Insights into the Cellular Uptake, Cytotoxicity, and Cellular

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Insights into the Cellular Uptake, Cytotoxicity and Cellular Death Modality of Phospholipid-Coated Gold Nanorods Toward Breast Cancer Cell Lines Nouf N. Mahmoud, Rana M. Abu-Dahab, Lama A. Hamadneh, Duaa Abuarqoub, Hanan Jafar, and Enam A. Khalil Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00470 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 10, 2019

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Molecular Pharmaceutics

1

Insights into the Cellular Uptake, Cytotoxicity and Cellular Death Modality

2

of Phospholipid-Coated Gold Nanorods Toward Breast Cancer Cell Lines

3 4

Nouf N. Mahmoud a *, Rana M. Abu-Dahab b *, Lama A. Hamadneh a, Duaa Abuarqoub c,

5

Hanan Jafar c,d, Enam A. Khalil b.

6 7

a Faculty

of Pharmacy, Al-Zaytoonah University of Jordan, Amman 11733, Jordan.

8

b School

of Pharmacy, The University of Jordan, Amman 11942, Jordan.

9

c

Cell Therapy Center, The University of Jordan, Amman 11942, Jordan.

10

d

School of Medicine, The University of Jordan, Amman 11942, Jordan.

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*Corresponding authors: E-mails: [email protected], [email protected].

13 14

Keywords

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Gold nanorods; phospholipid; breast cancer; cytotoxicity; apoptosis; necrosis; gene expression.

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Abstract

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Gold nanorods (GNR) have gained pronounced recognition in diagnosis and treatment of

19

cancers driven by their distinctive properties. Herein, gold-based nano-system was prepared by

20

utilizing a phospholipid moiety linked to thiolated poly ethylene glycol; 1,2-distearoyl-sn-

21

glycero-3-phosphoethanolamine-N-PEG-SH (DSPE-PEG-SH), as a surface decorating agent.

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The synthesized phospholipid-PEG-GNR displayed good colloidal stability upon exposure to

23

the tissue culture medium. Cytotoxicity of phospholipid-PEG-GNR was investigated toward

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MCF-7 and T47D breast cancer cells using Sulforhodamine B test, and the results revealed that

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phospholipid-PEG-GNR demonstrated high cytotoxicity to MCF-7 cells compared to T47D

1

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

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cells, and minimal cytotoxicity to human dermal fibroblasts. The cellular uptake studies

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performed by imaging and quantitative analysis demonstrated massive internalization of

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phospholipid-coated GNR into the MCF-7 cells in comparison to T47D cells. The cellular

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death modality of the cancer cells after treatment with phospholipid-PEG-GNR was evaluated

30

using mitochondrial membrane potential assay (JC-1 dye), gene expression analysis and flow

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cytometry study. The overall results suggest that phospholipid-modified GNR enhanced

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mainly the cellular apoptotic events in MCF-7 cells in addition to necrosis, while cellular

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necrosis and suppressing the cellular invasion are contributing to the cellular death modality in

34

T47D cell line. The phospholipid-coated GNR interact in a different manner with breast cancer

35

cell lines and could be considered for breast cancer treatment.

36 37

1. Introduction

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Gold nanoparticles (GNP), particularly gold nanorods (GNR) have a great potential in

39

diagnosis and treatment of cancers and microbial infections owing to their favorable optical

40

characteristics that rely on surface plasmon resonance 1-3. Moreover, the large surface area of

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GNP enables efficient conjugation and binding of diverse chemical moieties, that would

42

improve drug solubility, stability, and biocompatibility 4.

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Surface functionalization of GNP has pronounced and crucial effects on the nanoparticles’

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stability, solubility, cellular uptake and targeting capability, in addition to cytotoxicity

45

Numerous studies have evaluated the influence of conjugating diverse small ligands and

46

macromolecules to GNP on their journey within different biological systems 8-11.

