Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency

Abstract

Background: Physicochemical characteristics of liposome/DNA complexes influence transfection efficiency and affect each other in a very intricate way. The result of this is discrepancies in conclusions drawn about the individual influence of each one.

Methods: Aiming to elucidate the influence of liposome/DNA charge ratio and size on transfection efficiency and on each other, we used liposome/DNA complexes with charge ratio (+/-) in the range of 1-50 and extruded through membranes of 400, 200, and 100 nm. Plasmid DNA encoding green fluorescent protein was used to measure transfection efficiency by flow cytometry. Sizes of liposome/DNA complexes were measured by dynamic light scattering.

Results: Liposome size was reduced after extrusion but this was mainly driven by the charge ratio and not by the size of the membrane pores. Reduction of complex size at each charge ratio positively correlated with transfection efficiency. When the size of the complexes was approximately constant, increasing the charge ratio was found to promote transfection efficiency. Cationic lipid N-(1-(2,3-dioleoyloxy)propyl)N,N,N trimethylammonium chloride was used for modulation of positive charge and a cytotoxicity test showed that increasing its amount increases cytotoxicity.

Conclusion: It can be concluded that charge ratio dictates the size of the complex whereas overall size reduction and higher charge ratios promote transfection efficiency in vitro.

Keywords: liposome charge; liposome size; transfection efficiency.

Figure 1

Size of PC-DOPE-DOTAP/pEGFP complexes measured by dynamic light scattering. Notes: Results are expressed as z-average (av) or as size of major population from number distribution (nu). (A) Size of extruded complexes. (B) Size of unextruded complexes. Results are expressed as mean ± standard deviation of three separate experiments. Each sample was measured three times. Abbreviations: PC, phosphatidylcholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP, N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride; pEGFP, plasmid (encoding) enhanced green fluorescent protein.

Figure 2

Transfection efficiency of lipoplexes and electroporation in different cell types. Notes: Liposomal formulation: PC-DOPE-DOTAP/pEGFP complex, charge ratio 50, extruded through 100 nm membranes (light gray columns); electroporation (dark gray columns). Results are expressed as mean ± standard deviation of at least three separate experiments. Abbreviations: PC, phosphatidylcholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP, N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride; pEGFP, plasmid (encoding) enhanced green fluorescent protein.

Figure 3

Influence of composition, size, and charge ratio on transfection efficiency of complexes in 293T cells. Notes: Lipoplexes were prepared by hydration of thin lipid film with pEGFP solution (light gray columns), which were extruded through membranes with 100 nm pores (dark gray columns). Results are expressed as mean ± standard deviation of at least three separate experiments. Number in brackets denotes the charge ratio. Abbreviations: PC, phosphatidylcholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP, N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride; pEGFP, plasmid (encoding) enhanced green fluorescent protein; CHOL, cholesterol.

Figure 4

Cytotoxicity of complexes determined by MTT assay. Notes: Results are expressed as mean ± standard deviation of at least three separate experiments. Number in brackets denotes the charge ratio. Abbreviations: PC, phosphatidylcholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP, N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride; pDNA, plasmid DNA; CHOL, cholesterol.