Fundamental studies on the synthesis, characterization, stabilization, 3-D scaffolds, and trafficking mechanisms of nano-structured calcium phosphates (NanoCaPs) for non-viral gene delivery

Non-viral transfer of therapeutic genes into mammalian cells represents a potentially viable approach to (1) treat and cure acute and chronic genetically transferred congenital disorders and to (2) aid in tissue regeneration. Non-viral vectors have been praised for their potential to circumvent some of the limitations associated with viral vectors including immunogenicity, cytotoxicity and insertional mutagenesis. Among the various types of non-viral gene delivery vectors, nano-structured ceramic particles, particularly, particles of calcium phosphate (CaP) remain an attractive option because of their safety, biocompatibility, biodegradability, ease of handling as well as their adsorptive capacity for DNA. CaP-DNA complexes have been used in vitro since the 1970s and have recently been tested in vivo. However, despite CaPs' extensive use, concerns still remain regarding the synthesis and colloidal instability of this vector. Also, towards the development of a more efficient gene delivery agent, there is a need to understand the mechanisms involved in both the cellular uptake as well as in the subsequent intracellular processing of CaP-DNA complexes. Moreover, although significant advances have been made in the synthesis and design of tissue engineered constructs, the development of a safe, effective scaffold has yet to be realized. As such, the focus of this thesis has been to address these four concerns.;In this work, we begin by presenting a novel aqueous-based approach to synthesize nano-particles of CaP (NanoCaPs). Our results show that this approach generates nano-crystalline hydroxyapatite particles. When tested in vitro, transfection of these complexes resulted in higher, more consistent levels of gene expression when compared to particles synthesized via manual mixing. The optimized forms of these particles both effectively bound (90% efficient) and condensed (70% efficient) plasmid DNA (pDNA) and possessed negative zeta potentials of approximately -20mV.;We have also shown that issues regarding the colloidal instability of this vector can be addressed by lyophilization of the complexes with a lyoprotectant. Our results indicate that complexes lyophilized with sucrose can be stored for up to three weeks at 4°C without significant change in particle size or loss of transfection efficiency.;Also, we incorporated the NanoCaPs complexed with pDNA into a fibrin gel. Prolonged gene expression for up to 5 days in culture was observed using this gene delivery system (GDS). Fiber strand thickness was determined to be dependent upon both the fibrinogen/thrombin ratio as well as the CaCl 2 concentration used.;The uptake and intracellular processing of the NanoCaPs/pDNA complexes were investigated in HeLa and COS-7 cell lines. Our data demonstrated that the nano-particles were internalized via both the clathrin- and caveolae-mediated pathways. Also, our luciferase gene transfection results indicated that both pathways mediated gene delivery of the complexes. These results provide insight into the endocytic trafficking mechanisms involved in CaP-mediated gene delivery and thus will allow us to develop more efficient NanoCaPs based gene delivery vectors in the future.