Langmuir 1999, 15, 3707-3709
3707
The First Fluorescence Test of Vesicle Membrane Permeability without Chromatography Ju¨rgen-Hinrich Fuhrhop,*,† Claus Endisch,† Andrea Schulz,† James Turner, and Michael Eaton‡ Institut fu¨ r Organische Chemie, Freie Universita¨ t Berlin, Takustrasse 3, D-14195 Berlin, Germany, and Celltech Therapeutics Ltd., 216 Bath Road, Slough SL1 3EN, Berkshire, U.K. Received December 14, 1998. In Final Form: March 10, 1999 Endosome-type vesicle membranes containing 50% cholesterol were prepared in the presence of 10-6 M of a β-tetrapyridiniumporphyrin. Strong fluorescence at 640 nm was observed. Addition of a slight excess (1.5 × 10-6 M) of copper(II) meso-tetraphenylsulfonate totally quenches the fluorescence in the bulk water volume. The vesicle-entrapped pyridinium porphyrins still fluoresce. In distilled water the residual fluorescence diminishes by less than 10% within 6 h, and in buffered solution it is reduced to 50% within 1 h. In the presence of a harpoon-like disruptor, almost quantitative quenching is observed within seconds (dipalmitoylphosphatidylcholine) or 10 min (egg lecithin).
Introduction The escape of entrapped electrolytes from vesicles, in particular from the endosomes in humans, is an important process in the transport of nucleic acids and proteins through cell membranes.1 Membrane disrupters help,2 but tedious procedures are necessary to assay and optimize their efficiency. Charged water-soluble fluorescence dyes, for example, have been entrapped within the vesicle’s water volume, but to detect their release into the bulk water volume, the loaded vesicles have to be purified by gel chromatography or ultrafiltration.3,4 Loss of vesicles and of entrapped material is usually encountered if the columns are not carefully preconditioned for each probe and the procedures are time-consuming. The study of the kinetics of release processes is not feasible. Furthermore, endosome membranes and their models are also stiffened by large amounts of cholesterol, and careful adjustment of in- and outside buffer concentrations is mandatory in order to prevent vesicle damage during isolation processes, which again leads to extended optimization experiments.2 We report here on a sensitive fluorescence assay without any separation of vesicles from bulk media. A porphyrin isomer mixture of β-tetraethyl-β-tetramethylpyridiniumporphyrin (1; βTPyP) is used as a water-soluble fluorescence dye5 and copper meso-tetrasulfonatoporphyrinate (2; CuTPPS) as an efficient quencher. Both porphyrin electrolytes do not pass intact vesicle membranes within 6 h, if the solvent is pure water. We then apply buffers and the harpoon2 3 as disruptors to characterize their effects on the diffusion of the porphyrin electrolytes through the stiff vesicle membranes (Chart 1 and Figure 1).
Chart 1
1 and 2 have been described in ref 5, and the harpoon 3 is described in ref 3. Vesicles were obtained by 5 min of mild sonication. Fluorescence spectra were measured with a Perkin-Elmer MPF44B, excitation at 516 nm, and a fluorescence maximum at 640 nm (Figure 2). The transmission electron micrographs (TEM) were obtained on a Philips CM 12 by cryomicroscopy without any staining material.
Experimental Section
Results and Discussion
Egg lecithin (Fluka), dipalmitoylphosphatidylcholine (DPPC; Bechem), and cholesterol were obtained from Serva. Porphyrins
Both porphyrins βTPyP (1) and TPPS 2 are watersoluble to a concentration of 10-2-10-1 M, do not pass fluid vesicle membranes within several hours (Figure 2), and form heterodimers in solution without precipitation at concentrations 80% with lecithin vesicles.
lecithin or DPPC. A 1.5-fold excess of 2 in water is then added to quench the fluorescence of 1 in the bulk medium. The remaining fluorescence should directly indicate the amount of vesicle-entrapped porphyrin 1 (Figure 1). For the actual experiments the vesicles were prepared in 10-6 M solutions of 1 and were then mixed with a 10-2 M solution of 2 to give a final concentration of 1.5 × 10-6 M. Liquid-crystalline DPPC and fluid egg lecithin membranes, both rigidified by 50% cholesterol, were chosen as test systems. Such cholesterol-rigidified membranes are sensitive to the addition of membrane disrupting agents, and they are good models for the endosomes, which also contain about 50% cholesterol.6 As disrupters we used harpoon 3, as developed by Regen.2 The results realize all possible kinds of membrane disruption from immediate to slow to no action at all. In undisturbed vesicles the percentage of remaining fluorescence after addition of a 1.5-fold excess of 2 was always around 5% of the original bulk value. This is 5-10 times more than would be expected from the vesicle entrapment volume as calculated from the electron microscopic diameter of the vesicles and their concentra(6) Courtoy, P. J. Dissection of endosomes. In Intracellular trafficking of proteins; Steer, C. J., Ed.; Cambridge University Press: Cambridge, U.K., 1991.
