Uptake of Gold Nanoparticles by Intestinal Epithelial Cells: Impact of

Subscriber access provided by NEW YORK MED COLL

Article

Uptake of Gold Nanoparticles by Intestinal Epithelial Cells: Impact of Particle Size on their Absorption, Accumulation, and Toxicity Mingfei Yao, Lili He, David Julian McClements, and Hang Xiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03242 • Publication Date (Web): 27 Aug 2015 Downloaded from http://pubs.acs.org on August 28, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry 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.

Page 1 of 30

Journal of Agricultural and Food Chemistry

1

Uptake of Gold Nanoparticles by Intestinal Epithelial Cells: Impact of

2

Particle Size on their Absorption, Accumulation, and Toxicity

3

Mingfei Yao †, Lili He†, David Julian McClements†, §, Hang Xiao*, †, ‡

4

†

Department of Food Science, University of Massachusetts Amherst, Amherst, MA

5

‡

College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha,

6

Hunan, P. R. China

7

§

8

Saudi Arabia

Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah,

9

10

11 12

*Address correspondence to [email protected]; Tel: 413 545 2281; Fax 413 545 1262

13

14

15

16

1 ACS Paragon Plus Environment


17

18

ABSTRACT Inorganic nanomaterials have been increasingly utilized in many consumer products,

19

which has led to concerns about their potential toxicity.

At present, there is limited

20

knowledge about the gastrointestinal fate and cytotoxicity of ingested inorganic

21

nanoparticles. In this study, we determined the influence of particle size and

22

concentration of gold nanoparticles (AuNPs) on their absorption, accumulation and

23

cytotoxicity in model intestinal epithelial cells. As the mean particle diameter of the

24

AuNPs decreased (from 100 to 50 to 15 nm) their rate of absorption by the intestinal

25

epithelium cells increased, but their cellular accumulation in the epithelial cells decreased.

26

Moreover, accumulation of AuNPs caused cytotoxicity in the intestinal epithelial cells,

27

which was evidenced by depolarization of mitochondria membranes. These results

28

provide important insights into the relationship between the dimensions of AuNPs and

29

their gastrointestinal uptake and potential cytotoxicity.

30 31

Keywords: Gold nanoparticles; absorption; accumulation; toxicity; intestinal epithelial

32

cells; particle size: mitochondria potential.

33 34


Page 2 of 30

Page 3 of 30

35

36


INTRODUCTION Developments in the nanotechnology field during the past decade or so have led to

37

numerous applications of nanomaterial, such as in computer science, chemistry,

38

cosmetics, agrochemicals, and pharmaceuticals 1, 2. Nanotechnology has also been

39

utilized within the food industry to improve the microbiological safety, sensory attributes,

40

nutrition value, and shelf life of foods, as well as to develop innovative sensors to

41

monitor food quality and safety 3-5.

42

inorganic nanoparticles have been found in a wide range of food products, including the

43

nanoparticles based on silver, titanium dioxide, silica, zinc oxide, etc. 6.

44

nanoparticles may be intentionally added to food products as additives because of their

45

specific functional attributes (such as antimicrobial, whitening, or anticaking agents), or

46

they may be unintentionally added to foods (e.g., as a contaminant in an additive that is

47

supposed to contain larger particles).

48

nanoparticles within food products means that humans come into contact with them more

49

frequently 7. Hence, it is particularly important to establish whether there are any adverse

50

effects of ingesting inorganic nanoparticles in foods on human health, especially since

51

nanoparticles may have completely different absorption and toxicity patterns in

52

comparison to the larger particles.

53

Consequently, various types of nanomaterial such as

These

The increasing prevalence of inorganic

A number of studies suggest that ingestion of inorganic nanoparticles may have toxic 3 ACS Paragon Plus Environment


54

effects. It has been reported that ingestion of nanomaterials may promote inflammatory

55

bowel disease, DNA fragmentation, liver damage and pregnancy complications, as well

56

as result in abnormal intestinal barrier function and increased intestinal permeability. 8-11.

57

In particular, prolonged exposure to inorganic nanoparticles may have a detrimental

58

impact on the function of the intestinal membrane 12. However, detailed information

59

about the gastrointestinal uptake and cytotoxicity of ingested inorganic nanoparticles is

60

currently unavailable, and more studies are needed in this important area.

