The effect of positive and negative pH-gradients on the stability of small unilamellar vesicles of negatively charged phospholipids.
The stability of small unilamellar vesicles (SUV) made from negatively-charged phosphatidate by ultrasonication or pH-jump has been investigated. As criteria for the vesicle stability are used: (I) the bilayer integrity as judged from the permeability of the fluorescent probe carboxyfluorescein (CF) and (II) the susceptibility of the phospholipid vesicles to fusion as judged by gel filtration and freeze-fracture electron microscopy. Egg phosphatidate SUV (PA-SUV) whose internal cavity is in equilibrium with the dispersion medium are strictly speaking thermodynamically unstable by these criteria. They may, however, be regarded as stable from a practical point of view. CF-release is observed with a half-time of 14 days and also some vesicle fusion, particularly at low temperature (4 degrees C). The small effects observed, e.g., the small tendency of the vesicles to undergo fusion is probably due to the high surface charge density of PA bilayers. A main finding of this work is that the same positive pH-gradient which is used in the pH-jump method to drive the formation of SUV from large phosphatidic acid bilayer sheets has a stabilizing effect on the resulting PA-SUV. Stabilization is achieved by positive pH-gradients of about two pH-units or more with the pH of the external medium exceeding the pH of the vesicle cavity. Under these conditions, up to about 8 weeks no significant loss of entrapped CF and no fusion of SUV was observed both at 4 degrees C and room temperature. In contrast, a reverse or negative pH-gradient of several pH units applied to PA-SUV (with the external pH being lower than that of the vesicle cavity) destabilizes PA-SUV. Such a gradient can be shown to lead to a dramatic perturbation of the lipid bilayer packing as evident from a significant increase in CF permeability. The local perturbation of the phospholipid bilayer is accompanied by massive vesicle fusion which is prominent at low temperature (4 degrees C).
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Laboratorium für Biochemie, Eidgenössische Technische Hochschule Zürich, ETH-Zentrum, Switzerland.
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Hauser
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D004355:Drug Stability; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D005452:Fluoresceins; D008051:Lipid Bilayers; D010743:Phospholipids; C024098:6-carboxyfluorescein