Intracellular Uptake of Fluorescent Brightener 28 Loaded to Functionalized Gold Nanoparticles by T47D Breast Cancer Cells

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1 International Proceedings of Chemical, Biological and Environmental Engineering, Vol. 99 (2016) DOI: /IPCBEE V99. 1 Intracellular Uptake of Fluorescent Brightener 28 Loaded to Functionalized Gold Nanoparticles by T47D Breast Cancer Cells Zahrah Alhalili 1, Abeer Zaila 2, Barbara Sanderson 2 and Joe Shapter 1 1 Flinders Centre for NanoScale Science and Technology, School of Chemical and Physical Sciences, Flinders University, Adelaide, SA 5001, Australia 2 Medical Biotechnology, School of Medical Science and Technology, Flinders University, Adelaide, SA 5001, Australia Abstract. Gold nanoparticles functionalized with carboxylic terminated alkane-thiols and fluorescent brightener 28 (FB28) were synthesized for drug delivery and cellular imaging applications. The fluorescently labelled AuNPs were shown to be non-toxic. Confocal laser scanning microscopy (CLSM) showed an efficient internalization and uptake of AuNPs by T47D breast cancer cells within 1 hour of incubation. TEM results showed the localization of AuNPs in the cytoplasm. Keywords: gold nanoparticles, fluorescent brightener 28, cellular uptake. 1. Introduction The interactions between gold nanoparticles and mammalian cells and cellular uptake have been given significant attention due to active development of NPs for biomedical and pharmaceutical applications [1], [2]. The cellular uptake of AuNPs depends on different parameters including the particle s size, shape and surface functionalization [2]. Modification of the surface of gold nanoparticles is essential to further functionalization with biomolecules. Thiol ligands have been used extensively for surface modification [3], [4]. Gold nanoparticles have been utilized in diagnosis [5], photothermal treatment [6], drug delivery [7] and cell imaging [8]. Gold nanoparticles show distinctive physical, biological and chemical properties [9] no inherent cytotoxicity against human cells and are biocompatible. They can be synthesized in controllable sizes and shapes and their surface can be functionalized with different biomolecules [10], [11]. A staining agent can be loaded onto NPs to track, image and investigate their intracellular uptake to understand the interactions between those particles and cells thus helping to improve their effectiveness for diagnosis and treatment of diseases. Fluorescent brighteners, among them fluorescent brightener 28, are colourless dyes of the Calcofluor White M2R group. They are soluble in water and used to provide brightness and whiteness to paper and textile products and are included in laundry detergents to keep washed items looking bright and white [12], [13]. In biological applications, they are utilised to stain cell walls of plants and fungi [14]. Darken [14] was the first to report the biological application of Calcofluor White M2R in terms of its usage for observing fungi and bacteria cell walls [14]. Since this work, the fluorescent dye has been utilised to visualise cell walls of various sorts of fungi, bacteria, algae and plants [15]-[17]. For example, Davey et al. [13] described the fluorescent brighteners as stains for flow cytometric analysis of different types of microorganisms and spores [13]. Fluorescent brighteners absorb UV light and emit blue fluorescence [13]. The aim of this study was to investigate the cellular cytotoxicity of fluorescently labelled AuNPs to optimize the safe concentration of the fluorescent probe used for intracellular tracing and imaging of AuNPs. Corresponding author. Tel.: address: alha0167@flinders.edu.au 1

