Differences in the Aspect Ratio of Gold Nanorods that Induce Defects

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Differences in the Aspect Ratio of Gold Nanorods that Induce Defects in Cell Membrane Models Paula M. P. Lins, Valéria S. Marangoni, Thiers M. Uehara, Paulo B. Miranda, Valtencir Zucolotto, and Juliana Cancino-Bernardi* Nanomedicine and Nanotoxicology Group, Physics Institute of São Carlos, University of São Paulo, CP 369, CEP 13560-970 São Carlos, São Paulo, Brazil S Supporting Information *

ABSTRACT: Understanding the interactions between biomolecules and nanomaterials is of great importance for many areas of nanomedicine and bioapplications. Although studies in this area have been performed, the interactions between cell membranes and nanoparticles are not fully understood. Here, we investigate the interactions that occur between the Langmuir monolayers of dipalmitoylphosphatidyl glycerol (DPPG) and dipalmitoylphosphatidyl choline (DPPC) with gold nanorods (NR)with three aspect ratiosand gold nanoparticles. Our results showed that the aspect ratio of the NRs influenced the interactions with both monolayers, which suggest that the physical morphology and electrostatic forces govern the interactions in the DPPG−NR system, whereas the van der Waals interactions are predominant in the DPPC−NR systems. Size influences the expansion isotherms in both systems, but the lipid tails remain conformationally ordered upon expansion, which suggests phase separation between the lipids and nanomaterials at the interface. The coexistence of lipid and NP regions affects the elasticity of the monolayer. When there is coexistence between two phases, the elasticity does not reflect the lipid packaging state but depends on the elasticity of the NP islands. Therefore, the results corroborate that nanomaterials influence the packing and the phase behavior of the mimetic cell membranes. For this reason, developing a methodology to understand the membrane−nanomaterial interactions is of great importance.

1. INTRODUCTION Understanding how nanoparticles interact with biomolecules at bio−nano-interfaces is of great relevance to nanoparticle research. The composition of biological membranes greatly influences the cell uptake of nanoparticles, especially with variations in the fluid and dynamic arrangement of phospholipids in the membrane.1 Such changes in the molecular arrangement of phospholipids or chain orientation can be monitored via Langmuir monolayer experiments.2−4 These changes depend on the interactions between polar headgroups from the lipid and alkyl chain orientations, which produce different transition phases. Langmuir monolayer experiments are based on measurements of the surface pressure−area (π−A) isotherms by determining the decrease in the surface tension as a function of the area that is available for each molecule on the aqueous subphase.5,6 Important parameters can be monitored via Langmuir monolayer analyses, including monolayer formation and stability, the molecular area, the compressibility of the interface, and the occurrence of interactions between the molecules or nanomaterials that are present in the subphase with the monolayer. We have recently proposed that Langmuir monolayers are suitable models to investigate the interactions that occur between nanoparticles and cell membranes.2,3,7−10 This idea is based on the possibility © XXXX American Chemical Society

of monitoring changes in the membrane composition and the phase state. Simulation studies using Monte Carlo dynamics showed that changes in phospholipid domains modulate the diffusive signaling and transport of nanoparticles in cell membranes.10 The dipole−dipole interaction forces from nanoparticle surface charges and phospholipid polar headgroups could be a fundamental means to control membrane transport.10 Studies have indicated that nanomaterials can change the fluid-gel phase behavior of bilayers.11 Sanchez et al. showed that negatively charged nanoparticles promote membrane gelation, whereas positive nanoparticles induce fluidity because of electrostatic interactions with the headgroups.11 Peetla and Labhasetwar revealed that positive polystyrene nanoparticles significantly affect the surface pressure, which causes destabilization of the endothelial membrane models compared with the same nanoparticles with a negative charge.12 However, the influence of the aspect ratio of nanoparticles at bio−nanointerfaces has not yet been investigated in detail. Melbourne et al. showed that the aspect ratio of carbon nanotubes is a major Received: August 29, 2017 Revised: November 21, 2017 Published: November 22, 2017 A

DOI: 10.1021/acs.langmuir.7b03051 Langmuir XXXX, XXX, XXX−XXX

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Langmuir factor that influences the compression resistance and pressure of dipalmitoylphosphatidyl choline (DPPC) monolayers, which affects the interaction mechanisms.13 For this purpose, Langmuir monolayers of dipalmitoylphosphatidyl glycerol (DPPG) and DPPC were used to mimic a cell membrane to evaluate the ways it interacts with nanorods (NRs) with three different aspect ratios and gold nanoparticles (NP-cit). The influences of the aspect ratio, morphology, and charge of the nanomaterials on the interactions with the membrane were evaluated in terms of the surface pressure, molecular area, hysteresis, and compressibility. In addition, the nanomaterials were characterized using sum-frequency generation (SFG) spectroscopy. Although all techniques are wellestablished in the literature, the combination of using Langmuir isotherms and SFG spectroscopy to elucidate the interactions at the bio−nano-interface is more recent.3 As an advantage of the methodology described here, it was possible to investigate the effects of nanomaterials on the lipid monolayers, which include their insertion into the membrane and the average lipid chain conformation. Such detailed information is relevant for possible cellular uptake processes and highlights the influence of the phospholipid composition in this process.

