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Flow Cytometry–Based Quantification of Cellular Au Nanoparticles Jonghoon Park, My Kieu Ha, Nuri Yang, and Tae Hyun Yoon Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04418 • Publication Date (Web): 18 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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Analytical Chemistry

Flow cytometry scatter plots: side scattering (SSC-A) versus forward scattering (FSC-A) of HeLa cells in DMEM media (a, f) or exposed to (b) (−)CitAu40, (c) (−)CitAu60, (d) (−)CitAu80, (e) (−)CitAu100, (g) (+)bPEIAu40, (h) (+)bPEI Au60, (i) (+)bPEIAu80, and (j) (+)bPEIAu100 NPs. NP concentrations of 5 µg/mL were used for all exposed samples, and all media volumes were 10 mL. 454x152mm (96 x 96 DPI)

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Flow cytometry side scattering intensity (SSC-A) histograms of HeLa cells exposed to (a) (−)CitAu40, (b) Au60, (c) (−)CitAu80, (d) (−)CitAu100, (e) (+)bPEIAu40, (f) (+)bPEIAu60, (g) (+)bPEIAu80, and (h) (+)bPEIAu100 NPs. Black lines represent control cell samples, red lines represent cells exposed to citrate-coated Au NPs, and blue lines represent cells exposed to bPEI-coated Au NPs for 24 h. NP concentrations of 5 µg/mL were used for all exposed samples, and all media volumes were 10 mL. (−)Cit

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(a, b) ICP-MS results of cellular uptake of gold nanoparticles having various sizes and surface coating materials. The ICP-MS intensities were converted to (a) mass of NPs per cell and (b) number of NPs per cell by using an Au ion standard curve and cell counts. (c) Normalized side scattering signal (nSSC) from side scattering (SSC) histogram in Figure 2. Each mean SSC intensity (SSCi) was divided by SSCo of its control histogram. NP concentrations of 5 µg/mL were used for all exposed samples, and all media volumes were 10 mL. Error bars indicate the standard deviations of 3 replicate measurements. 246x68mm (96 x 96 DPI)



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2Ds plot of cellular Au NPs versus normalized SSC, including linear regression lines: (a) mass of NPs per cell and (b) number of NPs per cell. 126x138mm (96 x 96 DPI)


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Validations of estimate model equations. To determine the performance, measured c-Au NP amounts were compared with estimated values of (a) mass-based (pg/cell) uptake, using one equation for all particle sizes; (b, d) number-based (#/cell) uptake, using separate equations for each particle size; and (c, e) number-based (#/cell) uptake, using one equation incorporating particle size. (a), (b) and (c) plots show validation of all NPs used in this study, whereas (d) and (e) plots exclude Au40 NPs. Diagonal lines demonstrate complete agreement between measured and estimated results. 280x149mm (96 x 96 DPI)



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1

Flow Cytometry–Based Quantification of Cellular Au Nanoparticles

2

Jonghoon Park, My Kieu Ha, Nuri Yang, and Tae Hyun Yoon*

3

Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul, 04763, Republic of Korea

There has been a great deal of research regarding the cellular association of nanoparticles (NPs), although there are a few methods available yet for the quantitative measurements of cellular NPs. In this study, we propose a simple and quantita6 tive method to estimate the cellular uptake of Au NPs into cervical cancer cells (HeLa) based on their side scattering (SSC) intensi7 ties measured by flow cytometry (FCM). We have compared SSC intensities of HeLa cells exposed to eight different types of Au 8 NPs (40–100 nm size, with positive or negative surface charge) with the amount of cellular Au NPs measured by inductively cou9 pled plasma mass spectrometry (ICP-MS). Based on these comparisons, we have found linear correlations between the cellular Au 10 NPs and the SSC intensities and used them to estimate the amount of Au NPs associated with HeLa cells. Once the correlations 11 were found for specific cell lines and types of nanoparticles, this approach is useful for simple and quantitative estimation of the 12 cellular Au NPs, without performing labor-intensive and complicated sample preparation procedures required for ICP-MS ap13 proach. 4 ABSTRACT: 5 only