Figure 3. Cryo-transmission electron micrograph of multilayered vesicles made of egg lecithin containing 50% cholesterol in the presence of 10-5 M porphyrin 1. The perfect curvature at points of contact shows the stiffness of the membranes. Their thickness is 5 ( 1 nm.
tion. Some of the porphyrin is presumably not dissolved in the entrapped water volume but adsorbed to the inner vesicle surfaces and is not reached there by the copper porphyrin sulfonate. The measured entrapment volumes are therefore too large and not reliable. Nevertheless, release experiments are still possible, because the adsorbed porphyrin is also equilibrated with the bulk phase by membrane disrupting agents. The DPPC-cholesterol vesicle in distilled water releases less than 10% of the entrapped porphyrin within 6 h. In 10-2 M sodium acetate buffer, however, about 40% of the fluorescence is lost already after 1 h and then remains constant for 6 h. Flip-flop of surface-adsorbed porphyrins may be responsible for the initial loss and is obviously accelerated by electrolytes. In the more fluid egg lecithin vesicles also containing 50% cholesterol, this process continued over the whole observation period of 6 h. Addition of harpoon 3 with a bulky alkyl end2 accelerated the combination of porphyrins 1 and 2 by factors between 5 and 20 (Figure 2). In the DPPC vesicles, an immediate outburst was observed and more than 90% of the porphyrin was released within the first minute after addition of the harpoon. The release from the more fluid egg lecithin, whose composition is similar to that of endosomes,6 was
Letters
about 10 times slower. The kinetic curves were always reproducible within (5% and were obtained automatically on a scanning fluorescence spectrophotometer. A stoppedflow apparatus with a fluorescence detector to measure the fast release was not available to us. The applied aqueous solutions of both porphyrins (5 × 10-2 M) needed are stable for at least a year in the dark so that the vesicle test can be carried out many hundred times with a few milliliters of two standard solutions. One experiment typically consumed half an hour for the preparation of the samples and fluorescence measurements. About 200 experiments can be made with 1 mg of isomer mixture 1 and 1.5 mg of copper porphyrin 2. Comparative experiments with commercial mesotetramethylpyridinium porphyrin instead of 1 were not successful. Precipitation reactions occurred, and quantitative fluorescence quenching could not be achieved. More than a hundred of such permeability experiments have been performed, and the detailed results, especially pH-dependent release using new harpoons with acidic groups, will be reported in due course. Figure 3 shows a typical electron micrograph of a multilayered egg lecithin vesicle stiffened by 50% cholesterol in the presence of 10-5 M porphyrin 1. Multilayered vesicle membranes lead to the finding that fluorescence quenching sometimes levels off at values of about 6080%. The harpoon then obviously does not reach the inner
Langmuir, Vol. 15, No. 11, 1999 3709
membranes upon mixing. Systematic permeability studies on the same vesicle probe using different harpoons are, however, reliable and reproducible with the separationfree system described here. One has to accept the limitation set by the ill-defined adsorption of the porphyrins to membrane surfaces. This limitation is most serious with highly charged vesicle membranes. In such cases, dye adsorption to the membrane surface becomes a dominant factor and fluorescence quenching processes become irreproducible. Furthermore, the procedure is only reliable in serial tests, where only one or two parameters are changed. This latter limitation applies, however, for all membrane characterizations. The new method is more simple than the classic method of carboxyfluorescein release,2,7 because no filtration or gel chromatography is implied. Dozens of experiments are possible with one vesicle solution, which should be characterized at the beginning and the end of the experiments by TEM. In particular, it now becomes easy to determine the effects of osmotic stress and cholesterol and harpoon concentration with one probe and within 1 day. LA981712U (7) Ruiz, J.; Goni, F. M.; Alonso, A. Biochim. Biophys. Acta 1988, 937, 127.
