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Molecular Interactions Between Gold Nanoparticles and Model Cell Membranes: A Study of Nanoparticle Surface Charge Effect Peipei Hu, Wei Qian, Bing Liu, Cayla Pichan, and Zhan Chen J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b07565 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 18, 2016

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The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

Figure 1. Schematic of the general structure of a Au NP with a thoil group for anchoring, a PEG chain of 1k Da for biocompatibility, and a methoxy, amino, or carboxyl functional group as the terminal. 296x96mm (96 x 96 DPI)

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Figure 2. Molecular formula of Lipid DSPC and dDSPC. 259x89mm (96 x 96 DPI)


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Figure 3. TEM images of (a) AuNH20 (b) AuNH60 (c) AuCOOH20 and d) AuCOOH60. 171x169mm (96 x 96 DPI)



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Figure 4. SFG spectra collected from a dDSPC/DSPC lipid bilayer in the (a) C-D and (b) C-H stretching frequency ranges after the bilayer construction (black line), after (red line) replacing water with buffer (pH7.4) in contact with the bilayer, and after (blue line) the introduction of AuCOOH60 to the subphase. (c) Time dependent SFG signals detected at 2070 cm-1(C-D stretching, black line) and 2880 cm-1 (C-H stretching, red line) after the introduction of AuCOOH60 to the subphase. 433x132mm (96 x 96 DPI)


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Figure 5. ATR-FTIR spectra collected from the dDSPC/DSPC bilayer in (a) C-D and (b) C-H stretching frequency ranges after the bilayer construction (black), after replacing water with buffer (pH 7.4) in contact with the bilayer (red), and after the addition of AuCOOH60 to the buffer subphase (blue). The difference ATR-FTIR signals shown in (c) C-D stretching and (d) C-H stretching frequency ranges are the difference spectra of the lipid bilayer in contact with water and after replacing water with buffer. The difference ATRFTIR signals shown in (e) C-D and (f) C-H stretching frequency ranges are the difference spectra between those collected from the lipid bilayer before and after the addition of AuCOOH60 to the buffer subphase. 508x240mm (96 x 96 DPI)



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Figure 6. Time-dependent SFG signal intensity changes in (a) C-D and (b) C-H stretching frequency ranges when all four Au NP samples interacting with the dDSPC/DSPC bilayer at 2.00 x10-7 g/mL in water. The dots are experimental data and the lines are fitting results. 299x119mm (96 x 96 DPI)


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Figure 7. Time-dependent SFG signal intensity changes in (a) C-D and (b) C-H stretching frequency ranges when all four Au NP samples interacting with the dDSPC/DSPC bilayer at 2.00 x10-7 g/mL at pH 5. The dots are experimental data and the lines are fitting results. 299x119mm (96 x 96 DPI)



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Figure 8. Time-dependent SFG signal intensity changes in (a) C-D and (b) C-H stretching frequency ranges when all four Au NP samples interacting with the dDSPC/DSPC bilayer at 2.00 x10-7 g/mL at pH 7.4. The dots are experimental data and the lines are fitting results. 300x119mm (96 x 96 DPI)


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Figure 9. Time-dependent SFG signal intensity changes in (a) C-D and (b) C-H stretching frequency ranges when all four Au NP samples interacting with the dDSPC/DSPC bilayer at 2.00 x10-7 g/mL at pH 9. The dots are experimental data and the lines are fitting results. 300x119mm (96 x 96 DPI)



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TOC 439x270mm (96 x 96 DPI)


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Molecular Interactions between Gold Nanoparticles and Model Cell Membranes: A Study of Nanoparticle Surface Charge Effect Peipei Hu †, Wei Qian ‡, Bing Liu ‡, Cayla Pichan†, and Zhan Chen*† †

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor,

Michigan,48109, United States. ‡

IMRA America, Inc., 1044 Woodridge Ave., Ann Arbor, Michigan,48105, United States.

*Address Correspondence to [email protected] Tel: (734) 615-4189 (office): (734) 615-6628

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ABSTRACT Surface modification of nanoparticles (NPs) was reported to play a significant role in determining their interactions with cell membranes. In this research, the interactions between carboxyl or amine functionalized Au NPs and model mammalian cell membranes were investigated by a nonlinear optical spectroscopic technique, sum frequency generation (SFG) vibrational spectroscopy, supplemented by attenuated total reflection - Fourier transform infrared (ATR-FTIR) spectroscopy. In order to test the effects of surface composition on NP - membrane interactions, we altered the ratio of different functional groups on NP surfaces to create NPs with varied surface chemistry. In this study, a substrate supported 1,2-distearoyl-d70-sn-glycero-3-phosphocholine

(d70-DSPCor

dDSPC)/1,2-distearoyl-sn-glycero-3-

phosphocholine (DSPC) lipid bilayer was used as a model cell membrane. It was found that the interactions between Au NPs and the lipid bilayer induced the lipid bilayer to flip-flop. A higher surface coverage of carboxyl functional groups on Au NPs tends to induce lipid flip-flop at a faster rate. For amine functionalized NPs, a slower interaction rate was found in most cases for NPs with a higher surface coverage. Au NP solution pH also influences Au NP – lipid bilayer interactions. Our study provides a quantitative way to assess the interactions between a model mammalian cell membrane and Au NPs with different surface functional groups, amine vs. carboxyl groups at different pH of Au NP solution.


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INTRODUCTION Nanoparticles (NPs) have been widely utilized for many important biomedical applications including biosensing, cellular imaging, photothermal therapy, gene and drug delivery.1-21 Most of these applications require NPs to successfully reach desired target tissues and eventually enter into cells. In the literature, it has been extensively reported that size, shape and surface functionality of NPs are important parameters that affect NP-cell interactions.

22-38

In particular, one research focus has been to tune NP

surface functionality to increase the delivery efficiency of NPs to the target sites.

