Influence of phospholipid composition on cationic emulsions/DNA complexes: physicochemical properties, cytotoxicity, and transfection on Hep G2 cells

Figures & data

Table 1 Physicochemical properties of nanoemulsions and complexes

	Nanoemulsions		Complexes
	Mean diameter* (nm)	Zeta potential (mV)	Mean diameter* (nm)	Zeta potential (mV)
PC	231 ± 22	−36.8 ± 3.4†	334 ± 15	−54.0 ± 1.4
PC/SA	265 ± 14	+53.3 ± 4.8‡	298 ± 3	+52.1 ± 1.6
DSPC/SA	274 ± 27	+67.5 ± 2.3†	294 ± 13	+52.2 ± 3.0
DOPC/SA	272 ± 9	+67.0 ± 2.0	300 ± 17	+61.3 ± 4.5
DSPE/SA	270 ± 14	+67.3 ± 6.4	306 ± 23	+55.5 ± 3.5
DOPE/SA	279 ± 20	+67.5 ± 3.5†	314 ± 8	+57.7 ± 3.8

Notes:

* Results represent the mean ± standard deviation of three experiments;

† Difference between ζ-potential measurements before and after complexation with pDNA (Student’s T-test, P < 0.05);

‡ Difference among ζ-potential measurements of PC/SA compared to isolated phospholipids (One-way analysis of variance, Tukey post-hoc, P < 0.01).

Abbreviations: PC, phosphatidylcholine; SA, stearylamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

Figure 1 Transmission electron micrographs of the complexes formed between nanoemulsions/DNA phosphatidylcholine (A), phosphatidylcholine/stearylamine (B), and 1,2-distearoyl-sn-glycero-3-phosphocholine/stearylamine (C) in the charge ratio of 2.0. 100,000× magnification.

Figure 2 Migration of complexes formed between nanoemulsions/DNA plasmid phosphatidylcholine (A), phosphatidylcholine/stearylamine (B), 1,2-distearoyl-sn-glycero-3-phosphocholine/stearylamine (C), 1,2-dioleoyl-sn-glycero-3-phosphocholine/stearylamine (D), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine/stearylamine (E), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/stearylamine (F) in 1% agarose gel. M = 1 Kb DNA ladder; DNA plasmid = 0.1, 0.5, 1.0, 2.0, and 3.0 charge ratio of cationic nanoemulsions/DNA plasmid.

Figure 3 Migration of complexes formed between nanoemulsions/DNA plasmid phosphatidylcholine (A), phosphatidylcholine/stearylamine (B), 1,2-distearoyl-sn-glycero-3-phosphocholine/stearylamine (C), 1,2-dioleoyl-sn-glycero-3-phosphocholine/stearylamine (D), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine/stearylamine (E), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/stearylamine (F) in 1% agarose gel, after 30 minutes of incubation with DNase I. M = 1 Kb DNA ladder; DNA plasmid (a) = naked DNA plasmid, (b) = DNA plasmid + DNase I 0.1, 0.5, 1.0, 2.0, and 3.0 charge ratio of cationic nanoemulsions/DNA plasmid.

Figure 4 Viability of Hep G2 cells after incubation during 24 hours with nanoemulsions of phosphatidylcholine (⋄), phosphatidylcholine/stearylamine (○), 1,2-distearoyl-sn-glycero-3-phosphocholine/stearylamine (▵), 1,2-dioleoyl-sn-glycero-3-phosphocholine/stearylamine (▴), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine/stearylamine (□) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/stearylamine (■) complexed with DNA plasmid at different charge ratios.

Table 2 Quantification of GFP DNA detected in the transfection assay of Hep G2 cells with the cationic nanoemulsions/pDNA complexes at charge ratio of [+/−] = 2.0 by real-time PCR

Formulation	Amount of GFP DNA pg ± SD
PC*	0.94 ± 0.82
DSPC/SA	38.0 ± 9.4
DSPE/SA	17.0 ± 2.1
DOPC/SA	5.5 ± 0.85
DOPE/SA	4.0 ± 0.64
Lipofectamine 2000^®	49.9 ± 33.6†

Notes:

* The complex with control formulation PC was made with equivalent amount of internal phase of the charge ratio of [+/−] = 2.0;

† Difference in amount of GFP DNA between Lipofectamine 2000^® and DOPC/SA and DOPE/SA (Independent-Samples Kruskal–Wallis Test, P < 0.05).

Abbreviations: GFP, green fluorescent protein; PC, phosphatidylcholine; SA, stearylamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; SD, standard deviation.