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.

Autoxidation and effects of pro- and antioxidants in lyophilized red blood cell membranes.

Oxygen uptake and effects of pro- and antioxidants have been compared at 80 C in lyophilized red blood cell (RBC) bilayer membranes (ghosts). In this dry and relatively immobile system, catalytic metals have pronounced effects. When ghosts are prepared in the usual manner, in phosphate buffer, hypotonic saline, or deionized water (DI), oxygen uptake is extremely slow and limited unless: (a) catalytic metal, e.g. cobaltous ion, is supplied in the absence of metal-complexing buffers and (b) residual phosphate buffer is removed by repeated deionized water or hypotonic saline washes. Ghosts adsorb d-alpha-tocopherol strongly from 1% alcohol emulsion in buffer in amounts far above normal RBC concentrations, whereas synthetic antioxidant uptake seldom exceeds the normal tocopherol level. Uptake and effectiveness of antioxidants introduced by either perfusion or in the vapor phase, and resistance of ghosts to autoxidation introduced by either perfusion or in the vapor phase, and resistance of ghosts to autoxidation are discussed as they relate to protection of lyophilized membranes and freeze-dried foods.

Addition compounds of oxidizing tocopherol and soybean lecithin.

Evidence is presented for the formation of addition compounds from tocopherol and soybean lecithin, when adsorbed in monolayer on silica from a mixed solution in chloroform and oxidized at 80 C for 72 hr. Tocopherol was present at 7 mol % of the lecithin in the monolayer. The compounds produced are analogous to linoleic acid-tocopherol adducts previously reported by us. UV spectra of the mixed lecithin and tocopherol monolayers while in silica gel slurry in a solvent were obtained by the method previously reported by us for oxidized linoleic acid and tocopherol monolayers. The monolayer spectra show no evidence for tocopherol dimer. A minor amount of quinone is indicated, but the spectrum has a maximum at 287 nm with no other major peaks. The proposed addition compounds have been characterized by transesterification and hydrogenation, UV, IR, thin layer chromatography and gas chromatography retention behavior, mass spectrometry and elemental analysis. IR spectra of twice-chromatographed addition compounds show specific lecithin absorption bands (5.75 µ; 10.3 µ) together with much enhanced 3.4-3.5, 6.8, 7.25 and 9.14 µ peaks, the latter two being characteristic of α-tocopherol. The UV spectrum of the adducts showed λ max (CHCl) 3 287 nm, with shoulders at 276 and 300 nm. Hydrogenation removed the peaks at 287 and 276 nm, leaving a peak at 300 nm similar to that of the linoleic acid-tocopherol adduct. Esters of the adducts prepared by transesterification and hydrogenation were similar by all our chemical, chromatographic and spectral tests to the previously characterized esters of the linoleic acid-tocopherol adduct.