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Effect of targeted gold nanoparticles size on acoustic cavitation: An in vitro study on melanoma cells
Journal Pre-proofs Effect of Targeted Gold Nanoparticles Size on Acoustic Cavitation: An in vitro Study on Melanoma Cells Ahmad Shanei, Hadi Akbari-Zadeh, Neda Attaran, Mohammad Reza Salamat, Milad Baradaran-Ghahfarokhi PII: DOI: Reference:
S0041-624X(19)30720-6 https://doi.org/10.1016/j.ultras.2019.106061 ULTRAS 106061
To appear in:
Ultrasonics
Received Date: Revised Date: Accepted Date:
4 August 2018 10 November 2019 29 November 2019
Please cite this article as: A. Shanei, H. Akbari-Zadeh, N. Attaran, M. Reza Salamat, M. Baradaran-Ghahfarokhi, Effect of Targeted Gold Nanoparticles Size on Acoustic Cavitation: An in vitro Study on Melanoma Cells, Ultrasonics (2019), doi: https://doi.org/10.1016/j.ultras.2019.106061
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© 2019 Published by Elsevier B.V.
Effect of Targeted Gold Nanoparticles Size on Acoustic Cavitation: An in vitro Study on Melanoma Cells Ahmad Shanei1, Hadi Akbari-Zadeh1*, Neda Attaran2*, Mohammad Reza Salamat 1, Milad Baradaran-Ghahfarokhi3 1
2
Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Department of Medical Nanotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran
3
Department of Medical Radiation Engineering, Faculty of Advanced Sciences & Technologies, Isfahan University, Isfahan, Iran
*
Corresponding authors: Tel/Fax: +98-214-486-5154. E-mail addresses: [email protected] (N. Attaran),
[email protected] (H. Akbari-Zadeh).
Abstract When a liquid is irradiated with high intensities of ultrasound irradiation, acoustic cavitation occurs. Since cavitation can be fatal to cells, it is utilized to destroy cancer tumors. Considering cavitation onset and bubbles collapse, the required ultrasonic intensity threshold can be significantly decreased in the presence of nanoparticles in a liquid. The effects of gold nanoparticles size on acoustic cavitation were investigated in this in vitro study. For this purpose, ultrasonic waves were used at intensities of 0.5, 1 and 2 W/cm2 and frequency of 1 MHz in the presence of F-Cys-GNPs with 15, 23 and 79 nm sizes and different concentrations (0.2, 1 and 5 µg/ml) in order to determine their effects on the viability of melanoma cells. This was performed at different incubation times 12, 24 and 36 h. The viability of melanoma cells decreased at higher concentrations and sizes of F-Cys-GNPs. The lowest viability of melanoma cells was seen in those containing 79, 23, and 15 nm F-Cys-GNPs. This finding could be explained from the concept that the nucleation sites on the surface of GNPs increase with an increase in size of GNPs, which
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results in an increase in the number of cavitation bubbles. Acoustic cavitation in the presence of gold nanoparticles can be used as a way for improving therapeutic effects on the tumors.
Highlights
Using gold nanoparticles as a nucleation site to decrease the threshold intensity of the cavitation
Nucleation sites on the surface of gold nanoparticles increase with the increased size of nanoparticles
Acoustic cavitation in the presence of gold nanoparticles with the appropriate amount and size has been introduced as an approach to improve the therapeutic effects on tumors
Keywords Gold nanoparticle; Acoustic cavitation; Melanoma cells; Cancer cells targeting
1. Introduction In recent years, different applications of therapeutic ultrasound as an efficient way of tumor treatment have been successfully developed. This development is based on ultrasound waves’ interactions with tissues, which creates biological effects [1]. Acoustic cavitation is considered one of the most important bio effects of ultrasound, which is characterized by the formation, oscillation and collapse of bubbles in media irradiated with ultrasound waves [2–5]. In this regard, two types of cavitations exist; namely, stable cavitation and inertial cavitation. In the former, the bubbles oscillate around an equilibrium radius without collapsing, which happens during a considerable number of acoustic cycles. The latter case occurs at high ultrasound intensities and can be fatal to cancerous cells [2,6]. Currently, increasing ultrasound efficacy for therapeutic applications via reducing the threshold intensity for the occurrence of inertial cavitation and providing targeted treatment for the selected tissue are the main concerns of researchers [7].