2 2. Materials and Methods 2.1. Materials All chemicals and reagents were used as received without any further purification. Gold (III) chloride trihydrate, paclitaxel, (±)-α-lipoic acid (LA), 16- mercaptohexadecanoic acid (16-MHDA), Tween 20,fluorescent brightener 28 (FB28) were obtained from Sigma Aldrich, Australia. Trisodium citrate dehydrate was purchased from Ajax Finechem Pty Ltd, Australia Synthesis of FB28 Conjugated Functionalized Gold Nanoparticles Gold nanoparticles (AuNPs) were synthesised by sodium citrate reduction of HAuCl 4 method described by Grabar et al. [10]. Then, the synthesised citrate-stabilised gold nanoparticles colloids were first degassed with nitrogen (N 2 ) to avoid the oxidation of the alkane-thiol and then dispersed in phosphate buffered saline (PBS) ph 7.4 containing Tween 20 for approximately 1 h. A solution of lipoic acid was added to the mixture under nitrogen (N 2 ) at room temperature and stirred for 18 h followed by a solution of 16- mercaptohexadecanoic acid (16-MHDA) with further stirring for 18 h at room temperature. After that, a solution of FB28 probe was added to the mixture and stirred overnight. The resulting sample was centrifuged to obtain the FB28-AuNPs conjugate Cell Culture and Crystal Violet Assay Experimental Procedure Human ductal breast epithelial tumour cell lines (T47D) were used in the bioassays. The cell lines were grown in 1640 RPMI media, with 10% foetal bovine serum (FBS) and penicillin antibiotics to grow the cell lines. Cells were used every h after reaching 80% to 90% confluence. The cells were incubated in a completely humidified atmosphere at 37 C with 5 % CO 2. Cell concentration was estimated by Trypan blue dye exclusion counting involving diluting the cell suspension 1:1 with Trypan Blue dye (TB), then loading the stained cells into a chamber of a Neubauer haemocytometer. Viable and dead cells were counted at Mag 40x using a light microscope. The average number of cells per square was determined then multiplied by the dilution factor (x2) to obtain the viable cell concentration (x10 6 cells/ ml). To assess the relative cell survival after exposure to fluorescently labelled AuNPs, a crystal violet assay was carried out in 5 x 96-well flat-bottom plates, one as standard curve and the others as treatment plates (in four replicates). Serial dilutions of cells were prepared in 100 μl volume and cells were seeded. T47D was used at the concentration of cell/well in four replicates. Plates were incubated in a humidified 37 C, 5% CO 2 incubator for h. The following day, medium was removed and 50 μl/well of crystal violet stain was placed in standard curve plate and incubated for 10 min at room temperature, prior to washing with Milli-Q (MQ) water. Plates were then left to dry overnight. For treatment plates, cells were treated with 50 μl crystal violet mixed with 30 μl of four concentrations of fluorescently labelled AuNPs (initial concentration of FB28= mm). After 24 h, 50 μl of acetic acid was added to each well and the plates were incubated at room temperature for 10 min before reading with a spectrophotometer. 3. Results UV-Vis spectrophotometry of fluorescently labelled AuNPs (FB28-AuNPs hybrid) is shown in Fig. 1. The expected absorption peaks of the dye were observed both in the free molecules and after loading onto the nanoparticles. Loading onto the particle generally lead to broad AuNPs and FB28 relevant bands suggesting the molecules had been loaded to the AuNPs. The FT-IR spectrum of FB28 alone is illustrated in Fig. 2.a. The FT-IR spectra of FB28 alone shows the main characteristic peaks observed in fluorescent brightener [18]. A broad band at ~ cm -1 is attributed to O H and N H stretching vibrations. The peaks at ~1613, 1592,1538 and 1504 cm -1 are assigned to C=C and C=N stretching vibrations in the aromatic rings, respectively [19], [20]. The peak at ~1418 cm -1 is assigned to C H deformation of the alkyl group whereas the peak at 1362 cm -1 is due to OH bending. The absorbance bands at ~1309 and 1223 cm -1 are assigned to C N stretch in the aromatic rings. The band at ~1176 cm -1 is due to O=S=O asymmetric stretching vibrations in SO 3 H groups [21]. The band at ~1048 cm -1 is due to SO 3 symmetric stretching vibrations. The peaks at ~1081 and 1022 cm -1 are due to C O C deformation whereas the peaks at ~830 and 758 cm -1 are assigned to aromatic =C-H out of plane deformation 2

3 vibrations [20]. Fig. 2.b shows the main characteristic peaks of FB28 fluorescent dye after loading onto thiolated AuNPs. The observations from FT-IR indicate the presence of the fluorescent molecules on the thiolated AuNPs. Some characteristic peaks assigned to carboxylic terminated alkane-thiols were observed as well. Importantly, the C H stretching of the alkyl groups at ~2917 and 2849 cm -1, respectively were observed. The loading of FB28 onto thiol-aunps surface can be ascribed to the van der Waals electrostatic interactions between FB28 and carboxylic acids terminated alkane-thiols. Other peaks either assigned to of the staining agent or to carboxylic acids terminal alkane-thiols or AuNPs overlapped. Fig. 1: UV-Vis spectra of (a) FB28 alone and (b) FB28-AuNPs hybrid. Fig. 2: FT-IR spectra of (a) free FB28 and (b) FB28-AuNPs hybrid Cellular uptake of 30 μg/ml of free FB28 and the FB28-AuNPs hybrid was visualized by confocal laser scanning microscopy (CLSM). Images for T47D cells incubated with free FB28 solutions for 1 h, 6 h and 24 h show FB28 stained the cytoplasm but not the nuclei. Both free FB28 and FB28-AuNPs hybrid internalized in the cells. However, no big increase in fluorescence intensity in free FB28 was observed after incubation for different periods of time (1 h, 6 h or 24 h) (see Fig. 3.a,b and c). In contrast, in case of the FB28 on the AuNPs the fluorescence intensity increased with time (Fig. 3.d, e and f). After incubation for 1 h, low intensity fluorescence permeating throughout the membrane into the cytoplasm was observed in the cells (Fig. 3.d). After 6 h, no significant increase in fluorescence was noticed (Fig. 3.e). After 24 h of incubation, intracellular fluorescence was more intense and distributed across the cells compared to that observed after 1 h or 6 h of treatment (Fig. 3.f). The reason for this distribution could be because some of the fluorescent dye molecules are disconnected from the AuNPs after long times in the cells and the conjugate components are 3