61

Gold nanoparticles (AuNPs) have been widely used in the studies of the biological

62

fate and toxicity of inorganic nanoparticles because of their ease of synthesis, high

63

chemical stability, and unique optical properties 13, 14. Commercially, the main

64

applications of AuNPs are in the medical field, such as for drug delivery and

65

photodynamic therapy. However, AuNPs have also been used in cosmetics, beverages,

66

food packaging materials, and toothpastes, which leads to oral exposure of AuNPs in

67

humans 14-16. However, a review on the previous studies highlighted the current poor

68

understanding of the gastrointestinal uptake and potential cytotoxicity of AuNPs 17.

69

Therefore, the objective of this study was to determine the effect of nanoparticle size and

70

concentration on the intestinal absorption and cellular accumulation of AuNPs in the

71

model intestinal epithelial cells, and to assess the potential toxicity caused by the

72

exposure to AuNPs. 4 ACS Paragon Plus Environment

Page 4 of 30

Page 5 of 30

73


Differentiated human intestinal cells (Caco-2 cells) can be grown on a permeable

74

support to form a model intestinal epithelial monolayer. This model system can be used

75

to carry out systematic studies of the impact of nanoparticle characteristics (such as size,

76

surface chemistry, and concentration) on cellular responses such as absorption and

77

toxicity 12, 18.

78

by or transported across this type of epithelial monolayer 15. In this study, we utilized this

79

model to determine the influence of AuNPs dimensions (15 nm, 50, or 100 nm) on their

80

absorption, accumulation, and toxicity. The results of this study are important for

81

understanding the gastrointestinal fate of inorganic nanoparticles that may be ingested.

82

MATERIALS AND METHODS

83

Materials

Previous studies have shown that different nanoparticles can be absorbed

84

The following products were purchased from SigmaAldrich Chemicals (St. Louis,

85

MO, USA): 4% osmium tetroxide (OsO4), phosphatungstic acid (PTA), nitric acid, and

86

hydrochloric acid. AuNPs with different diameters (15, 50, and 100 nm) were purchased

87

from NanopartzTM (Loveland, CO). A mitochondrial membrane potential probe (JC-1 dye)

88

was purchased from Life Technologies (Thermo Fisher Scientific, Agawam, MA, USA).

89

Hank’s balanced salt solution, glutaraldehyde , cacodylate, formvar/carbon Cu grids 200

90

mesh, epoxy resin were purchased from EM Sciences (Hatfield, PA, USA).



91

92

Cell culture

Caco-2 cells (passage 55~65) were cultured in complete Dulbecco’s modified

93

essential medium (DMEM) containing high glucose, 10% fetal bovine serum (FBS), 1%

94

antibiotic, and 1% amino acids as we described previously 19. Cells were seeded at 3×105

95

cells/mL on transwells (Corning Inc., MA, USA) containing polyester filters (3 m pore

96

size and 4.7 cm2 surface area) and grown for 21 days. The transepithelial electrical

97

resistance (TEER) was monitored using a Millicell® ERS-2 epithelial voltammeter

98

(World Precision Instruments, Sarasota, FL). The TEER data is presented as resistance

99

per unit surface area Ω×cm2.

100

101

Transmission electron microscopy analysis

To visualize the AuNPs, a drop of liquid sample was allowed to air-dry onto a

102

carbon-coated 200 mesh copper grid, and then the sample was stained with 2% PTA.

103

Samples were visualized with a transmission electron microscope (TecaniTM 12 TEM,

104

FEI Company, Hillsboro, OR, USA). To visualize the uptake of AuNPs in intestinal

105

epithelial cells, suspensions of AuNPs (5 µg/mL, 15, 50, or 100 nm) were applied on

106

Caco-2 cell monolayers. After treatment for 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h, the media in

107

both the apical and basolateral sides were removed and the monolayers were rinsed with

108

Hank’s balanced salt solution (HBSS) that had been warmed to 37 ºC. The monolayers


Page 6 of 30

Page 7 of 30


109

were immediately fixed in 2.5% glutaraldehyde and 2% paraformaldehyde (0.1 M

110

sodium cacodylate buffer, pH 7.4) for 2 h at 37 ºC. They were then post-fixed in 1%

111

aqueous osmium tetroxide solution and then embedded in Epoxy resin. Thin sections

112

were obtained using an ultramicrotome with a diamond knife (DDK, Wilmington, DE).

