Kinetics Membrane Disruption Due to Drug Interactions of

Dec 18, 2008 - School of Chemistry, Physics and Earth Sciences, and Department of Clinical Pharmacology, Flinders University, Sturt Road, Bedford Park...
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Langmuir 2009, 25, 1086-1090

Kinetics Membrane Disruption Due to Drug Interactions of Chlorpromazine Hydrochloride Matthew R. Nussio,† Matthew J. Sykes,‡ John O. Miners,‡ and Joseph G. Shapter*,† School of Chemistry, Physics and Earth Sciences, and Department of Clinical Pharmacology, Flinders UniVersity, Sturt Road, Bedford Park, Adelaide, SA 5001 ReceiVed October 6, 2008 Drug-membrane interactions assume considerable importance in pharmacokinetics and drug metabolism. Here, we present the interaction of chlorpromazine hydrochloride (CPZ) with supported phospholipid bilayers. It was demonstrated that CPZ binds rapidly to phospholipid bilayers, disturbing the molecular ordering of the phospholipids. These interactions were observed to follow first order kinetics, with an activation energy of ∼420 kJ mol-1. Timedependent membrane disruption was also observed for the interaction with CPZ, such that holes appeared in the phospholipid bilayer after the interaction of CPZ. For this process of membrane disruption, “lag-burst” kinetics was demonstrated.

Introduction Understanding the effects of drugs on membranes is of considerable importance for current and future pharmaceutical research. Drug discovery and development has typically considered interactions between the ligand and molecular target (e.g., protein receptor), with the assumption that the membrane bilayer acts as a passive surrounding environment. In recent years, however, it has become apparent that membrane constituents, particularly phospholipids, interact with drugs and other hydrophobic chemicals.1 The ability of drugs to permeate membranes of the gastrointestinal tract and distribute to their site of action assumes further major importance in pharmacokinetics. Similarly, the nonspecific binding of drugs to incubation constituents, predominantly membrane phospholipids, decreases the concentration of free drug present in the experimental system, and this in turn leads to the underestimation of kinetic parameters (e.g., Michaelis constant and intrinsic clearance) in Vitro.2 Supported phospholipid bilayers (SPBs) can act as simple in Vitro models for physiological membranes. A hydration layer, typically with a thickness of around 1-2 nm, exists between the lower leaflet of the SPB and the solid substrate.3 The presence of this layer provides the bilayer with flexibility and fluidity, creating a dynamic system where lipid molecules can laterally diffuse freely. The ability to analyze SPBs by atomic force microscopy (AFM) has become an attractive technique for analysis of drug-membrane interactions. In a previous study from this laboratory, we demonstrated the ability of the antipsychotic drug chlorpromazine (CPZ) to increase the membrane fluidity of SPBs of dimyristoyl phosphatidylcholine (DMPC).4 It has also been reported that the macrolide antibiotic azithromycin5,6 causes a similar effect on dipalmitoyl phos* Corresponding author. Telephone: +61-8-8201-2005. Fax: +61-8-82012905. E-mail: [email protected]. † School of Chemistry, Physics and Earth Sciences. ‡ Department of Clinical Pharmacology. (1) Seydel, J. K.; Wiese, M. Drug-membrane interactions: analysis, drug distribution and modeling; Wiley-VCH: Weinheim, 2002. (2) McLure, J. A.; Miners, J. O.; Birkett, D. J. Br. J. Clin. Pharmacol. 2000, 49, 453–461. (3) Reimhult, E.; Ho¨o¨k, F.; Kasemo, B. Langmuir 2003, 19, 1681–1691. (4) Nussio, M. R.; Liddell, M.; Sykes, M. J.; Miners, J. O.; Shapter, J. G. J. Scanning Probe Microsc. 2007, 2, 41–45. (5) Berquand, A.; Mingeot-Leclerq, M. P.; Dufrene, Y. F. Biochim. Biophys. Acta 2004, 1664, 198–205.

