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Dynamic surface properties of PEG-coated CuS nanoparticles alter their interaction with cells as revealed by surface-enhanced infrared spectroscopy

Characterization of the dynamic changes of the basic surface properties of nanoparticles is of great significance to reveal the interaction mechanism between nanoparticles and cells; however, it is often neglected due to the limitations of existing analytical methods. This knowledge has been renewed by using surface enhanced infrared absorption spectroscopy (SEIRAS) to study the interaction between PEG-CuS nanoparticles and living cells attached to rGO-Au modified Au films. Based on the difference spectra of cell membranes and the associated water, we clearly revealed that Cu2+ ions produced by the degradation of PEG-CuS can coordinate with PEG, thus changing the interaction between nanoparticles and cells including how and how many nanoparticles enter the cells and the sequential photothermal effect, which breaks through the limitations of the present analytical methods.


Synthesis of graphene oxide (GO)
Graphene oxide (GO) was synthesized according to the modified Hummer's and Offeman's method as described everywhere 1 . Briefly, graphite (1 g) and 98 % H 2 SO 4 (23 mL) were mixed in a round-bottomed flask and stirred for 24 h, then NaNO 3 (0.5 g) and KMnO 4 (3 g) were slowly added with constant stirring for 30 min S4 and 5 min, respectively, in an ice bath. Then the temperature was kept at 35 ºС for 5 min, and 46 mL of distilled water was slowly added and stirred for 40 min. Finally, the flask was transferred to a 98ºС oil bath for 25 min, then 3.4 % H 2 O 2 (31 mL) was added to terminate the reaction to get brown yellow solution. The obtained solution was washed and dialyzed against water, then sonicated and centrifuged.

Preparation of rGO-Au-decorated Au Plasmonic Substrate.
The Au film decorated with rGO-Au (rGO-Au/Au) was electroless deposited onto the Si prism or wafer, which was polished with Al 2 O 3 slurry (1.0 μm) followed by washing thoroughly with deionized water. After immersing in a 40 wt % NH 4 F aqueous solution for 3 min, the rGO-Au/Au film was prepared by exposing the treated surface to a 1:1: To evaluate the enhancement factor (EF) of rGO-Au/Au plasmonic substrate, a reference spectrum of taking bare Si or rGO-Au/Au-coated Si prism was firstly recorded and then ~0.32 µg (~monolayer thickness) 2 of p-ATP ethanol solution was dropped on the surface of the bare Si and rGO-Au/Au-coated Si prism, respectively.
The sample spectra were recorded until total evaporation of ethanol by averaging 521 scans at a spectral resolution of 4 cm −1 .

Preparation and intracellular uptake of CuS NPs
The synthesis of CuS NPs in water was prepared according to the literature by using PEG as a stabilizer to S5 enhance biocompatibility, and prevent nonspecific interactions in physiological environments. 3 In a typical procedure, after the mixing of 25 mL of PEG-SH solution (1 mg/mL) and 25 mL of CuCl 2 (5 mM) aqueous solution, 1 mL of Na 2 S (0.25 M) was slowly added dropwise to the solution. The pale-blue CuCl 2 solution turned dark-brown immediately. After 10 min, the reaction flask was transferred to an oil bath (~90 ºС) with continuous stirring for 10 min until a dark-green solution was obtained. At last, the flask was cooled in an icewater bath to terminate the reaction. The as-synthesized PEG-coated CuS NPs was dialyzed with molecular weight cut-off (MWCO) of 30 kDa and stored at 4 ºС for later use.
To more directly explore the endocytic mechanism of CuS-PEG NPs internalized into HeLa cells, cells were pretreated with various inhibitors including dynasore (30 µg/mL, dynamin inhibitor ), chlorpromazine (CPZ, 10 µg/mL, clathrin inhibitor), filipin (10 µg/mL, caveolae inhibitor), and Cyt D (1 µmol/L, inhibitor of actin polymerization related phagocytosis and clathrin/caveolin independent endocytosis) for 1 h at 25°C or 4°C (energy independent pathway) detected using ICP-MS and ATR-SEIRS. Then, the cells were washed with PBS three times and cultured with CuS-PEG NPs at the concentration of 100 µg/mL for 2 h, the process of cell changes was monitored by ATR-SEIRS in real-time. And then cells were washed with PBS for three times, trypsinized, resuspended, counted, and centrifuged down, and the cell pellet was digested in 1 mL of aqua regia (V HCl :V HNO3 =3:1). Cell concentrations were determined by hemacytometry and the intracellular copper content was quantified using ICP-MS with parts per billion sensitivity. In control, cells were placed in 1 mL PBS without nanoparticles. 4, 5