4 dissociated from each other. The confocal microscopy results for T47D living cells treated with FB28- thiolated AuNPs hybrid revealed an increase of intracellular fluorescence over time as an indication of cellular uptake of AuNPs following the fluorophore s release. According to literature AuNPs are internalised the cells via a receptor-mediated endocytosis mechanism and trapped in small vesicles known as endosomes [22], [23]. Those endosomes then fuse with lysosomes for processing followed by transferring to the cell periphery for excretion [22], [23]. Different studies have confirmed the presence of AuNPs inside various sections of the endocytic pathway including endosomes and lysosomes [23], [24]. Fig. 3: Fluorescence (left) (λex = 405 nm, λem = 478), bright field (middle) and overlaid (right) images of T47D human breast cancer cells treated with free FB28 or FB28-AuNPs hybrid (a & d) after 1 h of incubation (b & e) after 6 h of incubation (c & f) after 24 h of incubation at room temperature. Original magnification was 63. Scale bars = 20 μm. Fig. 4: TEM images showing clusters and individual of AuNPs in cell cytoplasm of T47D cells exposed to 200 μl of FB28-AuNPs hybrid for 3-4 h. Scale bar = 500 nm. Transmission electron microscopy (TEM) was used to confirm the results obtained from CLSM in relation to intercellular uptake and localization of FB28-AuNPs hybrid. T47D cells treated with FB28- AuNPs hybrid (200 μl) were possibly taken up as groups of clusters and as individual particles as well. It was expected that AuNPs internalized the cells via endocytosis. The particles were mainly found in cytoplasm (Fig. 4). 4

5 Fig. 5: Cytotoxicity analysis of FB28-AuNPs hybrid in T47D human breast cancer cells. Results were tested by Oneway ANOVA and statistical significance showing * at P<0.05. The cellular cytotoxicity data shows the cells treated with FB28 containing samples show no significant reduction of cell relative survival indicating no cytotoxicity (Fig. 5). The results reveal the suitability of FB28 loaded thiolated AuNPs at any concentration tested to detect the cellular uptake and localization of AuNPs and suggesting potential use of this system in other biomedical applications. 4. References [1] P. Hu, et al., Molecular interactions between gold nanoparticles and model cell membranes. Phys Chem Chem Phys, (15): p [2] L. A. Dykman, and N. G. Khlebtsov, Uptake of engineered gold nanoparticles into mammalian cells. Chemical reviews, (2): p [3] M. Brust, et al., Synthesis of thiol-derivatised gold nanoparticles in a two-phase Liquid-Liquid system. Journal of the Chemical Society, Chemical Communications, 1994(7): p [4] J. Gao, et al., Colloidal Stability of Gold Nanoparticles Modified with Thiol Compounds: Bioconjugation and Application in Cancer Cell Imaging. Langmuir, (9): p [5] K. M. Mayer, and J. H. Hafner, Localized Surface Plasmon Resonance Sensors. Chemical Reviews, (6): p [6] L. C. Kennedy, et al., A new era for cancer treatment: gold-nanoparticle-mediated thermal therapies. Small, (2): p [7] B. Duncan, C. Kim, and V. M. Rotello, Gold nanoparticle platforms as drug and biomacromolecule delivery systems. Journal of controlled release : official journal of the Controlled Release Society, (1): p [8] C. S. Kim, et al., Cellular imaging of endosome entrapped small gold nanoparticles. MethodsX, : p [9] J. Lee, et al., Gold nanoparticles in breast cancer treatment: promise and potential pitfalls. Cancer letters, (1): p [10] X. Huang, and M. A. El-Sayed, Gold nanoparticles: optical properties and implementations in cancer diagnosis and photothermal therapy. Journal of Advanced Research, (1): p [11] P. Ghosh, et al., Gold nanoparticles in delivery applications. Advanced Drug Delivery Reviews, (11): p [12] H. Zollinger, Color chemistry: synthesis, properties and applications of organic dyes and pigments, 1991, VCH Publishers Inc.: New York. p [13] H. M. Davey, and D. B. Kell, Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms. Cytometry, (4): p [14] M. A. Darken, Applications of fluorescent brighteners in biological techniques. Science, (3465): p

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