Langmuir 2004, 20, 1393-1400
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Study of Molecular Interactions between a Phospholipidic Layer and a pH-Sensitive Polymer Using the Langmuir Balance Technique Franck Pe´triat,† Emmanuelle Roux,‡ Jean Christophe Leroux,‡ and Suzanne Giasson*,† Department of Chemistry and Faculty of Pharmacy, University of Montre´ al, Centre de Recherche en Science et Inge´ nierie des Macromole´ cules (CERSIM), Universite´ Laval, Que´ bec, G1K 7P4, Canada, and Canada Research Chair in Drug Delivery, Faculty of Pharmacy, Universite´ de Montre´ al, C.P. 6128 Succ. Centre-ville, Montre´ al, Que´ bec, H3C 3J7, Canada Received August 26, 2003. In Final Form: November 26, 2003 Molecular interactions between a terminally alkylated pH-sensitive N-isopropylacrylamide copolymer DODA-poly(NIPAM-co-MAA) and a monolayer of distearoylphosphatidylcholine (DSPC) at the air/water interface are investigated using the Langmuir balance technique. The compression isotherms of the copolymer monolayer at the air-water interface confirm that the copolymer undergoes a structural transition with a change in pH ranging from an extended coil state at neutral pH to a collapsed globular state at a pH corresponding to the pH of the polymer phase transition. Adsorption kinetics of DODA-poly(NIPAMco-MAA) in the DSPC monolayer is analyzed using a first-order kinetics model allowing an effective interaction area Ax between DSPC and DODA-poly(NIPAM-co-MAA) molecules to be evaluated. The results clearly indicate that the interaction area increases with a decrease in pH. The results also suggest that the penetration of the DODA-poly(NIPAM-co-MAA) within the phospholipid monolayer is enhanced by a decrease in pH which causes a change in the copolymer structure and an increase in specific attractive interactions between the copolymer and the phospholipid. Therefore, the copolymer can trigger the destabilization or rupture of the phospholipidic layer through a simple variation in its structure associated with a variation in molecular interactions when coupled or inserted within the membrane. This study greatly supports the prospects of the copolymer-functionalized liposomes as stable and tunable carrier systems for in vivo applications in drug delivery.
Introduction Since the last three decades, liposomes formed with naturally occurring phospholipids have been known as effective drug delivery systems.1 Considerable work has been carried out to provide more stability and/or functionality to these phospholipid vesicles.2 Many papers have reported liposomes whose release profiles can be controlled by environmental parameters such as temperature3-7 or pH.8-13 For example, fusogenic liposomes, which can * Corresponding author. E-mail:
[email protected]. † University of Montre ´ al and CERSIM at Universite´ Laval. ‡ Universite ´ de Montre´al. (1) Lasic, D. D. Novel applications of liposomes. Trends Biotechnol. 1998, 16(7), 307-321. (2) Woodle, M. C.; Lasic, D. D. Sterically Stabilized Liposomes. Biochim. Biophys. Acta. 1992, 1113 (2), 171-199. (3) Kim, J. C.; Bae, S. K.; Kim, J. D. Temperature sensitivity of liposomal lipid bilayers mixed with poly(N-isopropylacrylamide-coacrylic acid). J. Biochem. 1997, 121, 15-19. (4) Meyer, O.; Papahadjopoulos, D.; Leroux, J. C. Copolymers of N-isopropylacrylamide can trigger pH sensitivity to stable liposomes. FEBS Lett. 1998, 421 (1), 61-64. (5) Meyer, D. E.; Shin, B. C.; Kong, G. A.; Dewhirst, M. W.; Chilkoti, A. Drug targeting using thermally responsive polymers and local hyperthermia. J. Controlled Release 2001, 74 (1-3), 213-224. (6) Kim, J. C.; Kim, J. D. Release property of temperature-sensitive liposome containing poly(N-isopropylacrylamide). Colloids Surf., B 2002, 24 (1), 45-52. (7) Kono, K. Thermosensitive polymer-modified liposomes. Adv. Drug Deliv. Rev. 2001, 53 (3), 307-319. (8) Ellens, H.; Bentz, J.; Szoka, F. C., Jr. pH-induced destabilization of phosphatidylethanolamine-containing liposomes: role of bilayer contact. Biochemistry 1984, 23, 1532-1538. (9) Wang, C. Y.; Huang, L. Highly efficient DNA delivery mediated by pH-sensitive immunoliposomes. Biochemistry 1989, 28, 9508-9514. (10) Roux, E.; Lafleur, M.; Lataste, E.; Moreau, P.; Leroux, J. C. On the characterization of pH-sensitive liposome/polymer complexes. Biomacromolecules 2003, 4 (2), 240-248.
