Development of a slow non-viral DNA release system from PDLLA scaffolds fabricated using a supercritical CO₂ technique

Heyde, Mieke, Partridge, Kris A., Howdle, Steven M., Oreffo, Richard O.C., Garnett, Martin C. and Shakesheff, Kevin M. (2007) Development of a slow non-viral DNA release system from PDLLA scaffolds fabricated using a supercritical CO₂ technique. Biotechnology and Bioengineering, 98 (3), 679-693. (doi:10.1002/bit.21446).

Abstract

Polyamidoamine polymers (PAA) comprising methylene-bisacrylamide/dimethylethylene-diamine monomers were synthesized, complexed with DNA and incorporated into porous P(DL)LA scaffolds by using a supercritical CO₂ (scCO₂) technique. Scaffolds were made in a dry state consequently there was a need to lyophilize the complexes. A statistically significant reduction of the transfection efficiency was observed in the absence of trehalose when compared to the original complex after freeze-drying. Increasing concentrations (0-10% w/v) of trehalose were added to the complex prior to freeze-drying. Structure dependent differences in DNA binding were evaluated by gel electrophoresis and thermal transition analysis. TEM and PCS showed aggregate formation after freeze-drying without trehalose. Scaffolds were characterized by pore sizes of 173 ± 73 µm and a porosity of 71%. The transfection potential of the released DNA was investigated by seeding scaffolds with A549 cells and following firefly luciferase as a marker gene after 48 h exposure. Low but continuous levels of transfection were observed for PAA complexes during a 60-day study. Complexes made with Lipofectaminetrade mark gave initially higher levels of DNA release but no further expression was seen after 40 days. Uncomplexed DNA showed background levels of transfection. Culturing cells on 3D scaffolds showed a benefit in retention of transfection activity with time compared to 2D controls. Transfection levels could be increased when cells were grown in OptiMEM. This study demonstrated that PAA/DNA complexes incorporated into a P(DL)LA scaffold made by using scCO₂ processing exhibited a slow release and extended gene expression profile.

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Contributors

Author: Richard O.C. Oreffo

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