N=

2.303 × Abs Cext × l

where l is the length of the cuvette and Cext is the extinction coefficient value of 3.5 × 10−9 cm2. 2.2.2. Langmuir Monolayers. Surface pressure per area (and time) measurements were performed using a mini-Langmuir trough, from KSV-Nima Technology Ltd., made from poly(tetrafluoroethylene) and equipped with two barriers of the same material. The maximum surface area of the trough is 98 000 mm2. The surface pressure was measured with a Wilhelmy plate of wet filter paper suspended from a strain gauge. Before spreading the lipids, the surface of the trough was cleaned with chloroform and then with ethanol, followed by water, to remove the excess solvent. The subphase solution, with or without nanoparticles, was added to the trough, and the temperature was maintained at 21 ± 1 °C. The monolayers were produced by spreading 12 μL of DPPC or DPPG 1 g L−1 diluted in chloroform using a Hamilton syringe. These monolayers were left to equilibrate for 10 min before compression in the subphase without nanoparticles and for 40 min in the subphases containing NP-cit or NRs. In the hysteresis experiments, the barriers were compressed and decompressed at a speed rate of 10 mm/min until the surface pressure reached 50 mN m−1 for three successive cycles. For comparison, DPPC and DPPG monolayers were also investigated in the presence and absence of neat NP-cit (∼6 × 109 particles/mL), NR-A, NR-B, and NR-C concentrations of approximately 2 × 1010 particles/mL. The surface pressure for the air-subphase interface in the absence of a lipid monolayer remained at zero for a time greater than 10 min. The fact that the surface pressure did not change with time indicated that the nanoparticles did not facilitate significant surface activity. To analyze the in situ interactions of the DPPG and DPPC monolayers and nanoparticles, adsorption kinetics were performed on expanded monolayers for 40 min, followed by compression−decompression experiments. The in-plane elasticity (CS−1), or elasticity modulus, of the DPPG and DPPC monolayers was calculated using the expression CS−1 = −A(∂π/∂A) to study mechanical changes in the system, where A is the mean molecular area and π is the surface pressure. 2.2.3. Sum-Frequency Generation Spectroscopy. SFG spectroscopy was performed by in situ measurements using the same experimental procedures described by Uehara et al.3 To generate the SFG signal in the reflection geometry, visible and IR wavelengths overlap at the same spot (∼1 mm2) and time at the interface. SFG spectroscopy was used to investigate the molecular arrangement at the air−water interface of the Langmuir monolayers of DPPC and DPPG. These monolayers were produced in the presence of NP-cit, NR-A, and NR-C and yielded information regarding their alkyl chain conformation and ordering of their water molecules that interacted with the lipid/nanomaterial films by the OH and CH stretching vibrational modes. NRs (NR-A and NR-C) with a concentration of approximately 2 × 1010 particles/mL and gold nanoparticles, NP-cit, with a concentration of approximately 6 × 109 particles/mL were added to the subphase at this concentration before spreading the lipids. Particle concentrations were estimated based on the optical density of the surface plasmon resonance band of the particles. For NRs, the longitudinal plasmon peaks were used.17 A typical concentration for DPPC or DPPG was 0.5 mg/mL diluted in chloroform, and the volume spread on the surface of the aqueous subphase was 16 μL. The systems remained in standby for 40 min before compression. The trough barriers were moved with a constant speed of 8 mm/min until the surface pressure reached ∼35 mN m−1, and then, the surface pressure was maintained constant. SFG spectra were collected at a surface pressure of 35 mN m−1 in a frequency range of 2700−3800 cm−1, and the interval between data points was 10 cm−1.