phatidylcholine (DPPC) bilayer domains, producing a timedependent disruption of the molecular packing that eventually leads to the disappearance of gel domains. The incorporation of the anesthetic drug halothane has also been shown to give rise to structural changes in bilayer membranes.7,8 Further studies utilizing force spectroscopy revealed a difference in force measurements between pure DPPC bilayers and those containing halothane.8 Disruptive and solubilizing effects on lipid bilayers have also been observed with the antibiotic ciprofloxacin (CPX).9,10 The presence of CPX aggregates on the membrane surface have also been reported.9 In this work, we further characterized the physical changes in bilayer structures upon exposure to CPZ, in order to provide insights into the kinetics of drug-membrane interactions. CPZ, a cationic amphiphilic drug (CAD), is a dopamine-2 receptor antagonist used in the treatment of schizophrenia. Studies utilizing erythrocytes have reported that CPZ induces hemolysis by a colloid-osmotic mechanism.11 In addition, changes in the morphology of erythrocytes have been observed upon exposure of CPZ, inducing the formation of stomatocytes, spherostomatocytes, and sphereocytes.12 It was further reported that CPZ uptake does not saturate, a phenomenon that has also been observed in this laboratory with artificial liposomes.13

Materials and Methods Chemicals. 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) was purchased from Avanti Polar Lipids (Birmingham, AL). Chlorpromazine hydrochloride (CPZ; g98%), dimethyl sulfoxide (DMSO; ACS spectrophotometric grade, g99.9%), and HEPES (g99%) were purchased from Sigma-Aldrich (Sydney, (6) Berquand, A.; Dufrene, Y. F.; Mingeot-Leclerq, M. P. Pharm. Res. 2005, 22, 465–475. (7) Leonenko, Z. V.; Cramb, D. T. Can. J. Chem. 2004, 82, 1128–1138. (8) Leonenko, Z.; Finot, E.; Cramb, D. Biochim. Biophys. Acta 2006, 1758, 487–492. (9) Merino, S.; Domenech, O.; Diez, I.; Sanz, F.; Vinas, M.; Montero, T.; Hernandez-Borrell, J. Langmuir 2003, 19, 6922–6927. (10) Montero, M. T.; Pijoan, M.; Merino-Montero, S.; Vinuesa, T.; HernandezBorrell, J. Langmuir 2006, 22, 7574–7578. (11) Lieber, M. R.; Lange, Y.; Weinstein, R. S.; Steck, T. L. J. Biol. Chem. 1984, 259, 9225–9234. (12) Ahyayauch, H.; Gallego, M.; Casis, O.; Bennouna, M. J. Physiol. Biochem. 2006, 62, 199–206. (13) Nussio, M. R.; Sykes, M. J.; Miners, J. O.; Shapter, J. G. ChemMedChem 2007, 2, 366–373.

10.1021/la803288s CCC: $40.75  2009 American Chemical Society Published on Web 12/18/2008

Kinetics Membrane Disruption

Figure 1. AFM topography (2.0 × 2.0 µm; z-scale: 5 nm and phase (Z-scale: 10°)) of DMPC supported bilayers at temperatures of (A) 19 °C and (B) 23 °C. Schematics: Models of supported phospholipid bilayers indicative of partial melting at (A) upper and (B) lower bilayer leaflets.

