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Abstract
Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.
Highlights
To exert an optimal therapeutic effect, an administered drug must safely reach its target cell and the appropriate location within that cell
In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells
Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells
Summary
Materials—Cholesterol (CHOL), 2Ј,7Ј-[bis(carboxymethyl)amino] methyl-fluorescein (calcein), N-succinimidyl-S-acetylthioacetate (SATA), and sodium 3Ј-[1-(phenylaminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) were obtained from Sigma. For confocal laser-scanning microscopy analysis, cells that were incubated with liposomes were washed three times with 1 ml of phosphate-buffered saline before fixation with 2% (v/v) of formaldehyde in phosphatebuffered saline for 20 min at room temperature. Liposome Turbidity Assay—Liposomes composed of EPC and CHOL (molar ratio of 2:1) and with increasing amounts of PEG2000-DSPE (0, 2.5, 5, and 10 mol %) were added to a cuvette at a concentration of 300 or 1000 M in a final volume of 1.5 ml of HBS. After the addition of diINF-7 (lipid/peptide molar ratio of 50), peptide-induced aggregation of liposomes at the indicated pH values was determined by measuring the increase in optical density of the liposome dispersion with a spectrophotometer at 500 nm over a period of 8 –12 min at room temperature. Unilamellar liposomes of 150 nm in size containing calcein (0.8 mM) and CuSO4 (1 mM) in HBS were admixed at a concentration of 50
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