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Jul 6, 2015 - ABSTRACT: Sum frequency generation (SFG) vibrational spectroscopy was employed to study the interaction between memantine (a ...
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Qualitative and Quantitative Analyses of the Molecular-Level Interaction between Memantine and Model Cell Membranes Bolin Li,†,§ Hong-Yin Wang,†,§ Peiyong Feng,† Xiaofeng Han,† Zhan Chen,‡ Xiaolin Lu,*,† and Fu-Gen Wu*,† †

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China ‡ Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: Sum frequency generation (SFG) vibrational spectroscopy was employed to study the interaction between memantine (a water-soluble drug for treating Alzheimer’s disease) and lipid bilayers (including zwitterionic PC and negatively charged PG lipid bilayers) at the molecular level in real time and in situ. SFG results revealed how the memantine affected these lipid bilayers in terms of the lipid dynamics, average tilt angle (θ), as well as angle distribution width (σ). It was found that memantine could adsorb onto the zwitterionic PC surface but did not affect the flip-flop rate of the PC bilayer even in the presence of 5.0 mM memantine, indicating the negligible interaction between memantine and the PC bilayer. However, for the negatively charged PG bilayer, it was found that the outer PG leaflet could be significantly destroyed by memantine at a relatively low memantine concentration (1.0 mM), while the inner PG leaflet remained intact. Besides, the θ and σ of CD3 groups in the outer PG lipid leaflet were calculated to be ∼82.0° and ∼19.5° after adding 5 mM memantine, respectively, indicating that these CD3 groups were prone to lie down at the membrane surface (versus the surface normal) with the addition of 5 mM memantine while nearly standing up without the addition of drug molecules. These monolayer- and molecular-level results could hardly be obtained by other techniques. To the best of our knowledge, this is the first experimental attempt to quantify the drug-induced orientational changes of lipid molecules within a lipid bilayer. The present work provided an in-depth understanding on the interaction between memantine and model cell membranes, which will potentially benefit the development of new drugs for neurodegenerative diseases involving drug− membrane interaction.

1. INTRODUCTION The interaction between drugs and cell membranes is directly related to the drug delivery, segregation, distribution, efficacy, and resistance.1 Studying the drug−membrane interaction is critical to the better understanding of these drug actions, pharmacokinetics, and the design of new drugs.2 Given the complexity of real cell membranes, the model cell membranes, i.e., lipid monolayers and bilayers, have been commonly used as the alternatives of real cell membranes to investigate the lipid structures and their interactions with other molecules.3−9 Over the past decades, several computational and experimental techniques, such as molecular dynamics simulation,10,11 X-ray and neutron diffraction,12,13 solution and solid NMR,14,15 EPR,16 differential scanning calorimetry (DSC),17−23 and Fourier transform infrared spectroscopy (FTIR),17−23 have been widely employed to study model cell membrane systems and their interactions with a variety of different molecules. However, it is still a challenge to accurately probe the molecular-level structures or dynamics of the membrane surface and interface in real time and in situ, as well as quantitatively analyze the molecular orientation and orientation © 2015 American Chemical Society

distribution simultaneously. Fortunately, sum frequency generation (SFG) vibrational spectroscopy, with inherent interfacial selectivity and submolecular sensitivity, has been rapidly developed into a powerful technique to detect the surface and interfacial molecular structures. Moreover, the molecular orientation and orientation distribution information can be conveniently achieved by data analysis. SFG spectroscopy has been frequently used to investigate lipid monolayer or bilayer systems.24−40 In this study, we applied SFG spectroscopy to investigate molecular-level interaction between memantine and lipid bilayers supported on CaF2 substrates. Memantine (1-amino-3,5-dimethyl-adamantane, see its structural formula in Figure 1) has been approved by the US Food and Drug Administration (FDA) to be used for the treatment of moderate to severe Alzheimer’s disease.41,42 Recently, the key binding interaction between memantine and the N-methylD-aspartate (NMDA) receptor in the postsynaptic membrane has been investigated, and the results revealed that memantine Received: January 6, 2015 Published: July 6, 2015 17074

