Langmuir 1997, 13, 1869-1872
1869
Catalysis of Transbilayer Lipid Migration by Hydrophobically Modified N-Isopropylacrylamide Polymers Soumendu Bhattacharya,1a Robert A. Moss,*,1a Helmut Ringsdorf,*,1b and Joachim Simon1b,c Department of Chemistry, Rutgers University, New Brunswick, New Jersey 08903, and Institute of Organic Chemistry, University of Mainz, D-6500 Mainz, Germany Received October 15, 1996. In Final Form: February 4, 1997X Transverse lipid migration (flip-flop) of 2-F, a head-group-labeled dipalmitoylphosphatidylcholine (DPPC), in surface-differentiated 1:7 coliposomes with unlabeled DPPC (2-NF), is facilitated by added N-isopropylacrylamide (NIPAM) polymers at temperatures above the extended f contracted transition temperatures of the polymers (30-33 °C). The effectiveness of the polymers is sensitive to the identity of the substituents on their amide nitrogens; an octadecyl residue is particularly potent, whereas a butylpyrenyl substituent is ineffective. Evidence is also presented for interliposomal polymer transfer above the liposomes’ gel f liquid crystal transition temperature.
Interactions between polymers and liposomes are manifold and have many applications. Polymer/lipid supramolecular aggregates have been used as cytoskeleton models,2,3 and the control of membrane permeability has been mediated by hydrophobically modified polysaccharides,4 polymerizable lipids,5 or hydrophobic polyelectrolytes.6 The surface properties of liposomes have been modified by polymers that affect liposomal integrity7 or enable immunomimetic recognition.8 The use of polyethylene glycol polymers stabilizes liposomes, increasing their longevity under biologically relevant conditions.9 Alternatively, the liposomes themselves can be constructed of polymerized lipids.10 Even the morphology of liposomes can be modulated by the binding of polymers.11,12a Several years ago, we demonstrated that temperaturedependent morphological changes subsequent to the binding of hydrophobically modified N-isopropylacrylamide (NIPAM) polymers to exo/endo surface-differentiated dipalmitoylphosphatidylcholine liposomes could catalyze trans-bilayer lipid migration or “flip-flop”; i.e., the polymer functioned as a “mechanical flippase.”13 Here we report on the comparative catalytic efficiencies of NIPAM polymers that either lack additional hydrophobic residues or bear pendant octadecyl, cholesteryl, or pyrenyl residues. We describe the sensitivity of the catalytic effect on the polymer’s structural transition at its lower critical solution temperature (LCST) and present X
Abstract published in Advance ACS Abstracts, March 15, 1997.
(1) (a) Rutgers University. (b) University of Mainz. (c) Current address: Central Research and Development, Bayer, D-51368 Leverkusen, Germany. (2) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113. (3) Ringsdorf, H.; Sackmann, E.; Simon, J.; Winnik, F. M. Biochim. Biophys. Acta 1993, 1153, 335. (4) Sunamoto, J.; Sato, T.; Taguchi, T.; Hamazaki, H. Macromolecules 1992, 25, 5665. (5) Frankel, D. A.; Lamparski, H.; Liman, U.; O’Brien, D. F. J. Am. Chem. Soc. 1989, 111, 9262. (6) You, H.; Tirrell, D. A. J. Am. Chem. Soc. 1991, 113, 4022. (7) Thomas, J. L.; Tirrell, D. A. Acc. Chem. Res. 1992, 25, 336. (8) Kitano, H.: Kato, N.; Ise, N. J. Am. Chem. Soc. 1989, 111, 6809. (9) Lasic, D., Martin, F., Eds. Stealth Liposomes; CRC Press: Boca Raton, FL, 1995. (10) Review: Singh, A.; Schnur, J. M. In Phospholipids Handbook; Cevc, G., Ed.; Dekker: New York, 1993; pp 233-291. (11) Decher, G.; Kuchinka, E.; Ringsdorf, H.; Venzmer, J.; BitterSuermann, D.; Weisgerber, C. Angew. Makromol. Chem. 1989, 166/ 167, 71. (12) (a) Ringsdorf, H.; Venzmer, J.; Winnik, F. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 315. (b) Ringsdorf, H.; Venzmer, J.; Winnik, F. M. Macromolecules 1991, 24, 1678. (13) Bhattacharya, S.; Moss, R. A.; Ringsdorf, H.; Simon, J. J. Am. Chem. Soc. 1993, 115, 3812.