39-43

However, the

general design principle for these “smart” NPs is still under debate and not fully understood. One general idea for NP surface design is to add some protecting groups (usually polymers) to prevent NPs from nonspecific binding with proteins,44-47and some interacting groups (functional groups including hydroxyl, methoxy, amine and carboxyl, etc.) to facilitate the interactions with cell membranes for NP cellular uptakes.42-43, 48 It is generally believed that NPs with a positive surface charge demonstrate higher cellular uptake as well as higher cytotoxicity compared to those with a neutral or negative surface charge.49-51 However, controversial results reported sometimes, showing that more cell internalization of negatively charged NPs was observed than positively charged NPs.29, 36, 52-53To understand the reasons behind these observations, it is necessary to study how the inner and outer leaflets of a cell membrane respond to NPs with different surface functional groups during the entry of NPs into the cell, and how different lipid components. e.g., neutral and charged lipids, interact with NPs. In this study, a model system consisting of different functionalized Au NPs and a substrate supported phospholipid lipid bilayer serving as a model cell membrane was designed to study the effect of surface modification (e.g., using amine or carboxyl groups) of Au NPs on their interactions with model cell membranes. Au NPs were chosen because of their well-controlled shape and size and their easily modifiable surface.4, 54 Also, Au NPs with different surface charges have been widely studied in living cells and controversial results were found in the literature.55-56 We therefore hope that our studies can clarify some contradicting results. The scheme of the Au NPs we studied is shown in Figure 1. The Au



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NPs used for this study were coated with poly(ethylene glycol) (PEG) through the thiol group at one end of each PEG molecule (with a molecular weight of 1 kDa). The PEG molecules have different functionalities on the other end, including methoxy, amine, and/or carboxyl groups. Four kinds of surface modified Au NPs were studied here, 20% amine and 80% methoxy, 60% amine and 40% methoxy, 20% carboxyl and 80% methoxy, and 60% carboxyl and 40% methoxy. We also prepared Au NPs with 100% methoxy terminated PEG as control samples for zeta potential measurement. A substrate supported 1,2distearoyl-d70-sn-glycero-3-phosphocholine

(d70-DSPCor

dDSPC)/1,2-distearoyl-sn-glycero-3-

phosphocholine (DSPC) lipid bilayer was used in this study to serve as a model for mammalian cell membrane. Therefore, this study only investigates the interactions between Au NPs and zwitterionic (or neutral) lipids. Interactions between Au NPs with other types of lipids and more complicated model cell membranes or real cell membranes can be studied in the future. Sum frequency generation (SFG), a sub-monolayer surface/interface sensitive technique was used as the main analytical tool in this study. A second order nonlinear optical spectroscopy, SFG has intrinsic surface/interface sensitivity due to its selection rule.

57-58

It can provide chemical information of surfaces

and interfaces and also allows time-dependent measurements of molecular behaviors at the interface/surface in situ and in real time.59-61 Over the years, SFG has been widely used to study the interactions between lipid bilayers and many different materials/molecules including biomolecules, polymers and nanoparticles.62-67 For example, SFG has been used to probe lipid bilayer flip-flops 68-69 and we have examined how Au NPs with different sizes can affect such flip-flop.67 Here we studied the interactions between Au NPs with different surface functionalities and model mammalian cell membranes at different pH values using SFG and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopic techniques. It was shown that both Au NP surface functionalities and NP solution pH values affect the interactions between model cell membranes and Au NPs. It is worth mentioning that the Au NPs we used in this study are not regular commercially available Au NPs with citrate coatings. Here the Au NPs were fabricated using pulsed laser ablation of bulk gold target in ultrapure water by IMRA America, Inc.70 The Au NPs made by this method have “bare (free of 4 ACS Paragon Plus Environment

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capping ligands)” surfaces and are naturally highly negatively charged.

71

Unlike gold nanoparticles

produced by chemical methods, these laser-generated gold nanoparticles have a naturally negatively charged surface free of capping agents that allows their surface functionalization to be controlled. 72

SH

-NH2 (20% and 60%)+ OCH3 -COOH (20% and 60%)+ OCH3

PEG 1K

Au O-

Figure 1. Schematic of the general structure of a Au NP with a thoil group for anchoring, a PEG chain of 1k Da for biocompatibility, and a methoxy, amino, or carboxyl functional group as the terminal.

MATERIALS AND METHODS Materials DSPC and dDSPC with purities greater than 99% were purchased from Avanti Polar Lipid and were used as received. Their molecular formulas are shown in Figure 2. CaF2 right angle prisms used as the solid substrates to construct lipid bilayers for SFG experiments were purchased from Alto Photonics Inc. (Bozeman, MT, USA). The prisms were cleaned by toluene (Sigma Aldrich), Contrex AP detergent solution (Decon Laboratories), Milli-Q water, ethanol (Sigma Aldrich), deionized water, and Milli-Q water. Next they were treated by an oxygen plasma cleaner (PE-50, Plasma Etch Inc.) for two minutes. ZnSe total-internal-reflection crystals used for ATR-FTIR experiments were bought from Specac Ltd., Slough, England. Similarly, the crystals were rinsed with Milli-Q water, ethanol, deionized water, Milli-Q water and treated with oxygen plasma for 1 min. All the water used in this study was purified by a MilliQ water purification system with a minimum resistivity of 18.2 MΩ·cm. The bare Au NPs and Au NPs with different surface functional groups as we specified above were prepared by IMRA America, Inc. (Ann Arbor, MI) (for details, see synthesis of surface modified Au NPs, Figure S6, and Figure S7 in the



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Supporting Information) .70These Au NPs are referred to as AuNH20 (with 20% amine and 80% methoxy terminated PEG), AuNH60 (with 60% amine and 40% methoxy terminated PEG), AuCOOH20 (with 20% carboxyl and 80% methoxy terminated PEG), and AuCOOH60 (with 60% carboxyl and 40% methoxy terminated PEG).

