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MICROBIAL MEDIATED SYNTHESIS OF GOLD NANOPARTICLES:

PREPARATION, CHARACTERIZATION AND CYTOTOXICITY STUDIES

IRENA MALISZEWSKA*

Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wroclaw University of Technology, 50-370 Wroclaw, Wybrzeże Wyspiańskiego 27, POLAND

The present contribution focuses on the synthesis of metallic nanoparticles of gold using aqueous Au+3 ions with the cell-free filtrate of Trichoderma koningii. Fourier transform infrared spectroscopy (FTIR spectrum) suggested that proteins are mainly responsible for reduction of gold ions and long-term stability of the biogenic nanoparticles. The sucrose density gradient technique to separate the gold nanoparticles based on their size was demonstrated. The smallest spheres from 10 nm to 14 nm were concentrated in the 30%

fraction and their cytotoxicity was studied. The results suggested that the gold nanoparticles were taken up by the colon cancer cells via endocytosis and the filtrate protein is responsible for their noticeable toxicity against human cancer LoVo and LoVo/DX cells.

(Received June 16, 2013; Accepted August 20, 2013)

Keywords: biosynthesis, molds, gold nanoparticles, cytotoxicity, endocytosis

1. Introduction

Nanoscale metal materials have been exponentially developed in recent years because of their unique chemical and physical properties and important applications in chemical sensing, biolabeling, diagnostics and therapeutics [1, 2]. Owing to the wide range of applications offered by metal nanoparticles in different fields of science and technology, various protocols have been designed for their formation [3-4]. One of the approaches is the facile synthesis carried out by microorganisms. In the past three decades, it has been shown that several types of bacteria, yeast and fungi had a high ability to synthesize various metallic nanoparticles [5-6]. Among them, molds have been documented as an extremely good candidate in the synthesis of these nanoscale materials [7-8]. The capacity for metal nanoparticles formation was detected in Verticillum luteoalbum [9], Fusarium oxysporum [10], Colletotrichum sp. and Tricothecium sp. [11], Phaenerochaete sp. [12], Trichoderma koningii [13], Aspergillus foetidus [14], Aspergillus niger [15, 16], Penicillium sp. [17] and Alternaria alternate [18].

On the other hand, it is known that practical application of gold nanoparticles depends largely on their impact on human and environment health. Nowadays there is a wider debate about the possible risks associated with gold nanoparticles applications. During recent years different studies have been performed demonstrating that nanomaterials can affect biological behaviours at the cellular, subcellular and protein levels. According to Pan et al. [19] studies, smaller size particles have better ability to induce cytotoxicity as compared to bigger one. Moreover, the results obtained suggested that cationic particles are generally toxic at much lower concentrations than anionic particles, which they relate to the electrostatic interaction between the cationic nanoparticles and the negatively charged cell membranes [20]. Cytotoxicity also depends on the type of cells used. For example, 33 nm citrate-capped gold nanospheres were found to be noncytotoxic to baby hamster kidney and human hepatocellular liver carcinoma cells, but cytotoxic to a human carcinoma lung [21].

*Corresponding author: [email protected]

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In the present study, I used the cell-free filtrate of Trichoderma koningii [13] for bioreduction of the gold ions resulted in extracellular formation of very stable gold nanoparticles.

The main aim of this research was to examine in vitro toxicity of these biogenic nanostructures on human colon cancer cell line LoVo and multidrug resistance sub-line LoVo/DX . My studies importantly approach to understand the potential toxicity hazards of this biogenic material.

2. Materials and methods Reagents

All chemical agents including chloroauric acid (HAuCl4 x 4H2O) were obtained from (POCH) Poland.

Synthesis and characterization of the gold nanoparticles

Trichoderma koningii strain, isolated from the soil have been used in the study [13]. The basal medium used in experiments consisted of (%) : KH2PO4 0.7 ; K2HPO4 0.2; MgSO4 7H2O 0.01; NH4Cl 1.0; yeast extract 0.06; glucose 1.0. The Erlenmeyer flasks were inoculated with spores (105/mL) of Trichoderma koningii and incubated at 280 C with shaking (200 rpm) for 5 days. After the fermentation time, the biomass was filtered (Whatman filter paper No. 1) and then extensively washed with distilled water to remove any medium component. Fresh and clean biomass (10 g) was taken into the Erlenmeyer flask, containing 100 mL of Milli-Q deionised water (UV Ultrapure Water System, Burnstead, USA). The flask was agitated at 28 oC with shaking (100 rpm) for 48 h. The cell-free filtrate was collected by pre-filtration in Whatman No. 1 filter papers and filtered using Millex-GP filter (PES membrane, 0.22 μm). Chloroauric acid (1mM of final concentration) was mixed with the cell-free filtrate in an Erlenmeyer flask and agitated at 28 o C in dark. The control (without the cell-free filtrate) was also run along with the experimental flasks.

