(93a) Contribution of Sustained Release to In Vivo Gene Expression

Tissue engineering strategies are used to enhance the native ability of the body to heal itself. Many strategies are based on the use of polymeric scaffolds to provide mechanical support and promote cell infiltration, combined with the delivery of tissue inductive factors. Gene delivery from scaffolds has the potential to target one or more processes that limit regeneration by delivering DNA encoding for the proteins and factors needed at the local environment for tissue repair. Sustained release strategies have been postulated to enhance gene delivery; however, there has not been direct evidence to relate the release profile to the extent of gene expression. To address this issue, we used layered scaffolds made from PLG microspheres modified with cationic polymers in order to modify the release kinetics of naked DNA from the scaffolds. The layered scaffold increases DNA encapsulation and decouples the DNA delivery properties from the design that supports cell infiltration. Multiple cationic polymers, including PEI, PLL, and dopamine were used to modify the microspheres used to make the scaffolds, which could reversibly associate with the DNA to delay release from the scaffold. The microspheres were characterized for their zeta potential, particle size, and DNA binding capacity. DNA release studies were done using radiolabeled DNA for 14 days. Microspheres modified with dopamine were selected for further in vivo and in vitro studies since they were the most effective in terms of release kinetics and DNA encapsulation within the scaffold. In vitro studies using HEK 293T cells indicated that dopamine does not affect transfection. Dopamine modified and unmodified layered scaffolds loaded with a gene encoding for luciferase were implanted in the abdominal fat of mice for 3, 7 , 14 and 22 days. The release profile had little effect in the quantity and length of luciferase expression. Subsequent studies were done pre-releasing DNA from the scaffolds, and drying the DNA onto the scaffold, since it affects delivery kinetics. Trends similar to those obtained initially were obtained. In general, the studies suggest that the amount of DNA initially available for transfection is critical to achieve gene expression, and DNA available at later time points does not extend gene expression. These results have multiple implications in the future design of scaffolds for gene delivery, and the gene expression obtained from these scaffolds have potential for cell transplantation and future gene delivery applications.