(204b) Disassembly of Layer-by-Layer Films of Plasmid DNA and Reducible Tat Polypeptide

Authors: 
Blacklock, J., Wayne State University
Handa, H., Wayne State University
Soundara Manickam, D., Wayne State University
Mao, G., Wayne State University
Mukhopadhyay, A., Wayne State University
Oupicky, D., Wayne State University


DNA-containing thin films hold much promise for localized gene delivery with applications in the field of implantable materials, biomedical devices, and tissue engineering. Degradable cationic polymers can be bioengineered to sustain the release of DNA for controlled periods of time while functioning in an efficient and timely manner. By incorporating disulfide bonds in the structure of the polycations, degradation of the thin films can be controlled by reducing conditions. Multilayers of DNA and a reducible cationic polypeptide (PTAT) (PTAT/DNA)n have been created utilizing a layer-by-layer assembly. PTAT was prepared by an oxidative polymerization of TAT peptide derived from the transactivating transcriptional activator protein of HIV-1. The TAT peptide with the amino acid sequence CGRKKRRQRRRGC was polymerized via a mild oxidation of the terminal cysteinyl residues with dimethylsufloxide. Atomic force microscopy (AFM) and ellipsometric studies were performed in situ to evaluate the assembly and degradation of (PTAT/DNA)n thin films. (PTAT/DNA)n thickness was found to increase exponentially with the number of layers. The exponential growth is due to the rigidity of DNA as well as the increased presence of particulate complexes found after (PTAT/DNA)8.5 and the mobility of PTAT molecules within the multilayers. Other film features consistent with the exponential film growth mechanism included for example surface roughness increase with increasing number of deposited layers. AFM roughness studies showed that DNA layers have a higher roughness when compared to the PTAT layers. The hydrophobicity, evaluated by the contact angle measurement, shows a periodic variation during the layer-by-layer deposition. It was shown that PTAT-containing films disassemble readily in the reducing environment as well as in the presence of proteolytic enzymes. In contrast, control layer-by-layer films containing poly(L-lysine) were stable in the reducing conditions and exhibited slow degradation in the presence of proteases.