S0743-7463(96)00992-4 CCC: $14.00
evidence for interliposomal polymer transfer above the liposome’s gel to liquid crystal transition temperature (Tc). The five NIPAM polymers used in this study conform to structures 1a-e, where 1a is the NIPAM homopolymer
(average viscometric molecular weight, 382 000); 1b is a random copolymer of N-isopropyl- and N-octadecylacrylamide in a 200:1 molar ratio (∼370 000);12b 1c is an analogous copolymer bearing pendant 3-cholesteryl groups in place of the octadecyl moieties (27 800); 1d is a copolymer (∼1 100 000) carrying N-[4-(1-pyrenyl)butyl] substituents; and copolymer 1e bears 0.5% acrylamide residues that are substituted with both n-octadecyl and butylpyrenyl residues (390 000).14 A key property of the NIPAM polymers is a rapid (and reversible) extended f globular collapse above their LCSTs.12,15 The extended polymers are water soluble, but the solubility abruptly decreases as the polymers become globular above the LCST.16 As in our previous study,13 unilamellar 420-Å-diameter bilayer coliposomes of 1:7 functional (2-F) and nonfunctional (2-NF) dipalmitoylphosphatidylcholine (DPPC) were prepared by sonication at pH 6 (HCl) in 0.01 M BisTris buffer with 0.01 M added KCl.17 These coliposomes were surface differentiated by hydrolysis of the exoliposomal 2-F p-nitrophenyl benzoate moieties (pH 12/6 exo/ endo gradient, 30 min), followed by readjustment to pH 6,17 at which point the exoliposomal surfaces carried (14) (a) For detailed studies of NIPAM polymers, see: Simon, J. Doctoral Dissertation, Johannes Gutenberg University, Mainz, 1994. (b) For a recent review of NIPAM polymer applications, see: Snowden, M. J.; Murray, M. J.; Chowdry, B. Z. Chem. Ind. (London) 1996, 531. (15) Fujishige, S.; Kubota, K.; Ando, I. Polym. J. (Tokyo) 1990, 22, 15; J. Phys. Chem. 1989, 93, 3311. (16) The LCST values (°C) as measured by the dependence of turbidity on the temperature of aqueous polymer solutions were 31.8 for 1a, 30.3 for 1b, 31.0 for 1c, 33.0 for 1d, and 30.6 for 1e. (17) Synthetic and other experimental procedures have been published: Moss, R. A.; Okumura, Y. J. Am. Chem. Soc. 1992, 114, 1750.
© 1997 American Chemical Society
1870 Langmuir, Vol. 13, No. 7, 1997
Figure 1. % flip-flop of lipid 2-F in surface-differentiated 2-F/ 2-NF coliposomes at 35 °C as a function of time in the presence of 200 ppm of added NIPAM polymers. From top to bottom, the additives are 1b, 1a, 1c, and no polymer. The case of 1c is drawn with two straight line segments to emphasize its unusual behavior (see text). The % flip-flop values at zero time are e9% and reflect preparatory manipulation.
Figure 2. % flip-flop of lipid 2-F in surface-differentiated 2-F/ 2NF coliposomes at 35 °C as a function of time in the presence of 200 ppm of added NIPAM polymers. From top to bottom, the additives are 1b, 1e, 1d, and no polymer.
p-nitrophenol moieties from 2-F, while the endoliposomal 2-F lipids retained intact ester head groups. Aqueous solutions of the NIPAM polymers 1a-e were combined with the surface-differentiated coliposomes, establishing (molar) polymer/lipid ratios of 20-200 ppm, and the time courses of 2-F flip-flop were followed at several temperatures.17 We have already reported13 that there is little effect on flip-flop of added 1a or 1b at 25 °C, below both the LCSTs16 of polymers 1a and 1b and the liposomes’ Tc,18 where the liposomes are in their gel state and the polymers are in extended, water-soluble forms. At 35 °C, above the polymers’ LCSTs but below the liposomal Tc, the situation changes dramatically, as shown by the % flip-flop vs time data in Figures 1 and 2. Figure 1 compares the facilitating effects on lipid 2-F flip-flop of 200 ppm of polymers 1a and 1c, whereas Figure 2 presents analogous data for polymers 1d and 1e. Both figures also include data for polymer 1b and for flip-flop in the absence of polymer, because these situations mark the fastest and slowest (“benchmark”) cases. There is clearly a marked (18) The Tc is 40 °C17 and is unchanged in the presence of 20 ppm of polymer 1b.13
Letters
Figure 3. % flip-flop of lipid 2-F in surface-differentiated 2-F/ 2-NF coliposomes as a function of temperature after 2 h in the presence of 50 ppm of polymer 1b. The LCST of 1b is 30.3 °C.
sensitivity to the presence and identity of the pendant groups on the NIPAM polymers, and an approximate order of polymer effectiveness emerges: 1b > 1a ∼ 1e > 1c > 1d. Flip-flop rates increase with each of the added polymers except NIPAM-butylpyrene 1d. The extrapolated times required for 30% 2-F reequilibration (corresponding to half-reequilibration13) decrease from ∼12 h in the absence of polymer to 40 °C), however, do the liposomal leaflets fluidize sufficiently to permit withdrawal and reinsertion of the polymers’ C18 chains from donor to acceptor liposomes, i.e., an intraaggregate polymer transfer that can be detected by enhanced lipid flip-flop of 2-F within the acceptor liposomes. Clearly, the remarkable properties of NIPAM polymers14b can be adjusted by structural modification to produce a variety of effects in the modulation of liposomal dynamics.
(26) Although flip-flop is not markedly facilitated at 35 °C, there is a modest perturbation of the acceptor liposomes upon interaction with the donor liposomes. This is signaled by an initial “jump” in % flip-flop, visible in the polymer transfer experimental point recorded at 0.5 h in Figure 4. (27) Parallel transfer experiments using cholesteryl polymer 1c afford analogous results: flip-flop is not assisted at 35 °C but is clearly augmented at 45 °C.
Acknowledgment. We thank the U.S. Army Research Office (Rutgers University) and the Deutsche Forschungsgemeinschaft (University of Mainz) for financial support. LA960992F