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The collapse of these bubbles produces high temperature and pressure (5,000 K and 800 Pa). This great energy causes pyrolysis reaction that ultimately leads to the production of reactive oxygen species (ROS) [8,9], which can cause cell death by damage to DNA, protein, lipids, and mitochondria [10]. On the basis of a few reports, considering cavitation onset and bubbles collapse, the required ultrasonic intensity threshold can be significantly decreased in the presence of particles and dissolved gases in a liquid. This may provide nucleation sites for cavitation bubbles and is also responsible for increasing the quantity of bubbles when the liquid is irradiated by ultrasound [11,12]. Chen et al. stated that in the presence of the micro particles, cavitation increases due to an increase in the number of cavities in the liquid [13]. In 2005, Tuziuti et al. showed that the alumina particles increase the output of sono chemical reaction in an aqueous KI solution though it was mainly related to the amount and size of the particles [14]. They also found a relation between the acoustic cavitation and the oxidation reaction index of a KI solution during ultrasound irradiation [14]. On the other hand, in the past few years, usage of gold nanoparticles (GNPs) for biological and nano medical applications has been dramatically increased due to their excellent biocompatibility, inertness, stability, lack of cytotoxic effects, ability to be functionalized with various biochemical molecules, and strong spectroscopic capabilities [15–17]. Moreover, the size of GNPs can easily be controlled during the synthesis by adjusting the ratio gold salt to reducing agents in the chemical reaction [18]. Furthermore, in vivo studies have indicated that GNPs have the ability to be distributed in different organs and tissues within the body, and they can even penetrate subcellular organelles [19]. When GNPs are accumulated at the tumor site, a variety of methods can be used
for diagnosis and treatments of cancer cells [20,21]. In this study, the combination of ultrasound waves with a cavitation enhancing agent (GNPs) was studied as a means of increasing the efficacy of cancer treatment. Various approaches, such as folic acid targeting have been suggested for targeting cancer cells. Folic acid is water-soluble and exists in both cancerous and healthy cells due to the folic acid-receptor. It is one of the Bcomplex group vitamins and is essential for proper cell function, especially with an important 3
role in the DNA nucleotide synthesis and cell division. Therefore, the cells need it to continue their life, however, cancer cells require more folic acid due to their high growth rates [22]. It has been suggested that this receptor makes an appropriate targeting agent due to its particular characteristics such as relatively overexpression level in cancerous tissues, low expression in normal tissues, and its possible conjugation GNPs [23]. The uptake of nanoparticles in cancer cells depends on a variety of factors such as size, shape, and concentration [24]. The conjugation of folic acid to nanoparticles was performed for the same uptake of GNPs in cells at different sizes. Shanei et al. evaluated the effect of GNPs’ size on acoustic cavitation using chemical dosimetry method [25]. They showed that the nucleation sites on the surface of particles increase with the increased size of particles, which results in an increase in the number of cavitation bubbles [25]. In this study, the effect of cavitation due to the change in the GNPs’ size on the viability of melanoma cells was evaluated. For this purpose, GNPs with different sizes (13, 23 and 79 nm) and concentrations (0.2, 1 and 5 µg/ml) in combination with 1 MHz ultrasound (including 0.5, 1 and 2 W/cm2 for 3 min) at three different post-treatments (12, 24 and 36 h) times were considered.
2. Materials and Methods 2.1 Materials and characterization techniques All the chemical compounds used in this research were purchased from Merck (Germany), Fluka (Switzerland) and Sigma Aldrich (USA). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4.3H2O, 99.5% purity) and sodium citrate dihydrate (C6H5Na3O7.2H2O) were purchased from Merck. Folic acid (FA) was obtained from Sigma Aldrich, USA, and deionized water was also used in the experiments.
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UV–visible spectra were recorded by using a SPEKOL 2000 (Analytik Jena, UK) spectrophotometer. Transmission electron microscopy (TEM) was performed using a Zeiss EM 900 and it was used to investigate morphology and size of GNPs. The surface charge and hydrodynamic size were determined by a ZEN 3600 nanosizer (Malvern, UK) and Dynamic Light Scattering method (DLS), respectively.
2.2. Synthesis of gold nanoparticles in different sizes 2.2.1. Synthesis of <20 nm gold nanospheres 13 nm GNPs were synthesized by the citrate reduction of HAuCl4 [20,26,27]. The glassware was cleaned in aqua regia (3 parts HCl, 1 part HNO3), rinsed with H2O, and then oven-dried prior to use. A mixture containing 5 mL of HAuCl4 (0.2%, w/w) and 90 mL of water was prepared and refluxed under stirring. Next, 5 mL of sodium citrate trihydrate solution (1%, w/w) was quickly added, and after the color changed from pale yellow to deep red, the solution was refluxed for an additional 15 min. This process allowed the solution to cool down to room temperature. The nanoparticles made in this way typically have an average size around 13 nm.
2.2.2. Synthesis of 20 to 100 nm gold nanospheres Various sizes of nanoparticles, from 20 to 100 nm, were synthesized according to the method described by Frens [28]. For this step, 50 mL of 0.01% HAuCl4 solution was heated to boiling temperature while stirring in a 100 mL round bottom flask. Then, a few hundred µL of 1% of trisodium citrate solution was quickly added to HAuCl4 solution so that the color was changed from yellow to red or purple color within several minutes, depending on the sizes of the nanoparticles. The amount of citrate solution determines the size of the nanoparticles [29]. The 350 µL and 230 µL of 1% of trisodium citrate solution obtain GNPs with around 23 nm and 79 nm in diameters, respectively.