Australia). All other chemicals were of the highest commercial quality available and were used without further purification. All aqueous solutions were prepared with Milli-Q grade reagent water with resistance ≈ 18.2 MΩ. Aqueous solutions were also filtered through a 0.2 µm membrane filter; solutions comprising DMSO required a nylon membrane. Vesicle Preparation. Multilamellar vesicles (MLVs) were prepared by first dissolving aliquots of lipid in chloroform/methanol (3:1 v/v), followed by evaporation of the solvent under nitrogen. Lipid samples were further dried under vacuum for 2 h prior to being suspended in 10 mM Hepes buffer containing 150 mM NaCl and 2 mM CaCl2 (pH 5.5). Acidic pHs are used to solubilize the chlorpromazine hydrochloride which is a basic drug. The final concentration of lipids was 1 mM. Samples were then sonicated for 30 min. Sonication ensures MLVs are converted into large unilamellar vesicles (LUVs) which when subjected to extrusion give small unilamellar vesicles (SUVs) of well-defined size. During sonication, periodic vortex mixing was carried out prior to their extrusion (Avanti Mini-Extruder, Avanti Polar Lipids, Birmingham, AL) 20 times through a polycarbonate membrane filter of defined pore diameter, typically 100 nm. Extrusion was performed at temperatures higher than the transition temperature (TM) of the component phospholipids, as gel-state lipids are difficult to extrude at lower temperatures.14 Resultant SUVs yielded a homogeneous size distribution and were used for all further experiments. AFM Imaging. The imaging of SPBs was performed using a commercial AFM (Nanoscope IV, Digital Instruments, Veeco, Santa Barbara, CA). All images were obtained by means of in situ tapping mode using triangular Si3N4 cantilevers (Digital Instruments, Veeco) which had a spring constant of 0.15 N m-1. Formation of SPBs was achieved by depositing 100 µL of 100 nm SUV solution onto a freshly cleaved mica surface. The prepared surfaces were then incubated at temperatures greater than their gel-fluid phase TM for 2 h to promote SPB formation. Prior to imaging, bilayer surfaces were washed five times with a buffer containing no lipid or calcium salt. A Nanoscope heater controller (model: HS-1) was utilized in experiments to maintain and adjust the temperature.

Results and Discussion The characteristic domain formation of DMPC was initially investigated using AFM. Differences in topography and phase imaging were observed at temperatures close to 19 °C (Figure 1A). For the current study, a measured height difference of 0.44 ( 0.06 nm and a large phase difference (∼1.0°) were observed (14) MacDonald, R. C.; MacDonals, R. I.; Menco, B. P.; Takeshita, K.; Subbarao, N. K.; Hu, L. R. Biochim. Biophys. Acta 1991, 1061, 297–303.

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between the upper and lower domains of the membrane bilayer. The height difference measured between bilayer domains in the topographic image was consistent with the top leaflet being partially melted.15,16 Phase imaging provides further insights into the two observed membrane heights as the phase lag of the tip relative to the excitation signal is sensitive to surface properties, such as adhesion and viscoeleasticity. With respect to SPBs, phase contrasts are interpreted as a change in bilayer compactness.17-19 The strong phase difference observed between upper and lower domains in Figure 1A inferred that the top leaflet was partially melted, with the upper and lower domains of the top leaflet corresponding to gel (Lβ) and liquid-crystalline (LR) phases, respectively. The bottom leaflet was completely in the Lβ phase, owing to its stabilization by the mica surface.15 Previous studies on DMPC bilayers have demonstrated the phase of the top leaflet to decouple from the bottom leaflet.15,20 Decoupling occurs in the phase transition between each bilayer leaflet, since the solid substrate stabilizes the lipid solid phase.15,20 This is observed as a contrast in topography for temperatures above the TM of the component phospholipid. For the current study, the phase transition of the bottom leaflet of DMPC bilayers was observed at ∼23 °C (Figure 1B). In contrast to the observations at 19 °C, the top leaflet is completely in its LR phase and the bottom leaflet is partially melted. The observed height difference in the topography image (0.39 ( 0.07 nm) is now due to partial melting of the bottom leaflet.15 As expected, the topographic height difference observed in parts A and B in Figure 1 is very similar, as they are both due to the presence of two different domains in one leaflet (in the top leaflet in the case of Figure 1A and bottom for Figure 1B). Smaller phase differences (∼0.2°) between the surface features in Figure 1B confirm the presence of just the LR phase on the entire top leaflet of the bilayer. The temperatures observed for domain melting in the present study, however, differ significantly from those previously reported for AFM experiments.15,20 The temperature at which the membrane changes from the Lβ to the LR phase is dependent on the aqueous medium. Divalent cations (Ca2+ and Mg2+), for example, can affect the TM by ionic interaction with the phosphate group; the phospholipids interact more closely, decreasing their molecular mobility and, as a result, increasing the TM range.21-23 In contrast to previous studies, the work presented here does not utilize divalent cations. The pH of the buffer medium is also considerably lower, and there is 1% DMSO in the buffers in all experiments. Under these circumstances, the observation of a lower TM compared to that observed previously is expected. To confirm the transition temperature for these systems, a temperature(15) Feng, Z. V.; Spurlin, T. A.; Gewirth, A. A. Biophys. J. 2005, 88, 2154– 2164. (16) Tokumasu, F.; Jin, A. J.; Feigenson, G. W.; Dvorak, J. A. Ultramicroscopy 2003, 97, 217–227. (17) Leonenko, Z. V.; Carnini, A.; Cramb, D. T. Biochim. Biophys. Acta 2000, 1509, 131–147. (18) Evans, K. O. Int. J. Mol. Sci. 2008, 9, 498–511. (19) Tokumasu, F.; Jin, A. J.; Dvorak, J. A. J. Electron Microsc. 2002, 51, 1–9. (20) Garcia-Manyes, S.; Oncins, G.; Sanz, F. Biophys. J. 2005, 89, 4261– 4274. (21) Asai, Y.; Watanabe, S. Drug DeV. Ind. Pharm. 1999, 25, 1107–1113. (22) MacDonald, R. C.; Simon, S. A.; Baer, E. Biochemistry 1976, 15, 885– 891. (23) Garcia-Manyes, S.; Oncins, G.; Sanz, F. Biophys. J. 2005, 89, 1812– 1826. (24) Lee, A. G. Mol. Pharmacol. 1977, 13, 474–487. (25) Joshi, U. M.; Rao, P.; Kodavanti, P. S.; Coudert, B.; Dwyer, T. M.; Mehendale, H. M. J. Pharmacol. Exp. Ther. 1988, 246, 150–157. (26) Lullmann, H.; Wehling, M. Biochem. Pharmacol. 1979, 28, 3409–3415. (27) Takegami, S.; Kitamura, K.; Kitade, T.; Kitagawa, A.; Kawamura, K. Chem. Pharm. Bull. 2003, 51, 1056–1059. (28) Xia, Z.; Ying, G.; Hansson, A. L.; Karlsson, H.; Xie, Y.; Bergstrand, A.; DePierre, J. W.; Nassberger, L. Prog. Neurobiol. 2000, 60, 501–512.