To verify reduction of gold ions the solutions were scanned in the range of 200-800 nm in a spectrophotometer (Shimadzu, UV 3600). The size and morphology of the nanoparticles were analyzed with the transmission electron microscope TEM (Zeiss EM 900). The sample was prepared by placing a drop of the gold nanoparticles on a carbon-coated copper grid and subsequently drying in air before transferring it to the microscope. From electron micrographs the particle size was found for no less than 150 particles.

Separation of the gold nanoparticles

The separation of the gold nanoparticles was performed according to the method described by Kumar et al. [22] with minor modifications. In detail, we created a discontinous sucrose density gradient by layering dilute sucrose solutions upon one another in a centrifuge tube: 7 mL of 30%, 40%, 50% and 60% w/v sucrose. Finally 7mL of the gold nanoparticles synthesized by the cell- free filtrate obtained from 5-days biomass cultured in media contained NH4Cl as nitrogen source with intensive shaking (AuNPs) was loaded onto this gradient and centrifuged at 2320 x g at 10 oC for 1h. Fractions of the gradient were collected using a pipette, dialyzed (MWCO 8000-10000) against Milli-Q deionised water at room temperature and lyophilized (Freeze Dryer Modulyo, Edwards). These particles were made in a KBr pellet and the spectrum was recorded with FTIR spectrometer (Perkin Elmer 1600). The stability study of the gold nanoparticles was carried out at room temperature. The change in surface plasmon resonance of the nanoparticle dispersion was recorded up to six month using UV-vis spectroscopy. Zeta potential of the gold nanoparticles concentrated in the 30% sucrose fraction (AuNPs-30) was determined using Zetasizer 2000, Malvern Instruments.

Assessment of cytotoxic effect

Human colon adenocarcinoma cell line LoVo and multidrug resistance sub-line LoVo/DX used in our experiments were kindly provided by Joanna Wietrzyk, professor of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Science, Wrocław. The studied cancer cells were cultured in OptiMEM+RPMI 1640 medium (1:1) supplement with 5% heat-inactivated foetal bovine serum, 1% of 2 mM L-glutamine and 1% of 1mM sodium pyruvate, 100 IU/ml penicillin and 100 μg/ml streptomycin.

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cheme 1. Gene

old particles e human can l plates and i the wells w ml. After 24

scopy (Olym harvested by eous osmium

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ehan et al. [2 cold 50% tri or 1 hour, aft he plates left lowed to be i washed with The plates e (pH 10.5,

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As shown gold ions were reduced during exposure to the Trichoderma koningii cell-free filtrate. The nanoparticles formation was visually observed by the color of the reaction mixture changing from colorless to red. A characteristic of all gold colloids is the color which can vary between light red via purple-red to bluish-red [24]. This effect is caused by a surface plasmon resonance (SPR) described by the Mie theory [25]. The appearance of red color in solution containing the cell free filtrate of Trichoderma koningii and gold ions suggested the formation of colloidal gold nanoparticles in the medium. According to the Mie theory, spherical gold nanoparticles exhibit only one SPR band, usually in the region of 500-600 nm, whereas anisotropic particles show two or three bands. Figure 1 shows the UV-vis absorption spectrum recorded from the gold nanoparticles solution after 24 h of reaction. The results indicate that the reaction solution has an absorption maximum at about 524 nm attributed to the surface plasmon resonance band of the gold nanoparticles.

Fig. 1. UV-vis spectrum of the gold nanoparticles synthesized by the cell-free filtrate of Trichoderma koningii

For separation of gold particles synthesized the sucrose gradient technique was applied.

This method is often used to separate organelles or viruses by ultracentrifugation. It was possible to separate the nanoparticles based on their size by a density gradient of 30% to 70% sucrose using centrifuge with low speeds. Fractions of 3,5 mL were collected and monitored for separation by TEM technique. Figure 2A shows that AuNPs are symmetrical and spherical shaped, well distributed without aggregation in solution with average size is about 14±4 nm. Spheres from 10 nm to 14 nm were concentrated in the 30% fraction (Figure 2B) and spheres from 12 nm to 17 nm in the 40% (Figure 2C). Gold nanoparticles have not been observed in fractions 50%-70%.

0.0 0.2 0.4 0.6 0.8 1.0

200 300 400 500 600 700 800

Absorbance

[nm]

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Fig. 2. TEM images of gold nanoparticles synthesized by the cell-free filtrate of Trichoderma koningii: (A) before concentration (A); from fraction collected at 30% (B);

from fraction collected at 40% (C)

In comparison with other separation protocols such as electrophoresis [26, 27], diafiltration [27], chromatography [29], sucrose density gradient separation is easier to carry out and takes less time.