2.3. Conjugation of Cysteamine to Folic Acid
5
First, the folic acid N-hydroxysuccinimide active ester (FA-NHS) was synthesized by the previously reported method, as follows [20]: A solution of anhydrous dimethyl sulfoxide and triethylamine was stirred for 10 min. Then, 0.25 g folic acid was gradually added to this mixture and continuously stirred in the dark overnight. Next, folic acid was mixed with 0.1 g dicyclohexylcarbodiimide and 0.1 g Nhydroxysuccinimide, and the mixture was stirred for further 24 h. The by-product, dicyclohexylurea (DCU), was removed by filtration. DMSO and triethylamine were evaporated under vacuum. Second, for the preparation of cysteamine-folic acid conjugate, vacuum dried FA-NHS was dissolved into the mixture of DMSO and trimethylamine. 0.1 g cysteamine was then added to the mixture and stirred overnight. The resulting yellow solid cysteamine-folic acid conjugate was obtained by filtration and then with twice washing by ethylether.
2.4. Preparation of GNPs with Cysteamine-Folic Acid Conjugate The adhesion of nanoparticles into the organic molecules has been studied on several occasions. In this work, GNPs were conjugated with folic acid via cysteamine as a thiol linker. The cysteamine-folic acid was bound to the GNPs’ surface by a single coupling reaction to produce cysteamine-folic acid conjugated GNPs (F-Cys-GNPs). Conjugation of cysteamine-folic acid on the GNPs surface was carried out as follows [20]: The dried cysteamine-folic acid conjugate was gradually added to the solution of the prepared GNPs and the mixture was stirred for 4 h at room temperature in order to obtain cysteaminefolic acid conjugated GNPs (F-Cys-GNPs). Finally, the prepared F-Cys-GNPs were purified by centrifugation and double re-precipitation from distilled water. The steps for the procedure of preparing F-Cys-GNPs were shown in Figure 1 according to which, in the first step, the conjugation of cysteamine to folic Acid is carried out in the presence of dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), and in the second step, the cysteamine-folic acid compound is conjugated on the freshly prepared GNPs surface to obtain F-CysGNPs.
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Figure 1. Preparation of GNPs with Cysteamine-Folic Acid Conjugate: First step: the conjugation of cysteamine to folic Acid in the presence of DCC and NHS; Second step: the conjugation of cysteamine-folic acid on GNPs surface
2.5. Characterization techniques 2.5.1. Optical studies of particles Ultraviolet–visible (UV–Vis) spectroscopy is one of the important characterization techniques to study the optical properties of GNPs and GNPs conjugated with cysteamine-folic acid (F-Cys-GNPs). These spectra were recorded by using a SPEKOL 2000 (Analytik Jena, UK) spectrophotometer in a quartz cuvette with 1.0 cm path length. Fourier transform infrared spectroscopy is used to study functional groups on the surface of materials using the discrete energy levels for vibrations of atoms in these groups. Therefore, it was used to verify the conjugation of folic acid to GNPs. FTIR spectra of F-Cys-GNPs and FA were recorded by using Avatar 370 FT-IR Therma Nicolet spectrometer.
2.5.2.
Morphology and size studies of particles
Transmission electron microscopy (TEM) was performed using a Zeiss EM 900 and it was used to investigate the morphology and size of GNPs and its conjugation with cysteamine-folic acid. The samples 7
for TEM measurements were prepared by placing a droplet of the colloidal solution onto a carbon-coated copper grid and allowing it to dry in the air naturally. Based on the TEM images, the size distributions of the final product were determined by counting at least 300 particles. The surface charge and hydrodynamic size were determined by a ZEN 3600 nanosizer (Malvern, UK) and Dynamic Light Scattering method (DLS), respectively.
2.6. Cell culture Melanoma cancer cells were purchased from Iran Cell Bank of Pasteur Institute (Tehran, Iran). The cells were cultured in 25 cm2 culture flasks in Dulbecco’s modified Eagle’s medium (DMEM Gibco Laboratories, CergyPontoise, France) supplemented with 10 % fetal bovine serum (FBS GibcoLaboratories, CergyPontoise, France), 2 mM glutamine, 100 U penicillin per ml, and 100 mg streptomycin per ml (Gibco Laboratories, CergyPontoise, France). They were grown in a humidified cell incubator at 37°C under 5% CO2 atmosphere and 95% air. Melanoma cells separated from the flask surface when the cells filled 70% of the flask using Trypsin-EDATA and seeded in the plates after counting. Before applying treatment protocols, they were allowed to adhere and grow overnight in cell culture medium.