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Figure 2. AFM topographic (2.5 × 2.5 µm; z-scale: 5 nm) and phase (Z-scale: 10°) images of DMPC supported bilayer (A) before and (B-G) after exposure to a solution of 50 µM CPZ. Times shown are the times since introduction of the CPZ: (B) 7 min; (C) 16 min; (D) 23 min; (E) 30 min; (F) 36 min; and (G) 48 min. (H) Cross-sectional analysis of defect locations demonstrated a defect height of ∼1 nm and diameters equivalent to 40-60 nm. Complete set of data available in the Supporting Information.

Figure 3. Plot of area (µm2) as a function of time (s) for the (A) total defect area and (B) lower leaflet Lβ domain area.

dependent study demonstrating the melting of Lβ domains on the top and bottom leaflets can be viewed in the Supporting Information. Interestingly, the first signs of melting at the bottom leaflet began prior to the complete disappearance Lβ domains on the top leaflet. The previous studies using different buffer medium observed approximately 2 °C difference between the final stages of melting on the top leaflet and the onset of melting at the bottom leaflet. The observed contrasts in onset temperature for melting of Lβ domains between the work in this paper and previous studies reinforce the effects of buffer medium on the stability of bilayer membranes. Changes in molecular organization of the DMPC bilayer due to CPZ were investigated at ∼23 °C (Figure 2). Prior to drug interaction, a phase contrast of 0.2° between surface features confirmed the presence only the LR phase on the entire top leaflet (Figure 2A). As consecutive scanning of the AFM tip can potentially alter image contrast or create artificial features, the stability of the SPB was confirmed by continually scanning the same location for 45 min prior to drug interaction (Supporting Information). No changes in the bilayer are observed during these scans. Following the injection of 50 µM CPZ, time lapsed AFM images demonstrated a clear change in morphology. Images were collected 5 min after injection, as this was the time required to re-establish scanning. Consistent with work performed previously by this group,4 there was a progressive decrease in the lateral size of Lβ domains until they completely disappeared.

