Organosilicate nanoparticles as gene delivery vehicles for bone cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2004.Includes bibliographical references.(cont.) proliferation. The metabolic response of these cells after particle ingestion was also characterized in order to ensure that the osteoblasts retained their phenotype. The expression of various proteins involved in bone formation, such as alkaline phosphatase, osteocalcin, osteopontin and fibronectin was quantified. The results indicated that osteoblasts retained their phenotype after organosilicate nanoparticle ingestion. The levels of various cytokines expressed during inflammatory response remained low due to the biocompatibility of amorphous silica. An optimized Ca-SiO₂ nanoparticulate system was developed with maximum particle uptake that enhanced cell viability. A model gene delivery system was created by complexing these nanoparticles with plasmid DNA that encoded for green fluorescent protein (GFP). The effects of nanoparticle size, composition and surface charge on complex size, DNA binding affinity and subsequent GFP expression in osteoblasts were investigated in detail. In addition to primary and immortalized osteoblasts, we have studied the effect of this gene delivery system on two other control cell lines: fibroblasts (connective tissue cells) and hepatocytes (non-connective tissue cells). The Ca-SiO₂-DNA complexes displayed significantly higher transfection efficiencies in osteoblasts and fibroblasts relative to hepatocytes compared to lipofectamine-DNA complexes. In addition, Ca-SiO₂-DNA complexes enhanced osteoblast cell proliferation while achieving successful transfection ...While bone has a substantial capacity to heal itself, there are approximately 1 million fractures that occur in the U.S. annually that are difficult to heal. These include fractures that occur at sites of low vascularity, fractures that result in a large amount of tissue loss, and fractures that result from bone fragility syndromes such as osteoporosis. There has been a great deal of interest in the tissue engineering of bone in order to treat such fractures. One major aspect of the tissue engineering approach involves the addition of growth factors or proteins to synthetic grafts to accelerate bone regeneration. However, delivering these proteins at the appropriate times and therapeutic levels poses significant challenges. Alternatively, delivering the genes that encode for these proteins could offer a more effective treatment, since proper incorporation of the appropriate genes into cellular nuclei would allow the cells to manufacture authentic protein products. The motivation of this research was to design new materials for gene delivery to bone cells. Conventional non-viral vectors are plagued by toxicity and low transfection efficiencies. The purpose of this work was to design bioactive nanoparticles that could enter the osteoblast membrane without inducing toxicity. These materials were silicate-based, since doped silicates have been shown to possess osteogenic properties. A method to synthesize monodisperse, spherical organosilicate nanoparticles using a surfactant-stabilized sol-gel technique was developed. Surface dopants such as Ca, Mg and Na were found to influence cellular response to nanoparticles. In addition to particle composition, particle size was also found to have a significant effect on osteoblast uptake and cellby Suniti Moudgil.Ph.D