14

50 quantitative

In recent years, nanoparticles (NPs) have been applied in and pharmaceutical fields as delivery vehicles for 17 targeted drugs and nucleic acids, as well as in biomedical im1–3 18 aging and biosensing applications. As a result, there has 19 been increasing interest in the interaction of NPs with biologi20 cal systems. Quantification of cellular NPs has an essential 21 role in understanding how NPs interact with cells. Methods to 22 quantify cellular NPs can be either direct, by means of fluo4,5 23 rescence or electron microscopy, or indirect, by means of 24 spectroscopic measurement of total accumulated particle 6,7 25 mass. However, fluorescence microscopy is limited by the 26 difficulty in calibrating the fluorescent signal intensity due to a 8,9 27 lack of suitable standards. Transmission electron microscopy 28 (TEM) allows quantification of NPs in subcellular structures 10 29 with high-resolution images, but cannot be used to perform 30 real-time image acquisition. Spectroscopic techniques such as 31 inductively coupled plasma–mass spectroscopy (ICP-MS) can 32 detect and quantify NP elements inside cells, but this cannot 33 be done with live cells, and the sample preparation procedure 34 can be labor-intensive. 35 Recently, flow cytometry (FCM) has been used in many 36 studies to analyze intracellular NP uptake due to FCM’s sim37 plicity, sensitivity, and ability to measure in the single-cell 38 mode. In this technique, the side scattering (SSC) signal that is 39 scattered at a right angle to the incident laser beam is known 40 to be related to the inner complexity or granularity of the cells, 41 whereas the slightly deflected forward scattering (FSC) signal 11 42 is related to the size of the cell. Therefore, these light scatter43 ing data from the FCM measurements can provide useful in44 sight into the cellular association with engineered NPs. For 45 example, it was previously reported that the mean intensity of 46 SSC is proportional to the concentration of engineered NPs 12–15 47 inside the cells. However, FCM is limited to semi48 quantitative analysis of cell-associated NPs. 15

16 biomedical

In this study, we combined FCM with ICP-MS to develop a method to assess the amount of cell-associated 51 NPs. We used gold (Au) NPs because they are one of the most 52 widely used NPs and well known for its ability to be internal16 53 ized in many different types of cells, as well as their low 17,18 54 toxicity, which is advantageous in nanomedicine. Further55 more, the element Au exhibits the ability to resonantly scatter 19 56 light due to surface plasmon resonance, which makes it suit57 able for measuring light scattering using FCM. 58 Eight types of Au NPs with different sizes and surface coat59 ing material were exposed to HeLa cells. FCM-SSC intensities 60 of the cells were carefully monitored, and were compared with 61 cellular Au content measured by ICP-MS as well as the Au 62 NPs’ nominal core sizes to study the relationship between 63 these parameters. Based on these empirical relationships, we 64 propose a simple, easy, and quantitative method for estimating 65 the amount of cellular Au NP from their SSC intensities and 66 core size, which might contribute for better understanding of 67 NP interactions with cells. 49

INTRODUCTION

68

EXPERIMENTAL SECTION

69 Gold

nanoparticles In this study, we used Au NPs (NanoXact Gold Nano71 spheres, Nanocomposix, San Diego, CA, USA) having the 72 nominal diameters of 40, 60, 80, and 100 nm, and capped ei73 ther with positively charged branched polyethlyeneimine 74 (bPEI) or negatively charged citrate (Cit). The 8 types of Au (+)bPEI 75 NPs studied herein are abbreviated as follows: Au40, (+)bPEI 60 (+)bPEI 80 (+)bPEI 100 (−)Cit 40 (−)Cit 60 (−)Cit 76 Au , Au , Au , Au , Au , Au80, (−)Cit 100 77 and Au . 70

78 Physicochemical

characterization of Au NPs

Core size (diameter) distributions of Au NPs were calculat80 ed from image analysis of representative transmission electron 81 microscope (TEM, JEM-2000EX, JEOL Ltd., Japan) images 82 by using the image analysis protocol of the ImageJ software 79



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(ImageJ 1.41n, NIH, USA).11,13 In addition, the hy3 water sizes and surface charges in both deionized (DI) 4 5 Table 1. Core size, hydrodynamic size, surface charge and dispersion stability of citrate-coated and bPEI-coated Au NPs 6 used in this study. Confidence intervals given are the standard deviations of 3 replicate measurements. 1 package

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2 drodynamic

Hydrodynamic size (nm) Core size (nm)