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several times, dried with nitrogen gas, and finally cleaned with an oxygen plasma cleaner. 2.2. Sample Preparation. The Langmuir−Blodgett (LB) system (KN 2003, KSV NIMA Co., Ltd.) was applied to fabricate the lipid monolayer supported on a CaF2 prism. The procedures were briefly described as follows: One of right-angle faces of the prism was attached to a homemade sample holder and the other right-angle face was vertically dipped into water in the LB trough. The lipid solution was cautiously added onto the water surface using a microsyringe. After the evaporation of solvent, the lipid monolayer on the water surface was condensed by two Teflon barriers at a rate of 5 mm/min until a surface pressure of 34 mN/m was reached. Next, the CaF2 prism was perpendicularly lifted out of the water at a rate of 1 mm/min and a lipid monolayer supported on the CaF2 prism surface was finally formed. The Langmuir−Blodgett (LB) and Langmuir−Schaefer (LS) method were widely employed for the preparation of lipid bilayer,5−9 and the following procedures were commonly applied. First, a CaF2 prism with a lipid monolayer as previously prepared was fixed on a homemade holder. Next, a lipid monolayer with a 34 mN/m surface pressure was prepared on water in a Teflon container placed on a lifting platform. And then the platform was uplifted gradually so that the monolayer on water could contact with the monolayer on the CaF2 prism to form a lipid bilayer. 2.3. SFG Experimental. A commercial pico-second SFG system (EKSPLA) based on a pico-second Nd:YAG laser was employed in this experiment. The details regarding the SFG system have been extensively described elsewhere.44−48 In brief, the pico-second Nd:YAG laser generated a fundamental output beam (1064 nm wavelength) with a pulse width of ∼20 ps, which was doubled via the harmonic unit to generate the visible beam (532 nm). The frequency tunable infrared (IR) beam was generated by an optical parametric generation/amplification (OPG/OPA) and difference frequency generation (DFG) system based on LBO and AgGaS2 crystals. The incident angles of the visible beam and the IR beam were around 60° and 55° with respect to the surface normal, respectively. The sum frequency generation (SFG) signal was generated by overlapping the 532 nm visible beam with the tunable IR beam at the sample interface. The overlapped beam spot diameter was about 0.5 mm. The energies of visible beam and IR beam were approximately 35 μJ and 60 μJ, respectively. The SFG signals were collected by the PMT detector. The scanning interval was 5 cm−1. The intensity of SFG signal was normalized by the energies of the input visible and IR beams. The schematic of the SFG experimental geometry employed in this research was depicted in Figure 1. The SFG spectra were collected in the frequency range from 2800 to 3900 cm−1. The time-dependent SFG signal intensities of the CH3 symmetric stretching (ss) vibrational mode at 2875 cm−1, the CD3 symmetric stretching (ss) vibrational mode at 2070 cm−1, and/or the OH stretching vibrational mode of interfacial water at ∼3200 cm−1 were monitored in real time and in situ, respectively. All the SFG spectra were collected at room temperature (22 °C) using ssp (s-polarized sum frequency output, s-polarized visible input, and p-polarized infrared input) polarization combination except the SFG spectra of the DPPG/ dDPPG bilayer which used both ssp and ppp polarization combinations (as shown in the Supporting Information).

Figure 1. Schematic of the SFG experiment.

has a higher affinity with the receptor than amantadine.43 Direct interactions between memantine and the cell membrane were also reported. For example, the distribution and dynamics of memantine in a lipid bilayer were studied using the molecular dynamics (MD) simulations, demonstrating the preferred location of memantine within a lipid bilayer, which deepens the understanding of how memantine reaches its target within the cell.10 Up to now, very few attempts have been focused on the interactions between drugs and model cell membranes at the molecular level in real time and in situ. Furthermore, previous studies only focused on the variations of the drug structure or lipid bilayer from a qualitative perspective during the interaction process.6,7 Very few quantitative or even semiquantitative analyses have been performed on the orientation or orientation distribution of the lipid bilayer before and after the addition of drugs. The effect of drugs on the conformational or orientational changes of lipid molecules plays an important role in certain cell membrane functions.2 However, previous results have not fully elucidated the interaction between memantine and model cell membranes, and more efforts should be conducted to illuminate the interaction kinetics, molecular orientation, and orientation distribution of the lipid molecules for an in-depth understanding of this important biological process at the interface. To the best of our knowledge, the present work represents the first attempt to quantify the druginduced orientation variations of the terminal groups of lipid molecules within a lipid bilayer. The average tilt angle (θ) and angle distribution width (σ) of CD3 groups in the outer dDPPG leaflet for the DPPG/dDPPG bilayer before and after the addition of drugs were quantitatively analyzed.

