Graphene oxide (GO) is employed in a broad range of biomedical applications including antimicrobial therapies, scaffolds for tissue engineering, and drug delivery, among others. However, the inability to load it efficiently with double-stranded DNA impairs its use as a gene delivery system. To overcome this limitation, in this work, the functionalization of GO with cationic lipids (CL) is proficiently accomplished by microfluidic manufacturing. To this end, we use CLs 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and {3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]} cholesterol (DC-Chol) and zwitterionic dioleoylphosphatidylethanolamine and cholesterol to generate a library of 9 CL formulations with systematic changes in lipid composition. Combined dynamic light scattering, microelectrophoresis, and atomic force microscopy reveal that graphene oxide/cationic lipid (GOCL) nanoparticles (NPs) are positively charged and uniformly coated by one lipid bilayer. GOCL NPs are able to condense plasmid DNA into stable, nanosized complexes whose size and zeta-potential can be finely tuned by adjusting the DNA/GOCL weight ratio, Rw. Luciferase assay results show that positively charged GOCL/DNA complexes (Rw = 0.2) efficiently transfect HeLa cells with no appreciable cytotoxicity. In particular, the ternary GOCL formulation made of DOTAP, DC-Chol, and Cholesterol (GOCL8) is as efficient as Lipofectamine® 3000 in transfecting cells, but much less cytotoxic. Confocal microscopy clarifies that the high transfection efficiency of GOCL8 is due to its massive cellular uptake and cytosolic DNA release. Implications for nonviral gene delivery applications are discussed.
Microfluidic-generated lipid-graphene oxide nanoparticles for gene delivery
Di Santo R.;
2019-01-01
Abstract
Graphene oxide (GO) is employed in a broad range of biomedical applications including antimicrobial therapies, scaffolds for tissue engineering, and drug delivery, among others. However, the inability to load it efficiently with double-stranded DNA impairs its use as a gene delivery system. To overcome this limitation, in this work, the functionalization of GO with cationic lipids (CL) is proficiently accomplished by microfluidic manufacturing. To this end, we use CLs 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and {3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]} cholesterol (DC-Chol) and zwitterionic dioleoylphosphatidylethanolamine and cholesterol to generate a library of 9 CL formulations with systematic changes in lipid composition. Combined dynamic light scattering, microelectrophoresis, and atomic force microscopy reveal that graphene oxide/cationic lipid (GOCL) nanoparticles (NPs) are positively charged and uniformly coated by one lipid bilayer. GOCL NPs are able to condense plasmid DNA into stable, nanosized complexes whose size and zeta-potential can be finely tuned by adjusting the DNA/GOCL weight ratio, Rw. Luciferase assay results show that positively charged GOCL/DNA complexes (Rw = 0.2) efficiently transfect HeLa cells with no appreciable cytotoxicity. In particular, the ternary GOCL formulation made of DOTAP, DC-Chol, and Cholesterol (GOCL8) is as efficient as Lipofectamine® 3000 in transfecting cells, but much less cytotoxic. Confocal microscopy clarifies that the high transfection efficiency of GOCL8 is due to its massive cellular uptake and cytosolic DNA release. Implications for nonviral gene delivery applications are discussed.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
