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Study of pH-Sensitive Copolymer/Phospholipid Complexes Using the Langmuir Balance Technique: Effect of Anchoring Sequence and Copolymer Molecular Weight Franck Pe´triat and Suzanne Giasson* Department of Chemistry and Faculty of Pharmacy, Universite´ de Montre´ al, C.P. 6128 Succ. Centre-ville, Montre´ al, Que´ bec, Canada H3C 3J7 Received January 14, 2005 The behavior of three copolymers of N-isopropylacrylamide (NIPAM), methacrylic acid (MAA), and hydrophobic moiety was studied at phospholipid monolayer/subphase interfaces. The hydrophobic moieties, N-terminal dioctadecylamine (DODA) and random octadecylacrylate (ODA), were used as anchoring groups. The interactions between a 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) monolayer and the copolymers were studied using the Langmuir balance technique. The effect of subphase pH, distribution of anchors along the copolymer chain, and copolymer molecular weight on the nature of the interactions between the copolymer chains and the DSPC monolayer were investigated. A first-order kinetics model was used to analyze the copolymers adsorption at the DSPC monolayer/subphase interface and allowed the interaction area between the copolymer chains and the DSPC monolayer, Ax, to be determined. The interaction area appears to depend on the subphase pH and the copolymer molecular weight. On decreasing pH, the interaction area of high molecular weight copolymers increases significantly; this is consistent with the copolymer chain phase transition from an extended coil to a collapsed globule while pH is lowered. In the latter conformation, strong hydrophobic attractive interactions between the copolymer chains and the hydrophobic part of the DSPC monolayer favor the copolymer intercalation, which could eventually provoke the phospholipidic layer destabilization or rupture.
Introduction Over the past 20 years, liposomes have played a dual role as model membranes1 and as potential candidates for in vivo drug delivery. By incorporating hydrophobic or hydrophilic pharmacologically active molecules either in the liposome membrane or in its internal lumen, one can reduce the drug cytotoxicity relative to the nonspecific distribution at healthy cells, leading to a greater therapeutical index. This promising feature, combined with the infinite possibilities of formulations, with regards to the lipid composition, net charge, and size of the carrier, have attracted considerable interest for liposome-based drug delivery systems. However, conventional liposomes comprising phospholipids and cholesterol have demonstrated a rapid clearance from the systemic circulation by the macrophages of the mononuclear phagocyte system (MPS).2-4 This major concern associated with conventional liposomes has stifled their prospects as efficient in vivo drug delivery systems for the treatment of therapeutical targets beyond the MPS. To improve the systemic circulation lifetime of liposomal formulations, one approach consists of functionalizing the liposome membrane with polymers. Lipid derivatives of poly(ethyleneglycol) have been extensively used to formulate sterically stabilized liposomes (SSLs).5-8 Considering tumoral targets, such polymer/lipid drug delivery * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Marsh, D. Lateral pressure in membranes. Biochim. Biophys. ActasRev. Biomembr. 1996, 1286 (3), p 183-223. (2) Allen, T. M. Interactions of liposomes and other drug carriers with the mononuclear phagocyte system. Liposomes as Drug Carriers; Gregoriadis, G., Ed.; John Wiley and Sons: Toronto, 1988; pp 37-50. (3) Patel, H. M. Serum opsonins and liposomessTheir interaction and opsonophagocytosis. Crit. Rev. Ther. Drug Carrier Syst. 1992, 9 (1), 39-90. (4) Patel, H. M. Influence of lipid-composition on opsonophagocytosis of liposomes. Res. Immunol. 1992, 143 (2), 242-244.