NPs

(−)Cit

Au40

(+)bPEI (−)Cit

(−)Cit

0h

24 h

DI water

DI water

DMEM

DMEM

39.0 ± 16.6

37.3 ± 0.2

36.7 ± 0.3

55.6 ± 0.4

DMEM

DI water

DMEM

57.6 ± 2.9

−41.0 ± 1.6

−10.3 ± 0.3

0.65

0.99

−10.0 ± 1.3

0.97

1.04

45.8 ± 0.7

110.7 ± 1.1

129.1 ± 5.7

63.7 ± 0.8

55.6 ± 14.6

58.2 ± 0.6

57.1 ± 0.7

86.9 ± 0.6

82.8 ± 2.2

−46.0 ± 0.9

−10.2 ± 0.4

0.96

1.16

Au60

56.4 ± 6.8

62.2 ± 0.4

59.5 ± 0.5

99.3 ± 0.4

91.4 ± 0.8

33.6 ± 1.0

−12.2 ± 0.4

0.54

1.18

73.9 ± 16.5

77.5 ± 0.6

75.7 ± 0.9

113.7 ± 0.4

110.0 ± 0.7

−45.8 ± 0.5

−10.9 ± 1.4

0.86

1.17

80

83.0 ± 10.4

99.6 ± 0.9

97.6 ± 0.8

150.5 ± 0.7

156.5 ± 0.5

64.1 ± 1.7

−10.5 ± 0.3

0.92

0.97

100

91.0 ± 11.2

89.6 ± 0.9

88.9 ± 1.7

134.2 ± 0.2

128.3 ± 2.9

−44.2 ± 2.3

−9.6 ± 1.1

0.81

1.36

Au100

94.7 ± 7.8

100.9 ± 0.5

97.7 ± 0.7

137.1 ± 1.1

146.1 ± 4.8

38.7 ± 1.3

−12.2 ± 1.2

0.52

1.14

Au

and cell culture media at 0 h and 24 h were also measured using a particle size analyzer (Zetasizer Nano-ZS, Malvern 9 Instrument Ltd., UK). Au NPs suspensions in cell culture me10 dia were prepared by separately mixing each type of Au NPs 11 with Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 12 USA) containing 10% FBS (fetal bovine serum, Gibco) and 1% 13 penicillin–streptomycin (Gibco). UV-vis absorbance values, 14 measured by Optizen (Mecasys Optizen-2120UV, Daejeon, 15 Korea), in DI water and DMEM media at 24h were divided by 16 the values at 0h to determine dispersion stability. 7

8 by

17 Cell

DI water

47.4 ± 0.4

Au

(+)bPEI

24 h

41.1 ± 8.9

Au80

(+)bPEI

0h

Dispersion stability

60

Au

(+)bPEI (−)Cit

Au

40

Surface charge (mV)

culture and Au NP exposure

A HeLa cell line (ATCC CCL-2) was obtained from the Biological Resource Center and cultured in fresh 20 DMEM cell culture medium. After seeding, cells were incu21 bated at 37 °C in a 5% CO2 incubator (Forma Scientific, Wal22 tham, MA). HeLa cells were cultured for 48 h and then ex23 posed for 24 h to each type of Au NPs prepared; all were test24 ed at 3 and 5 mg/L concentrations, and bPEI-coated NPs were 25 additionally tested at 1 mg/L. 18

19 Korea

26 Flow

cytometry measurements

For flow cytometry measurements, after exposure to NPs, were washed three times with Dulbecco’s phosphate 29 buffered saline (DPBS, Welgene, Korea), trypsinized, centri30 fuged at 3,000 rpm for 1 min, and re-suspended in DPBS. 31 Then, these cells were analyzed using a flow cytometer (Canto 32 II, BD Biosciences, USA) equipped with a 488 nm argon laser. 33 FCS Express software (v4, De Novo Software, Canada) was 34 used for further analysis of FCM scattering data. 28 cells

measurements of cellular Au NPs

To determine the amounts of cellular Au NPs, ICP-MS 300D, PerkinElmer Inc., USA) measurements were 38 performed and compared with the results from FCM data. Re39 suspended cells were counted by using a hemacytometer, and 40 were centrifuged again for aqua regia exposure. Before ICP41 MS measurements, samples were diluted in 3% nitric acid + 42 solution. Au ion standard samples (PerkinElmer Inc., USA) 43 were used to prepare a calibration curve for quantitative meas44 urements. Based on this calibration curve, cellular Au contents 45 were estimated from the measured ICP-MS intensities. 36

37 (NexION

analysis and validation 47 FCM and ICP-MS results were compared to find a model 48 equation for quantitative estimation of cellular Au NPs. Before 49 establishing the model, SSC information on every sample was 50 normalized by dividing the SSC intensity of each sample to 51 the control’s mean SSC intensity (nSSC = SSC ⁄ SSC , where is the mean SSC intensity of cells exposed to Au NPs and 52 SSC is the mean intensity of the control cells). A training data 53 SSC 54 set containing nSSC values, core diameters of Au NPs, and 55 mass-based and number-based cellular uptake amounts was 56 used to develop model equations. An additional data set simi57 lar to the training data set was used to validate the model. The 58 amount of cellular Au NPs was estimated by inputting the 59 nSSC values to the model equations, and the cellular uptake of 60 Au NPs (c-Au NPs) measured by means of ICP-MS was com61 pared with the estimated c-Au NP amount. 62

RESULTS AND DISCUSSION

63 Physicochemical

characteristics of Au NPs

The physicochemical properties of Au NPs used in this 65 study were carefully characterized; Table 1 lists the character66 ization results, comprising core size, hydrodynamic size, sur67 face charge, and dispersion stability. The core size distribu68 tions of the Au NPs agreed well with the manufacturer’s spec69 ifications (TEM images of each Au NP are shown in Figure S70 1). There were no significant differences between the hydro71 dynamic sizes measured at 0 h and 24 h in the medium, indi72 cating that the Au NP suspensions remained stable without 73 any significant agglomeration for 24 h. However, the type of 74 media (e.g., DI water and DMEM medium) induced differ75 ences in the hydrodynamic sizes of Au NPs having the same 76 nominal core size from manufacturer. The hydrodynamic sizes 77 of Au NPs in serum containing DMEM media were larger 78 than those in DI water, which is probably due to the adsorp20 79 tion of serum proteins onto the surfaces of the Au NPs. In 80 addition to the effect of protein adsorption, agglomeration of 81 nanoparticles caused by the interactions between the surround82 ing proteins can also increase the hydrodynamic size. Addi83 tionally, the type of coating material and surface charge also 84 have significant influences on the serum protein adsorption, 85 particle agglomeration, and resultant 64