2. EXPERIMENTAL SECTION 2.1. Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl(d62)-sn-glycero-3-phosphocholine (DPPC-d62 or dDPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho(1′-rac-glycerol) (sodium salt) (DPPG) and 1,2-dipalmitoyl(d62)-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPG-d62, or dDPPG) were purchased from Avanti Polar Lipids and used without further purification. The DPPC (or dDPPC) and DPPG (or dDPPG) were dissolved in chloroform and a mixed solution of chloroform and methanol (v:v = 4:1), respectively. Their molecular structures are shown in Figure S1. Memantine hydrochloride was purchased from J&K Scientific Ltd., and its structural formula was shown in Figure 1. Rightangle CaF2 prisms were ordered from Chengdu YaSi Optoelectronics Co., Ltd. and employed as solid supported substrates. They were carefully washed prior to the deposition of the lipid monolayer: The prisms were soaked in toluene for 12 h, rinsed with ethanol and ultrapure water (18.2 MΩ·cm) 17075

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Figure 2. Time-dependent SFG ssp signal intensities collected at 2070 and 2875 cm−1 from the dDPPC/DPPC bilayer without (A) and with (B) the addition of 5 mM memantine into the subphase (adding memantine immediately after contacting to form the bilayer). Both of the spectra in Figure 2A and 2B were normalized to each other.

Figure 3. Time-dependent SFG ssp signal intensities collected at 2070 and 3200 cm−1 from the dDPPC/dDPPC bilayer after the addition of 2 mM (A) and 5 mM (B) memantine into the subphase, respectively.

3. RESULTS AND DISCUSSION 3.1. Interaction between Memantine and PC Bilayer. To determine whether and how the memantine molecules affect the PC bilayer, the supported dDPPC/DPPC bilayer was prepared (the inner lipid layer attached to the CaF2 prism surface consists of dDPPC, whereas the outer lipid layer on the water surface is composed of DPPC). Figure 2A shows the time dependent SFG signal intensities of the CH3 symmetric stretching (ss) vibrational mode at 2875 cm−1 in the DPPC layer and the CD3 symmetric stretching (ss) vibrational mode at 2070 cm−1 in the dDPPC layer, which were simultaneously monitored without the addition of memantine into the subphase. As shown in Figure 2A, once the inner dDPPC leaflet on the CaF2 prism contacted with the outer DPPC leaflet on water surface to form the dDPPC/DPPC bilayer, the SFG signal intensities of the dDPPC/DPPC bilayer collected at 2875 and 2070 cm−1 rose significantly, resulting from the enhancement of the interfacial Fresnel coefficient, as we have previously reported.49 As time went by, the transbilayer movement (flip-flop) of the lipid molecules in the dDPPC/ DPPC bilayer contributed to the gradual decrease in the net population difference for both DPPC and dDPPC molecules in two leaflets, leading to the decrease of the SFG signal intensities ICH3 (2875 cm−1) and ICD3 (2070 cm−1). Finally, Both ICH3 and ICD3 remained unchanged until around 2000 s, suggesting that the flip-flop process of the dDPPC/DPPC bilayer finished in a shorter time as compared with that of the dDSPC/DSPC bilayer.7 For comparison, the time-dependent SFG signal

intensities of ICH3 and ICD3 for the dDPPC/DPPC bilayer with the addition of 5 mM memantine were also monitored (Figure 2B). For a quantitative comparison, all the time-dependent curves in Figure 2 were fitted using a reported method6,7,50,51 (see details in the Supporting Information). The fitting results for the 2875 cm−1 revealed that the flip-flop rate (k) and the half-life (t1/2) of the neat lipid bilayer were (3.6 ± 0.8) × 10−4 s−1 and (9.6 ± 2.7) × 102 s, respectively. With the addition of 5 mM memantine, they became (3.4 ± 1.2) × 10−4 s−1 and (1.0 ± 0.6) × 103 s, respectively, which did not have a significant difference compared with those of the neat lipid bilayer without memantine. The results indicated that the memantine molecules did not have a significant effect on the flip-flop rate and the half-life of the dDPPC/DPPC bilayer even at a high memantine concentration of 5 mM, and there was no appreciable interaction between memantine and the dDPPC/ DPPC bilayer. The CH3 or CD3 SFG signal contributed from the lipid tails was commonly used for monitoring the structural change of lipid bilayers or the interactions of lipid membranes with other molecules.5−7,49−51 The SFG signal coming from the OH vibrational mode of the interfacial water was usually employed to study the interfacial adsorption behaviors.6,7,49,52 Here, to further confirm the adsorption and interaction between memantine and the PC bilayer, SFG experiments were performed for the dDPPC/dDPPC bilayer, and the SFG signal changes of CD3 vibrations after the addition of different concentrations of memantine were recorded. Figure 3A and 3B show the time-dependent SFG signal intensities collected at 17076

