Endocytosis and intracellular fate of liposomes using pyranine as a probe

Abstract

Lipid vesicles (liposomes) containing pH-sensitive fluorophores were used as probes for the study of liposome entry and intracellular fate. Pyranine [8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)] was entrapped in the liposome aqueous core during preparation to provide a means of detecting internalization into living cells. HPTS is highly water soluble and shows a strong pH-dependent shift in its fluorescence excitation spectrum. Fluorescence emission (FEM) is slightly pH dependent with excitation (lambda EX) at 350-415 nm but highly pH dependent with lambda EX at 450 nm. Liposomes bearing a net negative charge bound rapidly to CV-1 cells and underwent endocytosis. One hour after liposome addition, high FEM with lambda EX at 413 nm and low FEM with lambda EX at 450 nm suggest that most cell-associated liposomes had been internalized and resided at a mean pH of approximately 6.6. Collapse of cellular H+ gradients with NH4Cl or monensin treatment rapidly and reversibly increased FEM with lambda EX at 450 nm. Direct examination by fluorescence microscopy corroborates the fluorometric data on internalization; over time, FEM remained high with lambda EX at 350-405 nm but decreased with lambda EX at 450-490 nm, showing that all lipid vesicles were internalized within 40 min at 37 degrees C. Acidification of intracellular liposomes increased over 3 h, reaching a minimum value of approximately pH 5.5. HPTS persisted within acidic cellular vesicles for 2-3 days, and cytoplasmic dye was observed infrequently, suggesting that liposome fusion with cellular membranes seldom occurs. Material delivered to the endocytic pathway via lipid vesicles labeled an assortment of intracellular organelles of varying motility and morphology, including dynamic tubular structures whose lumen is acidic.

Boron neutron capture therapy demonstrated in mice bearing EMT6 tumors following selective delivery of boron by rationally designed liposomes

“…Furthermore, MAC increases the efficiency of overall liposome uptake by cells. This characteristic arises from the negative surface charge MAC imparts to the liposomes, a property that has been demonstrated to enhance cellular adhesion, leading to greater internalization via clathrin-mediated endocytosis (31)(32)(33)(34).…”

Intravenous liposomal benznidazole as trypanocidal agent: increasing drug delivery to liver is not enough

“…Firstly, the assay described in Section 3.2 accounted for the existence of phagocytic uptake of liposomal particles loaded with BNZ. Since upon excitation at 440 nm, punctual and pale emission indicates HPTS in acidic compartments (HPTS confined to endosomes/lysosomes), whereas homogeneously distributed strong emission indicates HPTS in a neutral compartment (cytosol) (Daleke et al, 1990;Straubinger et al, 1990), the assayed revealed the confinement of loaded hydrophilic marker inside the endo-lysosomal system. Therefore, for the small hydrophobic BNZ loaded in liposomes, it was expected that its permeation across the endosomal or lysosome membranes would only depend on its own chemical structure (Lloyd, 2000).…”

Synthesis andin vivomurine evaluation of Na4[1-(1′-B10H9)-6-SHB10H8] as a potential agent for boron neutron capture therapy

“…Analogous reactions also have been reported for the synthesis of alkoxy and amine derivatives (5,9,10 The utility of this class of compounds for BNCT has been investigated (4,5). Tumor-selective unilamellar liposomes, capable of delivering their contents intracellularly (11), were used as delivery vehicles for the compounds, which have little inherent tumor specificity. Although the liposomes successfully delivered the species to the tumor, the utility of the compounds for BNCT depends on the retention of the drug by the tumor.…”

“…In the 1 H NMR spectrum, one signal, at 1.3 ppm, corresponding to the protons on the three methyl groups of the tertiary butyl substituent, is present. In the 11 B NMR (note axis in Fig. 1), two terminal apical boron signals, at Ϫ5.2 and Ϫ8.9 ppm, appear as singlets in the proton-decoupled spectrum and as doublets in the proton-coupled spectrum.…”

“…The remaining up-field signals are assigned to the remaining equatorial boron atoms. The two-dimensional 11 B NMR spectrum ( Fig. 1) shows a cross-coupling peak between the two boron atoms of the intercage connection (11.6 and 7.9 ppm), as well as a cross-coupling peak between the signal corresponding to the boron atom bearing the sulfur substituent (Ϫ0.5 ppm) and one of the signals corresponding to an apical boron atom (Ϫ8.9 ppm).…”