Figure 4.

Nucleic acid incorporation into FLIP scaffolds. (a) Process of converting DNA/PEI particle-loaded bulk HA-AC gels to sHMP, again using the sieving process, and then annealing the microgels together into the microporous FLIP scaffold. (b) Retention of lyophilized DNA/PEI particles within FLIP scaffolds for different sieve sizes and the HA-NB coated particles. (c-e) Annealing and mechanical properties of FLIP scaffolds loaded with DNA/PEI particles. (c) Jamming study to obverse annealing time from Q/K peptides FXIII crosslinking. (d) Shear rheology on annealed gels following 1 hour incubation at 37°C to derive the storage (G’) and loss (G”) moduli. (e) Comparison of loaded and non-loaded annealed gels for Young’s modulus from microstrain compression testing. Values and error bars correspond to S.D, n=3. (f) Overview of 3D transfection process, with loading coated, lyophilzied DNA/PEI particles into bulk gels, converting to sHMP, and annealing into FLIP within a custom 3D cell culture chamber. Cells are then seeded on top or within the scaffold for exposure to the microgels and degradation to release the DNA/PEI particles. Transfection is measured based on GLuc and GFP reporter genes. (g) Lyophilized coated DNA/PEI particles and FLIP transfection in mouse mesenchymal stem cells. The dashed line represents the background levels from the cell-only negative control, while “bolus” is positive control transfected cells from fresh DNA/PEI particles when seeded into the gels (the equivalent of cells being exposed to all of the particles at once). The w/w HA/PEI represents different coating ratios by weight and are all samples that have been lyophilized and resuspended in the hydrogel HA-AC precursor solution. n=3-5, with one-way ANOVA and Tukey’s HSD (p<0.01). (h) Viability is also improved by the HA-NB coating, when compared to bolus transfection from fresh DNA/PEI particles as a control. (i) Comparison of FLIP from sHMP of different sieve size, loaded with HA-NB coated particles. (j) Time study of loaded FLIP scaffolds (HA-NB coated DNA/PEI particles, 2.5 μg/μL), of D1 MSCs, with GLuc expression monitored every 2-3 days post seeding, compared to a bolus transfection. Loaded FLIP gave sustained transfection levels over the month long study, while bolus dissipated over time, despite higher overall RLU levels. n=5, with one-way ANOVA and Tukey’s HSD (p<0.01). For all figures, p <0.05 (*), <0.01 (**), <0.005 (***), and <0.001 (****).