27

35 ICP-MS

46 Data

86


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1

1. Flow cytometry scatter plots: side scattering (SSC-A) versus forward scattering (FSC-A) of HeLa cells in DMEM media (a, f) or to (b) (−)CitAu40, (c) (−)CitAu60, (d) (−)CitAu80, (e) (−)CitAu100, (g) (+)bPEIAu40, (h) (+)bPEIAu60, (i) (+)bPEIAu80, and (j) (+)bPEIAu100 NPs. NP 4 concentrations of 5 µg/mL were used for all exposed samples, and all media volumes were 10 mL. 2 Figure

3 exposed

5 increase

48 non

6 Au

of hydrodynamic sizes in DMEM media. For those NPs with the same nominal size, the hydrodynamic size of (+)bPEI 7 the Au NPs was typically larger than that of the (−)CitAu 8 NPs, indicating that the hydrodynamic size of Au NPs was 9 affected by the surface charge. Since the serum proteins are 20 10 known to have slightly negative surface charge , we assume 11 that the adsorption of these serum protein may occur prefera(+)bPEI 12 bly on the positively charged surface of Au NPs and re(−)Cit 13 sulted in larger hydrodynamic sizes than those of the Au 14 NPs. Moreover, the type of coating materials may also have 15 significant influence on the protein adsorption and following 16 agglomeration process. In the case of citrate, this small triden17 tate anionic ligand may form relatively homogeneous and 18 complete coverage of the positively charged sites available on 19 the surface of Au NPs. In contrast, the bPEI, a large polyelec20 trolyte ligand with variable chain lengths, might form only 21 partial attachments on the NP surface due to the steric hin22 drance by the neighboring ligands. These partial adsorptions 23 of polyelectrolytic ligands will produce dangling polymer 24 chains with variable lengths on the surface of NPs, which 25 would cause significant differences in their surface charges, 26 hydrodynamic size, interactions with serum proteins, and fol27 lowing agglomeration process. For instance, zeta potentials (+)bPEI 28 observed for the Au NPs with four different core sizes (-)Cit 29 were quite different, while the four different Au NPs 30 showed quite similar zeta potential values in DI water. Addi(31 tionally, the difference in hydrodynamic size between the )Cit 32 Au NPs in DI water and DMEM media was relatively con33 stant for all four NPs with different core sizes, whereas the (+)bPEI 34 differences between the hydrodynamic sizes of Au NPs 35 in DI water and DMEM media were quite variable among the 36 NPs with different core sizes. Particularly, the size increments (+)bPEI 37 of Au40 NPs in DMEM media was the most dramatic 38 (3.14 fold-increase between the hydrodynamic size in DMEM 39 (24 h) and its core size), while only 1.54 fold-increase was (+)bPEI 40 observed for Au100 NPs (see Table 1). These observations 41 support our assumption that the type of coating materials also 42 have significant influence on the serum protein adsorption, 43 agglomeration of NPs, and resultant increments in hydrody44 namic sizes. 45 In DMEM media with 10 % FBS, highly negative and posi46 tive zeta potentials of Au NPs in DI water all were changed 47 into slightly negative values of about −10 mV. This phenome-

49 nation

can be explained by two hypotheses. One possible explais that serum proteins adsorbed onto the surface of NPs 50 formed protein coronas, turning the surface charge of Au NPs 51 into those of serum proteins in cell culture medium. This 52 agrees with the increment of hydrodynamic size observed for 53 Au NPs in DMEM. Another hypothesis is that the measured 54 zeta potentials might actually be derived from free serum pro55 teins in the suspensions. Given that the percentage of FBS in 56 DMEM was 10%, which is rather high, there was probably an 57 excessive amount of free serum proteins that could outweigh 58 the amount of Au NPs. Additionally, the serum protein con59 taining DMEM media seemed to stabilize the Au NP suspen60 sions. In DI water, the dispersion stability measured by optical 61 densities of Au NP suspensions varied significantly depending 62 on the size and surface charge of the NPs, but in DMEM me63 dia the dispersion stabilities were all approximately 1, indicat64 ing that these dispersions were quite stable and there were 65 little sedimentations of Au NP in DMEM even after 24 h. 66 Analysis