2. MATERIALS AND METHODS 2.1. Materials. DPPC and DPPG (sodium salt) were purchased from Avanti Polar Lipids. Analytical grade chloroform and methanol were purchased from J.T. Baker. Sodium citrate anhydrous (99.5%), HAuCl4 solution, PAH [poly allylamine hydrochloride], formic acid (98%), absolute ethanol, NH4OH (28−30%), and cetyltrimethylammonium bromide (CTAB, 99%) were purchased from Sigma-Aldrich. All solutions were prepared with ultrapure water with a specific resistivity of 18.2 MΩ cm. 2.2. Methods. 2.2.1. Synthesis of Gold Nanoparticles and Gold NRs. Citrate-coated gold nanoparticles (NP-cit) were synthesized according to the Turkevich method.14 Briefly, 14 mL of HAuCl4 0.5 mmol L−1 was heated until boiling under vigorous stirring, followed by the addition of 1 mL of 1% citrate solution. After 10 min, the color of the suspension changed to a reddish color. The particles were centrifuged and washed to remove excess citrate (10 000 g, 15 min). Gold NRs were synthetized based on the seed-growth method in the presence of the surfactant CTAB.15 First, 7.5 mL of CTAB 0.1 mol L−1 was mixed with 250 mL of HAuCl4 0.01 mol L−1 under agitation for 1 min. Then, 600 μL of a cold NaBH4 0.01 mol L−1 solution was continuously added with stirring for 10 min. The color of the solution changed from yellow to pale brown. This suspension was maintained at 25 °C for 2 h before use. The growth of the NRs was performed by mixing 3 mL of HAuCl4 0.01 mol L−1 with 47 mL of CTAB 0.1 mol L−1, followed by 350 mL of AgNO3 0.01 mol L−1 and 480 μL of ascorbic acid 0.1 mol L−1. The solution became colorless. Finally, 100 mL of the previously prepared gold seeds was added. The system was maintained at room temperature for at least 12 h and then centrifuged at 1500g for 2 min to remove excess crystallized CTAB. The supernatant was centrifuged at 8000g for 5 min, and the pellet was resuspended in ultrapure water and centrifuged again. Different volumes of the AgNO3 0.01 mol L−1 solution were used, which enabled the fabrication of gold NRs with distinct aspect ratios (NR-A, NR-B, and NR-C). The article morphologies and sizes were determined by transmission electron microscopy (JEOL JEM-2100, 200 kV). The particles were further characterized by employing UV−vis−NIR spectroscopy (Hitachi U-2900), and the surface charge was analyzed by zeta potential measurements (Nano-ZS, Malvern Instruments). The NR concentration (particles/mL) was calculated using the longitudinal absorption peak from the absorbance measurement and its correlation with the following equation16

3. RESULTS AND DISCUSSION The use of membrane models for nanoparticle interaction evaluation allows for investigating the molecular level at the bio−nano-interface in a dynamic way. For this purpose, three B

DOI: 10.1021/acs.langmuir.7b03051 Langmuir XXXX, XXX, XXX−XXX

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Figure 1. UV−vis−NIR and the respective TEM images of the (A) NP-cit and (B) CTAB-coated gold NRs with aspect ratios of 2.1, 2.8, and 3.4 for NR-A, NR-B, NR-C, respectively. Scale bar: 50 nm.

curves in Figure 2 were obtained by spreading DPPC and DPPG solutions on the subphase that contained the nanoparticles. For NR-A and NP-cit, the surface pressure remained near zero, which indicates no surface activity or interaction even after spreading both lipids. By contrast, for NR-B and NR-C, the surface pressure increased to 1 mN m−1 after DPPC and DPPG spreading, which showed some surface activity. Note that NR-A and NP-cit did not show surface activity when subjected to the surface pressure measurements. The increase in the surface pressure observed in Figure 2 for NR-B and NR-C for both DPPC and DPPG systems is related to the higher aspect ratios that these particles presented compared with those of the NR-A and NP-cit. Hence, the incorporation of NR-B and NR-C through the monolayers was easier, as evidenced by the Langmuir isotherms analyses shown in Figure 3. Interestingly, this increment in the surface pressure for the DPPC and DPPG monolayers occurred only for higher aspect ratio NRs, which suggests that the effect of the aspect ratio of the NRs on the interaction with the membrane was more pronounced than the effects related to the charge or morphology. Note that DPPC is a zwitterionic molecule, whereas DPPG has a negative net charge that comes from the hydroxyl and phosphate polar head groups. The increase in the surface pressure for NR-B in the first few minutes was more pronounced for the DPPC than for the DPPG monolayers, which exhibited a decrease in the surface pressure values during the same time of interaction. Although NR-B and NR-C had higher aspect ratios, their diameters were smaller than the other nanoparticles, which may have facilitated their incorporation through the membrane compared with NR-A and NP-cit. The surface pressure decreased in the presence of NR-A and NP-cit until reaching 0.25 mN m−1 from a maximum value of 0.5 mN m−1, which was independent of the nanoparticle concentration. The nanoparticles did not exhibit surface activity, which indicates that these nanoparticles probably removed molecules from the phospholipid monolayer because of their similar morphologies, despite their opposite surface charge. Similar effects have been reported by Torrano et al. for NP-cit and NP−PAH, which were allowed to interact with DPPC and DPPG monolayers.4 As an explanation, the authors

