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AutorPérez Méndez, Mercedes ; Rodríguez Martínez, Daniel ; Fayos, José
Palabras claveNon-viral vectors
Gene therapy
Fecha de publicación13-jul-2016
CitaciónsciBAC 2016 | Scientific Congress, in the frame of the 10th Annual Congress of the Spanish Federation of Biotechnologists.
ResumenCholesteric Liquid Crystal Polymers (ChLCP), synthetized in our laboratory through a stereoselective polycondensation reaction1 as multifunctional optically active materials, have been extensively characterized 2 by NMR, Raman spectroscopy, steady-state fluorescence, molecular modeling, and SAXS/WAXS. These ChLCP behave both as thermotropic and lyotropic, confering interesting macromolecular properties indicative of potential application on the biomedical and engineering field. They have shown to be biocompatible against macrophages and fibroblasts cellular lines, and able to interact with biomacromolecules such as lipids (both neutral and cationi) and nucleic acids, the structures of the complexes being identified by synchrotron radiation source 3, 4, 5. Cationic liposomial/surfactant systems based on our CLCP were developed which entrapped DNA plasmids, acting as non viral cationic vectors for gene therapy, which successfully transfected in several tumor cell lines 6, 7. Cationic functionalized ChLCP have been synthesized, dispersed in TAE (0,04M TRIS; 0,001M EDTA) and complexed directly with commercial DNA of increasing complexity: [Poly-A]; [Poly-C]; [Poly-G]; [PolydT]; [PolyC-PolyG]; [PolyAC-PolydT]; commercial calf thymus DNA and plasmid. Three different proportions ChLCP:DNA were prepared: (1:2), (1:1), and (2:1) respectively by mixing and digesting for 12h in a swinging shaker. Neutron scattering experiments, had shown sufficient contrast (scattering length density difference) between new cholesteric PTOBEE-Ammonium (1.5 to 1.9 x 1010 /cm2) and polynucleotide [PolyC-PolyG] (3.32 x 1010 /cm2) for contrast variation SANS experiments. This experiment was successfully performed at NIST 8. The structure of the cationic complexes has been studied by SAXS at the BM16 beamline at ESRF, at room temperature. A monochromatized beam at = 0,9795 Å was used. Two-dimensional data recorded by an image-plate detector was placed at 5975 cm from the sample. The program Fit2D was employed to evaluate the beam centre position and to generate a mask file. Binary data are normalized by the detector response and pixels are radially averaged into 1D. Silver behenate (d= 58.3 Å) was used to calibrate the angular axis. Structural analysis of the cationic complexes is proposed.
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