2.7. Ultrasound generator system Ultrasound irradiation was conducted with a therapeutic ultrasound unit (215X; a coproduct of Novin Medical Engineering Co, Tehran, Iran; and EMS Co, Reading, Berkshire, England) in a continuous mode at a frequency of 1 MHz with intensities of 0.5, 1 and 2 W/cm2 for 3 minutes. Acoustic calibration for the power of the device was performed in a degassed water tank using an ultrasound balance power meter (UPM 2000; Netech Corporation, Grand Rapids, MI) with uncertainty of ±1 mW. All quoted intensities were of spatial-temporal average in our experiments. An ultrasound transducer with a surface area of 7.0 cm2 was horizontally submerged at the bottom of a container filled with degassed water and was vertically irradiated to the cells. 8
2.8. Experimental design Various steps of the experiment are as follows: 1- Cell seeding: Melanoma cancer cells were grown in 96-well and 24-well plates. 2- Adding F-Cys-GNPs: The cells were treated with F-Cys-GNPs in different concentrations and sizes. After completion of 12 h incubation time, the supernatant medium of the well was completely discarded and then, the cancerous cells were washed with PBS (three times) to remove the extra nanoparticles. Afterwards, the fresh culture medium was added to them again. 3- Ultrasound irradiation (US): Ultrasound exposure time was set at 3 min for evaluation of different intensities of ultrasound wave. For the 96-well plate, the three adjacent wells of 6.6 mm diameter were simultaneously exposed to ultrasound. However, for the 24-well plate, a single well of 15.6 mm diameter was independently exposed to ultrasound. 4- Cell survival assay: The cell survival was investigated using MTT and trypan blue assay after 12, 24 and 36 h from the interventions. The steps applied to the cells were proportional to the treatment groups. The treatment groups are as follows: (A) Control group: With no treatment; (B) F-Cys-GNPs only: Treated only with F-Cys-GNPs (15, 23, and 79 nm) at different concentrations (0.2, 1 and 5 µg/ml); (C) US only: Treated only with ultrasound wave at 0.5, 1 and 2 W/cm2 for 3 min; (D) F-Cys-GNPs + US: Treated with an ultrasound wave in the presence of 15, 23, and 79 nm F-CysGNPs. Different concentrations of F-Cys-GNPs including 0.2, 1 and 5 µg/ml, and ultrasonic intensities, as well as 0.5, 1 and 2 W/cm2 were used at a frequency of 1 MHz on this treatment group. Table 1 shows the summary of intervention in different treatment groups. To avoid the variability inherent to the test used, all assays were done for three independent experiments.
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Table. 1: Type of intervention in different treatment groups; the interval between each intervention was 12 h. Interventions
Cell
Cell
Cell
Survival
Survival
Survival
Assay
Assay
Assay
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Cell
Adding F-
US
Seeding
Cys-GNPs
Irradiation
Groups Control
*
F-Cys-GNPs *
*
only Ultrasound only
*
F-Cys-GNPs + *
*
Ultrasound
2.9. Viability tests 2.9.1. MTT Assay The viability of cells was determined through MTT assay, which is a widely used test for cell viability and is based on the reduction of the yellow tetrazolium dye (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT)) to a purple water insoluble formazan in mitochondria of living cells [30–33]. Cells were cultured in a 96-well plate at the concentration of 5,000 cells in each well. After the incubation of cells for 12 h in these wells, they were exposed to different interventions (F-Cys-GNPs and ultrasound waves) as described in experimental group section. The cells were incubated again for three different times (12, 24 and 36 h). Then, after washing the cells with PBS, 90 µL of fresh medium was added to each plate. Next, 10 µL of MTT solution (50 mg/mL in PBS) was added to them and the plates were incubated for 4 h. In the end, the supernatant was removed and 100 µL of DMSO was added to the wells and incubated for 20 min in dark by shaking. The absorbance of the solution was read at 570 nm by using a microplate Elisareader (Bio-Rad, USA) to measure cell viability.
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The absorbance value for each group was converted into cell viability as follows: the average absorbance value of the control group was taken as 100%; the absorbance of each experimental group was expressed as a percentage of control group value.
(1)
2.9.2. Trypan Blue Exclusion Assay
The trypan blue exclusion assay is one of the common cell survival assays. With this test, living cells can be detected from dead cells by staining. In the living cell, the cell membrane controls the entry of substances into the membrane, but due to lack of this control in the dead cells, trypan blue can penetrate through the membrane and turn them to blue. This assay was done in 24-well plate with 50,000 cells per well.
In this study, the trypan blue assay was done as follows: Initially, the cells were detached from the surface of the 24-well plate; and after centrifugation, trypan blue was added to them. Finally, the counting process of the number of the dead and living cells was performed under a microscope using a hemocytometer.
