Bioreducible and acid-labile poly(amido amine)s for efficient gene delivery

Figures & data

Table 1 Synthesis and characterization of poly(amido amine)s

Polymers	Monomers (equiv)				Reaction time (h)	Ketal (mol %)		Mn (GPC)	Mw/Mn
	CBA	HMBA	KDA	HDA		In feed	In polymer
NonR	0	1	0	1	48	0	0	24900	1.36
AcidR	0	0	1	1	96	0	0	16800	1.54
RedoxR	1	0	0	1	48	0	0	26800	1.50
DualRl	1	0	1	0	96	100	100	19000	1.83
DualR2	1	0	0.75	0.25	96	75	73	21700	1.70
DualR3	1	0	0.50	0.50	96	50	48	23200	1.72
DualR4	1	0	0.25	0.75	96	25	26	25400	1.57

Abbreviations: CBA, N,N′-cystaminebisacrylamide; HDA, hexamethylene diamine; HMBA, hexmethylenebisacrylamide; KDA, [1,1′-(2,2′-(propane-2,2-diylbis(oxy)) bis(ethane-2,1-diyl))diurea]; GPC, gel permeation chromatography; NonR, non-responsive; AcidR, acid responsive; RedoxR, redox responsive; DualR, dual responsive.

Figure 1 Synthetic scheme of dual-responsive poly(amido amine)s.

Abbreviations: CBA, N,N′-cystaminebisacrylamide; HDA, hexamethylene diamine; HMBA, hexmethylenebisacrylamide; KDA, [1,1′-(2,2′-(propane-2,2-diylbis(oxy)) bis(ethane-2,1-diyl))diurea]; NonR, non-responsive; AcidR, acid responsive; RedoxR, redox responsive; DualR, dual responsive.

Figure 2 ¹H-nuclear magnetic resonance spectra of DualR1, DualR2, DualR3, DualR4, RedoxR, and AcidR in d₆-dimethylsulfoxide, and NonR in D₂O.

Abbreviations: NonR, non-responsive; AcidR, acid responsive; RedoxR, redox responsive; DualR, dual responsive.

Figure 3 Hydrolysis kinetics of DualR1 at pH of 5.0 and pH of 7.4.

Figure 4 DNA condensation efficiency of various DNA/poly(amido amine) polyplexes at different N/P ratios.

Abbreviations: NonR, non-responsive; AcidR, acid responsive; RedoxR, redox responsive; DualR, dual responsive.

Figure 5 (A) Particle sizes and (B) zeta potential of various DNA/poly(amido amine) polyplexes at different N:P ratios.

Figure 6 Agarose gel electrophoresis of various DNA/poly(amido amine) polyplexes.

Table 2 Cytotoxicity of poly(amido amine)s in three cell lines

Polymers	IC50 (μg/mL)
	HePG2	A549	MDA-MB-468
PEI	12.3 ± 9.7	10.2 ± 11.6	7.8 ± 6.7
NonR	27.9 ± 5.5	20.2 ± 7.6	9.2 ± 6.6
AcidR	227.4 ± 39.9	190.4 ± 90.2	116.2 ± 55.2
RedoxR	93.2 ± 13.6	42.6 ± 26.8	75.3 ± 30.4
DualR1	618.3 ± 499.5	492.0 ± 210.0	292.3 ± 82.9
DualR2	310.9 ± 74.3	319.0 ± 49.3	176.5 ± 70.7
DualR3	199.1 ± 54.2	161.4 ± 82.6	159.0 ± 77.2
DualR4	131.6 ± 23.8	93.5 ± 56.4	126.3 ± 79.0

Abbreviation: PEI, polyethylenimine.

Figure 7 Luciferase expression of DNA/poly(amido amine) complexes in HePG2 cells.

Abbreviations: PEI, polyethylenimine; RLU, relative light units.

Figure 8 Fluorescence microscopy of green fluorescent protein transfection of DNA/poly(amido amine) polyplexes.

Abbreviation: PEI, polyethylenimine.

Figure 9 Green fluorescent protein expression of DNA/PAA polyplexes on flow cytometry.

Abbreviations: PAA, poly(amido amine)s; PEI, polyethylenimine.

Figure S1 Synthetic scheme of [1,1′-(2,2′-(propane-2,2-diylbis(oxy) bis(ethane-2,1-diyl))diurea] (KDA).

Figure S2 ¹H nuclear magnetic resonance spectrum of compound 1 in DCCl₃.

Figure S3 ¹H nuclear magnetic resonance spectrum of compound 2 in d₆-acetone.

Figure S4 ¹H nuclear magnetic resonance spectrum of [1,1′-(2,2′-(propane-2,2-diylbis(oxy)) bis(ethane-2,1-diyl))diurea] (KDA) compound in DCCl₃.

Figure S5 ¹H nuclear magnetic resonance spectrum of N,N′-cystaminebisacrylamide (CBA) in d₆-dimethylsulfoxide.

Figure S6 ¹H nuclear magnetic resonance spectrum of hexmethylenebisacrylamide (HMBA) in d₆-dimethylsulfoxide.

Figure S7 ¹³C Nuclear magnetic resonance spectrum of branched poly(amido amine)s.

Figure S8 ¹³C Nuclear magnetic resonance spectrum of poly(amido amine)s.

Figure S9 Gel permeation chromatography of DualR1 before (up) and after (down) incubation with 20 mM DTT.

Figure S10 Luciferase expression in DNA/poly(amido amine) polyplexes for A549 cells.

Abbreviations: PEI, polyethylenimine; RLU, relative light units.

Figure S11 Luciferase expression in DNA/poly(amido amine) polyplexes for MDA-MB-468 cells.

Abbreviations: PEI, polyethylenimine; RLU, relative light units.