Biphasic protonation of hydrophilic cargo agents in unilamellar phospholipid vesicles: implications about cargo location.
The commonly used cargo agents for liposome entrapment, chromate and 5(6)-carboxyflourescein (CF), have been sequestered in small unilamellar vesicles composed of dipalmitoylphosphatidylcholine through preparations involving either sonication or extrusion methods. Once loaded, these water-soluble chromophoric cargo agents have been exposed to small quantities of externally applied acid solution, which decreases the pH from neutral to approx. 6. By monitoring photometrically the time profile of the protonation of the sequestered chromophores, it is evident that the uptake of protons by each cargo agent is biphasic. An immediate spectral change is followed by further change over 10-40 min, where the extent of protonation occurring in each time frame is approximately equal. The vesicles themselves are unaffected by the induced pH change. The leakages of both chromate and CF from loaded sonicated vesicles were monitored at both 25 degrees C and 45 degrees C. Overall, the leakage processes exhibited a deceleration over time. The biphasic protonation and decelerating leakage phenomena are together interpreted in terms of a mechanism of cargo loading involving an intercalation of the water-soluble agent along with water into the vesicle bilayer, rather than involving internal capture of the cargo inside the vesicles, or through electrostatic interactions with the bilayer surfaces. In addition, the measured extents of cargo loading are more consistent with calculated estimates of loading through bilayer intercalation than with those for internal capture.