Surface and Size Effects on Cell Interaction of Gold Nanoparticles with

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Surface and size effects on cell interaction of gold nanoparticles with both phagocytic and non-phagocytic cells Xiangsheng Liu, Nan Huang, Huan Li, Qiao Jin, and Jian Ji Langmuir, Just Accepted Manuscript • DOI: 10.1021/la401556k • Publication Date (Web): 01 Jul 2013 Downloaded from http://pubs.acs.org on July 5, 2013

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Surface and size effects on cell interaction of gold nanoparticles with both phagocytic and nonphagocytic cells Xiangsheng Liu, Nan Huang, Huan Li, Qiao Jin and Jian Ji* MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China. KEYWORDS: gold nanoparticles, surface charge, size, cell interaction; phagocytic/nonphagocytic cells

ABSTRACT: With the development of nanotechnology and its application in biomedicine, studies on the interaction between nanoparticles and cells have become increasingly important. To understand the surface and size effects on cell interaction of nanoparticles, the cellular uptake behaviors of two series of gold nanoparticles (AuNPs) with both positively and negatively charged surfaces and sizes range from ~16 to ~58 nm were investigated in both phagocytic RAW 264.7 and non-phagocytic HepG2 cells. The internalization of AuNPs was quantified by ICP-MS and the intracellular fate of NPs was evaluated by TEM analysis. The results showed that the AuNPs with positive surface charge have much higher cell internalization ability than those with negative surface charge in non-phagocytic HepG2 cells. However, the uptake extent of negatively

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charged AuNPs was similar with that of the positively charged AuNPs when in phagocytic RAW 264.7 cells. Among the tested size range, negatively charged AuNPs with a diameter of ~40 nm had the highest uptake in both cells, while the positively charged AuNPs did not show a certain tendency. Intracellular TEM analysis demonstrated the different fate of AuNPs in different cells, where both the positively and negatively charged AuNPs were mainly trapped in the lysosomes in HepG2 cells, but many of them were localized in phagosomes when in RAW 264.7 cells. Cytotoxicity of these AuNPs was tested by both MTT and LDH assays, which suggested NP’s toxicity is closely related to the tested cell types besides the surface and size of NPs. It demonstrates that cell interaction between nanoparticles and cells is not only affected by surface and size factors but also strongly depends on cell types.

1. INTRODUCTION

Inorganic nanoparticles, due to their unique physicochemical properties, have been demonstrated to be promising platforms for biomedical applications such as biosensing, phototherapy, imaging, and drug/gene delivery.1-9 Despite the novel proof-of-concept studies of innovative design strategies for targeted delivery of nanomaterials have attracted great attention; the fundamental studies on nano-bio interface should also be taken seriously.10-14 A recent trend in nanotechnology is investigating the interactions of nanomaterials with biological systems, known as nano-bio interactions.10,

15, 16

Physicochemical characteristics of nanoparticles will

significantly affect their circulation, biodistribution, cellular internalization, and trafficking in biological systems.17, 18 Understanding of the interactions between nanoparticles and biological systems, especially the biophysicochemical interactions at nano-bio interface, is important for the safe and effective use of nanomaterials.10-13,

15

Interactions between nanoparticles and cells

should be considered first as many applications require a firm control over nanoparticle-cell


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interactions. Extensive studies have been focused on probing predictive relationships correlating the properties of nanomaterials such as size19-27, shape19-21, 27, 28, chemical functionality25, 26, 29-31 and surface charge16,