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Our previous experiments using surface plasmon resonance (SPR)13 have also suggested a change in fluidity for DMPC bilayer membranes upon exposure to CPZ. Researchers have also demonstrated CPZ can directly affect the TM value of the phospholipids using differential scanning calorimetry.24 The progressive change in the morphology of the bilayer also indicates binding of CPZ to the bilayer membrane. We have previously demonstrated the ability of CPZ to bind to lipid bilayer vesicles utilizing SPR.13 In the SPR study, the drug did not dissociate completely from the membrane and it was believed that a drug-phospholipid complex had formed. The drug-phospholipid complex was thought to mimic phospholipidosis,25,26 a lipid storage disorder. It is assumed the observed change arises from disruption of lipid packing by CPZ. Previous work investigating the interaction of CPZ with phosphatidylcholine vesicles associated the increase in the motion of acyl chains with positive ∆S.27 The phenomena of binding of CADs, such as CPZ, to phospholipid bilayers is such that the hydrophobic ring structure partitions into the bulky hydrocarbon phase of the membrane, while the tertiary amine interacts with the hydrophilic portion of the lipid, typically via the phosphate group.28,29 Under these circumstances, we believe that CPZ disrupts the arrangement of the phospholipid chains.28 Interestingly, the height difference between the two domains also increased by ∼40% (from 0.39 ( 0.07 to 0.71 ( 0.09 nm) after the injection of CPZ. Previous work examining the interaction of halothane with single component DPPC and DOPC SPBs has shown that the anesthetic drug induces a thinning of the membrane bilayer.7 In the case of CPZ, this thinning will occur in the LR phase predominantly, as LR phases are more accessible to drug interactions, in accordance with our previous work performed using SPR.13 The height difference between the Lβ and LR phases thereby increases, and this is observed by the measured height change. Studies of the physiological effects of ethanol7 and propan-2-ol30 demonstrated a decrease in membrane thickness, and this was considered to be associated with lipid interdigitation. We believe the progressive disruption of the molecular packing of the Lβ phase was due to the melting of the Lβ phase domains in the bottom leaflet, as experiments were performed at ∼23 °C. This was confirmed by the low contrast in the phase images measured on the surface of the bilayer throughout the experiment. A previous model for the interaction of CADs haloperidol and spiperone with phospholipid bilayers has also demonstrated the ability of these drugs to distribute to both leaflets of the membrane after initial interaction with one leaflet face.31 Consistent with previous work, we observed small defects in the bilayer after the interaction of CPZ.4 Locations outside the scanned area also revealed many defects, confirming their presence is due to drug interaction (data not shown). The measured depth of these defects was on the order of 1 nm deep (Figure 2H) and is equivalent to the height of one leaflet of the phospholipid bilayer. Although enlarged by tip-induced broadening effects, the measured diameters of these defects were on the order of 40-60 nm. Due to the size of these defects, AFM is the only available technique capable of discriminating these surface locations from the entire bilayer surface. Previous studies utilizing fluorescence microscopy, however, have shown that the CAD spiperone can distort giant unilamellar vesicles, leading to observation of many membranous fragments formed on both (29) Chen, J. Y.; Brunauer, L. S.; Chu, F. C.; Helsel, C. M.; Gedde, M. M.; Huestis, W. H. Biochim. Biophys. Acta 2003, 1616, 95–105. (30) McClain, R. L.; Breen, J. J. Langmuir 2001, 17, 5121–5124. (31) Baciu, M.; Sebai, S. C.; Ces, O.; Mullet, X.; Clarke, J. A.; Shearman, G. C.; Law, R. V.; Templer, R. H.; Plisson, C.; Parker, C. A.; Gee, A. Philos. Trans. R. Soc., A. 2006, 364, 2597–2614.

