Access Type

Open Access Dissertation

Date of Award

January 2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemical Engineering and Materials Science

First Advisor

Guangzhao Mao

Second Advisor

David Oupicky

Abstract

This dissertation describes the research of bioreducible polymers for gene delivery. A series of bioreducible poly(amido amine)s (PAAs) were synthesized. They complex with DNA forming polyplex nanoparticles and layer-by-layer (LbL) thin films as gene delivery vectors. Atomic force microscope (AFM), especially in situ real time AFM, provides a microscopic view of DNA release dynamics. It is shown that the depolymerization of bioreducible polymer triggers DNA release via disulfide-thiol exchange reaction. The AFM images revealed a three-stage pathway beginning with a morphological change from metastable nanostructures into the more favorable toroid structure. Then toroids interact with each other by aggregation and fusion. Finally, DNA wormlike chains gradually unravel from the polyplex resulting in loose loops/tails that are held by a central compact core. Polyelectrolyte exchange induced DNA release shares a similar morphological pathway. The transfection efficiency difference could be correlated with DNA release dynamics and polymer structure. On the other hand, the degradation kinetics of PAA/DNA LbL films are modulated by insertion of a non-bioreducible polycation, poly(ethylenimine) (PEI), as a barrier layer and crosslinking of the LbL films using 1,5-diiodopentane. The PEI barrier layer is found to be effective in lowering the degradation rate of the film. Without the PEI barrier layer, the PAA/DNA films undergo fast bulk degradation with micrometer size particles released to the solution. The periodic insertion of the PEI layer changes the PAA/DNA degradation behavior to prolonged surface erosion. Transfection studies on PAA/DNA films with and without the PEI barrier layer are carried out in vitro and in vivo. The cell transfection is further improved by the incorporation of a fibronectin terminal layer and hyaluronic acid. The study of bioreducible polymers and DNA release at molecular level provides stratgies for developing non-viral gene delivery vectors.

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