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Coupling of GNP to phospholipid bilayer has been reported to offer stable and bio-compatible

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nano-platforms that display several biomedical applications. For examples, clusters of

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phospholipid-decorated GNR were employed as effective biosensors

50

functionalized gold nano-composites were developed as nano-carriers for chemotherapeutic

51

agents or to achieve synergistic anticancer activity 13, 14. Few reports have studied the cellular 2


12,

5-7.

while lipid-

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internalization and cytotoxicity of nanoparticles linked to a phospholipid moiety. For

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examples, Chithrani et al. have found that incorporation of GNP into liposomes has enhanced

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their cellular uptake into Hela cells 15. Similarly, the surface modification of gold nanospheres

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with a phospholipid, has increased their cellular uptake into breast cancer cells compared to

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PEGylated counterparts, however, no significant cytotoxicity was observed for both 16.

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The cellular membrane of cancer cells, such as breast cancer, exhibits significant alteration in

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phospholipids and fatty acids profiles compared to normal cells in order to provide enough

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resources to form new membranes

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mostly composed of zwitterionic phospholipids, high percentage of phosphatidylserine (PS)

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and phosphatidylethanolamine (PE) are presented in the outer cell membrane of cancers such

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as breast and colon cancers 19. Accordingly, designing ligands that may exert effect on cancer

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cell membrane’s lipid conformation, membrane fluidity and cell function is gaining growing

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attention 20.

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Different cell death modalities such as apoptosis and necrosis and other related signaling

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pathways were observed for GNP with different particle size, morphology, and surface

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properties 21. For example, the cellular viability of human dermal fibroblast and glioblastoma

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cell lines was investigated after treatment with GNP of several morphologies and

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functionalities. As a result, cancer cells and normal cells demonstrated different death

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modalities with apoptotic death for cancer cells and necrosis for normal cells 22. However, it

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was reported recently that GNR caused necrosis of the cancerous cells by a specific pathway

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related to the lysosomal-cathepsin B 23.

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In the current study, a simple and efficient gold-based nano-system was prepared where a

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phospholipid moiety linked to thiolated-poly ethylene glycol; 1,2-distearoyl-sn-glycero-3-

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phosphoethanolamine-N-PEG-SH (DSPE-PEG-SH) was exploited to decorate the surface of

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GNR in order to obtain phospholipid-decorated GNR (phospholipid-PEG-GNR). The

17, 18.

While the outer cell membrane of healthy cells is

3



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cytotoxicity and cellular uptake of phospholipid-PEG-GNR toward MCF-7 and T47D breast

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cancer cell lines, and normal human dermal fibroblasts were investigated and compared to that

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of PEGylated counterparts. The death modality of the cancerous cells when treated with

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phospholipid-PEG-GNR was explored by mitochondrial membrane potential assay (JC-1 dye),

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gene expression test and flow cytometry analysis.

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2. Experimental Part

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2.1 Materials and Instruments

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1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG-SH (DSPE-PEG-SH, MW~2000

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g/mole), Nanosoft Polymers, USA. Chloroauric acid; HAuCl4.3H2O (99.9%); silver nitrate;

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AgNO3 (99%); sodium borohydride; NaBH4 (99%); ascorbic acid (99%); sodium oleate

88

(NaOL);

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cetyltrimethylammonium bromide; CTAB (99%); gold standard for ICP (1000 ppm);

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Sulforhodamine B stain; Trichloroacetic acid; Tris powder, Sigma-Aldrich Chemicals, USA.