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Figure 4. Plot of natural log area (lower leaflet Lβ domains) versus time for the incubation of DMPC supported bilayers with (A) 50 µΜ (R[2] ) 0.99), (B) 125 µΜ (R[2] ) 0.94), and (C) 100 µΜ (R[2] ) 0.98) CPZ. AFM topographic (2.0 × 2.0 µm; z-scale: 5 nm) images of DMPC supported bilayer recorded in a solution of (D) 125 µM and (E) 100 mM CPZ at increasing incubation times. Circled: defects due to drug interaction. (F) Zoom (0.5 × 0.5 µm2) of commonly observed defects. Experiments utilizing 50 and 125 µΜ were performed at ∼23 °C, whereas incubations of 100 µΜ CPZ were performed at ∼25 °C.

sides of the membrane bilayer, highlighting the ability of CADs to disrupt the membrane bilayer.31 Researchers believed the interaction of CADs would induce micellelike structures capable of removing both drug and membrane components. We were able to observe the increase in abundance of defects over time. The defects were particularly evident by phase imaging, where these locations demonstrated a large phase difference compared to that of the entire bilayer surface. Utilizing flood analysis,32 the total defect area (a sum of the areas for each defect observed) as a function of time was calculated. Figure 3 presents plots for the time-dependent behavior of the observed total defect area (A), together with an overlay showing the decrease in bottom leaflet Lβ domain size as a function of time (B). It was observed that the increase in total defect area begins to plateau at ∼44 min. Comparing the time-dependent increase in total defect area with the decrease in Lβ domain size, it was evident that a lag period was also present for defect formation. This lag can be attributed to the fact that the defects occur through the removal of the top leaflet due to the interruption of lipid-lipid interactions by CPZ. It is likely that the removal of material from the leaflet will only occur after considerable membrane absorption of the drug while the observed induced phase changes can occur at much lower drug membrane loadings. Hence, there will be a time lag between the phase change and the appearance of defects. In fact, results for the interaction of haloperidol with phospholipid bilayers have suggested a lag period for membrane degradation33 and have compared this to previous work whereby the interaction of phospholipase A2 with phospholipid bilayers demonstrated “lag-burst” kinetic behavior.34-36 We believe a similar process is also occurring for the interaction CPZ. In parallel with membrane solubilization,31,33,37 a local increased concentration of CPZ may be capable of inducing micelle-like structures by (32) Horcas, I.; Fernandez, R.; Gomez-Rodriguez, J. M.; Colchero, J.; GomezHerrero, J.; Baro, A. M. ReV. Sci. Instrum. 2007, 78, 13705–13713. (33) Casey, D. R.; Sebai, S. C.; Shearman, G. C.; Ces, O.; Law, R. V.; Templer, R. H. Ind. Eng. Chem. Res. 2008, 47, 650–655. (34) Honger, T.; Jorgensen, K.; Stokes, D.; Biltonen, R. L.; Mouritsen, O. G.; Byron, R.; Edward, A. D. Methods Enzymol. 1997, 286, 168–190. (35) Leidy, C.; Linderoth, L.; Andresen, T. L.; Mouritsen, O. G.; Jorgensen, K.; Peters, G. H. Biophys. J. 2006, 90, 3165–3175. (36) Niesen, L. K.; Risbo, J.; Callisen, T. H.; Bjornholm, T. Biochim. Biophys. Acta 1999, 1420, 266–271. (37) Schreier, S.; Malheiros, S. V. P.; de Paula, E. Biochim. Biophys. Acta 2000, 1508, 210–234.

