| Description |
Many strides have been made towards utilizing gene therapy to treat genetic disorders in humans. Low gene expression and formulation stability during storage have hindered the use of nonviral carriers thus far. The purpose of this research was to develop a cationic PLGA-b-bPEI micelle-based delivery system to co-deliver genes and small therapeutic drugs to the cell nucleus that could also be lyophilized for long-term storage and reconstituted as needed by simply adding water. Several PLGA-b-bPEI copolymers were synthesized and characterized for their potential to form reconstitutable micelle-based gene therapeutic delivery systems that showed enhanced nuclear delivery over other current cationic nonviral delivery systems. The first section of this dissertation addresses the characterization of a reconstitutable charged polymeric micelle system to assess its suitability for gene therapeutics delivery. A PLGA-b-bPEI block copolymer was synthesized and characterized for buffering capacity, particle size, zeta potential, complexation ability, cytotoxicity, transfection efficiency and reporter gene expression, and retention of physicochemical and biological characteristics upon lyophilization and reconstitution. Results showed that micelles and micelle/pDNA complexes retained their physicochemical characteristics following lyophilization and reconstitution. Transfection levels were enhanced using reconstituted complexes compared to their fresh counterparts without significant cellular toxicity even with pDNA doses up to 20?g, and transfection increased linearly with increasing pDNA dose. The second section of this dissertation investigates the use of dexamethasone-loaded micelles for enhanced nuclear accumulation of a reporter gene. PLGA-b-bPEI polymers synthesized in the first section were used to prepare dexamethasone-loaded micelles and complexed with pDNA. Gene expression was significantly enhanced using dexamethasone-loaded micelle/pDNA complexes compared to blank micelle/pDNA complexes, particularly for complexation weight ratios less than 1. Dex-micelles achieved gene expression equivalent to the blank system using a fraction of the polymer (weight ratio 0.25 versus 1), indicating that dexamethasone increased nuclear accumulation of polyplexes. Gene expression was nearly equivalent to a bPEI control carrier, leading to future plans of co-loading the micelle system with other types of drugs and examining this system's suitability for specific applications such as treating patients suffering from asthma or other inflammatory diseases where dexamethasone treatment could be additionally beneficial for its anti-inflammatory properties. |
