ABSTRACT
All PPAA-chitosan-DNA nanoparticle formulations were tested for gene transfection efficiency in HEK293 and HeLa cells. Replicate wells of cells (n = 8) were incubated with the nanoparticle formulations for 4 h and assayed for luciferase gene expression 3 days post-transfection. The pre-addition of 0.1 fig PPAA to the nanoparticles enhanced luciferase gene expression in HEK293 cells approx. 1.75 times over the unmodified chitosan-DNA nanoparticles (Fig. 5). This was the only dose in the case of pre-addition that had some enhancing effect on transfection efficiency. The pre-addition of higher doses of PPAA (10, 50 fig) resulted in a significant negative effect on transfection efficiency, which may be due to particle precipitation mentioned earlier. In contrast, the post-addition of PPAA to the nanoparticles enhanced the luciferase expression in HEK293 cells at all PPAA doses compared to unmodified chitosan-DNA nanoparticles, with a broad peak at a dose of 10 fig of PPAA (Fig. 5). Expression levels after the post-addition of 10 fig PPAA were similar to that obtained with the pre-addition of 0.1 fig PPAA. Typically, unmodified
Formulation of chitosan-DNA nanoparticles with PPAA enhances gene expression 481
Figure 4. DNA release from ternary complexes after incubation in serum containing cell culture medium. For all gels, lanes 1, 7 and 13 are naked DNA controls, lanes 2-6 are post-addition PPAA nanoparticles in doses of 50, 10, 1, 0.1 and 0.01 /xg, respectively, lanes 8-12 are pre-addition PPAA nanoparticles in doses of 50, 10, 1, 0.1 and 0.01 fxg, respectively, lane 14 is chitosan-DNA nanoparticles with no PPAA added, (a) DNA release from formulations incubated in PBS for 3 h. Gels for all time points of PBS incubation appear similar to the gel at 3 h, with no additional DNA release. DNA release from nanoparticle formulations after incubation with MEM + 10% FBS is shown for (b) 3 h, (c) 24 h, (d) 72 h and (e) 10 days.