interacting with surrounding phospholipids and hence creating holes by the removal of membrane components. Any membrane components removed by this action would potentially also have CPZ bound. Hence, the number of defects possible must be linked to the total amount of drug available to interact with the membrane. As there is a finite amount of drug available, only a fraction of the membrane will be affected with these defects, as it is likely that, in the production of the defects, due to a local high concentration of drug, the drug is also removed from the system. The observed defects can be explained by a drug induced disruption of the membrane. This effect may be analogous to the hemolysis caused by CPZ under physiological conditions (arising from the rupturing of cell membranes).11 Electron cryomicroscopy studies have also suggested that propranol, another CAD, is capable of bilayer perturbation.26,38 Haloperidol and spiperone are known to cause membrane degradation by means of acid-catalyzed ester hydrolysis.31,33 The hydrolysis of membrane phospholipids by these CADs form lyso-lipids that are capable of translocating from the phospholid bilayer surface. It was established in our previous study that the effects of CPZ were concentration-dependent. However, it was not possible to determine unequivocally whether the process was zero- or firstorder. Experiments performed in the current study were able confirm our original assumption of first-order kinetics, as a plot of the natural logarithm of the area (lower leaflet Lβ domains) versus time was linear (R2 ) 0.99) (Figure 4A). As predicted, the slope of the line was comparable to that observed for experiments performed with 125 µM CPZ (R2 ) 0.94) (Figure 4B), as both were performed at the same temperature, and hence, their rate constants should be equivalent. The rate constant at 23 °C was equivalent to ∼1.038 ( 0.079 ms-1. At 100 µM CPZ, experiments were conducted at ∼25 °C, which was a slightly higher temperature than that in the previous work. As expected, bilayers initially demonstrated a complete LR phase on the top leaflet and the bottom leaflet was more extensively melted (Figure 4E) than that in the experiment shown in Figure 1B. A plot of the natural logarithm of the area (bottom leaflet Lβ domains) as a function of time revealed a linear relationship (R2 ) 0.98) with a different slope (k ) 5.916 ( 0.538 ms-1) from that observed (38) Scuntaro, I.; Kientsch, U.; Wiesmann, U. N.; Honegger, U. E. Br. J. Pharmacol. 1996, 119, 829–834.

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for experiments conducted at 23 °C (Figure 4C). While the rate constant should be sensitive to changes in temperature, the change in slope for these experiments conducted at ∼25 °C was significant. This suggests the interaction of CPZ with the membrane bilayer to be very sensitive to changes in temperature. Using the Arrhenius equation, the activation energy for CPZ to perturb the molecular organization of lower leaflet Lβ phase domains of DMPC was determined. This was considered the minimum energy required to break the interaction forces between many neighboring lipid molecules in the Lβ phase domain. The value obtained was ∼420 kJ mol-1. Previous work has established the activation energy for the uptake of the amphiphilic drug, doxorubicin, into LUVs to be a factor ∼3 less.39 However, the work presented in this paper is investigating the change in the molecular order of the phospholipids, not the uptake. The activation energy presented here corresponds to the energy required to break many interactions of neighboring phospholipids located at the bottom leaflet of the lipid bilayer. The stability of these interactions is governed by the electrostatic and van der Waals interactions between the phosphatidylcholine polar headgroups and hydrocarbon chains, respectively. It is also known that the bottom leaflet is stabilized by the mica surface. Hence, a large amount of energy is required to break these interactions. Previous research has also demonstrated the system enthalpy for the Lβ to LR phase transition of DMPC SPBs to be equivalent to 628 kJ mol-1.19 Although not a direct correlation to the measured activation energy for the current study, this large system enthalpy does imply there is a significant internal energy associated with the interactions between neighboring phospho(39) Harrigan, P. R.; Wong, K. F.; Redelmeier, T. E.; Wheeler, J. J.; Cullis, P. R. Biochim. Biophys. Acta 1993, 1149, 329–338.

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lipids in the Lβ phase. Considering the activation energy for the current study is of similar magnitude, the measured value of ∼420 kJ mol-1 seems reasonable in terms of the energy required to disrupt the molecular associations between DMPC phospholipids in their Lβ phase during CPZ exposure.

Conclusion The ability to access kinetic information for drug-membrane interactions using AFM was successfully demonstrated. Results revealed that CPZ is capable of distributing to the bottom leaflet of the SPB and disrupting the molecular ordering of the phospholipids. This effect followed first order kinetics, and the activation energy required to break the interaction forces between neighboring phospholipids of the Lβ phase domains was determined to be ∼420 kJ mol-1. Time-dependent membrane disruption was also clearly observed, where “lag-burst” kinetics was revealed. These experiments show that the methods used in the current study are useful for evaluation of drug-membrane binding interactions, for determination of kinetics of new drug candidates and for providing insights into processes that contribute to drug-membrane binding. Acknowledgment. This work is supported by the Australian Microscopy and Microanalysis Research Facility (AMMRF) and Flinders University. Supporting Information Available: Temperature-dependent topography and phase images for a DMPC supported bilayer as well as AFM images showing the DMPC layer is unaffected by continuous scanning. This material is available free of charge via the Internet at http://pubs.acs.org. LA803288S