25-27, 29, 30, 32-38

with cell interactions including biomolecular signaling,

biological kinetics, transportation, and toxicity. 10 Among all these factors, size and surface’s effects on cell interactions have been studied most. Chan et al. have shown that, for spherical gold nanoparticles stabilized by citric acid ligands, 50nm diameter is an optimal size to maximize the rate of uptake and intracellular concentration in mammalian Hela cells.19 A similar tendency of size effect was observed in the transferrin-coated gold nanoparticles in three different cell lines (STO cells, which are fibroblast cells, HeLa cells, which are ovarian cancer cells, and SNB19 cells, which are brain tumor cells).21 Except for cell uptake, the size effect is closely related to nanotoxicity.22, 23, 26 Chan et al. showed that 40- and 50-nm nanoparticles were found to alter cell functions most greatly within the 2-100 nm size range.23 Besides size effect, surface chemistry plays an important role on nanoparticle-cell interactions.16 Many studies demonstrated that the positively charged nanoparticles are more easily uptaken by cells than the neutral or negatively charged nanoparticles.27, 29, 30, 32-34, 38 The mammalian cellular uptake of gold nanorods with polyelectrolyte (PE) coatings can be tuned from very high to very low by manipulating the surface charge and functional groups of the PEs.30, 38 Mukherjee et al. demonstrate that surface charge of gold nanoparticles plays a critical role in modulating membrane potential of different malignant (ovarian cancer CP70 and A2780 cells) and nonmalignant (human bronchial epithelial cells (BECs) and human airway smooth muscle (ASM) cells) cell types and subsequent downstream intracellular events including cell uptake, localization of the nanoparticles and their biological functions.32 For cytotoxicity, Hussain et al. investigated the impacts for cellular processes of 1.5 nm Au NPs with different


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surface charges in a human keratinocyte cell line (HaCaT). Their results indicated that surface charge is a major determinant of how Au NPs impact, with the charged NPs inducing cell death through apoptosis and neutral NPs leading to necrosis. Surface charge even seems to be a more important factor than size for cell uptake and cytotoxicity in many cases. Xia et al. have examined the role of surface charge in internalization of gold nanoparticles by using an I2/KI etchant approach which allows a quantitative differentiation of surface adsorbed nanoparticles versus internalized nanoparticles. They showed that neutral and negatively charged nanoparticles being adsorbed much less on the negatively charged cell-membrane surface and consequently show lower levels of internalization as compared to the positively charged particles. Considering the polymer-coating causing some minor aggregation and size increase to Au nanospheres in this study, the results of 5-10 times higher uptake for the positively charged Au nanospheres than for the neutral and negatively charged Au nanospheres indicated that the type of surface charge seems to be a more important factor in determining the uptake by SK-BR-3 breast cancer cells.33 Recently, Iyer et al. have simultaneously compared the impact of size, charge, and functionalization of QDs alone or in combination on biological responses using primary normal human bronchial epithelial cells. Their results showed that positively charged QDs are significantly more cytotoxic compared to negative QDs and suggested that QD-elicited cytotoxicity is not a function of a single property but a combination of factors where charge > functionalization > size.26 As described above, in the vast majority of cases, the nano-bio interaction studies are mainly focused on the parameters of nanoparticles. However, cell types might play an equally important role in this process which may result in quite different uptake behavior of nanoparticles for different cells. In fact, the importance of cell types is often taken into account as many studies


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were performed in two or more kinds of cells.21, 23, 29, 32, 39 Both malignant and nonmalignant cell types were applied in some cases,21, 32 and not only cell lines but also primary cells were applied in these fundamental studies.26, 29 Although many cell types were utilized in the studies of size and surface effects, few work has declared that size and surface effects are significantly affected by cell types. Among the so many cell types, phagocytic and non-phagocytic cells are of great importance. In our recent study, we noticed that the uptake of negative 11-mercaptoundecanoic acid (MUA) capped quantum dots in non-phagocytic cells was much lower than that in phagocytic cells.40 The phagocytes are the most important cellular component of the innate immune system and are omnipresent in the body. Among all phagocyte types, macrophages are most efficient in internalization of body-foreign material, which mainly reside in liver and spleen tissues.29 The phagocytic cells of the MPS will quickly uptake the nanoparticles once administered into the blood due to their strong phagocytic activity.41 Most nanomaterials are design to target the non-phagocytic cells at a disease site of the body,27,

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but for some

applications the phagocytic cells of immune system are also desired.42 It is well-recognized that physicochemical properties such as size and surface charge strongly affect the interactions of nanoparticles with cells. Therefore, an understanding of the difference for these effects between phagocytic cells and non-phagocytic cells is of significant interest. In current study, we have simultaneously compared the impact of size and charge of nanoparticles alone or in combination on cell uptake using both phagocytic and non-phagocytic cells. Gold nanoparticles, which are widely used in biomedical applications,3-6, 9, 11, 43-47 were chosen as a model system because of the simplicity and reproducibility of the synthetic and surface modification techniques.23 Moreover, the AuNPs uptaken by cells can be quantified by ICP-MS with high detection sensitivity and be clearly observed in intracellular compartments by