113

The sections were stained with uranyl acetate followed by lead citrate. The image

114

samples were then viewed with the transmission electron microscope (TecaniTM 12, FEI

115

Company, Hillsboro, OR, USA).

116

ICP-MS analysis

117

After treatment with AuNPs, the cell monolayers and medium samples were

118

collected at the specific time period from the apical and basolateral sides of the transwell

119

as described above. A bicinchoninic acid (BCA) kit was used to quantify the protein

120

concentration of the monolayers in each well. Each sample was mixed with 0.5 mL of

121

aqua regia and then the sample was diluted to 10 mL with de-ionized water. A series of

122

standard solutions of Au were prepared under the same conditions. Inductively coupled

123

plasma (ICP) mass spectrometry (MS) measurements were performed on a mass

124

spectrometer (NexION 300X ICP, PerkinElmer, Cambridge, MA).

125

Calculation of number of gold nanoparticles

126

The number of gold nanoparticles in a given sample was calculated from the Au 7 ACS Paragon Plus Environment


127

concentration measured by ICP-MS. The number of gold atoms per gold nanoparticle (n)

128

can be estimated from the following expression 20:

=

129

Where, D is the diameter of a gold nanoparticle and d is the diameter of a single gold

130

atom, which is approximately 0.28 nm 21.

131

calculated from the measured Au concentration as follows:

=

Hence, the number of AuNPs can be

×

132

Where Cn = the number of AuNPs per unit volume, Cm = the measured mass of Au per

133

unit volume, and NA is Avogadro’s constant.

134

Determination of mitochondrial membrane potential

135

Aqueous suspensions of AuNPs (5 µg /mL, 15, 50, or 100 nm) were applied on to

136

well-differentiated Caco-2 monolayer (TEER > 200 Ω). Four hours after the treatment,

137

the apical and basolateral media were removed and the monolayers were rinsed three

138

times with warmed HBSS. A medium containing 10 µg/mL of JC-1 (mitochondrial

139

membrane potential probe) in warmed DMEM solution was added to the Caco-2 cell

140

monolayers. After the incubation for 15 minutes (37 ºC, 5% carbon dioxide, and 100%

141

humidity), the monolayers were washed twice with HBSS, placed onto a microscope 8 ACS Paragon Plus Environment

Page 8 of 30

Page 9 of 30


142

slide.

143

sealed and stored overnight at 4 ºC. The microstructure of the samples was observed

144

using a confocal fluorescent microscope (Olympus FV-1000, Waltham, MA). The JC-1

145

treated coverslips were excited at 488 nm, and the emission was recorded simultaneously

146

at 527 and 590 nm by independent detectors 22. Two-photon excitation of JC-1 labeled

147

Caco-2 cell monolayers allowed for the real-time analysis of mitochondria membrane

148

potential. The data were analyzed using the following expression as described

149

previously23:

150

After adding a fluorescent dye (DAPI) to the samples, the slides were rapidly

Ratio = 5 × Ired/Igreen

151

Here Ired and Igreen are the intensities of the signals in the red and green regions of the

152

spectra.

153

256 levels.

154

Statistical Analysis

155

The factor “5” is used to transform the ratiometric results into an 8-bit scale of

All values are expressed as means ± standard deviations (SD) unless stated otherwise.

156

The difference among samples were analyzed by ANOVA with significance level of p
50 nm > 15 nm (Figure

306

4A).

307 308

Quantification of the ratio of red/green florescence further

In summary, the absorption, accumulation, and transfer of AuNPs in the intestinal epithelial cells were highly dependent on their particle diameter (15, 50 or 100 nm). 17 ACS Paragon Plus Environment


309

AuNPs with intermediate sizes (50 nm) were the most efficiently transported across the

310

intestinal epithelium: uptaken from the apical side and excreted into the basolateral side.

