Figure 2.

Loading and transfection of lyophilized particles from bulk HA-AC hydrogels. (a) Overview of hydrogel formation and DNA/PEI particle loading, with acrylated HA crosslinked through Michael addition chemistry via an MMP-cleavable dithiol peptide. The precursor solution is mixed directly with the lyophilized or fresh DNA/PEI particles to encapsulate prior to crosslinking. DNA was stained with YOYO-1 dye and imaged on confocal microscopy. (b) Comparison of HA coatings, with non-modified HA, HA-AC, and HA-NB for their top performing conditions from DOE-optimization and 2D transfection studies. Bar plots correspond to nanoparticle size distributions, based on particle analysis in ImageJ (n=3). (c) Further optimization of HA-NB coated particles by altering the sucrose concentration to balance precursor solution viscosity and aggregation within the HA-AC material with increased particle loading. (d) Minimal release of loaded DNA/PEI particles (HA-NB coating, sucrose lyophilization) in PBS solution, with and without collagenase degradation. Plasmids were labelled with P32 for scintillation, while gels were labelled with a fluorescent dye for microplate measurements. (e) Similar to (d), but demonstrating controlled released from hyaluronidase degradation of gels containing DNA/PEI particles. (f) Transfection of cells in 2D cell culture using loaded gels degraded by collagenase and hyaluronidase to release the DNA/PEI particles. (g) Viability data for the transfection in (f). For both (f) and (g), samples were compared for effect of sucrose cryoprotection and HA-NB coating at their optimized conditions (HA/PEI w/w 5, 87.5 μg sucrose / μg DNA, and loading at 2.5 μg/μL gel).