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TEM as AuNPs with high density provide higher spatial resolution for TEM analysis. Citratecapped AuNPs with diameter from ~16 to ~58 nm were modified by negatively charged sodium 11-mercaptoundecanoic acid (MUA) or positively charged (10-mercaptodecyl)-trimethylammonium bromide (TMA). Their cell uptake behavior affected by size and surface charge were evaluated in both phagocytic and non-phagocytic cells, where RAW 264.7 macrophages and HepG2 cells were used as model phagocytic and non-phagocytic systems, respectively (Scheme 1). The cell uptake and intracellular distribution of the two series of AuNPs in two types of cells were investigated by ICP-MS and TEM analysis. Accompanied cytotoxicity of these nanoparticles for the cells were evaluated by both MTT and LDH assays. Our results suggested that the cell interactions with nanoparticles were not only affected by nanoparticles’ size and surface charge, the surface and size effects on cellular uptake behavior but also strongly depends on cell type.

Scheme 1. Schematic of the AuNPs with different charges, chemical structures of the charged ligands and models of phagocytic and non-phagocytic cells (not to scale).

2. EXPERIMENTAL SECTION


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Materials. (10-Mercapto-decyl)-trimethyl-ammonium bromide (TMA) was synthesized according to the literature as described before.48 11-Mercaptoundecanoic acid (95%, MUA) and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Lactate dehydrogenase (LDH) Kit was purchased from Applygen Technologies Inc.

Hydrogen

tetrachloroaurate

hydrate

(HAuCl4·4H2O),

trisodium

citrate

dihydrate

(C6H5Na3O7·2H2O) and other regents were purchased from Sinopharm Chemical Reagent Co., Ltd. All cell lines were purchased from China Center for Typical Culture Collection, and all reagents for the cell culture were used directly after purchased. All water was distilled and subsequently purified to Millipore Milli-Q quality. For nanoparticles synthesis, all glassware used was cleaned by freshly prepared aqua regia solution (HCl/HNO3, 3:1). For cell experiments, all the solutions and substrates were sterilized in advance. Synthesis of citrate protected AuNPs. Citrate-capped AuNPs (AuNP-Cit) with various sizes were synthesized according to the method developed by Frens with minor modifications.49 Briefly, 1.214 ml of 10 mM HAuCl4 was added to 50 ml of Milli-Q water and the solution was heated to boiling. Next, 5.1 ml, 3.4 ml, 2.55 mL and 1.7 ml of 10 mM citrate sodium were added to the solution to synthesize 16 nm, 26 nm, 40 nm and 58 nm AuNPs, respectively. The solution was refluxed for half an hour as a color change from dark blue to red was observed. After cooled to room temperature, these AuNPs were modified with positive and negative thiols. Synthesis of negative and positive thiols protected AuNPs. In the following discussion, we referred the negative carboxylic group (MUA) modified AuNPs as AuNP-MUA and the positive quaternary ammonium group (TMA) modified AuNPs as AuNP-TMA. An additional number was added in the front of the symbol to present the different sizes of nanoparticles. For more brief, the “AuNP” was omitted in case of comparing the nanoparticles of different sizes and