of SSC intensities of FCM

In flow cytometry, the intensity of side scattering (SSC) is 68 known as the parameter reflecting the inner density or granu69 larity of each cell, whereas the intensity of forward scattering 70 (FSC) is related to the cellular volume. Therefore, the SSC 71 data from FCM measurements have been applied to study 13,14 72 cell–NP association. For instance, Zucker et al. confirmed 73 the relationship between SSC intensities and the cellular asso74 ciations of TiO2 and Ag NPs from FCM results and dark field 75 images. Recently, we have also reported a study to assess cel76 lular SiO2 NPs semi-quantitatively by using FCM and X-ray 21 77 Fluorescence Spectroscopy. Building upon this previous 78 work, further FCM experiments were performed on HeLa cells 79 exposed to Au NPs with different core sizes, surface charges, 80 and hydrodynamic sizes. Figure 1 shows the SSC and FSC 81 results of FCM experiments using HeLa cells exposed to 82 5 µg/mL Au NPs having different core sizes and surface 83 charges. 84 The FSC intensities of the HeLa cells exposed to Au NPs 85 were similar to those of control cells. Because changes in FSC 86 intensity can arise from swelling or shrinking of cells due to 87 cell death processes (necrosis or apoptosis), our observation of 88 nearly constant FSC intensities agreed well with the non-toxic 89 properties of Au NPs under exposure conditions. 67



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1 (−)Cit 2 Figure 2. Flow cytometry side scattering intensity (SSC-A) histograms of HeLa cells exposed to (a) Au40, (b) (−)CitAu60, (c) (−)CitAu80, (−)Cit 100 (+)bPEI 40 (+)bPEI 60 (+)bPEI 80 (+)bPEI 100 3 (d) Au , (e) Au , (f) Au , (g) Au , and (h) Au NPs. Black lines represent control cell samples, red lines 4 represent 5 tions

cells exposed to citrate-coated Au NPs, and blue lines represent cells exposed to bPEI-coated Au NPs for 24 h. NP concentraof 5 µg/mL were used for all exposed samples, and all media volumes were 10 mL.

The SSC intensities, in contrast, showed systematic changes respect to the core sizes and surface charges of the Au (+)bPEI 8 NPs (Figure 2). Cells treated with Au NPs (Figures 2e–h) (−)Cit 9 showed greater SSC intensity than cells treated with Au 10 NPs (Figures 2a–d) when compared to control cells, under the 11 constant exposure concentration of 5 µg/mL and total media 12 volume of 10 mL. These surface charge–dependent increments 13 of SSC intensity indicated an increase in the inner complexity 14 of cells due to the association of Au NPs. 15 In addition to these surface charge–dependent trends, there 16 were remarkable core size–dependent changes. Cells treated (−)Cit 17 with Au NPs showed slight increments relative to control 18 peaks for increasing core size from 40 to 100 nm, whereas (+)bPEI 19 SSC intensities of cells treated with Au NPs increased 20 clearly with increasing core size up to 80 nm, but decreased 21 again for the core size of 100 nm (Figures 3e–h). These SSC 22 results suggested that there are optimal core sizes for cellular 23 association of Au NPs. 24 However, these FCM results on cellular association of NPs 25 were only qualitative. Recently, we also reported a FCM study 26 correlating SSC intensities with X-ray fluorescence (XRF) 27 signals but could only establish a semi-quantitative relation28 ship between cellular SiO2 NPs and FCM-SSC intensities, due 21 29 to the limitations of XRF technique. In the present study, to 30 develop a more quantitative method, we conducted inductively 31 coupled plasma mass spectrometry (ICP-MS) measurements 32 of cellular Au content and correlated them with SSC intensi33 ties of FCM measurements. 6

7 with

34 ICP-MS

measurements of cellular Au NPs

The c-Au contents (mass and number of Au NPs per cell, in 36 pg/cell and #/cell, respectively) in HeLa cells exposed to all (+)bPEI 37 Au NPs were found to be significantly larger than that of (−)Cit 38 Au NPs (Figures 3a–b), in agreement with previous FCM 22 39 results. Harush-Frenkel et al. conducted an endocytosis 40 study of Au NPs in HeLa cells and reported that the surface 41 charge of NPs affects their cellular endocytosis mechanism as 42 well as their cellular uptake capability. It is known that NPs 35

43 interact with cells through a protein corona layer. Calatayud et 23 44 al. suggested that due to the negative charge of serum pro(+)bPEI 45 teins, Au NPs may adsorb more proteins than (−)CitAu NPs; (+)bPEI 46 our finding of larger hydrodynamic size of Au NPs in 47 DMEM