positively charged gold NRs with different aspect ratios (NR-A, NR-B, and NR-C) and negatively charged gold nanoparticles (NP-cit) were used, and their interaction with DPPC and DPPG phospholipid monolayers was investigated considering their aspect ratio, morphology, and charge. Figure 1A shows the UV−vis spectrum of NP-cit and its respective TEM image. The typical plasmonic band at approximately 500 nm suggested small-sized particles. The size and morphology of the nanoparticles were confirmed by transmission electron microscopy, which revealed spherical particles with an average diameter of 15 nm. The characterizations of the gold NRs are shown in Figure 1B. In the UV− vis−NIR spectra, the band at approximately 500 nm is due to the coherent motion of the conduction band electrons along the transversal axes, and the second peak corresponds to the oscillation in the longitudinal direction.18−20 The average aspect ratio, which is the ratio between the length and width of the NRs, was determined from the TEM images, and the ratios were approximately 2.1, 2.8, and 3.4 for NR-A, NR-B, and NRC, respectively, which are consistent with their optical properties. The surface charges of the four nanoparticles were compared by zeta potential measurements (Table 1). The zeta potential of NP-cit was −31.8 mV, which suggests a stable and negatively charged nanoparticle, whereas all the NRs presented a highly positive surface charge. The final concentration of the nanoparticles and NRs in the studied system was ∼109 particles/mL, which is on the same order of magnitude of the concentration of particles that can accumulate within tumor tissues.21 The adsorption kinetics Table 1. Zeta Potential Values for NP-cit, NR-A, NR-B, and NR-C zeta potential (mV) NP-cit NR-A NR-B NR-C

−31.8 +39.2 +31.1 +38.2

± ± ± ±

3.1 2.3 3.5 1.9 C

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Figure 2. Adsorption kinetic interaction processes of the (a) DPPC and (b) DPPG monolayers with NR-A, NR-B, NR-C, and NP-cit at the air− water interface. Changes in the surface pressure with time were monitored for 40 min with 6 × 109 particles/mL of NP-cit or 2 × 1010 particles/mL of NR-A, NR-B, or NR-C in the subphase for each nanoparticle.

Figure 3. Surface pressure−area isotherms of the (a) DPPC and (b) DPPG monolayers with 2 × 1010 particles/mL of NR-A, NR-B, NR-C, or NP-cit with 6 × 109 particles/mL in the subphase. (*) The NP-cit isotherm was displayed up to 180 Å2 for a better comparison with the other curves, and for this reason, the surface pressure started at 4 mN m−1.

subphase at a surface pressure of 25 mN m−1 indicated a molecular area of 52, 82, 78, and 80 Å2 for NR-A, NR-B, NR-C, and NP-cit, respectively. A higher molecular area shift was not observed for NR-A in the subphase when compared with pure DPPC (55 Å2) at the same surface pressure. This result is probably due to the larger diameter that is exhibited by NR-A, which makes its incorporation in the DPPC monolayer difficult. Alternatively, the shift toward higher molecular areas observed in the DPPC monolayer due to the presence of NR-B and NRC was probably caused by their smaller particle diameters and lower surface activities, which enabled their interactions with time, as observed in Figure 2a. Additionally, bromide counter ions (Br−) of CTAB that were present on the NR surface could also reduce the repulsion effects. In NR-A, the counter ion did not effectively promote variations in the measurement, probably because the larger diameter of NR-A overcame this effect compared with NR-B and NR-C, indicating changes in the surface pressure versus the molecular area response. Indeed, the Br− counter ions may have screened the interactions between the quaternary ammonium of the CTAB on the surface of the NRs with the DPPC headgroup, whereas the bromide reduced the repulsion phenomena, increasing the probability of incorporating CTAB into the membrane. NR-B and NR-C had similar aspect ratios and diameters that might have indicated a sparser density, facilitating the incorporation of NRs in the surface via van der Waals interactions between CTAB and DPPC. Moreover, the plateau remained constant in all isotherms, which were

assumed that the adsorption process was instantaneous for the time scale of the experiment and was sufficiently long to achieve a quasistatic thermodynamic equilibrium.4 To understand the influence of the morphology, aspect ratio, and charge of the nanoparticles in the phospholipid monolayers, DPPC and DPPG were spread at the air−water interface on a dispersion containing NP-cit, NR-A, NR-B, and NR-C in the subphase and then compressed to the minimum area available in the Langmuir trough (see Figure 3). Figure 3 shows the surface pressure−area isotherms for the DPPC and DPPG monolayers, which feature a minimum area of 45 and 38 Å2, respectively, and a collapse pressure (not shown) of 68 and 60 mN m−1, respectively. According to Figure 3a, the DPPC isotherm (black line) shows a plateau region at 5 mN m−1 between 80 and 100 Å2, which corresponds to a liquid-expanded/liquid-condensed (LE−LC) phase.22 The plateau region is highly dependent on temperature; therefore, the plateau pressure is below 10 mN m−1 because of the temperature used in the experiment.23 The surface pressure of the isotherm with NP-cit shows a shift in comparison with pure DPPC, and the LE−LC transition disappears. This expansion, along with the absence of a plateau, indicated that the nanoparticles are at the interface, as previously observed in other studies.4 A monolayer expansion also occurred when NRB and NR-C were in the subphase, but in this case, the LE−LC phase transition was preserved (see Figure 3a). By contrast, NR-A did not change either the phase transition or the molecular area. A comparison of these nanoparticles in the D

