195. Tat Peptide Conjugates of Low Molecular Weight Polyethylenimine as Effective Non-Viral Gene Delivery Vectors

Abstract

Tat peptide derived from the arginine-rich basic domain of the HIV transactivator of transcription are capable of mediating intracellular delivery of various types of drugs, including large biomolecules and nanoparticulate drug carriers. For gene delivery, several recent publications reported that Tat peptide-mediated transfection in cultured cells is strongly dependent on the addition into the culture medium of chloroquine, a weak base that inhibits fusion of the endosome with the lysosome by buffering the lysosome interior. While effective in vitro, the chloroquine co-treatment is not a feasible approach for in vivo gene delivery application. We reasoned that protonable amines in polyethylenimines (PEIs) could be used to replace chloroquine. We developed a solid-phase chemical synthetic method to synthesize a peptide-polymer hybrid. PEI600 was conjugated to the last glycine residue of Tat peptide on a resin support right after solid phase peptide synthesis when the side chains of other peptide residues were still protected. The PEI600-Tat conjugates could bind to and fully condense plasmid DNA, as demonstrated in a DNA retardation assay and particle size measurement. The biophysical parameters of the PEI600-Tat/DNA complexes were similar to those of DNA complexes compacted with PEI 25kDa, the gold standard for nonviral gene carriers. Transfection experiments demonstrates that the use of PEI600-Tat conjugates was more effective than the use of the two compounds without chemical conjugation and that the P600-Tat conjugate and PEI 25kDa provided similar levels of transfection efficiency in cultured cells. Attractively, the newly developed conjugates maintain the desirable property of low cytotoxicity displayed by lower molecular weight PEI polymers and Tat peptides. Thus, a new polymer-peptide hybrid was prepared using a versatile methodology that couples synthetic polymers directly to resin-supported peptides. The developed hybrid biomaterial takes advantage of the unique features associated with the two original cationic materials and functions as a novel gene delivery vector with good biocompatibility.