It is well-known that the practical application of gold nanoparticles significantly depends on their time-dependent stability. To investigate the stability of gold nanostructures, the particles concentrated in the 30% sucrose fraction were stored at room temperature for the period of 6 months. Any precipitation is not observed even after 6 months of storage suggesting that these colloidal gold nanoparticles are extremely stable. Such long-term stability of the particles indicated that nanostructures are stabilized in the solution by the capping agent, which is likely to be protein secreted by Trichoderma koningii. FTIR spectroscopy measurements were carried out to identify the molecules that bound specially on the gold surface. Representative spectrum of the obtained nanoparticles shows the presence of absorption peaks located at about 3410 cm-1 , 1560 cm-1, 1350 cm-1 and 1065 cm-1 (Figure 3).

Fig. 3. FTIR spectrum of AuNPs-30

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Fig. 4. M

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Microscope ima

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nd at 1350 l known tha e amine gro icles by the s es are highly zeta potential properties.

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in the prese As can be see seems that th e more brigh s the interfere to resolve w uorescence fr d nanoparticl

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extracellular materials, particularly nanoparticles [33]. The mechanism of uptake can be dependent on many factors, such as, the physiochemical properties of gold nanoparticles (size and surface properties) and cell type. For example, Rejman et al. showed that the particles of sizes between 50 to 200 nm were taken up primarily by clathrin-mediated endocytosis, while particles of size 500 nm and above were taken up in a caveolin-dependent fashion [34]. Clathrin-mediated endocytosis occurs when gold nanoparticles accumulate on the cell membrane and clathrin-coated pits are formed to transport the NPs into the cell, resulting in the formation of endosomes.

Fig. 5. TEM images of Lovo cancer cells incubated with AuNPs-30: (A) after 2h; (B) after 24h The cytotoxic potential of CF, AuNPs and AuNPs-30 against human cancer cell line LoVo and sub-line LoVo/DX was examined using SRB method [23]. This well-known colorimetric assay estimates cell number indirectly by staining total cellular protein with the dye SRB (sulphorhodamine B is a bright pink aminoxanthine dye). In our study, the cells were treated with different concentrations of AuNPs, AuNPs-30 and CF for 72 hours. The proportions of surviving cells were then estimated and IC50 values (concentrations leading to 50% inhibition of viability) were calculated (Table 1). The data show that the cell-free filtrate strongly and specifically inhibited the proliferation of both cell lines (LoVo and Lovo/DX) with their IC50 values of

Table 1. Cytotoxicity of the cell-free filtrate and gold nanoparticles against colon cancer cell lines LoVo and LoVo/DX

IC50 [μg/ml]

LoVo LoVo/DX CF 1 14.15±2.2

AuNPs 2 33.04±4.9 AuNPs-30 3 186.5±7.3

4.01±1.7 28.88±2.9 146.0±6.9

1 The cell-free filtrate of Trichoderma koningii

2 The gold nanoparticles synthesized by the cell-free filtrate of Trichoderma koningii

3 The gold particles concentrated in the 30% sucrose fraction

14.15±2.2 µg/mL and 4.01±1.7 µg/ mL, respectively, indicating the presence of cytotoxic compounds in the filtrate of Trichoderma koningii. In SRB assay, AuNPs were able to suppress proliferation of Lovo/DX cells more effectively than LoVo, with their IC50 values of 28.88±2.9 μg/mL and 33.04±4.9 μg/mL, respectively. Moreover, the results obtained reveal that AuNPs-30 have no significant cytotoxic effect on the cancer cells tested. Calculated values of IC50 are 186.5±7.3 μg/mL and 146.0±6.9 μg/mL against LoVo and LoVo/DX, respectively (Table 1). It was quantitatively confirmed that the filtrate protein in the gold nanoparticles synthesized by Trichoderma koningii is responsible for their noticeable cytotoxicity. These results strongly

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highlight the importance of comparing the cell-free filtrate toxicity with the original nanoparticle solution as a precious control experiment to understand the origin of the nanoparticles toxicity.

Conclusions

The results presented support the hypothesis that gold nanoparticles can be prepared and separated in a simple, eco-friendly and cost-effective manner. The simply sucrose density gradient technique to separate the gold nanoparticles based on their size could be successfully applied.

Intracellular distribution of the smallest gold nanospheres has been studied with the general conclusion that these nanostructures are able to enter cancer LoVo cells and are trapped in vesicles, but are not able to enter the nucleus. Moreover, experimental results strongly indicate that the cell-free filtrate of Trichoderma koningii is responsible for the cytotoxicity of the gold nanoparticles against human cancer cell line LoVo and sub-line LoVo/DX. Even though these results may not accurately predict the in vivo toxicity it does provide a basis for understanding the mechanism of toxicity of nanoparticle uptake at the cellular level.

Acknowledgement

This work was supported by NCN (grant no. NN 507 5150 58). I would like to thank Agnieszka Błażejczyk, MSc student for experimental assistance.

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