Figure 2. Molecular formula of Lipid DSPC and dDSPC. SFG Data Analysis. SFG is a second-order nonlinear optical spectroscopy and has been widely used to study surfaces and interfaces.57-58, 73-75 Its relevant theoretical background has been reported elsewhere57 and will not be repeated here. SFG signal can only be produced from a medium with no inversion symmetry (under the electric dipole approximation). SFG signal intensity is related to the number of surface/interface molecules probed and their average molecular orientation. For a DSPC/DSPC lipid bilayer, due to the approximate inversion symmetry, only weak SFG C-H stretching signal can be detected. For a dDSPC/DSPC bilayer, both strong SFG C-H and C-D signals can be detected.57 For a partially mixed lipid bilayer (e.g., due to some flip-flop so that both leaflets contain some dDSPC and DSPC, but their distributions are not equal), SFG signal intensity is determined by the net lipid distribution between the inner and outer leaflets of an asymmetric lipid bilayer:

I SFG ∝ ( N inner − N outer ) 2

(1)


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Where Ninner and Nouter are the numbers of a certain type of (e.g., regular or deuterated) lipid molecules in inner and outer leaflets respectively. Here for a dDSPC/DSPC bilayer, SFG signals can be collected from the C-D (CD3 νs) symmetric stretch at 2070 cm-1 and C-H (CH3 νs) symmetric stretch at 2880 cm-1 from the deuterated and hydrogenated phospholipids, respectively. Initially, SFG signals are strongest for the two leaflets. However, the signals will decrease if the asymmetry of the lipid bilayer decreases (e.g., due to flip-flop caused by Au NP interaction). In this study, a decrease in SFG signal is expected for each leaflet if Au NPs can effectively interact with the lipid bilayer. Additionally, by monitoring C-D and C-H simultaneously as a function of time, quantitative information of interaction kinetics such as rate constant and half-life of SFG signal intensity change can be extracted by fitting SFG time-dependent signals using the following equation: 68-69

I CH 3 / CD3 (t ) = I max

−4 kt

+ Io

(2)

where Imax represents the maximum SFG intensity from the symmetric stretching vibrations of the terminal CH3 or CD3 groups of the lipid tails at the initial contact of the lipid bilayer, and Io is the intensity offset. k is the rate constant for SFG signal intensity change, which can be used to characterize the time-dependent change of the lipid bilayer behavior (e.g., lipid flip-flop). Furthermore, SFG signal intensity decay half-life (t1/2), which also can be used to characterize the time-dependent Au NP - lipid interaction, can be derived from k:

t1/ 2 =

ln(2) 2k

(3)

SFG Experiment A two-step method named Langmuir-Blodgett (LB) and Langmuir-Schaeffer (LS) method was used to prepare solid supported planar lipid bilayers for SFG studies.76 Details about constructing such lipid bilayers were reported extensively by our group previously and will not be repeated here.77-78 In this study, a surface pressure of 34 mN/m for lipid on water used for both inner and outer leaflet deposition. After the preparation of the supported lipid bilayer, the bilayer was in contact with water in a 2.0 mL small plastic reservoir. For the studies on the interactions between the lipid bilayer 7 ACS Paragon Plus Environment


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and Au NPs in water, Au NP stock solution was added to the water subphase to reach the desired concentration. For the studies on the interactions between the lipid bilayer and Au NPs in buffer of a certain pH, the water subphase in contact with the lipid bilayer was replaced by buffer while keeping the bilayer in contact with the aqueous phase during this process (by adding buffer with one pipette and removing water with another pipette continuously). For studies of Au NPs in buffer, three different pH values were used for buffer: 5.0, 7.4 and 9.0. The solutions of pH 5.0, 7.4 and 9.0 were prepared by using Britton-Robinson buffer consisting of a mixture of 0.04 M H3BO3, 0.04 M H3PO4 and 0.04 M CH3COOH titrated by 0.2 M NaOH. For SFG experiments, an Au NP stock solution was injected to the subphase to reach a concentration of 2x10-6 g/mL. A magnetic stirring bar was placed at the bottom of the 2 mL reservoir to ensure the homogeneous distribution of Au NPs in the subphase solution in contact with the lipid bilayer. All of the SFG experiments were conducted at room temperature (21oC) at which the studied lipid molecules were in gel phrase. The details of our SFG spectrometer were described in previous publications79 and will not be repeated here. In this study all the SFG spectra were collected in the ssp polarization combination (SFG signal beam is in s-polarization, the visible input beam is in s-polarization and the IR input beam is in p-polarization) from the lipid bilayer/Au NP solution interface. For this study, the pulse energy of the visible beam is 25 µJ and the pulse energy of the IR beam is 80 µJ at 2070 cm-1 and 150 µJ at 2875 cm-1. ATR-FTIR spectroscopy The dDSPC/DSPC lipid bilayer was constructed on a clean ZnSe totalinternal-reflection crystal by the LB-LS method and their interactions with Au NP solutions were measured by a Thermo Nicolet 6700 FTIR spectrometer (Waltham,WA, USA). The sample preparation procedures for ATR-FTIR studies are the same as those for SFG studies presented before.80 We collected ATR-FTIR spectra of the lipid bilayer before and after the interactions with different Au NPs in water or buffer with different pHs. All collected spectra were averaged over 64 scans. Similar to SFG experiments, all ATR-FTIR experiments were also carried out at room temperature (22°C). RESULTS AND DISCUSSION 8 ACS Paragon Plus Environment

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Characterization of functionalized Au NPs. The four samples of colloidal Au NPs with different surface modifications used in the present study were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). TEM images of all the NP samples are shown in Figure 3. Table 1 and 2 summarize the results of the NPs prepared in water solution including the sizes measured by TEM and DLS, and zeta potentials by DLS. According to TEM data, the sizes of the five different Au NPs (including four amine/carboxyl functionalized Au NP samples) are very similar, varying from 21.1±3.0 to 23.2±4.6 nm. The hydrodynamic sizes of the NPs obtained by DLS are also similar, in the range of 33.0±0.3 and 35.8±0.3 nm. It is reasonable that the hydrodynamic sizes are larger than the sizes measured using TEM. Therefore, both TEM and DLS results indicated that the Au NP samples with different functionalities used in this study were very similar in size.

