Gene delivery using injectable hydrogels can serve as a potential method for regulated tissue regeneration in wound healing. Our microporous annealed particle (MAP) hydrogel has been shown to promote cellular infiltration in both skin and brain wounds, while reducing inflammation. Although the scaffold itself can promote healing, it is likely that other signals will be required to promote healing of hard-to-treat wounds. Gene delivery is one approach to introduce desired bioactive signals. In this study, we investigated how the properties of MAP hydrogels influence non-viral gene delivery of polyethylenimine-condensed plasmid to cells seeded within the MAP gel. From past studies, we found that gene transfer to cells seeded in tissue culture plastic differed from gene transfer to cells seeded inside hydrogel scaffolds. Since MAP scaffolds are generated from hydrogel microparticles that are approximately 100 μm in diameter, they display local characteristics that can be viewed as two-dimensional or three-dimensional to cells. Thus, we sought to study if gene transfer inside MAP scaffolds differed from gene transfer to cells seeded in tissue culture plastic. We sought to understand the roles of the endocytosis pathway, actin and microtubule dynamics, RhoGTPases, and YAP/TAZ on transfection of human fibroblasts.