311

The smaller AuNPs (15 nm) were more rapidly absorbed by the intestinal epithelial cells

312

and more quickly spread throughout the interior of the cells.

313

AuNPs (100 nm) tended to accumulate within the intestinal epithelial cells since their rate

314

of excretion into the basolateral side was slow. The increasing accumulation of the total

315

mass of gold caused by AuNPs resulted in increasing mitochondria toxicity to the

316

intestinal epithelial cells. Overall, this study provided detailed information on the impact

317

of particle size on the absorption, accumulation and cytotoxicity of AuNPs in the

318

intestinal epithelium.

Conversely, the larger

319 320 321

ACKNOWLEDGMENTS This study was partially supported by grants from United States Department of

322

Agriculture.

323

REFERENCES

324 325 326 327 328 329 330 331 332 333 334 335 336 337

(1) Yun, Y.; Cho, Y. W.; Park, K., Nanoparticles for oral delivery: Targeted nanoparticles with peptidic ligands for oral protein delivery. Adv Drug Deliv Rev. 2013, 65, 822-832. (2) Fisichella, M.; Berenguer, F.; Steinmetz, G.; Auffan, M.; Rose, J.; Prat, O., Intestinal toxicity evaluation of TiO2 degraded surface-treated nanoparticles: a combined physico-chemical and toxicogenomics approach in caco-2 cells. Part Fibre Toxicol. 2012, 9. (3) Chaudhry, Q.; Scotter, M.; Blackburn, J.; Ross, B.; Boxall, A.; Castle, L.; Aitken, R.; Watkins, R., Applications and implications of nanotechnologies for the food sector. Food Addit Contam Part A Chem Anal Control Expo & Risk Assess. 2008, 25, 241-258. (4) Duncan, T. V., Applications of nanotechnology in food packaging and food safety: Barrier materials, antimicrobials and sensors. J Colloid Inter Sci. 2011, 363, 1-24. (5) Weiss, J.; McClements, D. J.; Takhistov, P., Functional materials in food nanotechnology. Food Australia. 2007, 59, 274-275. (6) Wang, H. F.; Du, L. J.; Song, Z. M.; Chen, X. X., Progress in the characterization and 18 ACS Paragon Plus Environment

Page 18 of 30

Page 19 of 30

338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376


safety evaluation of engineered inorganic nanomaterials in food. Nanomedicine-Uk. 2013, 8, 2007-2025. (7) Abbott, L. C.; Maynard, A. D., Exposure Assessment Approaches for Engineered Nanomaterials. Risk Anal. 2010, 30, 1634-1644. (8) Weber, C. R.; Nalle, S. C.; Tretiakova, M.; Rubin, D. T.; Turner, J. R., Claudin-1 and claudin-2 expression is elevated in inflammatory bowel disease and may contribute to early neoplastic transformation. Lab Invest. 2008, 88, 1110-1120. (9) Yamashita, K.; Yoshioka, Y.; Higashisaka, K.; Mimura, K.; Morishita, Y.; Nozaki, M.; Yoshida, T.; Ogura, T.; Nabeshi, H.; Nagano, K.; Abe, Y.; Kamada, H.; Monobe, Y.; Imazawa, T.; Aoshima, H.; Shishido, K.; Kawai, Y.; Mayumi, T.; Tsunoda, S.; Itoh, N.; Yoshikawa, T.; Yanagihara, I.; Saito, S.; Tsutsumi, Y., Silica and titanium dioxide nanoparticles cause pregnancy complications in mice. Nat Nanotechnol. 2011, 6, 321-8. (10) Lagopati, N.; Tsilibary, E. P.; Falaras, P.; Papazafiri, P.; Pavlatou, E. A.; Kotsopoulou, E.; Kitsiou, P., Effect of nanostructured TiO(2) crystal phase on photoinduced apoptosis of breast cancer epithelial cells. Int J Nanomedicine. 2014, 9, 3219-30. (11) Wang, J.; Zhou, G.; Chen, C.; Yu, H.; Wang, T.; Ma, Y.; Jia, G.; Gao, Y.; Li, B.; Sun, J.; Li, Y.; Jiao, F.; Zhao, Y.; Chai, Z., Acute toxicity and biodistribution of different sized titanium dioxide particles in mice after oral administration. Toxicol lett. 2007, 168, 176-85. (12) Thubagere, A.; Reinhard, B. M., Nanoparticle-Induced Apoptosis Propagates through Hydrogen-Peroxide-Mediated Bystander Killing: Insights from a Human Intestinal Epithelium In Vitro Model. Acs Nano. 2010, 4, 3611-3622. (13) Liang, M.; Lin, I. C.; Whittaker, M. R.; Minchin, R. F.; Monteiro, M. J.; Toth, I., Cellular Uptake of Densely Packed Polymer Coatings on Gold Nanoparticles. Acs Nano. 2010, 4, 403-413. (14) Ghosh, P.; Han, G.; De, M.; Kim, C. K.; Rotello, V. M., Gold nanoparticles in delivery applications. Adv Drug Deliv Rev. 2008, 60, 1307-15. (15) Frohlich, E.; Roblegg, E., Models for oral uptake of nanoparticles in consumer products. Toxicology. 2012, 291, 10-7. (16) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A., Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer. Nano lett. 2005, 5, 829-34. (17) Dykman, L. A.; Khlebtsov, N. G., Uptake of Engineered Gold Nanoparticles into Mammalian Cells. Chem Rev. 2014, 114, 1258-1288. (18) Hubatsch, I.; Ragnarsson, E. G. E.; Artursson, P., Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat Protoc. 2007, 2, 2111-2119. (19) Yao, M. F.; Chen, J. J.; Zheng, J. K.; Song, M. Y.; McClements, D. J.; Xiao, H., Enhanced lymphatic transport of bioactive lipids: cell culture study of polymethoxyflavone incorporation into chylomicrons. Food Funct. 2013, 4, 1662-1667. 19 ACS Paragon Plus Environment