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surface. For example, 16-MUA meant 16 nm AuNPs modified by MUA and 40-TMA meant 40 nm AuNPs modified by TMA. The thiols protected AuNPs were obtained from the citrate coated AuNPs by exchange of citrate molecules with thiols. This reaction was performed similarly according the literature,48 as a large excess equivalent of thiols was used. Briefly, an aqueous solution of MUA or TMA (50 mM, 0.2 mL) was added into citrate coated Au-NPs solution (10 mL). For MUA, the pH of the solution was adjusted to be pH~9 by 1 M NaOH, as MUA has better solubility in alkaline condition, and it’s also helpful for keeping the AuNPs stable through the ligand exchange reaction. For TMA, the pH of the solution was adjusted to be pH~4 by 1 M HCl, as the acidic condition is helpful for keeping the positive AuNPs stable through the ligand exchange reaction. After stirred at room temperature for 24 h, the modified AuNPs were purified by centrifugation and redispersed in water. All AuNPs of different sizes were modified by the same way except for the centrifugation speed as higher speed was needed for smaller particles. Characterization of AuNPs. UV-vis analysis was carried out with a UV-vis Shimadzu UV2505 spectrometer using 1-cm-path length quartz cuvettes. Spectras were collected within a range of 400-800 nm. When detected the spectra of AuNPs in cell culture medium, Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) without phenol red was used to exclude the strong absorbance of phenol red. The UV-vis spectra of modified AuNPs in water, phosphate buffered (PB, 10 mM, pH 7.4) solution and cell culture medium were measured. Transmission Electron Microscopy (TEM) analysis was performed on a JEM-1230EX TEM operating at 80 kV in bright field mode. For the determination of particle size, over 100 particles were counted in multiple pictures from different areas of the TEM grid. The modified AuNPs in water, phosphate buffered (PB, 10 mM, pH 7.4) solution and cell culture medium were measured by TEM. Dynamic Light Scattering (DLS) analysis was performed on


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90plus/BI-MAS (90 Plus Particle Size Analyzer, Brookhaven Instruments Co.). The hydrodynamic size of modified AuNPs in FBS contained cell culture medium was measured. The scattering angle was kept at 90° and the wavelength in the vacuum was set as 633 nm during the whole experiment. Zeta potential measurements were also performed on a Delsa™ Nano C Particle Analyser (Beckman Coulter Ireland Inc.). The zeta potential of modified AuNPs in FBS contained cell culture medium was measured. Cell culture. RAW 264.7 cells and HepG2 cells were cultured with regular growth medium consisting of high-glucose DMEM supplemented with 10 % fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin, and cultured at 37 °C in a 5% CO2 humidified environment. Internalization of AuNPs detected by ICP-MS analysis. Cellular uptake of the AuNPs was determined by inductively coupled plasma mass spectrometry (ICP-MS) quantitatively. To determine the AuNPs uptake amount quantitatively, the cells were seeded on a 24-well plate at certain density (1×105 cells per well for HepG2 and 2×105 cells per well for RAW 264.7 which volume is smaller than that of HepG2 cells) and cultured for 24 h, and then were incubated with AuNPs (added 20 uL AuNP solution into 480 uL fresh culture medium, final concentration of Au was 10 mg/L) for 12 h. In all the processes, the cells are cultured in the medium with 10% FBS. At determined time, the cells were washed five times with PBS, and then treated by aqua regia (HCl: HNO3 =1: 3, volume ratio) for 2 h. The treated solution was diluted to determine Au concentration by ICP-MS (Thermo Elemental Corporation of USA, XSeries Ⅱ). The Au amount per well data from ICP-MS analysis are presented as mean ± standard deviation (SD) for experiments repeated three times. Divided by the number of cells, the data of Au amount per cell was calculated.


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Intracellular fate of AuNPs detected by TEM analysis. For TEM cell sections analysis, the cells were seeded on a 6-well plate at certain density (5×105 cells per well for HepG2 and 1×106 cells per well for RAW 264.7) and cultured for 24 h. After cultured for 24 h, the medium was replaced with fresh medium, and then the cells were incubated with AuNPs with a Au atomic concentration of about 10 mg/L for 12 h. At determined time, the cells were washed five times with PBS and trypsizined, centrifuged, and then fixed with 2.5% glutaradyhyde. After 2 h fixation at 4 °C, the samples were washed with phosphate buffered saline (0.1 M, pH=7.0) three times. Then the samples were fixed with 1% perosmic oxide for 2 h at 4 °C. After being washed in water, the samples were dehydrated in an alcohol series, embedded, and sliced with the thickness between 50 to 70 nm. Cytotoxicity assays. Cytotoxicity was performed by standard MTT assay and LDH assay. The colorimetric MTT test assesses cell metabolic activity based on the ability of the mitochondrial succinate/tetrazolium reductase system to convert the yellow dye (MTT) to a purple formazan in living cells. The metabolic activity of the cell is proportional to the color density formed. To determine the relative cell viability by MTT assay, cells were plated at certain density (5×103 cells per well for HepG2 and 1×104 cells per well for RAW 264.7) in a 96-well plate and cultured for 24 h. The medium was replaced with fresh medium containing the AuNPs of varying concentrations (the Au atomic concentration was determined by ICP-MS). Cells cultured in nanoparticle-free media were used as a control. After treatment for 24 h, the wells were washed with PBS (to rule out the interference from MTT interacts with AuNPs in the suspension14) and the medium was replaced with 100 µL fresh medium, 20 µL MTT (5 mg/ml) was added to each well and the cells were further cultured at 37 °C for 4 h. The dark blue formazan crystals generated by the mitochondria dehydrogenase in live cells were dissolved with 150 µL dimethyl