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Figure 4. Compression−decompression isotherms of the (a) DPPC and (b) DPPG monolayers with NR-A, NR-B, and NR-C in the subphase.

with minimal hysteresis during the first cycle, which was probably because of the electrostatic repulsion of the system. Interesting results were found for the DPPC monolayer in the presence of NRs, as observed in Figure 4a. Even though NR-B and NR-C caused the DPPC monolayer to expand, hysteresis was only observed in the first cycle with a ΔmMA of approximately 3 and 0 Å2 for the other cycles at 25 mN m−1. The absence of ΔmMA indicates that the physical stress that results from the movement of the barriers was not sufficient to overcome the effects of charge and morphology on the interaction of NRs with the DPPC monolayer. Only a shift of 3 Å2 in the molecular area was found for NR-A and DPPC. This small molecular shift was attributed to the weak interactions between the NR-A and DPPC polar head groups that were probably broken during the compression−decompression cycles. The compression−decompression cycles of DPPG with the NRs revealed significant hysteresis with a gradual loss of stability along the cycles for all NRs, as shown in Figure 4b. For NR-A, the monolayer remained stable after the first decompressed cycle. The presence of NR-B decreased the surface pressure−molecular area after each cycle because of some lipid removal by the NR-B, indicating the instability of the monolayer after physical stress. The ability of the nanoparticles to expand and/or change the fluidity of the monolayers is consistent with the literature. Such expansion has been observed for carbon nanotubes and iron and gold nanoparticles.2−4 The physical states of the monolayers can be classified on the basis of CS−1 as follows: 12.5−50 mN m−1: LE, 50−100 mN m−1: liquid, 100−250 mN m−1: LC, and >250 mN m−1: solid films.26 The maximum CS−1 can be used to characterize the surface packing of the monolayers, which is important for verifying how nanoparticles affect the local arrangement of the lipids and can be extended to an in vitro discussion because a surface pressure of 30 mN m−1 in a monolayer is reported to be equivalent to the lateral pressure of a natural cell membrane.27 The equilibrium in-plane elasticity CS−1 values for the DPPC and DPPG systems with NRs and NP-cit were obtained from the isotherms in Figure 3 and are shown in Table 2. The DPPC and DPPG monolayers formed on the water subphases can be classified as liquid phases because CS−1 lies at 75.5 and 86.8 mN m−1. The DPPC monolayers that contained NP-cit in the subphase showed a change in their physical state from a liquid to an LC state. The existence of 6 × 109 or 12 × 109 particles/mL of NP-cit in the subphase caused alterations in the DPPC monolayer. The dipole of the DPPC headgroup

independent of the size of the NRs. CTAB influence on the surface pressure profile along 40 min without lipids and nanoparticles is shown in the Supporting Information Figure S1. For the DPPG monolayers, no increase in the molecular area was observed when NP-cit was introduced to the subphase, although a different behavior was observed between 50 and 160 Å2. This increase in the surface pressure indicates that the nanoparticles reduced the cohesion forces in the microdomains.24 The effect of NR incorporation was more pronounced in the DPPG than in the DPPC monolayers because of the attractive net interactions between the negatively charged DPPG and positive NRs. Comparing the molecular area values of pure DPPG (41 Å2) with the values observed after the incorporation of NR-A, NR-B, and NR-C in the subphase (91, 94, and 85 Å2, respectively) at 25 mN m−1, a ΔmMA of at least 50 Å2 was estimated, which is higher than that observed in the DPPC system (ΔmMA = 20 Å2). Gold NRs caused a significant shift in the DPPG surface pressure isotherms to larger areas per molecule (see Figure 3b), which means that the lipid monolayers expanded, even at high surface pressures (40−50 mN m−1). This result suggests that the NRs remained adsorbed on the packed DPPG monolayer because of the electrostatic attraction that was independent of their size. All NRs caused a significant shift in the surface pressure for high molecular areas in the DPPG monolayers, which showed a phase transition with a high inclination of the curve between 100 and 140 Å2. The existence of LE and LC phases in the DPPG isotherms may indicate that the NRs created a more fluid monolayer, and as a consequence, the phase transition occurred as a small plateau region in the isotherm. After increasing the NR aspect ratio from NR-B to NR-C, a prominent shift in the phase transition was also observed. Additionally, the early collapse of the DPPG membrane occurred with NR-B into the subphase. The latter is evidence that NRs were incorporated through the monolayer. Similar results had been reported in the literature.2,3,12,25 Because of the ability of nanoparticles to expand the DPPC and DPPG monolayers, compression−expansion isotherms were performed to check if the interactions between the NRs and lipids could be influenced by the physical stress of the barriers. The compression−expansion isotherms for NP-cit showed a narrow hysteresis with a molecular area variation of 3 Å2at 25 mN m−1after three consecutive cycles, which implies a stable DPPC monolayer (Figure S3 in the Supporting Information). The compression−decompression isotherm was similar to that of the DPPG monolayer with or without NP-cit E

