TRANSFECTION OF LUNG CELLS IN VITRO AND IN VIVO: EFFECT OF INTRALIPOSOMAL ANTIOXIDANTS AND bFGF

Luo X^1,2,3,4, Belcastro R^1,2, Cabacungan J^1,2,Hannam V⁵, Negus A², Wen Y⁵, Plumb J⁵, Hu J^1,2,5, Steer B⁵, Koehler DR⁵, Downey GP^5,6, Tanswell AK^1,2,3,7

¹CIHR Group in Lung Development and ²Lung Biology and ⁵Lung Gene Therapy Programmes, Hospital for Sick Children Research Institute and the Departments of ⁶Medicine, ³Paediatrics and ⁷Physiology, University of Toronto, Ontario, Canada; ⁴The Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China

Objective: To examine the role of reactive oxygen species, and the effect of antioxidant interventions, on transfection of lung epithelial cells with liposome:DNA complexes.

Methods: In vitro transfections were performed on primary cultures of distal rat fetal lung epithelial cells (RFL₁₉Ep). In vivo transfections were by airway instillation in mice.

Results: H₂O₂ was present in the culture medium of RFL₁₉Ep at a concentration of .0.25 µM, a concentration similar to that in normal breath condensate. Plasmid DNA complexed with liposomes was protected against DNA injury mediated by H₂O₂ but not by that mediated by the hydroxyl radical. Antioxidant interventions with 50 µM Trolox, 0.2 µM diphenyl-phenyldiamine and 0.1 µM phenanthroline demonstrated reduced intracellular plasmid degradation and enhanced (. 2-fold) reporter gene expression, suggesting an hydroxyl-radical mediated effect. Inclusion of bFGF (20 ng/ml) within liposomes used for transfection increased reporter gene expression .4-fold. Our results suggested that this was only partly due to the antioxidant properties of bFGF. Localization of fluorescent DNA to nuclear membranes was enhanced by the use of intraliposomal bFGF, consistent with enhanced delivery of transfected DNA as bFGF binds to nuclear receptors. Intraliposomal bFGF also significantly enhanced transgene expression .4-fold in mouse lungs following airway delivery.

Conclusions: Endogenous production of reactive oxygen species may pose a significant barrier to transfection using liposome:DNA complexes. This work was supported by the Canadian Institutes of Health Research

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