© Copyright 2001 American Chemical Society
DECEMBER 11, 2001 VOLUME 17, NUMBER 25
Letters Stabilization of Phosphatidylserine/ Phosphatidylethanolamine Liposomes with Hydrophilic Polymers Having Multiple “Sticky Feet” Melinda L. Hwang,† Robert K. Prud’homme,† Joachim Kohn,‡ and James L. Thomas*,§ Department of Chemical Engineering, Princeton University, Princeton, New Jersey 08544, Department of Chemistry, Rutgers University, 610 Taylor Road, Piscataway, New Jersey 08854-8087, and Department of Chemical Engineering, Columbia University, 500 West 120th Street, New York, New York 10027 Received July 18, 2001. In Final Form: September 25, 2001 The ability of three novel polymers to protect phosphatidylethanolamine/phosphatidylserine (1:1) vesicles against calcium-induced destabilization (dye leakage) has been investigated. The polymers are a telechelic, hydrophobically terminated poly(ethylene glycol) (PEG), a polymer of the macromonomer PEG8000-lysine-stearylamide, and a hydrophobically modified poly(acrylic acid). These polymers are predominantly hydrophilic but have multiple hydrophobic moieties which serve to anchor them in the lipid bilayers. Like singly anchored PEG, the two PEG-based polymers were able to provide effective stabilization at concentrations sufficient to produce a polymer “brush” on the liposome surface. Interestingly, the poly(acrylic acid) polymer did not provide full stabilization even at concentrations that should have produced brush coverage, possibly owing to a more expanded polymer conformation. The potential advantages of stabilization with polymers having multiple “sticky feet” are discussed.
Introduction It is now well established that water-soluble polymers, anchored at the surfaces of colloidal particles such as lipid membrane vesicles or liposomes, provide stability that permits these nanometer-scale complexes to be used as effective drug-delivery vehicles.1-3 Stabilization is thought * To whom correspondence should be addressed. E-mail: jlt32@ columbia.edu. † Princeton University. ‡ Rutgers University. § Columbia University. (1) Blume, G.; Cevc, G. Biochim. Biophys. Acta 1990, 1029, 91-97. (2) Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. FEBS Lett. 1990, 268, 235-237. (3) Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S. K.; Lee, K. D.; Woodle, M. C.; Lasic, D. D.; Redemann, C.; Martin, F. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11460-11464.
to result from the prevention of contact between the liposome and macrophages; such contact results in absorptive endocytosis and rapid circulatory clearance of unprotected liposomes.4 Contact may be prevented by inhibiting deposition of serum proteins (“opsonins”) that could mediate monocyte binding or, alternatively, by the steric barrier against membrane-membrane contact provided by the polymer chains. (Recent work supports the latter explanation: Johnstone et al. found no significant inhibition of serum protein binding by surface-grafted poly(ethylene glycol) yet still obtained good inhibition of cellular uptake.4) For maximal effectiveness, the grafted or anchored polymers must be at a high enough areal density to reach the “brush” regime, in which the random coil polymers begin to interpenetrate.5,6 Higher densities (4) Johnstone, S.; Masin, D.; Mayer, L.; Bally, M. Biochim. Biophys. Acta 2001, 1513, 25-37.
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can result in destabilization: the osmotic repulsion between polymer chains creates a lateral pressure at the liposome surface that eventually results in micellization.7 In principle, water-soluble polymers with multiple ”sticky feet”, rather than a single hydrophobic anchor, could provide some advantages over single anchors for steric stabilization of liposomes. A single anchor must be extremely hydrophobic; otherwise, dissociation of the polymers from the liposomes limits the lifetime of steric stabilization.5,8 However, with multiple attachment sites the probability (and rate) of dissociation becomes very small even with relatively weak anchoring energies.9 The presence of multiple sticky feet will also alter the conformation of the surface-bound polymer, which could result in surface stabilization at lower polymer coverage. We report here a preliminary investigation into the efficacy of three different polymers with multiple sticky feet for the steric stabilization of liposome membranes. The polymers studied are (1) a telechelic hydrophobically terminated poly(ethylene glycol), C 16-PEG-C16, (2) a polymer of the macromonomer PEG8000-lysinestearylamide, and (3) a hydrophobically modified poly(acrylic acid). Specifically, the ability of these polymers to inhibit the calcium-induced disruption of anionic liposomes has been explored. Calcium-induced leakage of contents from phosphatidylserine-phosphatidylethanolamine mixed liposomes has been shown to depend on membrane contact, which is an important criterion for steric stabilization. Moreover, the effects of calcium on liposomes stabilized with hydrophobically anchored poly(ethylene glycol) have been well characterized,5 providing a point of comparison for these novel multiply anchored polymers. Materials and Methods L-R-Phosphatidylserine
(PS) and L-R-phosphatidylethanolamine (PE) (bovine brain) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Calcein (fluorexon, indicator grade) was obtained from Aldrich Chemical Co. (Milwaukee, WI) and used as received. C16-PEG-C16, 1, Mw ) 40 kDal, was obtained from Rohm and Haas, Philadelphia, PA. The synthesis of the poly(PEG8000-lysine-stearylamide) (PEG-LS), 2, was described elsewhere.10,11 The weight-average and number-average molecular weights were Mw ) 113 kDal and Mn ) 74 kDal. Hydrophobically modified poly(acrylic acid), 3 (hm-PAA), Mw ) 124 kDal, was synthesized as described elsewhere:12 3 mol % of the acid groups have been amidated with 1-tetradecylamine.
(5) Holland, J. W.; Hui, C.; Cullis, P. R.; Madden, T. D. Biochemistry 1996, 35, 2618-2624. (6) Kenworthy, A.; Hristova, K.; Needham, D.; McIntosh, T. J. Biophys. J. 1995, 68, 1921-1936. (7) Hristova, K.; Needham, D. Macromolecules 1995, 28, 991-1002. (8) Parr, M. J.; Ansell, S. M.; Choi, L. S.; Cullis, P. R. Biochim. Biophys. Acta 1994, 1195, 21-30. (9) Fleer, G.; Cohen Stuart, M.; Scheutjens, J.; Cosgrove, T.; Vincent, B. Polymers at Interfaces; Chapman and Hall: London, 1993; Chapter 6.
Letters Liposomes were prepared following a modification of the protocol described by Holland et al.5 PS and PE were dissolved in 95:5 v/v benzene/MeOH at 25 g/L each to form a stock solution. For each preparation, 100 µL of stock solution was freeze-dried overnight at