systems can extravasate from the porous vessels feeding fast growing tumors and benefit the poor lymphatic drainage (enhanced permeation and retention effect9) to act as microreservoirs at the tumor site. Enhancing the cytoplasmic bioavailability of therapeutical agents remains a major concern in designing drug delivery systems. First attempts have been to formulate pH-sensitive liposomes. The membrane of such liposomal formulations combines non-bilayer-forming polymorphic lipids, such as unsaturated 1,2-dioleyl-sn-glycero-3-glycerophosphatidylethanolamine (DOPE), and mildly acidic amphiphiles, such as cholesterylhemisuccinate (CHEMS), that acts as the bilayer phase stabilizer.10-12 Upon acidification, the protonation of DOPE induces the destabilization of the liposomal membrane and the formation (5) Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S. K.; Lee, K.; Woodle, M. C.; Lasic, D. D.; Redemann, C.; Martin, F. J. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. PNAS 1991, 88 (24), 11460-11464. (6) Woodle, M. C.; Lasic, D. D. Sterically stabilized liposomes. Biochim. Biophys. Acta 1992, 1113 (2), 171-199. (7) Lasic, D. D.; Vallner, J. J.; Working, P. K. Sterically stabilized liposomes in cancer therapy and gene delivery. Curr. Opin. Mol. Ther. 1999, 1 (2), 177-185. (8) 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. (9) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: A review. J. Controlled Release 2000, 65 (1-2), 271-284. (10) Ellens, H.; Bentz, J.; Szoka, F. C. Proton- and calcium-induced fusion and destabilization of liposomes. Biochemistry 1985, 24, 30993106. (11) Mui, B.; Ahkong, Q. F.; Chow, L.; Hope, M. J. Membrane perturbation and the mechanism of lipid-mediated transfer of DNA into cells. Biochim. Biophys. ActasBiomembr. 2000, 1467 (2), 281292. (12) Peschka-Suss, R.; Skalko-Basnet, N. The association of plain and ligand-bearing neutral and pH-sensitive liposomes with various cells. J. Liposome Res. 2000, 10 (1), 43-59.
10.1021/la050120q CCC: $30.25 © 2005 American Chemical Society Published on Web 06/28/2005
Study of Copolymer/Phospholipid Complexes
of a fusion competent hexagonal (HII) phase.13 After internalization by endocytosis, this class of pH-sensitive liposomes can fuse with the endosomal membrane and partially release their content into the cytoplasm of the targeted cell. However, their moderate stability and low circulation time in biological fluids have limited their use for in vivo applications. Coating liposomes with polymers has given further impetus to phospholipid drug carriers by providing them a certain steric stability and a stimuli-responsiveness. Poly(NIPAM) and derivatives are the most encountered polymer candidates, since they exhibit an inverse solubility upon heating. Such polymers undergo an abrupt conformational transition from an extended coil to a globular shape right above their lower critical solubility temperature (LCST).14,15 The addition of hydrophilic comonomers such as MAA renders NIPAM copolymers pH-responsive and raises their LCST from 32 °C [for pure poly(NIPAM)] to temperatures above 37 °C, which allows in vivo applications. The anchoring of the copolymer to the external leaflet of the liposomal membrane can be achieved through hydrophobic interactions, hydrogen bonding, or electrostatic interactions.16-19 Both temperature-20-23 and pH-dependent24-27 polymer-coated liposomes have been intensively exploited as drug delivery systems, whose in vitro content release has been associated with the copolymer pH phase transition,26 though the exact mechanism is still not completely elucidated. The present study aims to characterize the interactions between a phospholipidic monolayer at the air/water interface and hydrophobically modified pH-sensitive poly(13) Allen, T. M.; Hong, K.; Papahadjopoulos, D. Membrane contact, fusion, and hexagonal (HII) transitions in phosphatidylethanolamine liposomes. Biochemistry 1990, 29, 2976-2985. (14) 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. (15) Schild, H. G. Poly(N-Isopropylacrylamide)sExperiment, theory and application. Progr. Polym. Sci. 1992, 17 (2), 163-249. (16) 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: Physicochem. Eng. Aspects 1999, 147 (1-2), 17-25. (17) 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. (18) Wang, Y. J.; Winnik, F. M.; Clarke, R. J. Interaction between DMPC liposomes and HM-PNIPAM polymer. Biophys. Chem. 2003, 104 (2), 449-458. (19) Ringsdorf, H.; Sackman, E.; Simon, J.; Winnik, F. M. Interactions of liposomes and hydrophobically modified poly-(N-isopropylacrylamides): An attempt to model the cytoskeleton. Biochim. Biophys. Actas Biomembr. 