatly acknowledged.
Fig. 4. Fluorescence Microscopy of DPPC MLV Liposomes stained with Nile Red.
Fig. 5. DPPC-MLVs TEM analysis. a) MLVs; b-c) Mg2+-T7-MLVs d-e) MLV-phage T7 f) MLV-Mg2+ and g) Mg2+-T7-MLVs.