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potassium bromide, Acros, Belgium. MCF-7 and T47D breast cancer cell lines, human dermal

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fibroblasts cell line, American Type Culture Collection (ATCC), USA. Roswell Park Memorial

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Institute tissue culture medium (RPMI 1640); Fetal bovine serum (FBS); Gentamycin,

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Penicillin and Streptomycin (Penstrep) (50µg/mL); Trypsin EDTA 0.2 % in phosphate buffer

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saline (PBS), Euroclone, Europe. Iscove's Modified Dulbecco's Medium (IMDM), Eurobio,

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France. 96-well plates, Greiner Bio-One, Germany. Acetic acid, 97%; Ethanol 70%, Tedia

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Company Inc., USA. 4′,6-diamidino-2-phenylindole (DAPI) stain; Annexin V/PI apoptosis kit,

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Molecular Probs, USA. Hydrochloric acid (HCl), Scharlau, Spain. Isopropanol, Gainland

99

chemical company, U.K. Nitric acid, Vickers, U.K. Paraformaldehyde, Fluka, Switzerland.

100

Trypan blue stain (0.04%), Gibco, USA. SYBR green PCR master mix, Bio-Rad, USA.

methoxy-polyethylene

glycol-thiol;

4

m-PEG-SH


(MW

~2000

g/mole);;

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SuperScript® VILO™ cDNA Synthesis Kit, Life Technologies, USA. RNeasy® plus Mini

102

kit, Qiagen, USA. RT2 First Strand Kit (Qiagen, USA),

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The following instruments and equipment were used in this study: Ultraviolet-visible (UV-vis)

104

spectrophotometer, UV-1800, Shimadzu, Japan. Nano UV-spectrophotometer, Quawell, USA.

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Size/zeta potential analyzer, Nicomp Nano Z3000 particle, USA. FEI Morgani 268

106

transmission electron microscope (TEM), 60 kV, Netherland. Hettich EBA 12 Centrifuge,

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Gemini BV, Netherlands. Confocal laser scanning microscopy, LSM 780, Carl Ziess,

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Germany. Inductively coupled plasma-optical emission spectroscopy (ICP-OES), Optima 2000

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DV, Perkin Elmer, USA. Fourier-transform infrared (FTIR) spectroscopy, Shimadzu FTIR

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Affinity spectrophotometer, Japan. Centrifuge, Labofuge I, Heraeus Christ, Germany.

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Thermogravimetric

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Fluorescence plate reader, GloMax1-Multi Detection System, Promega, USA. Inverted

113

microscope, Meiji, Japan. ELISA plate reader, Biotek Instruments, USA. Laminar air flow

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cabinet, ESCO Micro, Singapore. Microliter-pipettes and multichannel pipette, Gilson, France.

115

pH meter, Hanna Instruments, Italy. Plate shaker, Boekel Scientific 130000, USA. Vortex,

116

VELP Scientifica, Europe. Water bath, Buchi, Switzerland. Real time PCR (CFX 96), Bio-

117

Rad, USA. Fluorescein activated sorter; FACS Canto II, Becton Dickinson, Biosciences, USA.

Analyzer

(Mettler-Toledo,

Columbus,

OH,

TA3000

System).

118 119

2.2 Methods

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2.2.1 Chemical synthesis of GNR

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GNR was synthesized following a previous procedure with some modifications

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suspension was prepared as the following: 5.0 mL of CTAB solution (0.20 M) and HAuCl4

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solution (0.0050 M) were mixed together. After that, ice-cold NaBH4 was added and a solution

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with a honey-color was resulted. The seed suspension was stirred for 2 min and left at 25 °C.

5


24.

Seeds


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For growth suspension, amounts of CTAB and NaOL were transferred to hot water (250 mL,

126

∼50 °C) and cooled to 30 °C. Then, 18.0 mL of AgNO3 (4.0 mM) and 250 mL of HAuCl4 (1.0

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mM) were added to the previous solution, the pH of the solution was adjusted by HCl, and then

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it was incubated for 15 min at 30 °C. After that, 1.25 mL of Ascorbic acid (64.0 mM) and 0.8

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mL of seeds suspension were injected into the growth suspension. The suspension was

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incubated at 30 °C for 24 h, and then was cleaned twice by centrifugation at 10 000 rpm for 10

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min and the obtained GNR pellets were preserved at 4 °C.