Viability is also indicated by:
Viability =
× 100
(2)
2.10. Cellular uptake Inductively coupled plasma mass spectrometry (ICP-MS) was used in order to assess the F-Cys-GNPs uptake in melanoma cells. For this purpose, 5 × 105 melanoma cancer cells were cultured in a 6-well plate and they were intervened with the nanoparticles of different sizes. After 12 h incubation time, the noninternalized GNPs were discarded from the wells, and the cells were washed three times using PBS. Afterwards, the cells were detached from the plate bottom using Trypsin-EDTA and the cell suspensions 11
were centrifuged. The residue cells were initially counted with a hemocytometer and then were completely digested by adding a mixture of 37% hydrochloric acid (HCl, 1.5 ml) and 69% nitric acid (HNO3, 0.5 ml) to them. The melanoma cancer cells were sonicated in an ultrasonic bath at 70 °C for 3 h for complete digestion. Finally, the samples were diluted to a volume of 4 mL for cellular uptake analysis.
2.11. Statistical analysis All statistical analyses were done in the SPSS software (version 16.0, Chicago, IL). Mean values and standard deviations were calculated, and statistical significance of the differences between the studied groups was evaluated. All data were analyzed using one-way ANOVA and Tukey test after performing the normality test. A significant level of 0.05 was considered to compare the data.
3. Results and Discussion 3.1. F-Cys-GNPs and GNPs Synthesis and Characterization GNPs were chosen 13 nm in size in order to characterize the synthesized F-Cys-GNPs and verify the conjugation of folic acid to GNPs. UV-Vis spectrometry was utilized to characterize the synthesized FCys-GNPs (Figure 2). It is clear that the F-Cys-GNPs display a typical surface plasmon broadened band at 705 nm, indicating formation of GNPs. The unmodified GNPs show characteristic surface plasmon absorption at 520 nm. As shown in figure 2, this red-shift in the position of the absorption peak indicates that FA has interacted with GNPs. In addition, the absorption peak at 272 nm is likely ascribed to the attached FA moieties [34]. The adsorption of molecules on the surface of GNPs might also perturb the electron density into the metal nanoparticle and change the interaction with the light, a phenomenon which brings change in the SPR band (Figure 2) [28].
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Figure 2. UV-Vis spectra of GNPs and GNPs conjugated with cysteamine-folic acid
Figure 3 gives the Fourier transform infrared spectra (FTIR) of F-Cys-GNPs. The FA verified the conjugation of folic acid to GNPs (Figure 3). In this figure, the band at 1315 cm-1 corresponds to stretching vibration of -NH2 in folate. C=O stretching band in carboxyl acids was observed at 1627 cm-1 and 1677 cm-1. While the band at 1516 cm-1 belongs to the C=O bond stretching vibration of –CONHgroup. The bands between 3000 and 3500 cm-1 are related to the O-H stretching and NH-stretching vibration bands of folic acid and cysteamine [34]. The absence of absorption band at 2100-2300 cm-1 is because of –SH absorption on the surface of the GNPs. It shows that –SH coordinates with GNPs on the surface of the particles. The band at 2850-2927 cm-1 relates to asymmetric and symmetric C-H stretching vibrations of –CH2.
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Figure 3. FTIR spectra of F-Cys-GNPs (green line) and FA (blue line)
The hydrodynamic sizes of the GNPs and F-Cys-GNPs measured via DLS were 22.4 nm and 33.8 nm, respectively (Figure 4). Zeta potential measurements were also employed to confirm a good reaction. Figure 5, shows the surface potential of the GNPs (-12.5 mV) and F-Cys-GNPs (-28.5 mV).
(a)
(b)
Figure 4. Hydrodynamic sizes of (a) GNPs and (b) F-Cys-GNPs [35]
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(a)
(b)
Figure 5. Zeta potential measurements of (a) GNPs and (b) F-Cys-GNPs [35]
The size distribution and morphology of the synthesized GNPs and F-Cys-GNPs were characterized using TEM (Figures 6 and 7). It can be noted that the synthesized GNPs through three various amounts of 1% of trisodium citrate solution have spherical shapes around 15, 23 and 79 nm in diameter. Moreover, the FCys-GNPs obtained from 13 nm GNPs had a spherical shape with a mean diameter of around 15 nm (Figure 6).
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Figure 6. TEM images and size distribuations of (a) ~13 nm GNPs and (b), its conjugation with cysteamine-folic acid
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Figure 7. TEM images and size distributions of (a) ~23 nm GNPs and (b) ~79 nm GNPs
3.2. Effect of F-Cys-GNPs on Cell Survival At first, in order to investigate the combined effects of ultrasound irradiation in the presence of F-CysGNPs, it was necessary to evaluate and measure the cytotoxicity level of the nanoparticles on melanoma cancer cells in order to select the appropriate concentrations. The toxicity of the F-Cys-GNPs with 15, 23 and 79 nm sizes at different concentrations and during 12, 24 and 36 h incubation is shown in Figure 8.