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The Journal of Physical Chemistry C 2070 cm−1 (CD3, ss) and 3200 cm−1 (strong OH vibration) after the addition of 2 mM and 5 mM memantine into the subphase, respectively. The SFG signal intensity collected at 2070 cm−1 almost remained unchanged while the SFG signal intensity collected at 3200 cm−1 increased slowly and subsequently reached a plateau after the addition of 2 mM memantine (Figure 3A). While after the introduction of 5 mM memantine, the SFG signal intensity at 2070 cm−1 also stayed constant whereas the signal intensity at 3200 cm−1 increased abruptly and then remained unchanged (Figure 3B). These observations indicated that the memantine molecules did not disturb the dDPPC/dDPPC bilayer. However, the above change of the SFG signal intensity at 3200 cm−1 after the introduction of memantine shows that these drug molecules were indeed adsorbed onto the lipid bilayer surface, since they rendered the lipid surface positively charged, making the water molecules tend to be more orderly oriented at the interface.53−58 As seen in Figure 3, the SFG intensity collected at 3200 cm−1 increased to be much larger with the addition of a higher memantine concentration (5 mM) compared to that with the introduction of 2 mM memantine, which again suggested that memantine molecules were adsorbed onto the lipid surface. To further monitor the adsorption behavior of memantine molecules at the PC surface, we have also collected the SFG spectra from the dDPPC/dDPPC bilayer in the frequency range from 2800 to 3900 cm−1 after the addition of different concentrations (0 mM, 2 mM, and 5 mM) of memantine. As shown in Figure 4, the broad band from 2800 to 3000 cm−1

developed into a methodology to investigate the interfacial adsorption, interaction, and reaction by the Eisenthal and Geiger groups.56,59−67 Herein, there are two contributions (the second-order χ(2) and third-order χ(3)) to the SFG signal intensity of the interfacial water. Without the addition of memantine, the SFG signals of interfacial water mainly come from the second-order χ(2) contribution. With the addition of memantine, the third-order χ(3) could have significant contribution to the observed water signal within a Debye length scale resulting from the interfacial potential induced by the positively charged memantine. The increased SFG signal of water here provides solid evidence that memantine molecules have adsorbed onto the lipid surface. In Figure 4, no apparent SFG signals of memantine molecules are detected in the vibrational frequency range from 2800 to 3000 cm−1 for the dDPPC/dDPPC bilayer, implying that the memantine molecules were randomly oriented at the lipid surface even though they indeed adsorbed onto the lipid surface. 3.2. Interaction between Memantine and PG Bilayer. To illustrate the interaction between memantine and the negatively charged PG bilayer, a dDPPG/DPPG bilayer was prepared. The time-dependent SFG signal intensities of the CD3 symmetric stretching (ss) vibrational mode at 2070 cm−1 in the dDPPG leaflet and the CH3 symmetric stretching (ss) vibrational mode at 2875 cm−1 in the DPPG leaflet were simultaneously monitored without the introduction of memantine. As shown in Figure S2, upon the formation of the bilayer, the SFG signal intensities at 2875 and 2070 cm−1 both increased significantly due to the enhancement of the interfacial Fresnel coefficients49 and then became constant subsequently, indicating that the PG bilayer did not have a noticeable flip-flop process within 2 h. Figure 5 shows the time-dependent SFG signal intensities collected at 2070 and 2875 cm−1 before and after the addition of 1 mM and 5 mM memantine, respectively. Addition of 1 mM memantine led to a gradual decrease of the SFG signal intensity at 2875 cm−1 at the beginning (within 250 s). After that, the signal became stable with time, while the SFG signal intensity collected at 2070 cm−1 remained unchanged after the addition of drugs (Figure 5A). As a comparison, when 5 mM memantine was added into the subphase, the signal intensity at 2875 cm−1 dropped instantly within 30 s and then became constant afterward. The SFG signal intensity at 2070 cm−1 still did not change after the addition of drugs (Figure 5B). These results illustrated that the outer DPPG leaflet could be significantly disturbed by memantine molecules, while the inner dDPPG leaflet was seldomly affected by drugs. This disturbance effect exerted to the outer lipid leaflet was more pronounced at an elevated drug concentration. To investigate the effect of drugs on the water molecules residing on the membrane surface, and to confirm the disturbance effect of drugs on the PG bilayer, time-dependent SFG signal intensities at 2070 and 3200 cm−1 were collected from a symmetric dDPPG/dDPPG bilayer after the addition of 1 mM (Figure 6A) and 5 mM (Figure 6B) memantine, respectively. As seen in Figure 6, the SFG signal intensity collected at 3200 cm−1 decreased abruptly upon the addition of both 1 mM and 5 mM memantine, indicating that the positively charged memantine molecules strongly interacted with the negatively charged outer dDPPG leaflet, which neutralized the membrane surface and led to randomly oriented water molecules on the membrane surface. Because of the strong electrostatic interaction between memantine and PG molecules, the outer dDPPG leaflet was significantly disturbed (while the

