The overall goal for this project is to elucidate the mechanisms by which organic nanoparticles of controlled shape, controlled site-specific surface chemistry, tunable particle matrix composition and tunable modulus undergo endocytosis and to use these findings to engineer the intracellular release of siRNA within mammalian cells to achieve effective gene knockdown. This information, in combination with on-going efforts to understand the bio-distribution of shape controlled particles, will help to establish the rules for the rational design of nano-carriers for the effective in vivo delivery of siRNA. This will be accomplished using a unique particle synthesis method developed at the University of North Carolina called PRINT, Particle Replication in Non- wetting Templates. PRINT is an off-shoot of the emerging lithographic processes used to fabricate devices in the microelectronics industry. In aims 1 and 2, the effect of nanoparticle composition, size, shape, surface charge and ligand choice on the cellular uptake of non-targeted and targeted nanoparticles will be examined. The kinetics of cellular internalization of the nanoparticles and the effect of charge and the spatial arrangement and the density of the ligands on the particle surface will be investigated with regard to specific pathways for cellular internalization of particles. Aim 3 will explore the rational design of PRINT nanoparticles for non-targeted and targeted in vitro delivery of siRNA. As the PRINT particles enter a cell, the particulate carriers will release the siRNA based on a stimuli induced biological or chemical degradation mechanism. The effectiveness of intracellular delivery monitored by luciferase gene silencing will be evaluated as a function of particle matrix composition, particle size and shape and pathway of internalization. Aim 4 will explore rational design of PRINT nanoparticles for targeted in vivo delivery of siRNA. The PRINT nanoparticles with encapsulated anti-luciferase siRNA will be decorated with cell specific ligands (folate, transferrin) and intravenously injected into tumor bearing mice. The efficacy and efficiency of siRNA delivery to the tumor will be evaluated as a function of particle surface and matrix chemistries. Understanding the detailed interplay between particle surface chemistry and shape on effective transfection both in vitro as well as in vivo is of significant importance. PUBLIC HEALTH RELEVANCE: Small interfering RNA (siRNA) has the potential to revolutionize the treatment of a number of life threatening human diseases, particularly cancer, but one obstacle facing researchers and companies attempting to develop siRNA therapies, or any type of nucleic acid therapeutic, is efficient and specific delivery of the polyanionic molecules into the cells, tissues or organ systems of choice. Using a technique known as PRINTTM (Particle Replication in Non-wetting Templates), we are able to fabricate nanoparticles with precise control over the particle size, shape, composition, cargo and surface properties to create truly engineered drug therapies that can be used to overcome the obstacles facing researchers and to provide the tools for the rational design of nano-carriers for the effective delivery of therapeutics in vitro and in vivo.