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Covalent Tethering of Plasmid DNA for Substrate-Mediated Gene Delivery

Kory M. Blocker1, Kristi L. Kiick2, and Millicent O. Sullivan1. (1) Chemical Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, (2) Materials Science and Engineering, University of Delaware, 201 DuPont Hall, Newark, DE 19716

Gene delivery enables mammalian cell manipulation necessary for disease treatment, tissue development, and comprehension of biochemical functions and cellular response.1 Currently, non-viral gene delivery is accomplished by bolus delivery of cationic polymer-complexed DNA, which is limited by mass transport and deactivation processes. For tissue engineering applications, incorporation of DNA onto biomaterial scaffolds may avoid these limitations and increase cell transfection by maintaining a high concentration of DNA in the cellular microenvironment.2 Surface immobilization of DNA also allows spatial targeting of cells.2,1 Presently, DNA immobilization to a surface is dependent upon molecular interactions between DNA packaging materials and the surface, and requires careful design to support cellular uptake of the DNA.3 As an alternative, covalent binding of the DNA to a substrate via a labile peptide sequence would allow surface immobilization and greater control over cellular transfection. Our design focuses on chemical functionalization of plasmid DNA to form the vector-surface covalent bond. This is accomplished using a peptide nucleic acid (PNA) and peptide-based surface tethering system. PNA is a DNA analog that sequence-specifically hybridizes with DNA to form a highly stable conjugate but does not interfere with the DNA transcriptional activity. DNA-PNA-peptide (DPP) conjugates may be directly linked to a variety of biomaterial surfaces. The use of coupling peptides that include cell adhesive and matrix metalloproteinase-1 (MMP-1) degradable sequences4 enables release and uptake of the complexes in a cell-responsive manner.

Formation of the DPP conjugate was validated using agarose gel electrophoresis and peptide estimation assays. After formation, the conjugates were attached to self-assembled monolayer-functionalized gold model surfaces.5 Atomic force microscopy was used to validate attachment and to observe the structures of the attached conjugates. The MMP-1 sensitivity of the coupling peptide has been studied with reverse phase HPLC. We are currently exploring the kinetics of conjugate release upon exposure to enzyme. After further validation of the tethering system via surface analysis techniques as well as cellular transfection studies, this chemistry will be translated from 2D model scaffolds to 3D tissue engineering scaffolds, and will be employed to promote the correct cellular responses necessary for tissue engineering.6

1) Gersbach, C.A. et al. Biomaterials 2007; 28; 5121-5127.

2) Segura, T. et al. Journal of Controlled Release 2003; 93; 69-84.

3) Jang, J. et al. Journal of Biomedical Materials Research Part A 2005; 77A; 50-58.

4) Lutolf, M.P. et al. Advanced Materials 2003; 15; 888-892.

5) Pannier, A.K. et al. Acta Biomaterialia 2005; 1; 511-522.

6) Segura, T. et al. Biomaterials 2005; 26; 1575-1584.