Figure 4. SFG ssp spectra collected from the dDPPC/dDPPC bilayer in the frequency range from 2800 to 3900 cm−1 after the addition of 0 mM (a), 2 mM (b), and 5 mM (c) memantine into the subphase, respectively. These SFG spectra were collected ∼30 min after the addition of drugs to the formed bilayer.

comes from the water signals rather than memantine because the resonant CH peaks of memantine molecules should be narrow and notable (see Figure 7 and Figure S3) if memantine molecules are orderly oriented at the interface. The SFG signal intensities at 3200 cm−1 (strong OH vibration) and 3400 cm−1 (weak OH vibration) increase with the elevated concentration of memantine. This is because memantine molecules rendered the lipid surface positively charged, resulting in a positive electric field. Therefore, the water molecules preferred to be more orderly oriented at the lipid surface. Besides, the thirdorder χ(3) term also contributes to the SFG signal of water due to the presence of positive charge at the interface. For a charged interface, the contribution of the third-order χ(3) to the second harmonic generation (SHG) signal has been scaled and 17077

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Figure 5. Time-dependent SFG ssp signal intensities of the CD3 symmetric stretching (ss) vibrational mode at 2070 cm−1 in the dDPPG leaflet and the CH3 symmetric stretching (ss) vibrational mode at 2875 cm−1 in the DPPG leaflet were simultaneously monitored for the dDPPG/DPPG bilayer before and after the addition of 1 mM (A) and 5 mM (B) memantine, respectively.

Figure 6. Time-dependent SFG ssp signal intensities collected at 2070 and 3200 cm−1 from the dDPPG/dDPPG bilayer after the addition of 1 mM (A) and 5 mM (B) memantine, respectively.

inner leaflet remained undisturbed, as discussed above and shown in Figure 5) so that the inversion symmetry of the lipid bilayer was broken at the interface, resulting in the instant increase of the SFG signal at 2070 cm−1. Such a disturbance effect on the outer PG layer was more pronounced at a higher drug concentration (5 mM), and thus the increase of the SFG signal at 2070 cm−1 was more significant. These results further confirmed the strong interaction between memantine and the outer PG leaflet. To further verify the adsorption behavior of memantine molecules on the PG bilayer, we investigated the variation of SFG spectra for the dDPPG/dDPPG bilayer before and after the introduction of different concentrations of memantine. Figure 7 shows the SFG spectra collected from the lipid bilayer in the frequency range from 2800 to 3900 cm−1 without (a) and with the addition of 0.1 mM (b), 0.2 mM (c), 0.5 mM (d), 1 mM (e), and 5 mM (f) memantine, respectively. As shown in Figure 7, curve a, the two major peaks at 2880 and 2940 cm−1 were assigned to the stretching vibrations of the nondeuterated CH groups in the dDPPG head region. Because of the negatively charged dDPPG molecules at the interface, there is a significant χ(3) signal overlapping and/or interfering with the χ(2) signal before the addition of memantine. As shown in the curves b−f of Figure 7, after the addition of memantine, the overall SFG signal of water decreases with the decrease of the negative surface charge density, resulting from the neutralization by the positively charged memantine molecules. The two peaks at 2880 and 2940 cm−1 interfere with the peaks of memantine at 2865 and 2925 cm−1 and are gradually replaced by the latter two peaks of memantine, which gradually become

Figure 7. SFG ssp spectra collected from the dDPPG/dDPPG bilayer in the frequency range from 2800 to 3900 cm−1 without (a) and with the addition of 0.1 mM (b), 0.2 mM (c), 0.5 mM (d), 1 mM (e), and 5 mM (f) memantine into the subphase, respectively. These SFG spectra are collected ∼30 min after the addition of drugs to the formed bilayer and have been offset for clarify.

sharper and narrower with the increase of memantine concentration, indicating that the adsorbed memantine molecules gradually dominated the membrane surface. These peaks at 2840, 2865, and 2925 cm−1 could be attributed to the CH stretching vibrations coming from drug molecules, since their peak intensities all gradually increased with the increase of memantine concentration. These peaks should not come from an asymmetric bilayer formed by the disturbance of memantine to the outer layer of the dDPPG/dDPPG bilayer, since an 17078

