This proposal describes the synthesis of a general class of nanoparticle delivery vectors based upon hybrid biomolecule-gold nanoparticle complexes. The vectors are designed to transport therapeutic oligonucleotides across cell membranes to target cancer cell nuclei. Therapeutic oligonucleotides are synthetic single stranded nucleic acid molecules that are resistant to digestion by nucleases. A multifunctional approach has been developed which combines cell-specific recognition ligands, endosomal escape peptides, nuclear localization signals and controlled release of therapeutic oligonucleotides to cells. The ability to target biomolecule-nanoparticle complexes to specific cell types, through a relatively simple attachment of cell-specific ligands or peptides, provides the potential for diagnosis and treatment of cancer. A quantitative test for nuclear delivery of an oligonucleotide drug to the nuclei of HeLa cells is proposed as an assay for the efficiency of delivery of a therapeutic agent. The long term goal of this research is to develop a non-viral vector capable of delivering therapeutic oligonucleotides to cancer cells in vivo. Achieving this long term goal requires the following steps. 1. Formulate stable nanoparticle-bioconjugates that are resistant to aggregation and chemical exchange in biological fluids and cells. 2. Develop microscopy techniques to monitor nanoparticle trajectories and quantitate localization in cells. 3. identify the best combination of peptides for performing the functions of endocytosis, endosomal escape and nuclear uptake. 4. Characterize fundamental interactions of protein-peptide conjugates with nanoparticles including their susceptibility to exchange or replacement. 5. Develop strategies for covalent attachment of proteins and oligonucleotides to nanoparticles. 6. Determine of the efficiency of regulated in vitro protein expression using a well-characterized model system.