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2.2.2 Functionalization of GNR with DSPE-PEG-SH; phospholipid-PEG-GNR

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DSPE-PEG-SH dispersion (1.0 mL, 20.0 mg/mL) was mixed with 10.0 mL of GNR suspension

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and kept under stirring overnight. The modified GNR suspension was cleaned twice by

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centrifugation for 8 min at 8000 rpm, and the obtained GNR pellets were preserved at 4 °C.

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2.2.3 Functionalization of GNR with thiolated- polyethylene glycol; PEG-GNR

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PEG-SH solution (1.0 mL, 10.0 mg/mL) was mixed with 10.0 mL of GNR suspension and kept

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under stirring overnight. The modified GNR suspension was cleaned twice by centrifugation

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for 8 min at 8000 rpm, and the obtained GNR pellets were preserved at 4 °C.

141 142

2.2.4 Characterization of phospholipid-PEG-GNR and PEG-GNR

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All the synthesized GNR suspensions were characterized by optical absorption spectroscopy

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over the wavelength of 200-1100 nm, effective surface charge, hydrodynamic size and TEM

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imaging. Phospholipid-PEG-GNR were characterized also by Fourier-transform infrared

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(FTIR) spectroscopy. Sample discs of potassium bromide were used for measurement by FTIR

147

spectroscopy.

6


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2.2.5 Estimation of ligand density of DSPE-PEG-SH on GNR by thermogravimetric analysis

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(TGA)

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The grafting density of DSPE-PEG-SH ligand on the surface of GNR was estimated using

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Thermogravimetric Analyzer. A powder sample of phospholipid-PEG-GNR was obtained by

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lyophilization of highly concentrated suspension of phospholipid-PEG-GNR. The temperature

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during analysis was increased from 25 °C to 600 °C at the rate of 10 °C/min under nitrogen.

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Weight loss and accordingly the ligand density per nm2 of GNR were calculated as described

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previously 25.

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2.2.6 Colloidal stability of PEG-GNR and phospholipid-PEG-GNR suspensions upon mixing

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with the tissue culture growth medium

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A volume equals to 0.5 mL of PEG-GNR or phospholipid-PEG-GNR suspensions of different

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concentrations was mixed with an equivalent volume of the tissue culture medium (RPMI)

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without fetal bovine serum (FBS) to obtain GNR-tissue culture medium mixtures of different

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concentrations (0.076- 300 µg/mL), and they were incubated at 37 C for 24 h. The colloidal

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stability of the mixtures was verified by observing their colloidal color, optical absorption

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spectra and zeta potential immediately and after 24 h of incubation at 37 C. TEM imaging was

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performed for highly concentrated phospholipid-PEG-GNR mixed with the tissue culture

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media.

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2.2.7 Cytotoxicity of PEG-GNR and phospholipid-PEG-GNR towards breast cancerous cells

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and the healthy human dermal fibroblasts cell lines

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4.2.7.1 Cell Culture

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MCF-7 and T47D breast cancer cells were cultured in RPMI 1640 medium while human

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dermal fibroblasts were cultured in IMDM. The cells were supplemented with l-glutamine (1.0

7



172

%, 2.0 mM), FBS (10.0% v/v), Penicillin (100 U/mL), Streptomycin (100 μg/mL) and

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Gentamycin (1.0 mL) at 5 % CO2 and 99 % relative humidity at 37 °C. The cells were stained

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after confluency with trypan blue dye (0.04%) and counted by a hemocytometer.

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2.2.7.2 Antiproliferative assay

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A volume of 100 µL of cell suspensions of 5x103 cell/well for the cancer cells and 10x103

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cell/well for the human dermal fibroblasts were seeded in 96-well plates and incubated for 24

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h before addition of GNR suspensions (PEG-GNR, phospholipid-PEG-GNR). A volume of

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100 μL of each GNR suspension at five different concentrations (10.0, 2.5, 0.6 and 0.15 µg/mL)

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was added to the wells without addition of FBS to maintain the colloidal stability of GNR. The

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cellular viability using Sulforhodamine B test was estimated after incubating the plates for 72

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h. The experiment was done in triplicate.

