Figure 1.

DNA/PEI particle coating strategy overview. (a) DNA/PEI particles were formed through mixing DNA/PEI and subsequently coated using hyaluronic acid, either as its non-modified state, or modified with acrylamide (Ac) and norbornene (NB). These nanoparticles are then cryoprotected with sucrose before lyophilizing for concentration and storage. (b) Design of experiment approach to optimizing the coating formulation, from the N/P ratio of PEI to DNA, and the mass ratio of HA to PEI. Parameters were optimized for the particle size and charge to ensure successful transfection, with the dashed line in each plot corresponding to the best N/P ratio, at 20, which was used for all subsequent analyses. (c) Particle stability following reconstitution in buffer, as assessed for 5:1 w/w ratio HA-NB to L-PEI, 87.5 μg sucrose / μg pDNA, at 2.5 μg DNA / μL gel. Freshly prepared DNA/PEI particles were compared to those reconstituted in a 10 μg/mL solution. (d) Transfection of lyophilized coated particles in mouse mesenchymal stem cells. Note that the non-coated (zero mass ratio) condition is a positive control, consisting of freshly prepared DNA/PEI particles. All other mass ratio conditions (1, 3, and 5) are for the lyophilized and reconstituted particles. (e) Viability measured as metabolic activity from PrestoBlue assay for the 2D transfection in (e). For both (d) and (e), the dashed line represents the cell-only background level. X-axis corresponds to HA/PEI coating mass ratios for samples that have been lyophilized and resuspended in 150mM NaCl, except for the “0” control, which are non-coated, freshly prepared DNA/PEI particles as a positive control. N=3, with one-way ANOVA and Tukey’s HSD (p<0.01).