Improving transfection efficiency of ultrapure oligochitosan/DNA polyplexes by medium acidification

Figures & data

Table 1. Experimental trials and interactions transformed into code units.

X1	X2	X3	X1X2	X1X3	X2X3	X1X2X3	Response
−1	−1	−1	+1	+1	+1	−1
+1	−1	−1	−1	−1	+1	+1
−1	+1	−1	−1	+1	−1	+1
+1	+1	−1	+1	−1	−1	−1
−1	−1	+1	+1	−1	−1	+1
+1	−1	+1	−1	+1	−1	−1
−1	+1	+1	−1	−1	+1	−1
+1	+1	+1	+1	+1	+1	+1

Table 2. Experimental trials and response in terms of size and zeta potential for O15 and O25 UOC.

			X3 [NaCl]	Size (nm)		Zeta (mV)
Trial	X1 pH	X2 N/P	(mM)	O15	O25	O15	O25
1	6.2	10	10	89.43	337.8	19.5	29.7
2	7.2	10	10	555.9	204.6	3.99	9.2
3	6.2	60	10	135.1	101.7	21.8	21.6
4	7.2	60	10	144.5	159.2	10.6	10.8
5	6.2	10	150	182.6	88.76	14.9	15.1
6	7.2	10	150	333.1	369.4	0.174	6.28
7	6.2	60	150	113.2	244.2	15.8	22.4
8	7.2	60	150	208.3	154.2	5.8	2.87

Table 3. Effects and interactions on size and zeta potential of the UOC/pCMS-EGFP polyplexes.

	Size (nm)		Zeta (mV)
Effects and interactions	O15	O25	O15	O25
X1e	180.37	28.73	−12.86	−14.91
X2e	−139.98	−85.31	3.86	−0.65
X3e	−21.93	13.31	−4.80	−6.16
X1X2i	−128.12	−44.98	2.26	−0.25
X1X3i	−57.57	66.58	0.50	0.73
X2X3i	42.88	55.43	−0.60	2.60

X₁e, pH effect; X₂e, N/P effect; X₃e, [NaCl] effect; X₁X₂i, pH–N/P interaction; X₁X₃i, pH–[NaCl] interaction; X₂X₃i, N/P–[NaCl] interaction.

Figure 1 Acid–base titration profiles of O15 UOC, O25 UOC and G214 chitosan in 150 mM NaCl solution.

Figure 2 EGFP expression and cell viability in HEK293 cells after the transfection with O15 and O25 UOC/pCMS-EGFP polyplexes at N/P ratios from 10 to 60. Transfection data were normalized to Lipofectamine 2000™.

Figure 3 Size and zeta potential of O15 and O25 UOQ/pCMS-EGFP and G214/pCMS-EGFP polyplexes (N/P ratio of 20) at pH 7.4 and 6.5.

Figure 4 Cellular uptake of FITC-labeled pCMS-EGFP delivered by UOC polyplexes (N/P = 20). Percentage of FITC-positive cells treated with O15 and O25 UOC and G214 chitosan at pH 6.5 and 7.4 in HEK293 (A1) and ARPE19 (A2) cell lines. Fluorescent images of FITC-pCMS-EGFP uptake: O15 UOC/FITC-pCMS-EGFP at pH 7.4 in HEK293 (B1); O25 UOC/FITC-pCMS-EGFP at pH 7.4 in HEK293 (B2); G214/FITC-pCMS-EGFP at pH 7.4 in HEK293 (B3); O15 UOC/FITC-pCMS-EGFP at pH 6.5 in HEK293 (B4); O25 UOC/FITC-pCMS-EGFP at pH 6.5 in HEK293 (B5); G214/FITC-pCMS-EGFP at pH 6.5 in HEk293 (B6); O15 UOC/FITC-pCMS-EGFP at pH 7.4 in ARPE19 (B7); O25 UOC/FITC-pCMS-EGFP at pH 7.4 in ARPE19 (B8); G214/FITC-pCMS-EGFP at pH 7.4 in ARPE19 (B9); O15 UOC/FITC-pCMS-EGFP at pH 6.5 in ARPE19 (B10); O25 UOC/FITC-pCMS-EGFP at pH 6.5 in ARPE19 (B11); G214/FITC-pCMS-EGFP at pH 6.5 in ARPE19 (B12).

Figure 5 Transfection efficiency of O15 and O25 UOC/pCMS-EGFP polyplexes (N/P = 20) at different pH values in HEK293 (A1) and ARPE19 cells (A2). Transfection data were normalized to Lipofectamine 2000™. Flow cytometry histograms (FL1/events) comparing transfection efficiencies at pH values of 6.5 and 7.4: HEK293 cells transfected with O15 UOC/pCMS-EGFP polyplexes (B1); HEK293 cells transfected with O25 UOC/pCMS-EGP polyplexes (B2); ARPE19 cells transfected with O15 UOC/pCMS-EGFP polyplexes (B3); ARPE19 cells transfected with O25 UOC/pCMS-EGFP polyplexes (B4). Overlay of phase contrast image with fluorescent illumination (GFP channel) to evaluate transfection efficiency: HEK293 cells transfected with O15 UOC vectors at pH 7.4 (C1); HEK293 cells transfected with O15 UOC vectors at pH 6.5 (C2); HEK293 cells trasnfected with O25 UOC vectors at pH 7.4 (C3); HEK293 cells transfected with O25 UOC vectors at pH 6.5 (C4); ARPE19 cells transfected with O15 UOC vectors at pH 7.4 (C5); ARPE19 cells trasnfected with O15 UOC vectors at pH 6.5 (C6).

Figure 6 Cell viability and morphology of HEK293 and ARPE19 cells after transfection with O15 and O25 UOC vectors (N/P = 20) and Lipofectamine 2000™. Influence of pH on cell viability of HEK293 and ARPE19 cells by CCK-8 assay (A). Phalloidin and DAPI staining of HEK293 and ARPE19 cells after 4 h transfection; HEK293 cells transfected with O25 UOC vectors at pH value of 6.5 (B1); HEK293 cells tranfected with O25 UOC vectors at pH value of 7.4 (B2); HEK293 cells transfected with Lipofectamine 2000™ (B3); ARPE19 cells transfected with O25 UOC vectors at pH value of 6.5 (B4); ARPE19 cells transfected with O25 UOC vectors at pH value of 7.4 (B5); ARPE 19 cells transfected with Lipofectamine 2000™ (B6).