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sulfoxide to measure the absorbance at 570 nm by a microplate reader (MODEL 550, Bio Rad). The relative cell viability (%) = the absorption of treated well/ the absorption of control well*100%. Cell membrane integrity can be measured by lactate dehydrogenase (LDH) leakage assay. LDH retention was measured using a cytotoxicity detection kit according the instruction. To determine the relative cell viability by LDH assay, cells were plated at certain density (1×104 cells per well for HepG2 and 2×104 cells per well for RAW 264.7) in a 96-well plate and cultured for 24 h. The medium was replaced with fresh medium containing the AuNPs of varying concentrations. Cells cultured in nanoparticle-free media were used as a control. After treatment for 24 h, the wells were washed with PBS three times (to rule out the interference from LDH kit interacts with FBS and AuNPs in the suspension). Then 100 uL 1% triton X-100 (in PBS) was added to each well to lyse the cell membrane for 10 min. Then 10 µL of the lysed solution was transferred to a new 96well followed by the addition of 20 µL of substrate mix (reagent A and reagent B, 5:1). The plate was incubated for 15 min at 37 °C followed by the addition of 16.5 µL reagent C. After incubated at 37 °C for another 15 min, 50 µL of termination solution was added to each well. Absorbance was recorded at 450 nm by a microplate reader (MODEL 550, Bio Rad). The relative cell viability (%) = the absorption of treated well/ the absorption of control well*100%. Statistical analysis. Au amount per well data from ICP-MS analysis are presented as mean ± standard deviation (SD) for repeated triple. Statistical significance was assessed with Student’s ttest, and p values of < 0.05 were considered statistically significant.

3. RESULTS AND DISCUSSION


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Nanoparticle synthesis and characterization. Citrate-capped AuNPs (AuNP-Cit) with various sizes were synthesized according to the Frens’s method by adjusting the ratio of citrate to HAuCl4.

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The AuNP-Cit was characterized by UV-vis and TEM analysis. The representative

TEM images of AuNPs were shown in Figure 1 (top column), NP’s sizes were determined from TEM images to be about 16 nm, 26 nm, 40 nm and 58 nm for the four kinds of AuNPs (Table 1). The absorbance peak red-shifts in surface plasmon resonance of AuNPs in UV-vis spectra also indicated an increase of AuNP size (Figure 1, top right column). Exchange of citrate with MUA and TMA was performed in the same way for all the AuNPs as both ligands bear a thiol group with high affinity to gold surface, which formed self-assembled monolayer on AuNP surfaces. TEM images of these modified AuNPs showed the modification did not change the AuNP core sizes (Figure 1). UV-vis spectra of AuNPs did not show obvious change after modified by MUA and TMA indicated good dispersion stability of these modified AuNPs except for 58 nm AuNPs with TMA. The change of UV-vis spectrum for 58 nm AuNP-TMA was due to some aggregation and sedimentation of the AuNPs happened during the ligand exchange process as the TMA could not well stabilize this large AuNPs.