1993, 1153 (2), 335-344. (20) 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. (21) Kim, J. C.; Kim, J. D. Release property of temperature-sensitive liposome containing poly(N-isopropylacrylamide). Colloids Surf. B: Biointerfaces 2002, 24 (1), 45-52. (22) 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. (23) Hayashi, H.; Kono, K.; Takagishi, T. Temperature-dependent associating property of liposomes modified with a thermosensitive polymer. Bioconjugate Chem. 1998, 9 (3), 382-389. (24) 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. (25) 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. (26) 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. (27) Taillefer, J.; Jones, M. C.; Brasseur, N.; van Lier, J. E.; Leroux, J. C. Preparation and characterization of pH-responsive polymeric micelles for the delivery of photosensitizing anticancer drugs. J. Pharm. Sci. 2000, 89 (1), 52-62.
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(NIPAM) derivatives using the Langmuir balance technique. A release mechanism based on the copolymer conformational transition has been proposed in our recent work.28 In the present paper, the influence of copolymer molecular weight and distribution of anchor groups along the macromolecular chain is investigated in order to identify and better control some of the key parameters governing the release mechanism of pH-sensitive polymer functionalized liposomes. Materials and Methods Chemicals. Ultrapure water used throughout the experiments was collected from a MilliQ water purification system (Millipore Corp., Bedford, MA). Its resistivity was ca. 18.2 MΩ‚cm. The total oxygen carbon (TOC) values were less than 3 ppb. Phosphate buffers (10 mM, pH 4 and 7) were purchased from Laboratoire Mat Inc. (Montreal, Canada) and used for the Langmuir balance experiments. 4-Morpholinoethanesulfonic acid monohydrate (MES) (99.5% pure) was purchased from Sigma-Aldrich and used for the 90° light scattering analysis. Sodium chloride (99% pure) was purchased from BDH Inc. (Toronto, Canada). DSPC was obtained from Avanti Polar Lipids (Alabaster, AL) and used without any further purification. Other standards and usual chemicals were analytical grade Aldrich products. NIPAM Copolymers. Terminally alkylated copolymers of NIPAM, MAA, and DODA-poly(NIPAM-co-MAA) were prepared by free radical polymerization as described elsewhere.29 Two octadecyl chains are attached to one end of the macromolecular chain, providing active sites for the anchorage of the copolymer to phospholipidic layers (Figure 1A). A random copolymer of NIPAM, MAA, and ODA [poly(NIPM-co-MAA-co-ODA)] was also synthesized (Figure 1B).27 The number of anchor moieties (NODA) randomly distributed along the copolymer chain was calculated according to the following equation:
NODA ) (MnPolymerfODA)/(MwNIPAfNIPA + MwMAAfMAA + MwODAfODA) (1) where fi and Mwi are the molar fraction and the molecular weight of the comonomer and MnPolymer is the number-average molecular weight of the copolymer. According to eq 1, NODA ) 2. The weightaverage molecular weight (Mw) and polydispersity index (PI) were determined by gel permeation chromatography (GPC) in THF. Monodisperse polystyrene standards were used for calibration. Copolymers molar compositions were obtained by 1H NMR spectroscopy and acid-base titration (MAA content). For clarity, the different copolymers are noted herein as copolymers 1, 2, and 3 with their structural characteristics given in Table 1. Randomly (copolymer 3) and terminally alkylated copolymers (copolymers 1 and 2) differ either by the