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of 5 mM memantine. The normalized ssp and ppp spectra collected from the DPPG/dDPPG bilayer in the CD stretching frequency range were shown in Figure S5. In order to quantitatively analyze the orientation information on CD3 groups in the dDPPG leaflet, the ssp and ppp spectra were fitted (Figure S5) and the fitting results were listed in Table S1. The average tilt angle (θ) of CD3 groups in the outer dDPPG layer of the DPPG/dDPPG bilayer was calculated. For the CD3 groups, a C3v symmetry could be approximately applied for the orientation analysis.68−72 Therefore, eqs 1−6 could be applied for describing the connection between the macroscopic secondorder nonlinear susceptibilities and the microscopic molecular hyperpolarizability for an isotropic surface or interface:71,73

asymmetric PG bilayer (e.g., the dDPPG/DPPG bilayer) had the CH stretching vibration peaks at 2875 and 2945 cm−1.9 Besides, the assignment of these three peaks could also be confirmed by the SFG ssp spectrum of memantine at the air/ water interface (shown in Figure S3), in which these peaks were clearly observed. The strong SFG signals observed for drug molecules in the dDPPG/dDPPG bilayer suggested that the memantine molecules were indeed orderly oriented at the dDPPG/dDPPG surface. This was in stark contrast to that of the dDPPC/dDPPC bilayer, where the memantine molecules adopted a random orientation in the bilayer and no SFG signal of memantine molecules could be detected. The electrostatic interaction between the negatively charged PG leaflet and the positively charged memantine molecules played an important role in making memantine molecules adsorb onto the membrane surface and be orderly oriented under the effect of the electric field. Such a strong electrostatic attraction interaction, together with the hydrophobic interaction between the dimethyl-adamantane part of memantine and the hydrocarbon tails of lipids, prevented the flip-flop process of drug molecules within the lipid bilayer, which ensured that drugs were only distributed at the outer lipid layer. The adsorption of memantine molecules onto the PG membrane surface was also supported by the decrease of the signal intensity of the strong OH vibrational mode at around 3150 cm−1, which resulted from the disordered outer negatively charged PG leaflet and neutralized surface caused by positively charged drug molecules. The strong interaction between memantine and the outer PG leaflet is similar to that of amantadine.6 However, there are differences between the interactions of memantine and amantadine with PG lipids. For example, memantine at a low concentration (0.2 mM) did not exert a significant effect on the outer PG leaflet (Figure S4) even though it was indeed adsorbed onto the lipid surface (as shown in Figure 7). In contrast, a 0.2 mM amantadine was reported to have a significant influence on the outer PG molecules.6 Compared with amantadine, the only structural difference in memantine is two additional methyl groups located in the ring backbone (Figure 1), which makes memantine larger than amantadine. Owing to the steric hindrance effect, the larger molecular size of memantine leads to an increased difficulty for the drug molecule to overcome the unfavorable interaction between the hydrophobic moiety of the drug and the hydrophilic, charged lipid headgroups, and thus a higher drug concentration was required to have a significant disturbance effect of memantine on the PG membrane. Different from memantine, amantadine at a concentration of 0.2 mM could easily penetrate into the outer PG leaflet, have conformational and orientational rearrangements within the bilayer with the change of time, and finally be well fitted within the interfacial region of the membrane, while for memantine, 1 mM memantine could disturb the outer PG layer immediately and no further molecular rearrangements were observed. These striking differences point out the importance of the drug’s size in drug−membrane interactions. 3.3. Change of Average Tilt Angle and Angle Distribution Width of CD3 Groups for the DPPG/ dDPPG Bilayer before and after the Addition of 5 mM Memantine. To evaluate the disturbance effect of memantine molecules on the outer PG layer, a DPPG/dDPPG bilayer was constructed for investigating the conformational change of CD3 groups in the outer dDPPG layer without and with the addition

χxxz , ss = χyyz , ss =

1 Nsβ [cos θ(1 + r ) − cos3 θ(1 − r )] 2 ccc , ss (1)

χxzx , ss = χyzy , ss = χzxx , ss = χzyy , ss =

1 Ns 2

βccc , ss(cos θ − cos3 θ )(1 − r )

(2)

χzzz , ss = Nsβccc , ss[r cos θ + cos3 θ(1 − r )]

(3)

1 χxxz , as = χyyz , as = − Nsβaca , as(cos θ − cos3 θ) 2

(4)

χxzx , as = χyzy , as = χzxx , as = χzyy , as =

1 Nsβ cos3 θ 2 aca , as

χzzz , as = Nsβaca , as(cos θ − cos3 θ)