Figure 3. TEM images of (a) AuNH20 (b) AuNH60 (c) AuCOOH20 and d) AuCOOH60. Table 1. Size of functionalized Au NPs measured by TEM and DLS.



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Sample AuNH60 AuNH20 AuCOOH20 AuCOOH60

TEM (nm) 23.2±4.6 22.4±3.6 21.9±3.6 22.7±3.9

Water 35.8±0.3 33.0±0.3 34.1±0.5 33.7±0.4

pH5 41.3±0.1 34.4±0.4 36.4±0.2 40.0±0.4

DLS(nm) pH7.4 35.9±1.0 32.4±0.5 37.4±0.2 48.0±1.4

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pH9 35.8±1.6 32.7±0.3 33.9±0.2 36.6±1.4

Table 2. Zeta Potential and pH of functionalized Au NPs measured by TEM and DLS. Sample AuNH60 AuNH20 AuCOOH20 AuCOOH60

pH in H20 5.71 5.77 6.02 6.08

Water -43±1.0 -60±1.0 -70±1.0 -69±1.0

pH5 -20.1±2.7 -29.4±3.1 -40.9±2.2 -43.8±3.8

pH7.4 -33.1±6.2 -34.1±0.1 -45.3±5.6 -46.3±5.6

pH9 -52.7±5.7 -58.1±6.3 -58.7±2.8 -60.0±0.1

It may be surprising to see that the zeta potentials for all the Au NPs studied here are negative. As reported in the literature, Au NPs modified with amine functionality usually carry positive charges.

81-82

As we discussed before, the Au NPs we used are different from regular commercially available Au NPs. Here the Au NP cores were fabricated by pulsed laser ablation at IMRA America, Inc.70 During the pulsed laser ablation, Au NPs were partially oxidized by oxygen present in solution. These Au-O compounds were hydroxylated, followed by a proton loss to give a surface of Au-O-.71 Therefore the Au NPs before the addition of PEG coatings are negatively charged. Our zeta potential measurement indicated that the surface potential of Au NP cores is ~-85 mV in water. The zeta potential of Au NPs with 100% methoxyterminated PEG on their surface was measured to be ~-68 mV. This is understandable because zeta potential measures surface charge, and it is likely the PEG coating screens the negative core potential of the NPs partially, resulting in a more positive zeta potential. With a carboxyl-terminated PEG coating, the zeta potentials of AuCOOH20 and AuCOOH60 were measured to be ~69 mV and ~70 mV respectively, which are both reasonable. In water, carboxyl groups are present in the COO- form, therefore the more carboxyl groups on the surface, the more negative charge should be. Also, they should be more negative compared to Au NPs with 100% methoxy-terminated PEG coating, which were also observed in the experiment. In water, amine should be in NH3 + form, therefore Au NPs coated with amine-terminated


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PEG should be less negative compared to Au NPs with carboxy-terminated PEG. This was indeed observed from our zeta potential measurements since the zeta potentials for AuNH20 and AuNH60 were measured to be -60 mV and -43 mV respectively. The different zeta potentials measured from different samples of Au NPs with different surface functionalities indicated the presence of amine and carboxyl groups on the Au NP surface. As we reported previously, we could experimentally confirm that they are indeed present on the Au NP surfaces.70 The solution pH may alter the surface charge and even size of NPs. Here we measured the size and surface charge of Au NPs at different pH values in buffer solutions using DLS (Table 1 and 2). The measured zeta potential follows the general trend that at a certain Au NP solution pH, the AuCOOH60 is the most negative, the AuCOOH20 is the second most negative, followed by AuNH20, and finally AuNH60. For a particular type of Au NP, at pH 5.0 the zeta potential is least negative, at pH 9.0 it is most negative, and at pH 7.4 it is in between. Such observations can easily be interpreted by the equilibrium of protonation and deprotonation of these functional groups at different pHs. It was reported that the pKa for amine (NH3+) and carboxylic group (COO-) are ~8.5-9.5 and ~2-3.5 respectively.

83

Therefore, at the

same pH, Au NP surfaces with highest carboxyl percentage have the highest COO- concentration, and those with the highest amine percentage have the highest NH3+. Because the Au NP cores are very negative, all the Au NPs studied here are negatively charged, regardless of the surface coatings. A similar phenomenon was reported previously. 49, 83 It was observed that pH had little effect on the sizes of mixed methoxy and amine-terminated Au NPs while the sizes of mixed methoxy and carboxyl-terminated Au NPs had a tendency to increase at pH 5.0 and 7.4. This phenomenon implies the possible aggregation of AuCOOH20 and AuCOOH60 under physiological and acidic conditions. We do not know how to interpret the aggregation effect of AuCOOH20 and AuCOOH60. In our previous study, we found that the Au NP size does not affect the interactions with DSPC lipid bilayer.58 Here we will not include the possibility of Au NP aggregation in the following discussion. 11 ACS Paragon Plus Environment


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Characterization of Au NP – dDSPC/DSPC interactions SFG was used to study the molecular interactions between a dDSPC/DSPC lipid bilayer and Au NPs with different surface functionalities at a concentration of 2.00 x10-6 g/mL. Figure 4a and 4b show SFG spectra collected from the dDSPC/DSPC lipid bilayer right after the construction by the LB-LS method in contact with water, after replacing water with buffer at pH 7.4, and after the addition of AuCOOH60 into the buffer subphase in contact with the bilayer. The SFG signals of C-D (1950-2300 cm-1) and C-H (2750-3000 cm-1) stretching frequency ranges were detected from the inner (proximal) and outer (distal) leaflets respectively. In the C-D spectra (Figure 4a), three characteristic peaks were detected from the inner leaflet. The peak centered at 2070 cm-1 arises from the CD3 symmetric stretching mode. The broad peaks at around 2130 cm-1 and 2215 cm-1 come from the CD3 Fermi resonance mode and CD3 asymmetric stretching mode respectively.78,

84-85

In the C-H

stretching spectra, two peaks at 2880 and 2945 cm-1 are from the C-H symmetric stretching mode and Fermi resonance of methyl groups respectively. 86 Both C-D and C-H spectra (Figure 4a and 4b) indicate that strong SFG signals were generated after the construction of lipid bilayer. It is worth mentioning that previous SFG studies from us and other groups have proved that there is no observable SFG signal change for both leaflets from a pure dDSPC/DSPC lipid bilayer since no flip-flop takes place between the two leaflets.