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Figure 8. Melanoma cell viability incubated at the different sizes and concentrations of F-Cys-GNPs for (a) 12, (b) 24 and (c) 36 h
In this study, it was found that the viability of melanoma cells was not affected at the used sizes and 0.2, 1 and 5 µg/ml concentrations of F-Cys-GNPs during the incubation, whereas, under our experimental conditions, F-Cys-GNPs in 10 µg/ml concentration showed detectable cytotoxicity. Until now, there have been a large number of researches concerning GNPs cell cytotoxicity in the living systems. However, there are two conflicting conclusions among the previous investigations. A small group of investigators insisted that GNPs are generally non-toxic while another group of scientists demonstrated the existence of toxicity in their researches [36–39]. The emergence of various opinions is mainly due to the variations in related study factors such as GNPs’ size, shape, surface charge and coating material that can lead to diverse results when determining GNPs’ interactions with biomolecules, cell lines and tissues. According to the published reports, as expected, other factors such as administration routes and doses applied, the time of exposure and examination, assays for assessing cells’ survival, methods for detecting the concentration of GNPs in specific sites and distribution of particles over cells play an important role in corresponding conclusions. Patra et al. have reported that cell type is dependent on response to GNPs [36], while Pan et al. have demonstrated an opposite opinion, indicating that the cytotoxicity of GNPs depends on their size [40]. It has been reported that smaller particle sizes (1 to 2 nm) were found to be highly toxic, while larger sizes were nontoxic [40]. It is necessary to mention that in this study, the 15, 23 and 79 nm nanoparticles were utilized. Chen et al. studied the toxicity of a wide range of injected GNP sizes which were spherical in shape with a diameter of 3 to 100 nm in mice [41]. They found that at the dose they used, the smallest size (less than 5 nm) and the largest size (50 to 100 nm) of GNPs were not toxic. However, they stated that the intermediate size range of 8 to 37 nm had lethal effects on mice. They reported that the systematic toxicity of this size was linked to major organ damage in the liver, spleen, and lungs. While, in the same 19
study, the same size nanoparticles were not toxic on Hela cell line [41]. This study has demonstrated a large inconsistency between the in vitro and in vivo results, and highlights the notion that in vitro experiments may not lead to the same results revealed during in vivo studies. Nevertheless, in vitro studies are a critical and first step for evaluating treatment outcomes of new modalities. Figure 9 shows the internalization value of F-Cys-GNPs in different sizes of them with concentration 5 µg/ml in melanoma cancer cells. These results show that the nanoparticle uptake in 15 and 23 nm does not significantly differ from each other (P> 0.05); however, the absorption of nanoparticles in 79 nm is slightly smaller than that in other two sizes. Considering the small size of the gold particles, it can be anticipated that the cells would internalize the nanoparticle conjugates from the extracellular environment through endocytotic processes. This judgment has been performed on the basis of the reports confirming that the entrance of the GNPs into the cells was carried out by endocytosis [42]. On the other hand, folic acid is one of the most important cells targeting candidates [43]. Cells need it to function properly, while cancer cells require more folic acid due to their high growth rate [22]. The folic acid attached to the GNPs is the best help to absorb in the melanoma cells. The Folate receptor in the cells is the best target for folic acid [44], and F-Cys-GNP can internalize into the cell by absorbing folic acid. In addition, the enhanced penetration and retention effect (EPR effect) stated that molecules with a certain size such as nanoparticles can penetrate the cancerous cells due to cells’ defect like lymphatic drainage. Indeed, this principle predicted that the nanoparticle tendency is more accumulated in cancerous cells than in healthy cells [45].
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Figure 9. Gold content of cells after 12 h of incubation with different sizes of F-Cys-GNPs
3.3. The Ultrasound Irradiation Effect on Cell Survival in the presence of F-Cys-GNPs with Different Sizes The biological effects of ultrasound can be described among the most effective ways of treating cancers. Combining these waves with some sensitizers can improve these effects. In this study, ultrasonic waves were used at intensities of 0.5, 1 and 2 W/cm2 [46] and frequency of 1 MHz in the presence of F-CysGNPs with 15, 23 and 79 nm sizes to determine their effects on the viability of melanoma cells. This was performed at different incubation times 12, 24 and 36 h. The optimum size and concentration of F-CysGNPs were found to be 79 nm and 5 µg/ml, respectively. Figure 10 shows the percentage viability of the melanoma cell line at incubation times of 12, 24 and 36 h for various experimental groups.
