A modified protocol for efficient DNA encapsulation into pegylated immunoliposomes (PILs).
Effective delivery of transgenes to the brain through a non-invasive route has great prospects for treating diseases in the central nervous system (CNS). Slightly anionic pegylated immunoliposomes (PILs) have been shown to be effective in reaching the CNS, but efficient DNA encapsulation into the liposomes used for this purpose is technically difficult and hard to reproduce. We here use an improved protocol for DNA encapsulation of pegylated immunoliposomes based on ethanol-mediated DNA condensation. We introduce a dialysis step following DNA encapsulation to remove ethanol and show that this step is necessary to ensure complete nucleolytic removal of non-encapsulated DNA. The uptake of the pegylated immunoliposomes into human cells was documented by live-cell confocal imaging, and specific targeting to the human insulin receptor was shown by inhibiting clathrin-mediated endocytosis.
Skjørringe
Tina
T
Kennedy Center, Gl. Landevej 7, 2600 Glostrup, Denmark.
Gjetting
Torben
T
Jensen
Thomas G
TG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology; D001921:Brain / Q000166:cytology; D002460:Cell Line; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D004247:DNA / Q000008:administration & dosage*; D042783:Endothelial Cells / Q000166:cytology; D000431:Ethanol / Q000737:chemistry; D005456:Fluorescent Dyes; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000276:immunology*; D018613:Microscopy, Confocal; D011092:Polyethylene Glycols / Q000737:chemistry*; D011972:Receptor, Insulin / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D005456:Fluorescent Dyes; D008081:Liposomes; D000431:Ethanol; D011092:Polyethylene Glycols; D004247:DNA; C435941:INSR protein, human; D011972:Receptor, Insulin