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For Sulforhodamine B assay, trichloroacetic acid (40%) was utilized to fix the cells in the

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plates. After that, the plates were incubated at 4˚C for 1 h and then washed with cold water. To

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each well, SRB stain was added, kept for 30 min, then washed with acetic acid (1.0%). To each

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well, a volume of 100 μL of Tris base buffer (10.0 mM, pH 10.5) was added. The absorbance

188

was recorded at 570 nm by ELISA plate reader.

189 190

2.2.8 Cellular uptake of PEG-GNR and phospholipid-PEG-GNR into breast cancer cells:

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2.2.8.1 Quantification of PEG-GNR and phospholipid-PEG-GNR uptake into breast cancer

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cells by ICP-OES

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A density of 2 ×106 of MCF-7 and T47D cells were seeded per 25-cm2 flask in 12.0 mL tissue

194

culture medium (RPMI) and allowed to attach for 48 h (in three replicates). Then, the PEG-

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GNR and phospholipid-PEG-GNR suspensions in tissue culture medium without FBS were

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immediately applied to the cells to obtain concentrations of 20.0 µg/mL and incubated for 3 h.

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The cells were trypsinized after two washing steps with PBS, centrifuged at 1400 rpm for 30

198

mins at 4°C and the obtained cell pellets were mixed with aqua regia (HNO3 and HCl; 1:3) in

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a water bath (70 °C) for 3 h. After that, the digested samples were diluted with milli-Q water

200

up to 4.0 mL and filtered by 0.22 µm Teflon syringe filter. Untreated cells presented the control.

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Using a validated ICP-OES analytical method, the concentration (mg/L) and the percentage of

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the internalized gold into cells were quantified at a wavelength of 242.795 nm using a

203

calibration curve of gold standard for ICP (0.2–10.0 ppm). The experiment was done in

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triplicate.

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2.2.8.2 TEM Imaging of MCF-7 treated with phospholipid-PEG-GNR or PEG-GNR

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suspensions

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MCF-7 cells were seeded into 25-cm2 flasks at a density of 2 ×106 cells per flask in RPMI

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tissue culture medium and allowed to attach for 24 h (in three replicates). Then, the

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phospholipid-PEG-GNR or PEG-GNR suspensions (20.0 µg/mL) in tissue culture medium

212

without FBS were immediately applied to the cells. The medium was withdrawn after 3 h of

213

incubation, and the cells were washed with PBS and trypsinized, then centrifuged at 1400 rpm

214

at 4 °C. The cell pellets were collected after removing the supernatant and fixed in

215

glutaraldehyde solution (3%) and phosphate buffer (pH 7.4). The cell pellets were then washed

216

in PBS and fixed in a buffer solution of osmium tetra-oxide for 2 h. After that, pellets were

217

dehydrated in ethanol solutions and kept overnight in epoxy/propylene oxide 1:1 (v/v). Then,

218

pellets were embedded in epoxy resin and sections of 70-nm thin were obtained using ultra-

219

microtome. The sections were fixed onto TEM grids (Formvar-coated) and imaged by TEM.

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Cells not exposed to GNR treatment were used as control in the experiment.

221

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2.2.8.3 Imaging of MCF-7 treated with phospholipid-PEG-GNR or PEG-GNR by confocal

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laser scanning microscopy

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MCF-7 cells were seeded onto round cover slips in 12-well plate at a density of 2 ×105

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cells/well in RPMI tissue culture medium and allowed to attach for 24 h. PEG-GNR and

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phospholipid-PEG-GNR in tissue culture media without FBS (20.0 µg/mL) were immediately

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applied to the cells and incubated for 3 h after which the media and treatments were removed,

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and the wells were washed with PBS. The cells were fixed for 30 min in paraformaldehyde

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(4%) at 4°C and washed with PBS three times. After that, the cover slips were gently removed

230

and slowly flipped over clean slides covered with 50.0 µL of DAPI stain. Cells not exposed to

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GNR suspension are considered as control in the experiment. The cells were imaged at

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excitation/emission wavelengths of 532 nm/750 nm for gold and to 360 nm/ 460 nm for DAPI.