Immobilization of Cells on rGO-Au/Au Film
Before culturing HeLa cells, the home-made poly(trifluorochloroethylene) cell and necessary experimental tools were sterilized by 70% ethanol and UV irradiation. 2 mL of cell suspension (5×10 5 cell/mL) was seeded on the poly-L-lysine layer (0.1 mg/mL PLL) modified rGO-Au/Au film. After incubation for 24 h, the IR cell was mounted on SEIR spectrometer. After washing three times with PBS, cells were exposed to 100 μg/mL of S6 PEG-CuS NPs by adding 25 µL PEG-CuS NPs aqueous solution (4 mg/mL) into a volume of 1 mL of PBS. 6 CuS NPs induced SEIRA spectra were obtained by taking cells in PBS as a reference spectrum. After incubation with 100 μg/mL PEG-CuS NPs for 2 h, the cells were washed three times with PBS and followed by being irradiated for 20 min using the 980 nm diode laser with power of 1 and 1.5 W/cm 2 . As the control experiments, 25 µL H 2 O, supernatant of CuS NPs (20 day) and 5 mM CuCl 2 were used to replace CuS NPs and added into the ATR chambers. Unless noted specifically, the control experiments were performed following the same protocol and each measurement was repeated in triplicate.

Cell culture and viability test
Human epithelial cervical cancer cells (HeLa) were routinely cultured in the fresh Dulbecco's modified After the FTIR experiment, cell viability on the rGO-Au/Au substrate was roughly assessed using top-view microscope for cell monolayer integrity and morphology, and the trypan blue exclusion test to stain dead cells.
Cells were trypsinized and resuspended in trypan blue (0.4 %, g/ml) for 3 min. Cell viability was calculated under 10×magnification using a hematocytometer. The percentage of cell viability was calculated as follows: N viable cells / N total cells ×100%.

Simulation
To simplify, the ellipsoid gold nanoparticle (R 1 =30 nm, R 2 =10 nm) obtained from AFM image and a rGO layer of 4 nm thick inferred from Raman data were considered, and simulation was performed by using 3D finite- A schematic illustration of the procedure for fabricating GO using modified Hummer's method 1 was shown in Figure S1. The surface structure and morphology of GO were further confirmed by TEM and AFM, the size of the synthesized GO is relatively uniform (~400 nm) and the height profile is ~1 nm thickness, corresponding to S9 a typical thickness of a two-dimensional single-layer GO sheet. The UV-vis spectrum exhibited a characteristic absorption peak at 230 nm and a shoulder peak at 300 nm, attributing to the electronic transition of π-π* of C＝ C bonds and n-π* of C＝O bands, respectively. 12 The oxygen content of as-prepared GO was ~31% based on XPS measurement. The ATR-FTIR spectrum of GO showed that the ν O-H stretching vibration (a broad 3450 cm -1 ), ν C＝O stretching of -COOH or C＝O groups (1739 cm -1 ), the resonance at 1622 cm -1 can be assigned to the bending vibrations of the adsorbed water molecules, and it contains a hidden 1600 cm -1 peak from skeletal The prepared HeLa cells were collected by centrifugation (1000 rpm, 10 min) and washed four times with 0.9 % NaCl solution to remove growing medium. A total of 50 L of the resulting cell pellet was introduced onto the surface of Si crystal, and ATR-FTIR spectra of HeLa cells were collected after dried by ultrapure nitrogen flux to remove any excess water and by taking the bare Si as a background ( Figure S4). In Figure S4, we can see that this situation should mainly come from the influence of ν(N-H) band, while it isn't same with our spectra, in which the absorption peaks of 3250 cm -1 and 3680 cm -1 appear simultaneously (Fig. 3). In turn, this can prove that our absorption peaks should mainly come from interfacial water interference. As we can see, the peak range between 1645 and 1490 cm -1 during the interaction of CuS-old NPs and HeLa cells was very board, so the movement of the maximum peak position may be a change in different chemical bonds. Therefore, the Gaussian curve-fitting analysis was carried out by using the OPUS 7.5 software in the interactive mode. Figure S5 shows that three deconvoluted components at ~1600, 1560 and 1543 cm -1 , which are attributed to δ(N-H) or ν asym (COO − ) and amide II, respectively [19][20][21] . The absorption peaks of carboxyl and amino groups are derived from the coordination of copper ions. As time goes on, the coordination effect increases gradually. So, the shift of the peaks represents the gradual coordination of copper. S15 Figure S6 Figure S6. Interaction of CuS-old NPs and HeLa Cells assessed by SEIRA spectra with and without inhibitor conditions. (A-E) CuS-PEG NPs-induced structure changes of HeLa Cells with and without inhibitor dynasore, CPZ, Filipin and Cyt D in SEIRA spectra by taking cells incubated rGO-Au/Au substrate immersed in PBS as a reference at 2 h. (F) The uptake degree quantified using the ratio of the peak intensity at ~3683 cm −1 to ~3255 cm −1 .