(5) (6)

where θ is defined as the average tilt angle of CD3 groups (the angle between the CD3 molecular principal axis and the surface normal); r is the ratio of βaac,ss/βccc,ss for the CD3 “ss” mode, which could be calculated to be 2.30.74−76 Besides, the ratio of βaca,as/βccc,ss could be calculated to be 1.73 based on the single bond additivity approach.71,77,78 All the interfacial Fresnel coefficients could be calculated based on a previously reported method.49 As is known, the effective second-order nonlinear susceptibility ratio between the “ss” mode in ssp spectra and the “ss” mode in ppp spectra can be used for describing the orientation information on CD3 groups at the interface.79,80 So the ratio of (2) χ(2) ef f,ssp,ss/χef f,ppp,ss can be expressed as χeff2 , ssp , ss χeff2 , ppp , ss

=

5.28⟨cos θ ⟩ + 2.08⟨cos3 θ ⟩ 7.53⟨cos θ ⟩ − 4.09⟨cos3 θ ⟩

(7)

here the brackets “⟨⟩” mean the ensemble average. Usually, the interfacial CD3 groups do not have the same tilt angle, so the ensemble θ of CD3 groups can be modeled by a Gaussian distribution accounting for the distribution of the possible molecular tilt angles according to the literature,81−83 which can be described as shown in eq 8: ⎡ (θ − θ )2 ⎤ 0 ⎥ f (θ ) = C exp⎢ − 2σ 2 ⎦ ⎣

(8)

here C is the normalization constant, and σ is the angle distribution width. Therefore, cos θ and cos3 θ can be expressed as eqs 9 and 10 when the Gaussian distribution is used. 17079

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∫ cos θf (θ) sin θdθ

⟨cos3 θ ⟩ =

between the glycerol moieties of the PG headgroups and the van der Waals forces between the neighboring PG tails. Therefore, the formed well-ordered DPPG/dDPPG bilayer ensured a narrow angle distribution width (σ) of CD3 groups. If we assume a very narrow tilt angle distribution width of CD3 groups (σ = ∼0°) of the lipid bilayer without the addition of memantine (the result of assuming an angle distribution width of ∼20 is shown in Figure S7), the effective nonlinear susceptibility for the ssp polarization combination (|χeff,ssp,ss,without|) can be calculated to be ∼4.98. To quantitatively determine the average tilt angle and its distribution width of CD3 groups after the addition of 5 mM memantine, the ratio of the effective nonlinear susceptibilities before and after the addition of memantine (|χeff,ssp,ss,without/χeff,ssp,ss,with|) could be calculated to obtain the relatively accurate average tilt angle and its distribution width based on the results in Figure 8. Figure 9

(9)

∫ cos3 θf (θ) sin θdθ

(10) (2) χ(2) ef f,ssp,ss/χef f,ppp,ss

Figure 8 shows the dependence of on the θ and σ of CD3 groups in a DPPG/dDPPG bilayer assuming a

(2) Figure 8. Dependence of χ(2) ef f,ssp,ss/χef f,ppp,ss on the average tilt angle (θ) and the angle distribution width (σ) of CD3 groups in a DPPG/ dDPPG bilayer assuming a Gaussian distribution. The red and blue horizontal lines indicate the experimental value of χef(2)f,ssp,ss/χef(2)f,ppp,ss obtained from fitting the SFG spectra of Figure S5 without and with the addition of 5 mM memantine into the subphase, respectively.

Gaussian distribution. The red and blue horizontal lines (2) indicate the experimental value of χ(2) ef f,ssp,ss/χef f,ppp,ss obtained from fitting the ssp and ppp spectra (in Figure S5) without and with the addition of 5 mM memantine, respectively. Without drugs, the experimentally measured datum (∼1.3) satisfied the orientation distribution width between a δ-distribution width (σ = 0°) and a relatively larger angle distribution width σ (σ = 50°), so the average tilt angle of CD3 groups was between ∼40° with a δ-distribution width (σ = 0°) and ∼0° with an angle distribution width of 50°. In contrast, with the addition of 5 mM memantine, the experimentally measured datum (∼0.9) met with the orientation distribution width between a δdistribution width (σ = 0°) and a relatively smaller angle distribution width of 20°, and thus the average tilt angle of CD3 groups was between ∼60° with a δ-distribution width (σ = 0°) and ∼90° with an angle distribution width σ (σ = 20°). These results proved that both the average tilt angle (θ) and angle distribution width (σ) of CD3 groups changed in the DPPG/ dDPPG bilayer upon the interaction with 5 mM memantine, indicating the disturbance effect of the interfacial order of dDPPG molecules as depicted in Figure S6. As shown in Figure S5 and Table S1, the relative SFG intensity of CD2 groups became larger than that of CD3 groups for both ssp and ppp spectra after the introduction of 5 mM memantine, leading to the appearance of more gauche defects in the lipid tail region. These results further confirmed that dDPPG molecules in the DPPG/dDPPG bilayer lost the interfacial order after interacting with 5 mM memantine. We believe that the outer dDPPG molecules were quite ordered before the addition of memantine, so the tilt angle distribution width (σ) of CD3 groups in the dDPPG leaflet should be narrow. For the DPPG/dDPPG bilayer, the dDPPG and DPPG molecules were in the gel state under the experimental conditions,6,7 in which the lipid tails were orderly and tightly packed through the hydrogen bonding interaction