in the present work agrees with this result. Thus, largprotein corona formed around (+)bPEIAu NPs could help these (−)Cit 49 NPs enter cells more efficiently, compared to the Au NPs. 50 Apart from this, there is a specific interaction between cells 24 51 and the serum proteins adsorbed on NPs. Karmali et al. re52 ported that different proteins are adsorbed on NPs with differ25 53 ent surface charges. Gessner et al. observed that positively 54 charged NPs prefer to adsorb onto proteins having isoelectric 55 point (pI) less than 5.5, such as albumin, whereas negatively 56 charged NPs favor the adsorption of proteins with pI greater 57 than 5.5, such as IgG. Therefore, in our case, some of the pro(+)bPEI 58 teins adsorbed on Au NPs were probably more favored (−)Cit 59 by the receptors on HeLa cells than those adsorbed by Au (+)bPEI 60 NPs, increasing the internalization of Au NPs relative to (−)Cit 61 Au NPs. Fleischer et al.26 provided another explanation, 62 that different internalization pathways might be responsible 63 for the different uptake levels of Au NPs. They reported that 64 the protein structure was retained after adsorption to anionic (−) 65 NPs, making the NP–protein complex compete with other 66 proteins for cellular binding sites, whereas the denaturation of (+) 67 proteins following adsorption to cationic NPs made the NP– 68 protein complex bind to specific scavenger receptors. This (+)bPEI 69 might lead to greater uptake of Au NPs compared to (−)Cit 70 Au NPs. 71 Furthermore, cellular uptake of both (+)bPEIAu NPs and (−)Cit 72 Au NPs tends to depend on the size of NPs as well. For 73 both NP types, the uptake tendency in mass (i.e. pg/cell) does 74 not simply increase or decrease with NP size; rather, there is 75 an optimal size of maximum uptake. Similar with our observa6 76 tions, Chithrani et al. reported that cellular uptake of Au NPs 77 depends on the size of citrate-coated Au NPs and reported 78 maximum uptake at 50 nm among the core sizes studied (i.e., 79 14, 30, 48 er

80


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1

3. (a, b) ICP-MS results of cellular uptake of gold nanoparticles having various sizes and surface coating materials. The ICP-MS were converted to (a) mass of NPs per cell and (b) number of NPs per cell by using an Au ion standard curve and cell counts. (c) ) was divided 4 Normalized side scattering signal (nSSC) from side scattering (SSC) histogram in Figure 2. Each mean SSC intensity (SSC of its control histogram. NP concentrations of 5 µg/mL were used for all exposed samples, and all media volumes were 10 mL. 5 by SSC 6 Error bars indicate the standard deviations of 3 replicate measurements. 2 Figure

3 intensities

74, and 100 nm). Osaki et al.30 also reported that there was 8 an optimal size (~ 50 nm) of semiconductor nanoparticles to 9 enter cells via receptor-mediated endocytosis. However, the (+)bPEI 10 cellular association of Au NPs in number (i.e. #/cell) had (11 a different tendency from the mass/number trend of cellular )Cit 12 Au NPs. Particularly, the cellular (+)bPEIAu40 NPs had an 13 unexpectedly high number (Figure 3b). We think that it is also 14 partially related to its unusually high hydrodynamic size in 15 DMEM medium, which was discussed in previous section. 16 Large hydrodynamic size accompanies higher degree of pro17 tein adsorption and receptor-ligand interactions, which may 27 18 facilitate enhancement in receptor-mediated endocytosis. (+)bPEI 19 Unusually high hydrodynamic size observed for Au40 20 NPs in this study may also imply heavy agglomeration of NPs 21 with asymmetric structures and larger surface area, which may 22 also help receptor-ligand interactions and lead to higher cellu(+)bPEI 23 lar association of Au40 NPs.28 24 To allow quantitative comparison of variations in SSC re ” 25 sults, SSC intensities were normalized as “nSSC = SSC /SSC is the mean SSC intensity of cells exposed to Au NPs, 26 (SSC 27 and SSC is the mean SSC intensity of the control cells). Simi28 lar to the observations from ICP-MS measurements, nSSC 29 patterns, especially for positively charged Au NPs, also 30 reached a maximum at a certain NP size (Figure 3c). HeLa (+)bPEI 31 cells exposed to Au NPs displayed significant size32 dependent variation of nSSC, whereas the same dose and ex(−)Cit 33 posure of Au NPs resulted in smaller nSSC changes. 34 However, the size leading to maximal nSSC is a little different 35 from the size leading to maximal uptake. This may be because 36 the intensity of scattered light is proportional to the size of the 21,29 37 submicroscopic particles. Even for cells that have incorpo38 rated the same number of NPs, SSC signals from cells with Au 39 NPs 100 nm in size will be stronger than those with Au NPs 40 40 nm in size, so the nSSC values of larger NPs are higher, 41 although the uptake of the larger NPs might not be maximal. It 42 is expected theoretically that the intensity of scattered light 43 will be proportional to the sixth power of the particle diameter, 44 but in our experimental data, only nSSC values of cells treated (+)bPEI 45 with Au NPs show steady increments with increasing 46 particle size, and these increments do not follow a sixth power 47 proportional relation. The difference between theory and ex48 perimental observation may arise because the light scattering 49 is not isotropic, whereas the detector in the flow cytometer is 7 50,

50 fixed,

so the detected signal is only a fragment of the total and does not follow the theory exactly. 52 Apart from particle size, other factors such as cell line, par53 ticle shape, coating material and fabrication quality may also 30 54 have certain impacts on the degree of cellular uptake of NPs. 55 Albanese et al. also performed ICP-MS quantification of cel28 56 lular uptake of Au NPs. Compared to our result (Figure 3b), 57 they reported much higher uptake number in HeLa cells for 58 26 nm, 49 nm and 98 nm citrate-capped Au NP aggregates, 59 approximately 2000 – 3000 NPs per cell, compared to 180 – 60 1500 citrate-capped Au NPs per cell in this study. Alkilany et 61 al. also used ICP-MS to quantify cellular uptake of self62 synthesized Au nanorods with three different coating materials 20 63 (CTAB, PAA and PAH) in HT-29 cells after 24 h exposure. 64 They reported completely different uptake number of each 65 nanorod type: 45 ± 6, 270 ± 20 and 2320 ± 140 nanorods per 66 cell for CTAB-coated, PAA-coated and PAH-coated Au nano67 rods, respectively. As can be seen from these two examples, 68 cellular uptake of NPs is highly dependent on the types of 69 cells and characteristics of NPs and the quantification results 70 in this study are not universally applicable. 51 signal

