Type of Document Dissertation Author Sizovs, Antons URN etd-05162012-160222 Title Development of Carbohydrate-based Diblock Polymers for Nucleic Acid Delivery Degree PhD Department Chemistry Advisory Committee
Advisor Name Title Theresa M. Reineke Committee Chair Carlier, Paul R. Committee Co-Chair Gibson, Harry W. Committee Member Madsen, Louis A. Committee Member Riffle, Judy S. Committee Member Keywords
- Gene Delivery
Date of Defense 2012-05-04 Availability unrestricted AbstractThe delivery of nucleic acids remains the major obstacle for nucleic acid-based therapies such as gene therapy and gene silencing therapies based on RNA interference. In this dissertation we have developed and studied nucleic acid delivery vehicles based on cationic diblock glycopolymers that contain glucosamine and trehalosamine.
Practical procedures were developed to synthesize 2-methacrylamido-2-deoxy glucose and 6-methacrylamido-6-deoxy trehalose starting with commercially available carbohydrates and utilizing trimethylsilyl protecting group chemistry. These monomers were polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization to yield glycopolymers with the desired lengths and low polydispersity indexes. Glycopolymers were chain-extended with aminoethylmethacrylamide to produce cationic diblock copolymers.
The ability of cationic diblock copolymers to bind nucleic acids was demonstrated with gel electrophoresis and heparin exclusion assays. Complexes of the synthesized polymers with nucleic acids were studied with dynamic light scattering to reveal nanoparticles of 100-250 nm that were stable in the presence of serum proteins. Quartz crystal microbalance experiments showed that serum proteins adsorb on polytrehalose coated gold surfaces and it was suggested that these interactions may help mask the polytrehalose coated nanoparticles from potential actions of the immune system. Polytrehalose was also shown to suppress water crystallization similarly to trehalose by lowering the energies associated with the water/ice phase transition. The property was utilized to freeze-dry siRNA containing polyplexes which could be re-dissolved in water after lyophilization to yield nanoparticles.
The polyplexes formulated with cationic diblock copolymers were shown to efficiently enter cervical cancer cells (HeLa cell line) and glioblastoma cells (U-87 cell line) and to deliver their nucleic acid cargo. Polyglucose-containing polymers were efficient mediators of exogenous gene expression in HeLa cells, and polytrehalose- containing polymers were effective in promoting the target gene down-regulation via RNA interference by delivered siRNA.
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