Labeled vesicles

Fig. 5 Polypeptide vesicles demonstrate the ability to utilize the EPR effect, (a) Chemical structure of the amphiphilic block polypeptide PSar-b-PMLG. (b) Fluorescence image using fluorescently labeled PEG. Fluorescence is not observed in the cancer site although accumulation is observed in the bladder, (c) Fluorescence image using ICG-labeled vesicles, showing evidence of vesicle accumulation due to the EPR effect. Adapted from [41] with permission. Copyright 2008 American Chemical Society...

Figure 10.11 The use of ferritin as a label for the mechanism of growth of vesicles (adapted from Berclaz et al, 2001a b). Schematic representation of the possible vesicle formation and transformation processes when oleate, and oleic acid, are added to pre-formed vesicles which have been labelled, (a) The situation if only de novo vesicle formation occurs, (b) Growth in size of the pre-formed and labeled vesicles which may lead to division, either yielding vesicles that all contain marker molecules (case i, a statistical redistribution of the ferritin molecules) or also yielding vesicles that do not contain markers (case ii). Compare all this with Figure 10.9.

Fresta, M., and G. Puglisi. 1996. Application of liposomes as potential cutaneous drug delivery systems. In vitro and in vivo investigation with radioactively labelled vesicles. J Drug Target 4 95. 

Membranes fusion can be studied using the energy-transfer mechanism. In fact, membrane vesicles labeled with both NBD and rhodamine probes are fused with unlabeled vesicles. In the labeled vesicles, upon excitation of NBD at 470 nm, emission from rhodamine is observed at 585 nm as a result of energy transfer from NBD to rhodamine. The average distance separating the donor from the acceptor molecules increases with fusion of the vesicules, thereby decreasing the energy-transfer efficiency (Struck et al. 1981). 

Chloride flux assay. Synaptic vesicles were prepared from brains of male ICR gice (20-30 g Blue Spruce Farms, Altamont, NY) (15) Assays of °C1 uptake involved preincubation (10-20 min) of vesicles with carrier solvent (ethanol or acetone, 0.5-1 yl) or insecticide, followed by incubation (4 sec) with bCl with or without GABA or added insecticides and isolation of labelled vesicles by rapid vacuum filtration. Detailed descriptions of the assay are published elsewhere ( 15,1 7) Abamectin stock solutions were prepared in absolute ethanol in silanized glass vials Abamectin in ethanol (1 yl) was added to give concentrations of 3 nM-3 yM in a final volume of 200 yl of vesicle suspension during preincubation or 3 yM in a final volume of 200 yl of bCl uptake medium. 

Figure 10 Internal trapping assays can be used to prove that transport really occurs across the membrane of intact vesicles. Transporters are added in small portions well below EC50 to labeled vesicles (a) with or (b) without internal inactivators. Without inactivators, activity will gradually increase and reach the value obtained when all transporters are added at once (b vs c). With inactivators, transporters are continuously trapped intravesicularly, and no activity is observed even at high total concentrations (a vs c).

Another strategy used vesicles constituted of catanionic surfactants resulting from the association of ionic surfactants of opposite charges. A triple-chained catanionic surfactant (TriCat) vras recently obtained via a spontaneaous acid-base reaction in water between AThexadecylamino-1-deoxylactitol and bis(hydro)g dodecyl)phosphinic acid, followed by pH measurements until stabilization (Fig. 16). A fluorescent double-chained catanionic amphiphile molecule (FluoCat) was also synthesized in order to label vesicles. In aqueous medium, TriCat-containing catanionic systems with or without the addition of FluoCat (TriCat and Tri-Cat/FluoCat 19/1) spontaneously self-assembled into stable vesicles, characterized by a narrow size distribution that did not exceed 200 nm in... 

---