67, 80, 87

This means that any observed SFG signal change after the addition of the Au NPs to

the subphase must come from the interaction between the lipid bilayer and Au NPs. Figure 4 also shows that the SFG signals collected from the lipid bilayer remain the same even after replacing water with pH 7.4 buffer. This means that there is no lipid flip-flop or disruption by changing the water subphase to buffer solution. By contrast, the intensity of characteristic peaks for both leaflets gradually decreased to almost zero after the introduction of AuCOOH60 to reach a concentration of 2.00 x10-6 g/mL in the subphase. SFG signal intensity decrease may be caused by a reduction of molecules probed or a change in orientation of probed molecules or both. Therefore here the observed SFG signal intensity decrease can be caused by lipid disruption (e.g., orientation change of the end lipid molecules), flip-flop (indicated by eqn.1), and lipid removal (reduction of the molecules probed). The SFG signal decrease rate for CD and CH can provide some information to distinguish between lipid disruption, lipid flip-flop, and lipid 12 ACS Paragon Plus Environment

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removal. Usually, different rates for CD and CH signal changes of the outer and inner leaflets are expected for lipid disruption or removal, while the same rate very likely shows the lipid flip-flop. To examine the interaction kinetics between Au NP and the lipid bilayer and see whether the CD and CH signal changes have the same rate, we collected time-dependent SFG signals at CD3 symmetric stretching (~2070 cm-1) and CH3 symmetric stretching (2880 cm-1) frequencies to monitor how the inner and outer leaflets respond to the interaction of AuCOOH60. As shown in Figure 4c, SFG signals dramatically decreased as a function of time after the lipid bilayer came into contact with the NP solution and eventually reached an equilibrium (close to zero). The experimentally observed time-dependent signal intensity curves were fitted by eqns. 2 and 3. Similar results on the decay half-life were derived for the inner and outer leaflets: 89 ± 7s and 91 ± 4s respectively. The well-overlapped, experimentally measured, time-dependent signal changes for the inner and outer leaflets shown in Figure 4c clearly indicate similar decay rates. This shows that interactions with Au NPs likely lead to flip-flop for the lipid bilayer.

Figure 4. SFG spectra collected from a dDSPC/DSPC lipid bilayer in the (a) C-D and (b) C-H stretching frequency ranges after the bilayer construction (black line), after (red line) replacing water with buffer (pH7.4) in contact with the bilayer, and after (blue line) the introduction of AuCOOH60 to the subphase. (c) Time dependent SFG signals detected at 2070 cm-1(C-D stretching, black line) and 2880 cm-1 (C-H stretching, red line) after the introduction of AuCOOH60 to the subphase. ATR-FTIR was employed to address whether lipid removal occurred during AuCOOH60dDSPC/DSPC bilayer interactions. A decrease in ATR-FTIR signal is indicative of lipid removal due to the interaction between Au NPs and lipid bilayer. ATR-FTIR spectra of the dDSPC/DSPC lipid bilayer in 13 ACS Paragon Plus Environment


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contact with water, pH 7.4 buffer, and AuCOOH60 added to the pH 7.4 buffer were collected in the C-D and C-H stretching frequency ranges (Figure 5a and 5b). For the dDSPC/DSPC lipid bilayer in contact with water, strong ATR-FTIR peaks were found for the CD2 symmetric and asymmetric stretching modes of the inner leaflet at 2086 cm-1 and 2192 cm-1. Also, the peaks of the CH2 symmetric and CH2 asymmetric stretching vibrations of the outer leaflet centered at 2850 cm–1 and 2917 cm-1 were observed.88-89 Figure 5 shows that all three ATR-FTIR spectra overlapped with each other very well, indicating that the addition of AuCOOH60 to the subphase in contact with the bilayer did not cause the lipid signal to decrease. Therefore there is no lipid removal due to the interactions of Au NPs. The differences in the ATR-FTIR spectra between those collected before and after the addition of AuCOOH60 (Figure 5e and f) confirm the above conclusion. This result is well correlated to the SFG conclusion that flip-flop is induced by the interaction of AuCOOH60 with the model mammalian cell membrane dDSPC/DSPC bilayer.

Figure 5. ATR-FTIR spectra collected from the dDSPC/DSPC bilayer in (a) C-D and (b) C-H stretching frequency ranges after the bilayer construction (black), after replacing water with buffer (pH 7.4) in contact with the bilayer (red), and after the addition of AuCOOH60 to the buffer subphase (blue). The difference ATR-FTIR signals shown in (c) C-D stretching and (d) C-H stretching frequency ranges are the difference spectra of the lipid bilayer in contact with water and after replacing water with buffer. The difference ATR-FTIR signals shown in (e) C-D and (f) C-H stretching frequency ranges are the difference spectra between those collected from the lipid bilayer before and after the addition of AuCOOH60 to the buffer subphase. 14 ACS Paragon Plus Environment