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Langmuir Table 2. Equilibrium In-Plane Elasticity, CS−1 (mN m−1), of the DPPC and DPPG Monolayers at 30 mN m−1 with NRs and NP-citb DPPC nanoparticle/concentration

C1

0 NP-cita NR-A NR-B NR-C

75.5 126.3 89.5 109.8 92.6

the DPPC membranes with 2 × 1010 or 4 × 1010 particles/mL in all the NRs, there was an increase in CS−1 values, which suggested that nanoparticles adsorbed on the DPPC membrane, making the lipids more condensed. Because the positively charged NR and DPPC dipoles repelled each other and their isotherms shifted toward high molecular areas (except for NR-A), the in-plane elasticity results suggest that the effect of the diameter, as discussed before, and the tendency of the Br− counter ions to adsorb on the polar headgroup of DPPC forming an electrical double layerallowed the NRs to electrostatically interact with the DPPC monolayer. Additionally, positive NRs could have interacted with DPPC (even with a dipole pointing downward) mediated by the presence of Br− counterions. This phenomenon could justify the expansion in most cases (except NR-A) and the consequent changes in elasticity. The DPPG/NR systems showed a decrease in their CS−1 values as a function of the concentration. This result indicates that NRs penetrated within the membrane, which made then more fluid, moving from a liquid to an LE state. Interestingly, the in-plane elasticity values of NR-B and NR-C decreased to a higher extent compared with that of NR-A. This finding can be attributed to the longer length of NR-B and NR-C compared with NR-A and to the excess of nanomaterials, which implies a higher concentration at the interface. 3.1. Sum-Frequency Generation Spectroscopy. To better understand the interaction of the lipids at the air− water interface with the NRs, SFG spectroscopy was used to elucidate the possible ways in which NRs and NP-cit interacted with the phospholipid monolayers, which led to the changes observed in the isotherms from Figure 3. In particular, SFG spectroscopy allows probing those interactions by measuring the changes in the vibrational spectrum of water interacting with a Langmuir film29,30 and the CH stretches of the lipid acyl chains, which are sensitive to the average conformation of the hydrophobic tails of phospholipids.29 Figure 5a represents the SFG spectra of DPPC, DPPC/NP-cit, DPPC/NR-A, and DPPC/NR-C. Figure 5b illustrates the spectra of DPPG, DPPG/NP-cit, DPPG/NR-A, and DPPG/NR-C. In Figure 5, all spectra with or without nanoparticles are dominated by resonances at 2879 and 2945 cm−1 and were assigned to the symmetric stretching of CH3 of the alkyl chains of DPPC and DPPG and its Fermi resonance with the

DPPG C2

C1

C2

179.6 91.1 198.6 186.5

86.8 114.8 81.1 40.3 40.8

82.9 65.7 63.8 69.3

a

NP-cit concentration is different from those of the NRs; see above. Concentrations used for NP-cit: 6 × 109 (C1) and 12 × 109 particles/ mL (C2). Concentrations used for the NRs: 2 × 1010 (C1) and 4 × 1010 particles/mL (C2)

b

positive end of the quaternary ammonium groupcould interact with the negative charge of NP-cit, causing condensation in the monolayer. Increasing the CS−1 values by incorporating particles in the hydrophobic regions may have increased the overall monolayer expansion that was observed in the isotherms, which induced the formation of lipid-rich and particle-rich microdomains. Therefore, the CS−1 values demonstrated a more condensed lipid phase for the NP-cit and lipid systems compared with that of the DPPC monolayers without the particles, which is similar to the results observed in the isotherms of Figure 3a. A smaller condensing effect was observed for the DPPG monolayers with a small increase in CS−1 for 6 × 109 particles/mL NP-cit, which was probably because of the electrostatic repulsion between the lipids and NPs. No significant change was observed in the DPPG monolayers in a subphase with a higher concentration of NPcit, which may have been a result of the increase in the ionic strength in the subphase. This result corroborated the isotherms that did not show any monolayer expansion at high pressures but only a slight modification in their slope. The latter result can be attributed to the repulsion of the negatively charged NP-cit and DPPG molecules because incorporating a small amount of nanoparticles within the phospholipid monolayer should have reduced CS−1, making the monolayer less rigid.2,4,28 As expected, the in-plane elasticity of the DPPC and DPPG monolayers was affected in a different manner by the NRs. For

Figure 5. (a) SFG spectra of the monolayers: DPPC, DPPC/NP-cit, DPPC/NR-A, and DPPC/NR-C. (b) SFG spectra of the monolayers: DPPG, DPPG/NP-cit, DPPG/NR-A, and DPPG/NR-C. The following concentrations were utilized: NP-cit at 6 × 109 and NRs at 2 × 1010 particles/mL. F