position of anchors along the polymer chain (1, 3) or their weight-average molecular weight (1, 2). pH-Phase Transition of Copolymers. The pH-phase transition, where the copolymer collapses, was determined using 90° light scattering (Brookhaven Instruments Corp., Holtsville, NY) at 532 nm and 37 °C. A freshly prepared copolymer aqueous solution (45 µg/mL) in MES-buffered saline (MES 60 mM, NaCl 75 mM) was kept stirred overnight at room temperature. The pH of 10 mL aliquots was adjusted using a concentrated sodium hydroxide solution, and the samples were filtered through 0.45 µm pore-size filters. Measurements were taken after a 5 min incubation time at 37 °C. The resulting normalized intensities (I/Imax) were plotted as a function of pH and the phase transition pH was determined as the intercept of the steep rising part of the plot with the abscissa axis. Experiments were duplicated. Compression Isotherms. The Langmuir balance technique was described in detail elsewhere.28 Surface pressure-interfacial (28) Petriat, F.; Roux, E.; Leroux, J. C.; Giasson, S. Study of molecular interactions between a phospholipidic layer and a pH-Sensitive polymer using the langmuir balance technique. Langmuir 2004, 20 (4), 13931400. (29) 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.
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Figure 1. Formulas of (A) terminally and (B) randomly alkylated poly(NIPAM) derivatives. Copolymers 1 and 2 are terminally alkylated and differ only by their weight-average molecular weight, whereas copolymer 3 is similar to copolymer 1 with regard to the molecular weight, molar composition, and number of C18 anchoring sequence but differentiates by the distribution of the anchors along the macromolecular chain. Table 1. Characteristic Properties of the Copolymers molar composition compd
Mw; PI
anchors
NIPAM
MAA
ODA
1 2 3
29 300; 2 8 200; 2,2 28 500; 2,1
N-terminal N-terminal random; N ) 2
95.2 97.3 93.7
4.6 2.4 4.9
0.2 0.3 1.4
area (Π-A) isotherms for pure materials were all recorded using the same procedure. Prior to each experiment, the Teflon tray and the Wilhelmy plate were thoroughly cleaned with methanol and rinsed several times with ultrapure water. The bath was then filled with approximately 500 mL of freshly prepared 10 mM phosphate buffer solution (pH 4 or 7). The 10 mM phosphate buffer/air interface was swept with the mobile barrier until complete compression, and any surface-active contaminant was removed by air suction. The process was repeated until the surface pressure upon complete compression was less than 0.1 mN/m. The barrier was then moved to its fully opened position and either a solution of DSPC or copolymer in chloroform (1 mg/mL) was deposited dropwise at the interface with a 50 µL microsyringe. The solvent was allowed to evaporate for a period of 10 min, and the DSPC or the copolymer molecules at the 10 mM phosphate buffer/air interface were compressed in a discontinuous pathway at a speed rate of 1 cm/min using 20 cm2/min compression steps alternating with 60 s of relaxation. This process was used to avoid the possible formation of heterogeneous compression regions and to highlight monolayer phase transitions. All the compression isotherms were recorded at 22 °C. Adsorption of the Copolymers at the DSPC Monolayer/ Water Interface. First, the trough was thoroughly cleaned as previously described and filled with 500 mL of 10 mM phosphate buffer subphase (pH 4 or 7). A DSPC solution in chloroform (1 mg/mL) was deposited dropwise at the contaminant-free 10 mM phosphate buffer/air interface. After the solvent evaporation, the DSPC monolayer was compressed to the desired surface pressure. The surface pressure constancy was ensured via the mobile barrier control, and a 5 min period was allowed for the
monolayer relaxation. The mobile barrier control was set to its maximum rate (45 cm/min) to avoid limiting the adsorption phenomenon. A certain volume of a 5 mg/mL copolymer solution was then injected using a 250 µL microsyringe through the DSPC monolayer into the subphase bulk to reach a final concentration of 0.5 or 2.5 mg/L (
