Lipid-DNA systems: An Approach to Biodistribution assays

NADIA CHIARAMONI; LUCÍA SPERONI; MARÍA C. TAIRA; SILVIA DEL V. ALONSO-ROMANOWSKI

Baltimore, USA

Congreso; Biophysical Society 48th Annual Meeting; 2004

  Abstract: The main objetive of this work was to develop a sensitive technique to study liposome biodistribution. To avoid the use of radioisotopes a PCR technique was implemented. VP7 is a protein of the outer envelope of the rotavirus capside. A cDNA fragment of VP7 was inserted into pGEMT  cloning vector and used as plasmid DNA probe. The viral dsRNA of the bovine rotavirus strain UK  was extracted and a RT-PCR reaction was performed to obtain the VP7 cDNA. The plasmid-DNA probe was encapsulated into several liposomal formulations containing cationic, non-charged or polymerizable lipids. The Lipid:DNA ratio was 300:1. Several assays were performed to  characterize the formulations like optical microscopy, electronic microscopy, and osmotic volume determinations. The non-charged liposomes containing DNA were twicw smaller than the liposomes without. The cationic and polymeric liposomes with DNA were bigger than the formulations without DNA. The cationic formulations showed higher efficiency of encapsulation, followed by non-charged liposomes and polymeric ones. Based on these studies the cationic liposomes are proposed as DNA carriers and polymerized liposomes also but subjected to interfacial modifications. The toxicity of the formulations was controlled in vitro through the determination of the peroxidation index and peroxidation induction on mitochondria and microsomes. The level of toxicity of liposomes with cationic lipids was similar as the polymerized ones and comparable more toxic than those containing EggPC and/or EggPC-Cholesterol. In order to test the plasmid extraction method, female Balb-c mices 6-8 month old were injected with several amounts of plasmid DNA. After 1 hour blood was extracted from retro-orbital vein, and after 24 hour the animal was euthanized and the total blood was extracted. Plasmid was separated with Phenol, Chloroform and isoamylic alcohol. Results shown that after one hour, only the biggest inncolation (5 mg) was  detected by PCR. After 24 hours, even 5 ug of Innoculated DNA was detected by PCR.