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Figure 6. Scheme of the different kinds of interaction modes between the condensed lipid monolayers and nanomaterials discussed in this study. The explanation for each cartoon is described below.

symmetric CH3 bending mode, respectively.31 Because the tails of DPPC and DPPG have a centrosymmetric arrangement of CH2 groups, they should not contribute to the SFG spectra if the saturated hydrocarbon chains are in the all-trans conformation, whereas gauche conformations break the inversion symmetry of the CH2 groups and create active stretches in the SFG spectra. Therefore, it is possible to infer from Figure 5 that both the DPPC and DPPG monolayers were highly organized with alkyl chains in the all-trans conformation to form compact monolayers on the subphase.31 The SFG spectra of the DPPC and DPPG monolayers in the presence of the gold nanoparticles and gold NRs revealed weak CH2 symmetric stretching at 2850 cm−1 (similar to the neat monolayers), which indicated that they remained highly organized at the air−water interface with well-packed all-trans alkyl chains. Because no significant CH2 stretching from the alkyl chains appeared in the spectra, it was possible to infer for all cases the highly organized conformation and orientation of the phospholipid film in a compact structure on the aqueous subphase (LC phase). Moreover, the broad band between 3000 and 3600 cm−1 represents O−H stretching of water molecules that were oriented in the subphase because of the interactions with the monolayer. For DPPC (Figure 5a), the weak interactions with the headgroup dipoles led to a weak orientation of surface water molecules, which yielded a weak band that is centered at ∼3250 cm−1. In the DPPG monolayer (Figure 5b), which had negatively charged polar headgroups that induced a high surface electric field, a high orientation of water molecules at the interface was produced that led to a prominent broad band of OH stretching. A small but noticeable effect on the water organization underneath the DPPC monolayer in the presence of all investigated nanomaterials could be observed in the SFG spectrum. The effect is more pronounced for NP-cit, where not only the signal increases but also the spectral shape is different. This result confirms that the nanomaterials adsorbed at the interface, and this effect could therefore modify the interlipid interactions and the phase behavior. In combination with the isotherms in Figure 3a, which show the expansion of the DPPC

monolayer at high pressures in the presence of NP-cit, NR-B, and NR-C, we conclude that in these cases, the nanomaterials were inserted as microdomains within the membrane, increasing the average area per lipid molecule (as shown in the isotherms) but keeping the alkyl chains of the phospholipids in the all-trans conformation and well-oriented toward the air side, from which only CH3 stretching modes were detected in the SFG signal. In other words, the uniform (single-phase) expansion of the monolayer could be ruled out from the SFG spectra because it would have led to a marked change in the CH stretching modes.29 For DPPC interacting with NP-cit, the shape of the isotherm also changes, suggesting that besides NP insertion within the lipid film, there is also a significant interaction with the lipid headgroup. In Figure 3b, a shift toward a higher molecular area per surface pressure was observed for the π−A isotherms of DPPG that contained NRs. Moreover, the SFG spectra in Figure 5b predominantly show CH3 stretching, indicating that the chain conformation and orientation remain ordered, which is typical of condensed monolayers. Therefore, the incorporation of NRs in the monolayers occurred in a nonuniform fashion with the formation of lipid-rich and nanoparticle-rich domains. The OH stretching regions also exhibited more significant changes because of the (negatively) charged nature of the DPPG monolayer. In the presence of NP-cit, an increase in the water band was observed, which also interfered with the CH resonances at 2879 and 2945 cm−1 and affected their intensities. This increase in the OH stretching results from the adsorption of negatively charged NP-cit, which led to a higher net surface electric field. For the positively charged NRs, their incorporation should have had the opposite effect, reducing the surface electric field and water contribution in the SFG spectra. However, this effect is not evident in the spectra with NR-A or NR-C, even though the isotherms suggest significant incorporation of these NRs into the monolayer. Conversely, a closer inspection of the line shape of the spectra shows that in the presence of NRs, the interference of the OH stretching with the CH stretching was different, which suggests that the OH mode amplitude had an opposite sign. This result indicates that G