21
22
Figure 10. Viability percentage of the melanoma cell line at (a) 12, (b) 24 and (c) 36 h incubation times in various experimental groups
There was no significant difference between the group incubated with 5 µg/ml F-Cys-GNP, size of 79 nm, and the control one at incubation times of 12, 24 and 36 h (P >0.5). Statistical comparison of the results showed a significant difference in cell viability between the ultrasound (1 and 2 W/cm2) irradiated groups and the control groups for incubation times of 12, 24 and 36 h (P <0.05), whereas, there was no significant difference in the cell viability between the ultrasound irradiated group at 0.5 W/cm2 intensity and the control for the studied incubation times (P > 0.3). Ultrasound irradiation appears to be an appropriate method for damaging malignant cells due to the intrinsic cellular responses. Indeed, it could generate microbubbles that fluctuate in response to the ultrasonic waves [47] that will collapse under enough intensity to produce the inertial or stable cavitation [48]. The collapse of these bubbles causes the production of ROS [8], which can ultimately cause cell death by damaging the cell organelles [10]. Moreover, significant synergistic effect was seen when ultrasound irradiation was employed in the presence of F-Cys-GNPs (P < 0.02). Statistical analysis showed that there was a significant difference in cell viability between the US irradiated cells in the presence of F-Cys-GNPs and other groups for 12, 24 and 36 h incubation times (P< 0.02). In this regard, some studies have shown that the presence of GNPs in a liquid phase can increase the possibility of cavitation and the bubbles collapse [12]. In a research on the correlation between the decrease in the cavitation threshold and the particles addition, Tuziuti et al. showed that the existence of particle in a liquid provides a nucleation site for cavitation bubble [14]. Sazgarnia and Shanei have evaluated acoustic cavitation in TA solutions containing GNPs by spectrofluorometry method. They reported that the fluorescence signal for TA solution containing GNPs is higher than the TA solution without GNPs in different intensities [12]. This finding could have been related to two types of actions: (1) GNPs acted as cavitation nuclei, that is, the nanoparticles may have
23
acted as the sites for cavitation and increased the cavitation rate; (2) increased collapse of cavities could have been another feasible process. Figure 10 (a, b and c) shows that any increase in incubation time will be associated with a decrease in the cell viability value in the US, and the US in the presence of F-Cys-GNPs groups. The effect of F-Cys-GNPs concentration on the melanoma cell’s viability in the field of 1 MHz ultrasound waves at the intensity of 2 W/cm2 and 36 h incubation time is presented in Figure 11. In this case, the size of F-Cys-GNPs was adjusted in 79 nm.
Figure 11. Effects of F-Cys-GNPs concentration on the melanoma cell’s viability in the field of 1 MHz ultrasound waves at intensity of 2 W/cm2 and 36 h incubation time
As can be seen in Figure 11, the viability of melanoma cells is reduced by increasing the concentration of F-Cys-GNP. On the basis of our results, in the case of 79 nm F-Cys-GNP at 0.2 µg/ml concentration and 2 W/cm2 intensity, the cancerous cell survival has not had a significant difference with ultrasound group
24
(P >0.4). However, increasing concentration (1 and 5 µg/ml) in these combination has provided a significant difference from ultrasound group (P<0.05). Statistical comparison of the combined group results at 2 W/cm2, intensity and different concentrations show a significant difference in all groups containing F-Cys-GNP when compared with each other (P<0.05) (Figure 11). The viability of melanoma cells is reduced by increasing the F-Cys-GNP concentrations. This is mainly due to a fact that, with an increase in the concentration of F-Cys-GNPs, the sum of surface area also increases, which could contribute to providing nucleation sites of active bubbles for cavitation. This cavitation can be fatal to cells and is used to destroy cancerous tumors. Such findings were also confirmed in similar studies using chemical dosimetry method. Shanei et al. investigated the effects of gold nanoparticle size on acoustic cavitation using chemical dosimetry method [25]. They showed that the recorded fluorescence signal intensity increased at higher concentrations of GNPs. On the basis of those results, a significant difference in the recorded fluorescence signal intensity between TA solutions containing 35 nm GNPs in different concentrations of GNPs and TA solution without GNPs with 2 W/cm2 intensity was observed. The effect of the size of GNPs on the viability of melanoma cells in the field of 1 MHz ultrasound waves at the intensity of 2 W/cm2 for 36 h incubation time is presented in Figure 12. In this case, the concentration of F-Cys-GNPs was adjusted on 5 μg/ml as the lowest ratio of the viability of melanoma cells was obtained with this concentration, as observed in the previous case of F-Cys-GNP dependence.
25
Figure12. Effects of F-Cys-GNPs size on the melanoma cell’s viability in the field of 1 MHz ultrasound waves at intensity of 2 W/cm2 and 36 h incubation time
It can be observed that at finer sizes of the F-Cys-GNPs, the viability of melanoma cells is significantly higher and decreases with the increased size of the F-Cys-GNPs. On the basis of our results, the lowest viability of melanoma cells was seen in cells containing 79, 23, and 15 nm F-Cys-GNPs. This finding could be explained from the concept that the nucleation sites on the surface of GNPs increase with an increase in size of GNPs, which results in an increase in the number of cavitation bubbles. Such findings were also confirmed in similar studies using chemical dosimetry method [25]. On the basis of the results obtained by Shanei et al., the recorded fluorescence signal intensity increased at a higher size of GNPs [25]. In this study, to investigate the effect of the ultrasound intensity on cell destruction, comparison of viability of the melanoma cell line in the experimental groups in the field of 1MHz ultrasound waves at intensities of 0.5, 1, and 2 W/cm2 for incubation time 36 h was studied.