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Figure 1. TEM images and UV-vis spectra of citrate capped AuNPs (AuNP-Cit, top), AuNPs modified with 11-mercaptoundecanoic acid (AuNP-MUA, middle) and (10-mercapto-decyl)trimethyl-ammonium bromide (AuNP-TMA, bottom). Table 1 presented DLS and zeta-potential measurement results of these modified AuNPs in cell culture medium DMEM (supplemented with 10% fetal bovine serum). The hydrodynamic diameter of all AuNPs were larger than their original sizes indicated some serum proteins adsorbed on the NP surfaces or some aggregation of AuNPs happened in the medium. The hydrodynamic size of MUA coated AuNPs in DMEM with 10% FBS slightly decreased even with the TEM size of single AuNPs increased. The reason could be that the MUA coated AuNPs with different size might adsorb different proteins and form different aggregates in the cell culture media. From the TEM images of MUA coated AuNPs in DMEM with 10% FBS, it was observed that the large 58 nm AuNPs only formed small clusters with two or three NPs together while the 16 nm AuNPs formed some large aggregates with many NPs together (Figure S1). It


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can also be observed that the zeta potential of TMA capped AuNPs change from positive (~30 mV) to negative (~-20 mV) before and after incubation in cell culture medium. Due to the positive charge of the quaternary ammonium groups, AuNP-TMA dispersions exhibited strongly positive surface potentials of about 30 mV. After incubation in cell culture medium, the adsorption with serum protein on NP surfaces leads to negative surface zeta-potentials of about 20 mV. For MUA capped AuNPs, their zeta-potentials became less negative after incubation in DMEM which may be due to the adsorption of serum proteins. Similar change of zeta-potential after incubation in cell culture medium was also observed for other nanoparticles.24, 38 No matter modified with positive or negative ligands, the AuNPs exhibited negative surface potentials when incubated in serum contained medium. The different zeta-potential values for the two series AuNPs were mainly due to their different surfaces and sizes would adsorb different proteins25, 50 and may also be affected by their size and aggregation states. The positive surfaces prefer to adsorb negative proteins resulted in an opposite negative potential; the negative surfaces seem to adsorb positive proteins resulted in a less negative potential. Besides the electrostatic attraction, hydrophobic interaction will strongly affect the protein adsorption. The AuNP-MUA might adsorb negative proteins by hydrophobic interaction as well which may be accounted for their final negative zeta-potential values. Table 1. AuNPs physicochemical properties Physical diameter [nm] a) 16.4 ± 1.6 26.5 ± 6.4 40.1 ± 5.5 58.4 ± 7.1

Ligand MUA MUA MUA MUA

Hydrodynamic diameter from DLS [nm] b) 131.1 135.8 129.4 97.2

16.4 ± 1.6 TMA 115.2 26.5 ± 6.4 TMA 140.0 40.1 ± 5.5 TMA 132.7 58.4 ± 7.1 TMA 172.7 a) The values were calculated from TEM images

Zeta potential ζ[mV] b) -11.26 -9.15 -10.64 -9.85

Zeta potential ζ[mV] c) -30.80 -30.92 -41.76 -52.65

Zeta potential ζ[mV] d) -16.87 -21.49 -24.83 -28.79

-14.29 30.20 N.A. -14.77 24.59 N.A. -18.70 29.94 N.A. -18.94 28.19 N.A. of unmodified AuNPs by software Image J;


b)

measurements

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were carried out in DMEM with 10% FBS (pH~7.4); c) in Milli-Q water, and d) in PB solution (pH~7.4). N.A., not available due to serious aggregation of AuNP-TMA in PB solution (pH~7.4). These values were means of triple measurements.

As shown in Figure 2, the UV-vis spectra of AuNP-MUA in PB solution (10 mM, pH 7.4) and cell culture medium DMEM were similar (Figure 2a and c). Although the spectrum shape was a little different from that in origin solution (Figure 1), the spectra without significant red-shift still indicated well dispersion of AuNPs in these solutions. While the UV-vis spectra of AuNP-TMA in PB solution (10 mM, pH 7.4) changed greatly indicates serious aggregation of AuNPs (Figure 2b). When only consider the pH effect, these positive TMA capped AuNPs can only be stable in acidic conditions as the electrostatic repulsion is not strong enough to stabilize them in neutral or basic solution.48 Zeta-potential may give an evidence to the surface charge of the AuNP-TMA, however, due to the serious aggregation of NPs, their zeta-potential measurements turned out a puzzle result which all exhibited negative values at pH 7.4 (data not shown). Differently, the UVvis spectra of AuNP-TMA in cell culture medium changed in much less extent compared in PB solution indicated much better stability in the serum contained condition (Figure 2d). Some aggregation of AuNPs was observed for all AuNPs when incubated in serum contained cell culture medium, but the aggregated extent of AuNP-TMA was much less serious than that in PB (pH 7.4) solution (Supplementary data, Figure S1 and S2). Stark et al. showed the cerium oxide suspensions in cell culture medium undergo protein adsorption, which results in comparable low surface charge density and favors rapid agglomeration when compared in ultrapure water.24 For this study, the protein adsorption favored the colloidal stability of AuNPs, especially for AuNPTMA, in cell culture medium which contain certain ionic and protein concentrations at pH 7.2~7.4. It demonstrated that the protein adsorption on AuNPs not only changed the outmost surface of NPs but also improved the stability of NPs in biological media. It was reported that both size and surface properties were found to play a very significant role in determining the