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Langmuir the adsorption of the NRs was sufficient to not only cancel but also overcompensate the surface charge of the DPPG monolayer, producing a net positive charge that again yielded a strong OH stretch intensity but with a different line shape, as observed in the SFG spectra of the negatively and positively charged lipid monolayers.30 Notably, elasticity changes in monolayers are frequently discussed in the current literature2,4,32 as reflecting changes in the monolayer state because of nanoparticle−lipid interactions. However, as discussed above for the cases of DPPG interacting with the NRs, the monolayer exhibited marked expansion, but the SFG spectra showed that the lipid chains were conformationally ordered, indicating the coexistence of nanoparticle-rich and lipid-rich regions (with highly organized chains). Therefore, the lower elasticity observed for this inhomogeneous system (which would suggest a more fluid lipid phase with more disordered chains) cannot be directly associated with changes in the lipid organization because the elasticity was the combination of the elasticities of both the lipid and nanomaterial domains. 3.2. Interaction Models between the Lipid Monolayer with NRs and NP-cit. Several factors have been reported to affect the ability of nanoparticles to incorporate or adsorb on phospholipid monolayers at the air−water interface. Some of these components include the size and surface charge, density of the particles, and existence of aggregates or agglomerates.9,12,33 Some of these interactions are driven by dipoles or van der Waals forces, DLVO forces (Derjaguin−Landau− Verwey−Overbeek theory), which are a combination of electrostatic and van der Waals interactions, or non-DLVO forces that consider the size, charge, and morphology of nanosystems. They yield effective interactions that are referred to as attractive/repulsive, electrical double layer, hydration, and hydrophobic forces.34 Nevertheless, we can infer from our results that the aspect ratio had more influence on the fluidity and elasticity of the zwitterionic membrane than the charge itself. In the negatively charged monolayer, the electrostatic effect was predominant, but the aspect ratio and shape of the nanoparticles also played a significant role. From our results, it is possible to recognize the different types of interactions between the nanomaterials and each lipid monolayer (in the condensed state), as illustrated in Figure 6. 3.3. DPPC Monolayer Versus NP-cit, NR-A, NR-B, and NR-C. i. NP-cit becomes incorporated in the DPPC monolayers because of electrostatic (dipole−charge) interactions, which may affect the monolayer, making it less fluid and more solid/compact. (Scheme 1). ii. NR-A did not affect the DPPC monolayers because of their larger diameter, which did not promote the incorporation of particles into the monolayer. This finding may also be associated with the electrostatic repulsion of the particles. In this case, the counter ions were not sufficiently effective to promote variations in the measurement, probably because the diameter overcame this effect compared with NR-B and NR-C (Scheme 3). iii. NR-B and NR-C incorporated within the DPPC monolayer more easily compared with NR-A because of their smaller diameters. The Br− counter ions from CTAB may also have induced this process, which facilitated their interaction with DPPC. Significant

monolayer expansion was observed, but the acyl chains of the phospholipids remained ordered and oriented toward the air, indicating the formation of lipid-rich and NR-rich domains (Scheme 5). 3.4. DPPG Monolayer Versus NP-cit, NR-A, NR-B, and NR-C. i. NP-cit did not increase the molecular area of the DPPG monolayers, and only a difference in the domains was observed, which may indicate that NP-cit changed the cohesion forces in the microdomains, mainly because of electrostatic repulsion (Scheme 2). ii. NRs were incorporated within the DPPG membrane, mainly because of electrostatic attraction. The lipids occupied a larger average area per molecule, but the acyl chains of the phospholipids remained ordered and oriented toward the air, indicating the formation of lipid-rich and NR-rich domains, as in the case of DPPC and NR-B and NR-C. The NR diameter also influenced the phase transition of the monolayers, in which NR-A maintained the microdomain profile of DPPG, and NR-B and NR-C induced a plateau region (Schemes 4 and 6).

4. CONCLUSIONS We used surface techniques to demonstrate the molecular interactions of DPPG and DPPC phospholipids as membrane models with gold NRs and gold nanoparticles. Our results showed that the differences in the diameter of the NRs and the charge of the nanomaterials/monolayers influenced the way they interacted, which demonstrated that electrostatic forces governed the DPPG−NR system, whereas the van der Waals forces ruled the DPPC−NR system. Moreover, the size of NRA, NR-B, and NR-C influenced the expansion isotherms for both the DPPC and DPPG membranes. However, this expansion was not accompanied by conformational disorder of the lipid tails, which suggests that phase separation (domain formation) occurred between the lipids and nanomaterials at the interface. Consequently, changes in the in-plane elasticity for the lipid monolayers, which are usually related to phase transitions between liquid and LE states, could no longer be simply interpreted because the elasticity was a combination of the elasticities of both the lipid and nanomaterial domains. Additionally, the compression−expansion isotherm cycles showed that the van der Waals forces were related to stable monolayers at the air−water interface and the electrostatic force promoted unstable monolayers. Therefore, our results corroborate the idea that some nanomaterials can induce defects in the packing of mimetic cell membranes, and for that reason, this methodology for understanding the membrane− nanomaterial interface is of great importance for nanomedicine.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b03051. CTAB influence on the surface pressure profile and compression−decompression isotherms (PDF) H

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or jcancinobernardi@gmail. com. Phone: +55 16 3373 9875. Fax: +55 16 3371 5381. ORCID

Paulo B. Miranda: 0000-0002-2890-0268 Valtencir Zucolotto: 0000-0003-4307-3077 Juliana Cancino-Bernardi: 0000-0002-9584-2248 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to FAPESP, CAPES, and CNPq for financial assistance. J.C.-B. thanks the FAPESP process 2012/ 03570-0, and P.M.P.L. thanks the FAPESP process 2012/ 15630-7. The authors thank LME/LNNano for technical support during the electron microscopy measurements.

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