Plasmid DNA as a probe for biodistribution of liposomes

NADIA S. CHIARAMONI; LUCÍA SPERONI; RICARDO A. GARGINI; MARÍA C. TAIRA; SILVIA DEL V. ALONSO-ROMANOWSKI

Paraiba, Brasil

Congreso; Pharmatech 2003; 2003

Plasmid DNA as a probe for biodistribution of liposomes. CHIARAMONI ns, SPERONI l, gARGINI ra, taira MC and Alonso-Romanowski S Universidad Nacional de Quilmes, Departamento de Ciencia y Tecnología, Laboratorio de BioMembranas. Roque Sáenz Peña 180 Bernal, (1876) BsAs. Argentina. salonso@unq.edu.ar Introduction. Nowadays to determine the stability of liposomal formulations is mandatory to use radioisotopes. This method is very expensive and toxic for the users. To achieve this purpose a cDNA fragment of VP7 was used as plasmidic DNA probe. VP7 is a protein of the outer envelope of the rotavirus capside. DNA containing liposomes were successfully used in DNA immunisation. Aims: The main objective of this work was to develop a sensitive technique for liposome biodistribution in vivo. Materials and methods: Lipids were all from Avanti Polar Lipids, all other reagents were from Sigma. In order to obtain the probe a RT-PCR was developed using a dsRNA from bovine rotavirus strain UK as template. With the RT-PCR product, a PCR reaction was performed and cloned into pGemT. Positive clones were sequenced and confirmed with gene Bank data-base. Preparation of plasmid DNA-containing liposomes : Several formulations were tested as DNA carriers. Positive results were obtained with SoyPC:DMPE:Chol (2:2:1, molar ratio) and the lipid/DNA ratio was 300:1. The mixture obtained was freeze-dried by lyophilization, suspended in PBS buffer and the non-encapsulated DNA was separated by centrifugation. Agarose-gel electrophoresis: The samples of liposome-entrapped were analyzed by agarose gel electrophoresis, to determine the retention of DNA by the liposomes. Measurement of osmotic volume and vesicle size: the osmotic volume was measured spectrophotometrically at different concentra-tions [1].and the dynamic liposome size, with or without DNA, was measured by light scattering. Biodistribution studies: they were performed in groups of six animal models (Balb/C mice) that were inoculated with DNA, DNA/liposomes and buffer (control) [2]. Results: Encapsulation of Plasmid-DNA into liposomes: A) Absorbance measurements and B) Agarose gel electrophoresis Figure 1: A) Absorbance spectra, green line: liposomes, red line: DNA-liposomes. B) Gel electrophoresis:Lane 1 centrifugation pellet: plasmid DNA/liposomes. Lane 2 centrifugation supernatant Osmotic Volume determination: Figure 2: Left Osmotic volume of liposomes A1:10.137. Right: Osmotic volume of plasmid-DNA/ liposomes A2:4.695. A2/A1=0.46. Conclusion: These studies demonstrate that DNA is efficiently encapsulated into liposomes, liposome/DNA system is half the osmotic volume size of the liposomes without DNA and the distribution study gave us an idea of the stability and life circulation of this particular liposome formulation as proposal for biodistribution studies  in vivo. Acknowledgements. This work was supported by grants from UNQ and CONICET, Argentina. [1]. LI Viera, S Alonso-Romanowski, V Borovyagin, MR Feliz and EA Disalvo. (1993) Biochim.Biophys.Acta, 1145,  157-167 [2] MM Fabani, RA Gargini, MC Taira, R. Iacono, and S. Alonso-Romanowski  (2002). J. Liposome Research 12 (1), 13-27.