be destabilized at the low pH values of the cellular endosomes, have been shown effective for the cytoplasmic delivery of large membrane-impermeable molecules such as plasmid DNA.14,15 The specific functions of liposomes are usually fulfilled by selecting appropriate phospholipids/amphiphiles or by incorporating specific functional groups or molecules to the liposome surface. It is known that the incorporation of polymers into or at the surface of liposomes is one of the most effective ways to functionalize them. In designing polymer-functionalized liposomes, two important properties are generally considered: (i) stability in vivo, i.e., the ability to maintain their physical integrity in the blood and resist clearance by the mononuclear phagocyte system (MPS) and (ii) specificity, i.e., the ability to interact only under specified conditions or at the specified target. It has been shown that coating liposomes with a hydrophobically modified polymer such as poly(ethylene glycol) (PEG) increases their stability by reducing protein adsorption (11) Leroux, J. C.; Roux, E.; Le Garrec, D.; Hong, K. L.; Drummond, D. C. N-isopropylacrylamide copolymers for the preparation of pHsensitive liposomes and polymeric micelles. J. Controlled Release 2001, 72 (1-3), 71-84. (12) Francis, M. F.; Dhara, G.; Winnik, F. M.; Leroux, J. C. In vitro evaluation of pH-sensitive polymer/niosome complexes. Biomacromolecules 2001, 2 (3), 741-749. (13) Drummond, D. C.; Zignani, M.; Leroux, J. C. Current status of pH-sensitive liposomes in drug delivery. Prog. Lipid Res. 2000, 39 (5), 409-460. (14) Zelphati, O.; Szoka, F. C., Jr. Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. Pharm. Res. 1996, 13 (9), 1367-72. (15) Zelphati, O.; Szoka, F. C., Jr. Mechanism of oligonucleotide release from cationic liposomes. Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (21), 11493-8.
10.1021/la035583f CCC: $27.50 © 2004 American Chemical Society Published on Web 01/20/2004
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and provides them with long circulation times.2,16 The increase in the in vivo half-life is explained by the fact that PEG-modified liposomes are protected by the PEG coating layer from rapid clearance by the MPS. Poly(N-isopropylacrylamide) (PNIPAM) and its derivatives5-7,11-13,17-19 are also widely employed as polymer coatings for liposome formulations. Even though PNIPAMcoated liposomes have not demonstrated stealth properties as good as their PEGylated counterparts,20 they exhibit responsiveness to temperature and pH. Indeed, PNIPAM undergoes in water a volume phase transition near its lower critical solution temperature (LCST) at 32 °C. Below the LCST, PNIPAM is soluble in water and adopts a hydrated extended coil shape, whereas above 32 °C the polymer is insoluble and exhibits a dehydrated collapsed globular shape.21 It has been shown that the release of contents from PNIPAM-coated liposomes correlates with this coil-to-globule phase transition;22 i.e., the release of entrapped therapeutic agents is enhanced above the LCST. Several groups, including ours, have recently provided pH sensitivity to liposomes by coating them with a hydrophobically modified copolymer of NIPAM and methacrylic acid (MAA). The presence of MAA groups raises PNIPAM LCST above 37 °C and provides the copolymer with pH-responsive properties. The anchoring of the copolymer to the liposomal membrane can be ensured by hydrophobic forces, hydrogen bonding, and/or electrostatic interactions20,23-27 depending on the polymer structure and nature. Our group used as hydrophobic anchoring sequences either a randomly positioned octadecylacrylate (ODA)11 or a terminal moiety of dioctadecylamine (DODA).28 With random ODA chains, the copolymer is strongly anchored at the liposomal surface, whereas the (16) Torchilin, V. P.; Omelyanenko, V. G.; Papisov, M. I.; Bogdanov, A. A.; Trubetskoy, V. S.; Herron, J. N.; Gentry, C. A. Poly(ethylene glycol) on the liposome surface: on the mechanism of polymer-coated liposome longevity. Biochim. Biophys. Acta 1994, 1195, 11-20. (17) Kono, K.; Yoshino, K.; Takagishi, T. Effect of poly(ethylene glycol) grafts on temperature-sensitivity of thermosensitive polymer-modified liposomes. J. Controlled Release 2002, 80 (1-3), 321-332. (18) Kirchmeier, M. J.; Ishida, T.; Chevrette, J.; Allen, T. M. Correlations between the rate of intracellular release of endocytosed liposomal doxorubicin and cytotoxicity as determined by a new assay. J. Liposome Res. 2001, 11 (1), 15-29. (19) Zignani, M.; Drummond, D. C.; Meyer, O.; Hong, K.; Leroux, J. C. In vitro characterization of a novel polymeric-based pH-sensitive liposome system. Biochim. Biophys. Acta 2000, 1463 (2), 383-394. (20) Yamasaki, A.; Winnik, F. M.; Cornelius, R. M.; Brash, J. L. Modification of liposomes with N-substituted polyacrylamides: identificationof proteins adsorption from plasma. Biochim. Biophys. Acta 1999, 1421, 103-115. (21) Maeda, M.; Higuchi, T.; Ikeda, I. Change in Hydration State during the Coil-Globule Transition