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Similar experimental results from SFG and ATR-FTIR studies were obtained for all samples. It was observed that for all samples, the ATR-FTIR data did not show the occurring of lipid removal (Figure S1-S4). No ATR-FTIR signal decrease may be because part of the lipid bilayers was destroyed by the Au NPs and some lipid molecules may attach to the surfaces of the Au NPs while the Au NPs were still staying on the lipid bilayer surface. If this was the case, the decay rates of the SFG signals collected from the two lipid bilayers would not be the same. Likely the first leaflet would be destroyed faster. Also, the Au NPs have PEG chains on the surface, which resist lipid deposition. As presented above, SFG C-H and C-D stretching signals from the dDSPC/DSPC bilayer had similar decreasing rate or decay half-life due to its interaction with the added Au NPs to the suphase. We therefore believe that the interactions between Au NPs surface-functionalized with either carboxyl or amine groups and the dDSPC/DSPC bilayer at different pH conditions all lead to the flip-flop of the lipid bilayer. SFG Studies on Surface Modification Effect at Different pHs As we presented above, various types of Au NPs with different surface modifications interact with model mammalian cell membrane via lipid flipflop. In this section, the effects of the functional groups on the surface of Au NPs on their interaction with lipid bilayer were studied by SFG under different pH conditions. It is necessary to discuss the structure of the lipid before we present the results on lipid – Au NP interactions. Generally, a lipid molecule is composed of a headgroup and two hydrophobic tails. In our study, we chose the dDSPC/DSPC lipid bilayer since phosphocholine (PC) is the main component in the mammalian cell membrane. As depicted in Figure S5, PC, usually considered as a zwitterionic headgroup, consists of a negatively charged phosphate (pKa AuNH60 > AuCOOOH20 > AuNH20. In water, the lipid contains a negatively charged phosphate and a positively charged choline. On the other hand, COOH (pKa=2-3.5) groups should be deprotonated to COO- while NH2 groups (pKa=8.5-9.5) would be protonated to NH3+ in pure water on Au NP surfaces.83 It is easy to understand that the AuCOOH60 induced the fastest lipid flip-flop, because Au NP surface carboxyl groups should be negatively charged in water. As we discussed before, the lipid bilayer has positively charged choline groups at the outmost position to interact with Au NPs, therefore AuCOOH60, having the highest amount of negatively charged groups on its surface as evidenced by its most negative zeta potential, should interact with the lipid bilayer most strongly, leading to the fastest lipid bilayer flip-flop. It is also easy to understand that the flip-flop rate has the order of AuCOOH60 > AuCOOOH20 > AuNH20, because the surface coverage of negatively charged COO- groups on AuCOOH20 is less compared to AuCOOH60, thus leading to a slower flip-flop rate. But comparing with AuNH20, which has positively charged amine groups on the surface, AuCOOH20 induced faster lipid flip-flop than AuNH20 because it has negatively charged COO- groups on its surface. If we only consider the interactions between Au NPs and the positively charged choline groups on the lipid, AuNH60 should lead to the slowest lipid flip-flop, because it has the highest surface coverage of positively charged groups


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with the least negative zeta potential. However, this was not what we observed. Interestingly, we observed that AuNH60 leads to a faster lipid flip-flop than AuCOOH20 and AuNH20. This observation suggested that the stronger interaction between the positively charged NH3+ groups on AuNH60 and the negatively charged phosphate groups on the lipid plays a role in the lipid flip-flop induced by AuNH60. Therefore, we believe that both the sign and number of surface charges on Au NPs affect their interactions with the dDSPC/DSPC lipid bilayer and because of this property, we observed in our experiments that the flip-flop rates of the lipid bilayer induced by Au NPs in water follow the order of AuCOOH60 > AuNH60 > AuCOOOH20 > AuNH20. That is, AuCOOH60 leads to the fastest lipid flipflop, which is followed by AuNH60, because they have the highest surface coverage of charged groups. Then AuCOOH20 and AuNH20 should lead to the third and fourth fastest flip-flop respectively because AuCOOH20 has negatively charged surface group COO- while AuNH20 has positively charged surface group NH3+.

a)

b)

Figure 6. Time-dependent SFG signal intensity changes in (a) C-D and (b) C-H stretching frequency ranges when all four Au NP samples interacting with the dDSPC/DSPC bilayer at 2.00 x10-7 g/mL in water. The dots are experimental data and the lines are fitting results. Table 3. Decay half-lives in C-D and C-H stretching ranges of SFG signals collected from the dDSPC/DSPC bilayer when interacting with Au NP with different surface modifications in water. Functional Group NH60 NH20

C-D (t1/2) (s) 213±7 399±3

C-H (t1/2) (s) 217±5 384±19 17



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COOH20 COOH60

287±16 171±4

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275±2 159±4

We then studied the effect of pH on the interactions between the lipid bilayer and Au NPs. Figure 7 shows the time-dependent SFG C-D and C-H stretching signals during the interactions between the four NPs and the dDSPC/DSPC bilayer with the Au NP solution of pH 5.0. The decay half-lives of these timedependent SFG signals are displayed in Table 4 after fitting the time-dependent SFG intensity curves. Again, the CD and CH stretching signals have the same decay rate for each Au NP, showing the lipid flipflop (confirmed by the ATR-FTIR data). Figure 7 and Table 4 show that the lipid flip-flop rates are much slower for all the Au NPs at pH 5.0 compared to the water cases presented above. The rate follows the order of AuCOOH60 > AuCOOH20 >AuNH20 > AuNH60, as expected. At pH 5.0, the dDSPC/DSPC lipid bilayer should be positively charged instead of zwitterionic. The structure of the quaternary ammonium ions makes the choline group stay positively charged at different pHs. However, some negatively charged phosphate groups become neutral by accepting H+ in the solution under acidic conditions. For this reason, the overall lipid molecule is positively charged. For Au NPs, COOH terminal groups are less deprotonated (less negative) and NH2 terminal group are more protonated (more positive) compared to those in pure water, confirmed by the measured zeta potential data. The measured zeta potential decreased from AuNH20 to AuNH60 to AuCOOH20 to AuNH69 (Table 2). Apparently, more negatively charged Au NPs would interact more strongly with the lipid bilayer, leading to faster flip-flop. Here the Au NPs are either less negatively charged or more positively charged than those in water, therefore their interactions with the dDSPC/DSPC bilayer are much weaker than the water case, leading to much slower lipid flip-flop. It is easy to understand the induced lipid flipflop rate order at pH 5.0 presented above: The interactions between the positively charged lipid bilayer and NH2-terminated NPs are not favored because they have the same charge sign, leading to lower flipflop rate. More positively charged AuNH60 has the slowest flip-flop rate and AuNH20 has the second slowest lipid flip-flop rate. The negatively charged COOH/COO- terminated particles should more 18 ACS Paragon Plus Environment