377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415

(20) Gu, Y. J.; Cheng, J. P.; Lin, C. C.; Lam, Y. W.; Cheng, S. H.; Wong, W. T., Nuclear penetration of surface functionalized gold nanoparticles. Toxicol Appl Pharm. 2009, 237, 196-204. (21) Coulter, J. A.; Jain, S.; Butterworth, K. T.; Taggart, L. E.; Dickson, G. R.; McMahon, S. J.; Hyland, W. B.; Muir, M. F.; Trainor, C.; Hounsell, A. R.; O'Sullivan, J. M.; Schettino, G.; Currell, F. J.; Hirst, D. G.; Prise, K. M., Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles. Int J Nanomed. 2012, 7, 2673-2685. (22) Wadia, J. S.; Chalmers-Redman, R. M. E.; Ju, W. J. H.; Carlile, G. W.; Phillips, J. L.; Fraser, A. D.; Tatton, W. G., Mitochondrial membrane potential and nuclear changes in apoptosis caused by serum and nerve growth factor withdrawal: Time course and modification by (-)-deprenyl. J Neurosci. 1998, 18, 932-947. (23) Keil, V. C.; Funke, F.; Zeug, A.; Schild, D.; Muller, M., Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria. Pflug Arch Eur J Phy. 2011, 462, 693-708. (24) Polte, J.; Ahner, T. T.; Delissen, F.; Sokolov, S.; Emmerling, F.; Thunemann, A. F.; Kraehnert, R., Mechanism of gold nanoparticle formation in the classical citrate synthesis method derived from coupled in situ XANES and SAXS evaluation. J. Am. Chem. Soc. 2010, 132, 1296-301. (25) Datta, S. R.; Brunet, A.; Greenberg, M. E., Cellular survival: a play in three Akts. Genes Dev. 1999, 13, 2905-27. (26) Jiang, W.; Kim, B. Y. S.; Rutka, J. T.; Chan, W. C. W., Nanoparticle-mediated cellular response is size-dependent. Nat Nanotechnol. 2008, 3, 145-150. (27) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M., Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater. 2009, 8, 543-557. (28) Yang, P. H.; Sun, X. S.; Chiu, J. F.; Sun, H. Z.; He, Q. Y., Transferrin-mediated gold nanoparticle cellular uptake. Bioconjugate Chem. 2005, 16, 494-496. (29) Wang, S. H.; Lee, C. W.; Chiou, A.; Wei, P. K., Size-dependent endocytosis of gold nanoparticles studied by three-dimensional mapping of plasmonic scattering images. J Nanobiotechnology. 2010, 8, 33. (30) Sonavane, G.; Tomoda, K.; Sano, A.; Ohshima, H.; Terada, H.; Makino, K., In vitro permeation of gold nanoparticles through rat skin and rat intestine: Effect of particle size. Colloid Surf B. 2008, 65, 1-10. (31) Bajak, E.; Fabbri, M.; Ponti, J.; Gioria, S.; Ojea-Jimenez, I.; Collotta, A.; Mariani, V.; Gilliland, D.; Rossi, F.; Gribaldo, L., Changes in Caco-2 cells transcriptome profiles upon exposure to gold nanoparticles. Toxicol Lett. 2015, 233, 187-199. (32) Taggart, L. E.; McMahon, S. J.; Currell, F. J.; Prise, K. M.; Butterworth, K. T., The role of mitochondrial function in gold nanoparticle mediated radiosensitisation. Cancer 20 ACS Paragon Plus Environment

