Erschienen:
[Erscheinungsort nicht ermittelbar]: eScholarship, University of California, 2018
Sprache:
Englisch
Entstehung:
Hochschulschrift:
Dissertation, eScholarship, University of California, 2018
Anmerkungen:
Beschreibung:
The efficient and sustained delivery of therapeutic genes in vitro and in vivo has a wide range of applications in studying biology and in developing therapies for treating disease or repairing tissue. Porous hydrogels have been widely used as three-dimensional scaffolds for both cell culture and tissue repair due to their ability to mimic the structural and biochemical properties of native tissue. In addition, therapeutics such as DNA-loaded nanoparticles called polyplexes can be delivered from these scaffolds to infiltrating cells; however, the main challenge plaguing such efforts has been insufficient transgene expression from the delivery of the transgene. It is hypothesized that this is due to significant aggregation of inherently charged polyplexes upon encapsulation in the hydrogel. To mitigate this issue, our laboratory has previously developed a method called caged nanoparticle encapsulation to load polyplexes in porous hyaluronic acid hydrogels at high concentrations; however, low transgene expression has been observed both in vitro and in vivo, prompting the search for alternative methods to enhance non-viral gene delivery from hydrogels. In considering this problem, we first developed two methods of loading polyplexes within porous hydrogel scaffolds and examined mechanisms of polyplex uptake. In the first method, we addressed the hypothesis that the high surface charge of polyplexes triggers aggregation by developing a PEGylated variant of the DNA-complexing polymer polyethyleneimine (PEI) to decrease the surface charge of polyplexes. While this technique did result in decreased polyplex aggregation upon encapsulation in the hydrogel, it also exhibited decreased internalization and transfection efficiency due to the decreased surface charge. In the second method, polyplexes were loaded into hydrogels by coating onto the hydrogel pore surfaces instead of encapsulation to improve DNA availability to infiltrating cells. This presentation resulted in long-term sustained transgene expression in vitro over a period of 30 days, which we concluded was due to the occurrence of re-transfection events. Lastly, we studied how material properties of a new promising class of injectable porous scaffolds known as microporous annealed particle (MAP) scaffolds can affect transgene expression. We found that building block size, stiffness, cell adhesion peptide concentration and presentation, and induced integrin specificity affect transgene expression upon polyplex transfection of cells cultured within MAP scaffolds, and that transfection of cells cultured in MAP scaffolds occur via different mechanisms that that of cells cultured two-dimensionally on tissue culture plastic. These findings provide further insight on how MAP scaffold design can be optimized for enhancing gene delivery.