of Aqueous Solutions of Poly(Nisopropylacrylamide) as Evidenced by FTIR Spectroscopy. Langmuir 2000, 16 (19), 7503-7509. (22) Hayashi, H.; Kono, K.; Takagishi, T. Temperature-Dependent Associating Property of Liposomes Modified with a Thermosensitive Polymer. Bioconjugate Chem. 1998, 9 (3), 382-389. (23) Polozova, A.; Winnik, F. M. Contribution of Hydrogen Bonding to the Association of Liposomes and an Anionic Hydrophobically Modified Poly(N-isopropylacrylamide). Langmuir 1999, 15 (12), 4222-4229. (24) Winnik, F. M.; Adronov, A.; Kitano, H. Pyrene-labeled amphiphilic poly-(N-isopropylacrylamides) prepared by using a lipophilic radical initiator: Synthesis, solution properties in water, and interactions with liposomes. Can. J. Chem. 1995, 73 (11), 2030-2040. (25) Roux, E.; et al. Polymer based pH-sensitive carriers as a means to improve the cytoplasmic delivery of drugs. Int. J. Pharm. 2002, 242 (1-2), 25-36. (26) Wang, Y. J.; Winnik, F. M.; Clarke, R. J. Interaction between DMPC liposomes and HM-PNIPAM polymer. Biophys.Chem. 2003, 104 (2), 449-458. (27) Polozova, A.; Yamazaki, A.; Brash, J. L.; Winnik, F. M. Effect of polymer architecture on the interactions of hydrophobically modified poly-(N-isopropylamides) and liposomes. Colloids Surf., A 1999, 147 (1-2), 17-25. (28) Roux, E.; Stomp, R.; Giasson, S.; Pe´zolet, M.; Moreau, P.; Leroux, J. C. Steric stabilization of liposomes by pH-responsive N-isopropylacrylamide copolymer. J. Pharm. Sci. 2002, 91 (8), 1795-1802.
Pe´ triat et al.
Figure 1. Chemical structure of DODA-poly(NIPAM-coMAA).
copolymer with a single end-terminal DODA group confers an optimal mobility to its polymeric chain and subsequent long circulating properties to the liposomes. This study aims to better understand the interactions of terminally alkylated (using DODA end groups) pH-sensitive PNIPAM with lipid membranes under a variation of pH, more specifically at pH 4 and 7 to observe both the collapsed and extended conformations of the copolymer. To achieve this goal, we investigated the nature of the molecular interactions between the NIPAM copolymer and a phospholipidic monolayer at the air/water interface using the Langmuir balance technique. A possible mechanism correlating the controlled release of therapeutic agents from the liposomal internal core to the polymer conformational changes with pH variation is proposed. Materials and Methods Chemicals. Water was purified by a MilliQ water purification system (Millipore Corporation, Bedford, MA) and its resistance was greater than 18 MΩ/cm. 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) was obtained from Avanti Polar Lipids (Alabaster, AL). Sodium chloride was purchased from BDH, Inc. (Toronto, ON, Canada). Buffer solutions (pH 4 and 7) were purchased from Laboratoire Mat, Inc. (Montreal, Qc, Canada)). Other standards and usual chemicals used were analytical grade Aldrich products. All the chemicals were used without any further purification. NIPAM Copolymers. The terminally alkylated polymer DODA-poly(NIPAM-co-MAA) (Figure 1) was prepared and characterized as described elsewhere.28 Its weight-average molecular weight (Mw) and polydispersity index were 29000 and 1.99, respectively. NIPAM, MAA, and DODA molar fractions were 0.952, 0.046, and 0.002 as determined by 1H NMR spectroscopy and acid-base titration (MAA content). The volume phase transition at 37 °C determined by light scattering occurred in a narrow window around pH 5.6.4,28 Langmuir Balance Technique. The Langmuir-Blodgett (LB) trough used is from Nima Technology (Coventry, England). The Teflon tray (300 × 200 × 5 mm) was equipped with a controllable dipping well. To ensure subphase temperature control, the trough was connected to an isotemp refrigerated circulator from Fischer Scientific. The temperature control during a run was 0.1 °C. A controllable Teflon barrier was used in order to control the total air/water interface area available for the amphiphilic molecules. Surface pressure was measured using a Wilhelmy balance with precut filter paper plates (10 mm diameter; 0.15 mm thick; chromatography paper-Whatmans Chr1). The LB trough was leveled by means of four adjustable legs and was placed on an antivibration table. The whole setup was located within a laminar flow cabinet to avoid contaminant deposition at the air/water interface. Prior to each experiment, the Teflon bath and Wilhelmy plate were thoroughly cleaned with methanol and rinsed several times with ultrapure water. The Wilhelmy balance was calibrated with a standard weighting set. Compression Isotherms. All surface pressure-area (π-A) isotherms were recorded using the following procedure. First, the Teflon bath was filled with a 10 mM phosphate buffer solution. To ensure that the air/water interface was contaminant free, the surface was swept upon complete compression with the mobile barrier and any residual surface active components were removed by air suction. The cleaning process was repeated until the measured surface pressure was nearly negligible (