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2.2.9 Cellular death modality of MCF-7 and T47D breast cancer cells after treatment with

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phospholipid-PEG-GNR

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2.2.9.1 JC-1 Assay of mitochondrial membrane potential

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MCF-7 and T47D cells were seeded onto 24-well plates and incubated in 5% CO2 at 37 °C for

237

24 h after which they were treated with phospholipid-PEG-GNR (20.0 µg/mL) for 24 h. After

238

media removal, JC-1 dye was added to the cells, and they were incubated for 20 min. After

239

that, the dye was withdrawn, and the cells were washed with PBS. The uptake of JC-1 was

240

quantified using by the fluorescence plate reader. Excitation and emission wavelengths of 540

241

nm and 570 nm respectively were used to measure the aggregates of JC-1, while monomers of

242

JC-1 were measured at excitation/emission wavelengths of 485 nm/535 nm, respectively. The

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percentage of cells showing changes in cells mitochondrial membrane potential was estimated

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as a ratio of red/green fluorescence from triplicate.

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2.2.9.2 Gene expression analysis in MCF-7 and T47D cells pre-treated with phospholipid-

247

PEG-GNR by quantitative real time-polymerase chain reaction (PCR)

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Total mRNA isolation from treated MCF-7 and T47D breast cancer cells with phospholipid-

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PEG-GNR of concentration of 0.5 µg/mL for MCF-7 and 5.0 µg/mL for T47D cells for 72 h

250

was performed using RNeasy® plus Mini kit based on the kit’s protocol. Nano UV-

251

spectrophotometer was utilized to measure the concentration and integrity of mRNA.

252

SuperScript® VILO™ cDNA Synthesis Kit was used to produce cDNA according to the kit’s

253

manufacturer. CFX96 Touch TM Real-Time PCR Detection System was utilized to perform

254

quantitative real time-PCR analysis using SYBR green PCR mastermix., with 1.0 µL starting

255

cDNA and primer sets. The analysis was performed in triplicate.

256 257

2.2.9.3 Evaluation of human apoptosis signaling pathway in MCF-7 cells pre-treated with

258

phospholipid-PEG-GNR by quantitative PCR array (qPCR)

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RNA was extracted from MCF-7 cells pre-treated with phospholipid-PEG-GNR (7.6 µg/mL

260

for 24 h) by using Trizol-hybrid method (Qiagen, USA). Then, cDNA was synthesized by

261

converted 0.5 μg total RNA by using RT2 First Strand Kit. Then, cDNA samples were diluted

262

and amplified with the RT2 SYBR® green master mix of (PAHS-004ZD, RT² Profiler™ PCR

263

Array Human Apoptosis, Qiagen, USA) following the kit’s instructions, and loaded into the

264

96-well array. The amplification conditions were as follow; 95 °C for 10 min, then 40 cycles

265

of 95 °C for 15 sec and 60°C for one min. Samples were run on CFX 96 C1000 system. Data

266

were analyzed automatically following the SABiosciences company SABiosciences company

267

(Qiagen, USA) web portal; www.SABiosciences.com/pcrarraydataanalysis.php by using ΔΔCt

268

method. The expression levels of the genes were normalized to the following housekeeping

269

genes; Beta-2-microglobulin (B2M), Hypoxanthine phosphoribosyl transferase 1 (HPRT1),

270

and actin beta (ACTB). For data analysis, the differential expression levels of the apoptotic

11



271

genes were estimated by Student’s t-test (two-tailed, unpaired). A cut-off point of 2.0 was used

272

as a threshold to determine the statistical significance of the upregulated or downregulated

273

genes (P

Insights into the Cellular Uptake, Cytotoxicity, and Cellular

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