How to study dendriplexes II: Transfection and cytotoxicity.
This paper reviews different techniques for analyzing the transfection efficiencies and cytotoxicities of dendriplexes-complexes of nucleic acids with dendrimers. Analysis shows that three plasmids are mainly used in transfection experiments: plasmid DNA encoding luciferase from the firefly Photinus pyralis, beta-galactosidase, or green fluorescent protein. The effective charge ratio of transfection does not directly correlate with the charge ratio obtained from gel electrophoresis, zeta-potential or ethidium bromide intercalation data. The most popular cells for transfection studies are human embryonic kidney cells (HEK293), mouse embryonic cells (NIH/3T3), SV40 transformed monkey kidney fibroblasts (COS-7) and human epithelioid cervical carcinoma cells (HeLa). Cellular uptake is estimated using fluorescently-labeled dendrimers or nucleic acids. Transfection efficiency is measured by the luciferase reporter assay for luciferase, X-Gal staining or beta-galactosidase assay for beta-galactosidase, and confocal microscopy for green fluorescent protein. Cytotoxicity is determined by the MTT test and lactate dehydrogenase assays. On the basis of the papers reviewed, a standard essential set of techniques for characterizing dendriplexes was constructed: (1) analysis of size and shape of dendriplexes in dried/frozen state by electron or atomic force microscopy; (2) analysis of charge/molar ratio of complexes by gel electrophoresis or ethidium bromide intercalation assay or zeta-potential measurement; (3) analysis of hydrodynamic diameter of dendriplexes in solution by dynamic light scattering. For the evaluation of transfection efficiency the essential techniques are (4) luciferase reporter assay, beta-galactosidase assay or green fluorescent protein microscopy, and (5) cytotoxicity by the MTT test. All these tests allow the transfection efficiencies and cytotoxicities of different kinds of dendrimers to be compared.
Shcharbin
Dzmitry
D
Institute of Biophysics and Cellular Engineering of NASB, Minsk, Belarus. d.shcharbin@mail.ru
Pedziwiatr
Elzbieta
E
Blasiak
Janusz
J
Bryszewska
Maria
M
D000818:Animals; D001681:Biological Assay; D001692:Biological Transport; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D050091:Dendrimers / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics; D009696:Nucleic Acids / Q000737:chemistry / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D050091:Dendrimers; D009696:Nucleic Acids; D009838:Oligodeoxyribonucleotides; D049452:Green Fluorescent Proteins; D012313:RNA; D049409:Luciferases, Firefly; D001616:beta-Galactosidase