Figure 9. Theoretical curve of the |χeff,ssp,ss,without/χeff,ssp,ss,with| for the CD3 groups of dDPPG molecules (assuming a δ-distribution width (σ = ∼0°) without the addition of memantine) in the DPPG/dDPPG bilayer as a function of the possible θ with σ deduced from Figure 8. The blue line indicates the measured value obtained from the fitting results listed in Table S1.

shows the calculated |χeff,ssp,ss,without/χeff,ssp,ss,with| for the CD3 groups in the DPPG/dDPPG bilayer as a function of the possible average tilt angle with angle distribution width deduced from Figure 8. The measured ratio was ∼7.2 for the CD3 groups obtained from the fitting results in Table S1. Figure 9 shows that this measured value (the blue line) was in the range between ∼59.4° with a δ-distribution width and ∼86.1° with a distribution width (σ) of ∼19.9°. The intersection point between the measured value (∼7.2) and the calculated theoretical curve corresponded to the average tilt angle of ∼82.0° with an angle distribution width of 19.5°, indicating the CD3 groups of dDPPG molecules were prone to lie down at the surface with the addition of 5 mM memantine, which was caused by the strong electrostatic interaction between positively charged memantine molecules and negatively charged dDPPG molecules. The results further confirmed that the molecular order of dDPPG molecules could indeed be seriously destroyed by drugs, which thus led to a significant decrease of the SFG signal.

4. CONCLUSION In conclusion, SFG spectroscopy was employed to investigate the interaction between memantine and the model cell membrane in real time and in situ. Both zwitterionic PC and negatively charged PG bilayers were prepared, acting as the model cell membranes. The results showed that the memantine 17080

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molecules interacted with the PC and PG lipids quite differently. There was no significant interaction between memantine and the PC bilayer, and memantine did not change the flip-flop rate of the PC bilayer below the drug concentration of 5 mM. Unlike the PC bilayer, the memantine molecules could significantly interact with the outer PG leaflet at the drug concentration of 1 mM or higher due to the strong electrostatic interaction between the memantine and the PG molecules. It is important to note that the inner PG layer could not be disturbed by memantine, as depicted in Figure S6. By analyzing the average tilt angle (θ) and angle distribution width (σ) of CD3 groups in the DPPG/dDPPG bilayer before and after the addition of 5 mM memantine, we can see that the CD3 groups tended to lie down at the surface with respect to the surface normal after the addition of drugs. The present work gave for the first time a combined qualitative and quantitative molecular-level analysis of the interaction between drug and the model cell membrane. These results can deepen our understanding of memantine’s influences on cell membranes and can contribute to the efforts devoted to understanding the drug’s pharmacodynamics and accelerating the development of new drugs for treating neurodegenerative diseases.

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ASSOCIATED CONTENT

S Supporting Information *

The calculation of the flip-flop rate, the molecular structures of DPPC, dDPPC, DPPG, and dDPPG, the SFG ssp spectrum of memantine at the air/water interface, the time-dependent SFG signal intensities collected at 2070 and 2875 cm−1 for the dDPPG/DPPG bilayer without and with the introduction of 0.2 mM memantine, the normalized ssp and ppp spectra collected from the DPPG/dDPPG bilayer before and after the addition of 5 mM memantine, the fitting results for the normalized ssp and ppp spectra of the DPPG/dDPPG bilayer before and after the addition of 5 mM memantine, the schematics illustrating the effect of memantine on the order of the PC and PG bilayer, and the result of assuming an angle distribution width of ∼20°. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.5b05747.

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Article

AUTHOR INFORMATION

Corresponding Authors

*(Fu-Gen Wu) E-mail: [email protected]. *(Xiaolin Lu) E-mail: [email protected]. Author Contributions §

The first two authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21303017 and 51173169), the Natural Science Foundation of Jiangsu Province (KB20130601), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the Fundamental Research Funds for the Central Universities (2242015R30016), the Scientific Research Foundation of Graduate School of Southeast University (YBJJ1412), and the Graduate Students’ Scientific Research Innovation Project of Jiangsu Province Ordinary University (CXZZ13_0122 and KYLX15_0164). 17081

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