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nanoparticle coronas on the different particles of identical materials.25,

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51

In fact, what the

biological cell actually “sees” when interacting with a nanoparticle dispersed in a biological medium likely matters more than the bare material properties of the particle itself,52, 53 which will further influence the cell interaction with the nanoparticles.

Figure 2. UV-vis spectra of MUA and TMA capped AuNPs in PB solution (10 mM, pH 7.4) and cell culture medium (DMEM, 10% FBS, pH 7.4). Quantification of AuNPs uptake in phagocytic and non-phagocytic cells. To investigate the cell uptake behavior of NPs affected by size and surface charge in both phagocytic and nonphagocytic cells, the two series AuNPs with four kinds of sizes and two kinds of surfaces were incubated in HepG2 cells (as model non-phagocytic cells) and macrophage RAW 264.7 cells (as model phagocytic cells), respectively. The different AuNPs were incubated with cells in same Au concentration, and the cell uptake was quantified by ICP-MS with high detection sensitivity described as Au content in cells. As shown in Figure 3a, for non-phagocytic HepG2 cells, the uptake of positive AuNP-TMA was much higher than that of negative AuNP-MUA in all tested


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size range. It indicated that the original surface charge plays a significant role on uptake of AuNPs by HepG2 cells. For size effect of MUA capped AuNPs in non-phagocytic HepG2 cells, it can be observed that the 40 nm AuNPs exhibited the highest uptake among the test sizes. This tendency agreed with previous report for citrate-, transferrin- and Herceptin-coated AuNPs in various cells.19, 21, 23 For TMA capped AuNPs, the HepG2 cells had the highest uptake for the 58 nm AuNPs and the second highest uptake for the 26 nm AuNP, while had lower uptake for the 40 nm AuNPs as 16 nm AuNPs. The higher uptake of 58 nm AuNP-TMA was not only affected by the size, as described above the 58 nm AuNP-TMA have is not so stable as other smaller NPs (Table 1 and Figure 2d), the aggregation and sedimentation effects may be another main reasons for their high uptake.39,

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Compared with the surface effect, size seems to be a less important factor in

determining the uptake by non-phagocytic HepG2 cells here. When incubated phagocytic RAW 264.7 cells with the two series of AuNPs, the uptake of positive AuNP-TMA was still higher than that of negative AuNP-MUA but decreased in extent, especially the uptake of AuNP-TMA was even a little less than that of AuNP-MUA when in size 40 nm (Figure 3b). The size effect of both AuNP-MUA and AuNP-TMA in phagocytic RAW 264.7 cells was similar with the tendency in non-phagocytic HepG2 cells. Furthermore, when focus on the surface charge effect; it can be observed that the uptake of AuNP-TMA was 4-10 times higher than that of AuNP-MUA in HepG2 cells, whereas the uptake of AuNP-TMA was similar with that of AuNP-MUA in RAW 264.7 cells (Figure 3c). When focus on the cell type effect, the uptake in RAW 264.7 cells was 3-6 times higher than that in HepG2 cells for AuNP-MUA, while the uptake in RAW 264.7 cells was even lower than that in HepG2 cells for AuNP-TMA (Figure 3d). Considering the uptake ability is also affected by the


