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

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 CO(2) (scCO(2)) 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 microm 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 scCO(2) processing exhibited a slow release and extended gene expression profile.

Bone tissue engineering therapeutics: controlled drug delivery in three-dimensional scaffolds

“…Recently, impregnation of three-dimensional porous scaffolds using carbon dioxide (scCO 2 ) has been used to develop alternative clean processes for the preparation of drug-loaded polymeric matrices when the drug is soluble in scCO 2 and the polymer chosen can be swollen by the supercritical fluid (Mooney et al 1996;Harris et al 1998;Kikic & Sist 2000;Duarte et al 2007;Heyde et al 2007). Impregnation using supercritical fluid technologies has the advantage of using a supercritical fluid with high diffusivity in the polymer chosen in addition to its high solubility and plasticizing capability (Berens et al 1992;Kazarian 2000;Duarte et al 2007).…”

Tissue engineering: strategies, stem cells and scaffolds

“…Once optimized, changing the inserted gene to alter the growth factor produced would allow a range of factors to be produced; however, uptake rates and toxicity are still major issues to this promising technique (Heyde et al 2007). …”

Sterically stabilized self-assembling reversibly cross-linked polyelectrolyte complexes with nucleic acids for environmental and medical applications

“…In our work, we have extensively investigated the linear PAAs as cationic polymers for DNA delivery [7][8][9][10][11]. This is a family of biodegradable polymers produced from a pair of co-monomers, giving a range of possible charge and structural characteristics [12].…”