Nanomedicine: Targeted nanoparticles for the delivery of biosensors and therapeutic genes

Nanomedicine is a concept that embodies a multidisciplinary approach to treating disease that enables individual cells to treat themselves in response to a pathogenic stimuli. The goal of this dissertation was to design and test the three major components of nanomedicine: targeted nanoparticle gene delivery; pathogen activated biosensor; and inducible therapeutic genes. These components were to be tested individually, in vitro. To this end, three nanoparticle systems were developed: layer by layer, semiconductor based, and magnetic nanoparticles. Layer by layer constructed nanoparticles were capable of targeted gene delivery via sugar coated outer layers. Inner layers contained alternating charged species, including DNA. Non-targeted gene delivery via lipid coated nanoparticles was found to be more efficient, but also more sensitive to nanoparticle cluster size. Semiconductor based nanocrystals were used as fluorescent markers during targeting studies. These <20nm particles could be found within cells even without targeting molecules, but targeting with arginine rich peptides and antibodies showed the greater cellular penetration. Magnetic nanoparticles were easy to purify and bioconjugate. Amplified DNA fragments containing a 5' biotin were easily bioconjugated to the surface of these particles (∼50nm). Lipid coating the DNA tethered magnetic nanoparticles greatly facilitated transfection. Gene expression from these nanoparticles was similar to transfected DNA fragments. Additional experiments demonstrated that the DNA tethered magnetic nanoparticle could be extracted and purified from an individual cell. Hepatitis C virus biosensors were able to detect and report the presence of a subgenomic replicon. A reactive oxygen species sensitive promoter based biosensor was obtained and a stable cell line produced. This cell line was found to be sensitive to chemical induction of reactive oxygen species. A targeted ribozyme gene therapy was constructed and tested in vitro. This ribozyme was targeted to the Hepatitis C virus internal ribosome entry site. The ribozyme cassette was cloned downstream of a tetracycline response element repeat. This construct appeared to cleave a Hepatitis C virus internal ribosome entry site that was transcribed in vitro. Later studies with a subgenomic Hepatitis C virus replicon showed promising results.