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volume of cells (RAW 264.7 is smaller than HepG2), the uptake ability of RAW 264.7 cells for AuNP-TMA should still be stronger than HepG2 cells (data given in uptake amount per well, Figure S3).When focus on the size effect, the uptake difference of AuNP-TMA to AuNP-MUA between HepG2 and RAW 264.7 was the least for 40 nm AuNPs due to the uptake in this size was high for AuNP-MUA but relative low when for AuNP-MUA (Figure 3c). Moreover, the ratio for uptake of AuNP-MUA in RAW 264.7 to HepG2 was highest for 16 nm AuNPs, but the ratio for uptake of AuNP-TMA was similar for all sizes (Figure 3d). The size effect is more depending on surface charge of NPs while less depending on the cell types, where the negatively charged nanoparticles exhibit a more obvious size tendency for cell uptake. Usually, one possible explanation for the higher uptake of positively charged nanoparticles is the faster rate of concentration increase of nanoparticles on the cell surface due to the high affinity of NP to the negatively charged cell membrane by electrostatic interactions.10, 16-18, 30, 32, 33

. When positively charged nanoparticles are attached to a negatively charged cell surface, the

cell membrane will try to maintain the original charge distribution by getting rid of the attached nanoparticles through endocytosis or other pathways. Under this condition, it is thought that the positively charged nanoparticles will be internalized more easily by the cells than negatively charged or neutral nanoparticles.33 Considering the zeta-potential of AuNP-TMA switched to be negative when incubated in cell culture medium due to protein adsorption, AuNP-TMA had a positive surface charge upon preparation are no longer cationic in the cellular media. Protein adsorption to the nanoparticle surface can mediate the uptake of the nanomaterial via receptormediated endocytosis.55 The ultimate surface charges between MUA and TMA coated AuNPs after protein adsorption are different, which implied the proteins adsorbed on the two types of AuNPs are different. The difference in protein adsorption may bring about different biological


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impacts of NPs such as toxicity, endocytic mechanisms and efficiency.25, 53, 55, 56 Therefore, there are at least two possibilities for the higher uptake of AuNP-TMA in non-phagocytic HepG2 cells. One is that the protein corona formed on AuNP-TMA may facilitate the uptake of the nanoparticles by HepG2 cells more effectively.16, 18 Another possibility is that the local positively charged groups on the AuNP-TMA still plays an important on the high uptake of NPs. Even though, the surface positive charge on NPs was screened by the adsorbed proteins, when the nanoparticle-corona complex adhered on cell’s surface, the electrostatic attraction between membrane and local positively charged groups on nanoparticles will favor adhesion onto the cell’s surface, leading to faster uptake.32, 33 Interestingly, when for the macrophage RAW 264.7 cells, the negatively charged AuNPs exhibited comparable cellular uptake with positively charged AuNPs. The reason could be that the protein corona on AuNP-MUA also facilitates the endocytosis by macrophages such as phagocytosis, though it did not favor the uptake by non-phagocytic cells. For the difference of AuNP-MUA and AuNP-TMA between the two types of cells, one possibility is that the corona formed on AuNP-MUA contained enough proteins that are recognized by macrophages RAW264.7 but little proteins that can mediate endocytosis by non-specilized HepG2. Differently, the corona formed on AuNP-TMA contained enough proteins that can facilitate uptake by both types of cells. This effect strongly enhanced the uptake difference between phagocytic and nonphagocytic cells for negatively charged NPs. It is important to remember that, in the presence of serum contained cell culture medium, the nanoparticle’s surface charge is quickly covered by a corona made up of multiple proteins.52,

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These protein coronas called as opsonins would

quickly be recognized by phagocytic cells and internalized into cells no matter the original surface charge is negative or positive.41 To unveil the exact principle of the problem, extensive


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studies are needed to indentify the composition of protein corona on different NPs, 25, 52 53, 57 the mechanisms of endocytosis55,

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and the existence of electrostatic attraction between protein-

coated NP and cell membrane by comprehensive analysis in future.

Figure 3. ICP-MS measurements for Au contents per cell of HepG2 (a) and RAW 264.7 (b) after incubation with different AuNPs at Au concentrations of 10 mg/L for 12 h, error bars represent mean ± SD (n = 3), *p

Surface and Size Effects on Cell Interaction of Gold Nanoparticles with

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