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favorably interact with the lipid bilayer. AuCOOH60 induces faster flip-flop than AuCOOH20 since AuCOOH60 NPs have more negatively charged groups.

Figure 7. Time-dependent SFG signal intensity changes in (a) C-D and (b) C-H stretching frequency ranges when all four Au NP samples interacting with the dDSPC/DSPC bilayer at 2.00 x10-7 g/mL at pH 5. The dots are experimental data and the lines are fitting results. Table 4. Decay half-lives in C-D and C-H stretching ranges of SFG signals collected from the dDSPC/DSPC bilayer when interacting with Au NP with different surface modifications at pH 5. Functional Group NH60 NH20 COOH20 COOH60

C-D (t1/2) (s) 2530±49 1553±38 911±24 888±39

C-H (t1/2) (s) 2553±16 1615±30 909±50 894±28

We then studied the interactions between Au NPs and the dDSPC/DSPC lipid bilayer with a Au NP solution at pH 7.4 in buffer, which contains a mixture of 0.04 M H3BO3, 0.04 M H3PO4 and 0.04 M CH3COOH titrated by 0.2 M NaOH. The pH condition here (7.4) is similar to the case in the water study (pH not far from 7). Therefore if the interactions detected here are similar to those in water, then the pH effect is dominating. If the interactions are different from the water case, then likely the ions in the buffer solution play a role. Figure 8 illustrates the SFG time dependent C-D and C-H signals collected during the interactions between the four types of functionalized Au NPs and the dDSPC/DSPC lipid bilayer at pH 7.4. Table 5 shows the decay half-lives of SFG signals obtained from fitting. At this pH value, the order



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of lipid flip-flop rates induced by the NPs with different types/amounts of terminal functional groups is AuCOOH60 > AuCOOH20 >AuNH20 >AuNH60, which is the same as that observed when the pH is 5.0, as discussed above. Therefore, the above discussion for the case of pH 5.0 can be applied here. At pH 7.4, the lipid should be zwitterionic, but the positively charged choline group is in the outmost position, therefore the Au NPs with more negatively charged surface groups should more favorably interact with the lipid bilayer, leading to faster flip-flop. Oppositely, the more surface positively charged Au NPs interact with the lipid bilayer less favorably, leading to slower lipid flip-flop. This leads to a lipid flip-flop order of AuCOOH60 > AuCOOOH20 >AuNH20 >AuNH60. It is easy to understand that at pH of 7.4, the lipid flip-flop rates induced by Au NPs are much faster than the situations of pH of 5.0, because at pH of 7.4, the Au NPs with surface negatively charged groups (COO-) are more negative, while the Au NPs with surface positively charged groups (NH3+) are less positive, therefore the interactions between Au NPs and the lipid bilayer at pH of 7.4 are stronger compared to the situations at ph of 5.0, leading to faster lipid flip-flop. It is interesting to observe that although the pH values for the pH 7.4 and water cases are not very different, the induced lipid flip-flop rates are quite different, showing that the ions in the pH of 7.4 buffer play a role in Au NP – lipid bilayer interactions. In the buffer solution, both negatively and positively charged ions exist (Na+, H+, OH-, H2PO4-, HPO42-, PO43-, H2BO3-, HBO32-, BO33-, CH3COO-). The negatively charged ions in the buffer may not strongly interact with the quaternary ammonium ions in choline, due to the possible steric effect. Yet the positively charged ions may interact with the phosphate group with negative charges, making the entire lipid bilayer slightly positive. This means when negatively charged AuCOOH60 and AuCOOH20 particles interact with the bilayer, the interactions are stronger at pH 7.4 than the above discussed water case, leading to faster flip-flop compared to the water case. The higher the negative charge is, the stronger the interactions are, leading to faster flip-flop. Therefore, AuCOOH60 has the fastest flip-flop, and ACOOH20 has the second fastest flip-flop. However, because of the (partial) neutralization of the phosphate group in the DSPC bilayer, the strong interaction between


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the dDSPC/DSPC and AuNH60 in the water case does not exist anymore. Therefore, the induced lipid flip-flop of the dDSPC/DSPC bilayer by AuNH60 is slower at the pH of 7.4 compared to the water case.

a)

b)

Figure 8. Time-dependent SFG signal intensity changes in (a) C-D and (b) C-H stretching frequency ranges when all four Au NP samples interacting with the dDSPC/DSPC bilayer at 2.00 x10-7 g/mL at pH 7.4. The dots are experimental data and the lines are fitting results. Table 5. Decay half-lives in C-D and C-H stretching ranges of SFG signals collected from the dDSPC/DSPC bilayer when interacting with Au NP with different surface modifications at pH 7.4. Functional Group NH60 NH20 COOH20 COOH60