29

71 Quantitative

estimation of cellular Au NPs

To find the quantitative relationship between the SSC inten73 sities of FCM and the amount (number or mass) of c-Au NPs 74 from ICP-MS measurements, normalized SSC intensities were 75 calculated and compared with the c-Au NPs obtained from 76 ICP-MS measurements. The relation between FCM and ICP77 MS data was fitted with a linear function under the assumption 78 that nSSC has a value of unity for the control sample (i.e., 79 nSSC = 1 when c-Au = 0). 80 The cellular Au NP in mass (i.e., pg/cell) was plotted 81 against nSSC intensity, showing a linearly proportional rela82 tionship between these parameters (Figure 4a); over the tested 83 concentration range this relationship can be linearly fitted with 84 the following regression equation: 85 c-Au (pg/cell) = 10.40 × (nSSC − 1) (Eq. 1) 86 This regression yielded the R2 value of 0.8730. However, no 87 clear size-dependency was observed in this plot, although SiO2 88 NPs with different core sizes yielded distinct slopes in their 21 89 regression equations. There are at least two reasons that may 90 roughly explain the dissimilar trends between Au and SiO2 91 NPs regarding the relationships between the nSSC and their 72



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 core

36

2 nm)

sizes. First, agglomeration of smaller Au NPs (e.g., 40 and resulting similarities in hydrodynamic sizes under 3 real exposure conditions lead to similar amount of Au NPs 4 associated with cells, despite of differences in their core sizes. 5 Second, the core size range of Au NPs considered in the pre6 sent work (i.e., 40 - 100 nm with 20 nm intervals) was much 7 narrower than that used in the previous work for SiO2 NPs (i.e., 8 30, 200, and 300 nm). Because of the small interval of the core 9 diameters, the regression curves for these Au NPs with differ10 ent sizes might be too similar to distinguish each other. The 11 number-based cellular Au NPs (i.e., #/cell) was also estimated 12 and plotted in Figure 4b. Interestingly, linear equations with 13 distinct slopes were found in their relationships between the 14 number-based cellular Au NPs and normalized SSC intensities. 15 The slopes of the linear regression curves decreased as the 16 core sizes of Au NPs increased (Eq. 2a, 2b, 2c and 2d for (±) 40 60 80 17 Au , Au , Au and Au100 NPs, respectively), which agreed 18 well with the trend observed in our previous study on SiO2 21 19 NPs. 20 c-Au (#/cell) = 12842 × (nSSC – 1) (Eq. 2a) 21 c-Au (#/cell) = 6885 × (nSSC – 1) (Eq. 2b) 22 c-Au (#/cell) = 2006 × (nSSC – 1) (Eq. 2c) 23 c-Au (#/cell) = 632 × (nSSC – 1) (Eq. 2d) 24 The slopes were fitted exponentially with Au NP core di25 ameter and combined with the linear equation for number26 based cellular Au NPs, which resulted in the following equa27 tions: 28 slope = −2179 + 55044 × e−0.032 × Core diameter (Eq. 2e)

37 nSSC

c-Au (#/cell) = (−2179 + 55044 × e−0.032 30 (nSSC − 1) (Eq. 3) 29

× Core diameter)

