OVCAR-3 cells internalize TAT-peptide modified liposomes by endocytosis.
For cytosolic delivery of liposomes containing macromolecular drugs, such as proteins or nucleic acids, it would be beneficial to bypass endocytosis to prevent degradation in the lysosomes. Recent reports pointed to the possibility that coupling of TAT-peptides to the outer surface of liposome particles would enable translocation over the cellular plasma membrane. Here, we demonstrate that cellular uptake of TAT-liposomes occurs via endocytosis rather than plasma membrane translocation. The coupling of HIV-1 derived TAT-peptide to liposomes enhances their binding to ovarian carcinoma cells. The binding was inhibited by the presence of heparin or dextran sulfate, indicating that cell surface proteoglycans are involved in the binding interaction. Furthermore, living confocal microscopy studies revealed that binding of the TAT-liposomes to the plasma membrane is followed by intracellular uptake in vesicular structures. Staining the endosomes and lysosomes demonstrated that fluorescent liposomal labels are present within the endosomal and lysosomal compartments. Furthermore, incubation at low temperature or addition of a metabolic or an endocytosis inhibitor blocked cellular uptake. In conclusion, coupling TAT-peptide to the outer surface of liposomes leads to enhanced endocytosis of the liposomes by ovarian carcinoma cells, rather than direct cytosolic delivery by plasma membrane translocation.
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands. m.m.fretz@pharm.uu.nl
Marjan M
MM
Fretz
Gerben A
GA
Koning
Enrico
E
Mastrobattista
Wim
W
Jiskoot
Gert
G
Storm
D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D015696:Gene Products, tat / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000493:pharmacokinetics*; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010051:Ovarian Neoplasms / Q000473:pathology*; D011509:Proteoglycans / Q000378:metabolism
D005456:Fluorescent Dyes; D015696:Gene Products, tat; D008081:Liposomes; D011509:Proteoglycans