Page 20 of 30

Page 21 of 30

416 417 418 419 420 421 422


Nanotechnol. 2014, 5, 5. (33) Wang, L. M.; Liu, Y.; Li, W.; Jiang, X. M.; Ji, Y. L.; Wu, X. C.; Xu, L. G.; Qiu, Y.; Zhao, K.; Wei, T. T.; Li, Y. F.; Zhao, Y. L.; Chen, C. Y., Selective Targeting of Gold Nanorods at the Mitochondria of Cancer Cells: Implications for Cancer Therapy. Nano lett. 2011, 11, 772-780.

Figure Captions

423

Figure 1. Morphology of the initial gold nanoparticle suspensions (nominal diameters of

424

15, 50 and 100 nm) used in this study. All images were taken under transmission electron

425

microscopy as described in the Method.

426

Figure 2. The absorption of AuNPs by intestinal epithelial cells. Caco-2 cell monolayers

427

were incubated with AuNPs (15 nm, 50 nm, or 100 nm) for various time periods (up to 24

428

hour). After thorough wash, Caco-2 cell monolayers were processed for TEM

429

(transmission electron microscope) analysis to visualize AuNPs absorbed by the cells.

430

AuNPs are highlighted by the circles. Arrows indicate the direction from apical to

431

basolateral side across microvilli. Different magnifications were used in different images.

432

Scale bar represent 500 nm. Representative images were selected from replicates of at

433

least 3.

434

Figure 3. Transport of AuNPs across intestinal epithelium. Change in the concentration of

435

gold measured in the basolateral compartment after incubating Caco-2 cell monolayers

436

with AuNPs in the apical compartment of the transwells.

437

different concentrations of gold nanoparticles were used (shown in figure): 2.5, 5 and 10

438

µg/mL in the apical medium.

439

Figure 4.

440

amount of gold measured in the Caco-2 monolayers after incubating Caco-2 cell

Suspensions containing

Accumulation of AuNPs within the intestinal epithelium. Change in the



441

monolayers with 5 µg/mL of AuNPs in the apical compartment of the transwells.

442

Amount of gold was expressed as either particle mass (A) or particle number (B) in the

443

Caco-2 cell monolayer.

444

Figure 5. The effects of AuNPs on the mitochondrial membrane potentials of intestinal

445

epithelial cells.

446

100 nm) for 4h separately. Mitochondria toxicity of AuNPs was visualized by confocal

447

microscope using a red fluorescent dye JC-1 (A). JC-1 exhibits potential-dependent

448

accumulation in mitochondria, indicated by a fluorescence emission shift from green to

449

red.

450

of dye aggregates that fluoresce red, whereas cells with low mitochondria membrane

451

potential (damaged) remain monomeric JC-1 that fluoresce green. The red/green ratio

452

was quantified using image J (B). Representative images were selected from replicates of

453

3. Different letters indicate statistical difference among the three groups at the given time

454

point (P

Uptake of Gold Nanoparticles by Intestinal Epithelial Cells: Impact of

Recommend Documents