C-D (t1/2) (s) 335±33 186±8 136±12 89±7

C-H (t1/2) (s) 335±31 180±25 144±12 91±4

Figure 9 shows the time-dependent SFG CD and CH stretching signals collected during the Au NP – lipid bilayer interactions with the Au NP solution at pH 9.0. The decay half-lives of the lipid bilayer flip-flop induced by the different surface functionalized NPs are displayed in Table 6. Figure 9 and Table 6 show that the flip-flop rate induced by different Au NPs has the order of AuCOOH60 > AuNH20 >AuCOOH20 > AuNH60. It is believed that the lipid bilayer remains zwitterionic in buffer at pH 9.0. As discussed above in the pH 7.4 case, non-favorable interactions between negatively charged ions in the buffer and quaternary ammonium ions in choline make the outmost layer in lipid positively charged. Therefore, at both pH 5.0 and 7.4, Au NPs with more negatively charged surface groups should 21 ACS Paragon Plus Environment


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interact more strongly with the lipid bilayer, leading to faster lipid flip-flop. This can be used to explain the flip-flop order of AuCOOH60 > AuCOOH20 > AuNH60. However, it is interesting to observe that the flip-flop rate induced by AuNH20 is higher than AuCOOH20. This may be because at pH 9.0, substantial amounts of amine surface groups are in the neutral form instead of the positive ion form, leading to a different interaction than the “pure” electrostatic force. It is interesting to observe that for all Au NPs, the induced flip-flop rates at pH 9.0 are slower than at pH 7.4. It is likely that at pH 9.0, the positive ions in the buffer have weaker interactions with phosphate groups in the lipid molecules (they can interact with hydroxyl groups in the buffer more), weakening the charge screening effect on the phosphate groups on lipid. Therefore, AuNPs with negatively charged surface groups like AuCOOH60 and AuCOOH20 have less interactions with the lipid bilayers compared to the pH 7.4 case, leading to slower lipid flip-flop. We believe that the similar flipflop rate for AuNH20 and different flip-flop rates for AuNH60 at pH 7.4 and 9 are due to the synergistic effect between the decrease in charge screen of lipid phosphate at pH 9.0 and the increase in neutral surface NH2 groups on AuNPs.

a)

b)

Figure 9. Time-dependent SFG signal intensity changes in (a) C-D and (b) C-H stretching frequency ranges when all four Au NP samples interacting with the dDSPC/DSPC bilayer at 2.00 x10-7 g/mL at pH 9. The dots are experimental data and the lines are fitting results.


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Table 6. Decay half-lives in C-D and C-H stretching ranges of SFG signals collected from the dDSPC/DSPC bilayer when interacting with Au NP with different surface modifications at pH 9. Functional Group NH60 NH20 COOH20 COOH60

C-D (t1/2) (s) 580±26 182±22 236±21 167±10

C-H (t1/2) (s) 582±39 185±7 256±10 151±17

CONCLUSIONS In this work, we investigated the molecular interactions between Au NPs with different surface coatings and a model mammalian cell membrane, substrate supported dDSPC/DSPC lipid bilayer. It is worth mentioning that SFG results could not provide distribution of the Au NPs in the lipid area probed. The results are the total contribution from the Au NPs probed. The Au NP solution concentration chosen was based on the detectable flip-flop rates of the lipid. Higher concentrations lead to the lipid flip-flops which are too fast to observe. The lower concentrations lead to the flip-flops which are too slow for practical experiments. Also, this concentration is in the range studied in the literature on Au NP applications. As reported in a review article, the concentrations used in Au NP cytotoxicity studies ranged from nM to mM.91 The tested concentration in this study is about 5µM. In this research, it was found that the Au NP and the lipid bilayer interaction led to lipid flip-flop as evidenced by SFG and ATR-FTIR experiments. We also examined the Au NP – lipid bilayer interactions as a function of Au NP solution pH. Generally, the Au NPs with more negative surface charges (e.g., higher surface coverage of COOH groups) caused faster lipid flip-flop. This is understandable because for a dDSPC/DSPC bilayer, the outmost functionality on the bilayer in contact with Au NPs is the positively charged choline group; the more favorable interaction between the positively charged choline group and negatively charged Au NP leads to a faster lipid flip-flop. Exceptional cases were observed that do not follow the above general rule. For example, in water, AuNH60 leads to faster lipid flip-flop than AuCOOH20 and AuNH20, due to the overall higher surface coverage of positively charged amine groups on AuNH60, which can more strongly interact with the lipid phosphate group, causing a faster lipid flip-flop. At pH 9.0 of the Au NP solution,



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some amine surface groups can become neutral, which may complicate the Au NP – lipid bilayer interactions. It is worth mentioning that here the “total charge” of the NPs plays an important role in the Au NP – lipid bilayer interaction, which may not be due to the specific interactions of amine or carboxyl groups. The Au NPs have the same core charge, and the different overall charges are caused by the different surface charges of amine or carboxyl groups. Therefore the different interactions between Au NPs covered with amine or carboxyl groups and the lipid bilayer are likely due to the different charge effect (caused by different surface groups with varied coverages), instead of specific interactions between the lipid bilayer and the amine or carboxylic groups. We want to emphasize that this study alone may not be enough for understanding the effect of the surface modification of Au NPs on their interactions with cell membranes, which requires examining the interactions between various types of surface-modified Au NPs and more complicated lipid bilayers that more accurately mimic native cell membranes in the future. Nevertheless, this study provides quantitative measurements for assessing the effect of the surface modification of Au NPs on the Au NP – lipid bilayer interactions at the molecular level. Continued success in such studies will aid in the development of Au NPs with desired biomedical properties for various important applications. Acknowledgement This research is supported by the University of Michigan and IMRA America, Inc. P. Hu thanks the University of Michigan Rackham Graduate School for the Rackham Merit Fellowship.

Conflict of interest: The authors declare no competing financial interest. Supporting Information Available: Additional ATR-FTIR spectra of the interactions between the dDSPC/DSPC bilayer and AuCOOH60 in water, at pH5.0 and 9.0 as well as AuNH20 at pH7.4, schematic of the lipid structure, and synthesis of surface modified Au nanoparticles. This material is available free of charge via the Internet at http://pus.acs.org. 24 ACS Paragon Plus Environment

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