×

To confirm the performance of the linear regression model, values from the validation set were used to estimate the 38 amount of c-Au NPs, and the results were then compared with 39 the c-Au NP quantities measured by ICP-MS. A linear rela40 tionship with unit slope would indicate good agreement be41 tween the amount of c-Au NPs estimated from FCM-SSC and 42 measured by ICP-MS; this relationship is plotted in Figure 5. 43 The correlation for mass-based Au NP content did not show 44 clear linear trend, with many deviations from the diagonal line, 2 45 resulting in the R value of 0.6918 (Figure 5a). Regarding 2 46 number-based content, the R value was 0.9036, indicating 47 high correlation between the measured and estimated amounts 48 (Figure 5b). The difference in the validation performance of 49 mass- and number-based cellular Au NP contents suggests that 50 the number-based content may be more amenable to quantita51 tive analysis based on nSSC values. Alternatively, we can use 52 Eq. 3 to estimate the amount of c-Au NPs. The linear relation 53 between the estimated values and the ICP-MS results had the 2 54 R value of 0.9342 (Figure 5c). This value is improved com2 55 pared to the R values of the relations shown in Figures 5a and 56 5b, indicating that the estimated uptake amount is closer to the 57 measured amount. This means that using the equation for 58 number-based cellular uptake that incorporates the particle 59 size may improve the model’s performance. 60 Although Figure 5b and 5c showed relatively good correla40 61 tion, it seemed that data from Au NPs still show significant 62 deviation from the diagonal line. As previously mentioned, 40 (+)bPEI 63 Au NPs, especially the Au40 NPs, have shown unusual 64 behaviors, such as high surface charge in DI water, large hy65 drodynamic size in DMEM media, and higher numbers in 66 cellular Au NPs compared to the other Au NPs. Therefore, we 40 67 assumed that data from Au NPs might be outliers and tried 68 additional validation for the dataset without data from 40 nm 69 Au NPs, to see if there would be any improvement in the cor70 relation between estimated and measured cellular uptake. It 71 turned out that the correlation was significantly improved. For 72 the estimation of number-based cellular Au NPs using four 73 independent equations (Eq. 2a, 2b, 2c and 2d, see Figure 5b 2 74 and 5d), R value was improved from 0.9036 to 0.9647, while 75 only a subtle increment was observed for the number uptake 76 estimation using one single equation (Eq. 3, Figure 5c and 5e), 77 from 0.9342 to 0.9440. These results suggested that the linear 78 regression work better for the Au NPs with core size larger 79 than 40 nm. 80

CONCLUSIONS

In this study, surface charge– and size- dependent cellular of Au NPs by HeLa cells were investigated by (−)Cit 83 using FCM and ICP-MS techniques. Compared to Au NPs, 84 significant enhancements in cellular association were observed (+)bPEI 85 for Au NPs, which confirmed that cellular association of 86 Au NPs is strongly dependent on their surface charge and pre87 ferred for positively charged NPs, rather than negatively 88 charged NPs. We also found that the measured SSC intensities 89 of FCM is linearly related to the cellular association of Au 90 NPs as well as core size of Au NPs. Based on our observations, 91 empirical equations describing the relationship between these 92 three parameters were proposed. Using these equations, we 93 demonstrated the feasibility of estimating the number of cellu94 lar Au NPs from the FCM-SSC intensities and the core diame95 ter of the Au NPs. Although current applications of this ap96 proach should be limited to use with HeLa cells exposed to Au 97 NPs having well-defined core diameters over restricted linear 81

82 associations

31

4. 2Ds plot of cellular Au NPs versus normalized including linear regression lines: (a) mass of NPs per 34 cell and (b) number of NPs per cell. 32 Figure 33 SSC,

35 Validation

of model performance

Page 12 of 14


Page 13 of 14 1 ranges,

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2 method

Analytical Chemistry we believe that this simple, easy, and quantitative of estimating cellular NPs can be further expanded to

3 various

cell lines and types of NPs, as well as to wider size

4 ranges.

5

5. Validations of estimate model equations. To determine the performance, measured c-Au NP amounts were compared with estivalues of (a) mass-based (pg/cell) uptake, using one equation for all particle sizes; (b, d) number-based (#/cell) uptake, using sepa8 rate equations for each particle size; and (c, e) number-based (#/cell) uptake, using one equation incorporating particle size. (a), (b) and (c) 40 9 plots show validation of all NPs used in this study, whereas (d) and (e) plots exclude Au NPs. Diagonal lines demonstrate complete 10 agreement between measured and estimated results. 6 Figure 7 mated

11

36 ciate

ASSOCIATED CONTENT

12 Supporting

37 Yoon

Information

38

13 The

Supporting Information is available free of charge on the 14 ACS Publications website. 15

images of (-)CitAu and (+)bPEIAu NPs, FCM and ICP-MS data 17 for cellular uptake, training and testing data for fitting and validat18 ing regression equations (DOCX) 16 TEM

REFERENCES

39 (1) 40 41 (2) 42 43 44 (3) 45

19

46 (4)

AUTHOR INFORMATION

20 Corresponding

47 48 (5)

Author

49

21 *Nanoscale

Characterization & Environmental Chemistry Lab., 22 Department of Chemistry, College of Natural Sciences, Hanyang 23 University, Seoul, Republic of Korea. E-mail: [email protected] 24 Author

51 (6) 52 54

25 JHP

performed the experiments. JHP, MKH and NY conducted 26 data analysis. JHP, MKH, NY and THY participated in the manu27 script writing. All authors have given approval to the final version 28 of the manuscript.

55 56 (8) 57 58 (9) 59 (10) 60

29 Notes 30 The

50

53 (7)

Contributions

61 (11)

authors declare no competing financial interest.

62 63 (12)

31

64

ACKNOWLEDGMENT

32 This

65 (13)

work was supported by the Industrial Strategic Technology 33 Development Program (10043929, Development of “User34 friendly Nanosafety Prediction System”), funded by the Ministry 35 of Trade, Industry & Energy (MOTIE) of Korea. We also appre-

66 67 (14) 68 69 70 (15) 71

Desy Maulina for proofreading of manuscript and Julie J. for TOC graphics.

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