26
Figure 13. Cell viability in various experimental groups in the field of 1MHz ultrasound waves at intensities of 0.5, 1, and 2W/cm2 for incubation time of 36 h
Figure 13 shows that the increase in ultrasound intensity decreases the cell viability value in the US, and the US in the presence of F-Cys-GNPs groups. Statistical comparison of the results showed that the viability of melanoma cells in the groups containing 15, 23 and 79 nm F-Cys-GNPs with ultrasound irradiation were significantly different in comparison to that without F-Cys-GNPs at 0.5, 1 and 2 W/cm2 intensities (P < 0.05). Moreover, significant differences are observed in the cell survival with an increase in the size of nanoparticles at special ultrasound intensity (P<0.05). As expected, cell viability for higher intensities was decreased. The ultrasound irradiation parameters such as intensity irradiation were effective in cavitation production and in turn, in the cell viability.
27
Table. 2: Results of cell survival percentage in the presence of F-Cys-GNPs with different concentrations (0.2, 1, 5 µg/ml) and sizes (15, 23, 79 nm) at 2 W/cm2 intensity and 36 h intervention time by using trypan blue assay F-Cys-GNPs with
F-Cys-GNPs with different sizes
different concentrations
15 nm
23 nm
79 nm
0.2 µg/ml
54.11 ± 2.90
50.14 ± 3.14
46.43 ± 4.85
1 µg/ml
47.80 ± 4.27
42.22 ± 4.24
33.61 ± 3.89
5 µg/ml
40.13 ± 4.66
22.49 ± 3.15
18.45 ± 3.50
Another assay was used to evaluate cell survival in order to justify our data. The viability of the cells after radiation was also tested using the trypan blue exclusion method. The results of this assay at 2 W/cm2 intensity and 36 h post-treatment are shown in Table 2. All of the results obtained with both techniques (trypan blue and MTT assays) showed good consistency with each other without any significant differences. Like MTT assay, the results indicated that optimum size and concentration of the F-Cys-GNPs are 79 nm and 5 µg/ml, respectively. In addition, with an increase in size and concentration of nanoparticles, cell survival decreases probably due to an increase in cavitation amount and its effect on the cells. MTT assays measure the cell mitochondrial function as one of the important organelles in the cell and the easiest target for ROS damage. Damage to this organelle can reduce the cellular survival and the consequence of this damage was investigated with the trypan blue assay. Free radicals produced in the presence of endogenous hydrogen peroxide can cause cellular oxidative stress, which is believed to be one of the key mechanisms of cytotoxicity leading to cell death.
Conclusion
28
This study was performed to investigate the effect of different GNPs sizes’ in combination with various US intensities on Melanoma cancer cells’ viability. This combination effect was measured at various nanoparticles concentrations using MTT and trypan blue assays 12, 24 and 36 h after intervention. Our result revealed that increasing the GNPs sizes’ can enhance their therapeutic effect in combination with US irradiation. In addition, sonosensivity effect on Melanoma cancer cells depended on nanoparticles concentration and US intensity. In addition, these results suggest that better therapeutic effects can be taken if the uptake of larger sizes nanoparticles in the cell can be increased and the side effects can be reduced by localizing the treatment to cancerous cells.
Acknowledgements
This manuscript is a part of a research project supported by Isfahan University of Medical Sciences by scientific code 195043. The authors would like to thank from the staff of the Central Laboratory of Isfahan Medical School who has kindly cooperated in this research. Also, we would like to thank the Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran, for the help in the synthesis of nano samples.
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34
Effect of Targeted Gold Nanoparticles Size on Acoustic Cavitation: An in vitro Study on Melanoma Cells Ahmad Shanei1, Hadi Akbari-Zadeh1*, Neda Attaran2*, Mohammad Reza Salamat 1, Milad Baradaran-Ghahfarokhi3 1
2
Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Department of Medical Nanotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran
3
Department of Medical Radiation Engineering, Faculty of Advanced Sciences & Technologies, Isfahan University, Isfahan, Iran
*
Corresponding authors: Tel/Fax: +98-214-486-5154. E-mail addresses: [email protected] (N. Attaran),
[email protected] (H. Akbari-Zadeh).
Highlights
Using gold nanoparticles as a nucleation site to decrease the threshold intensity of the cavitation
The nucleation sites on the surface of gold nanoparticles increase with the increased size of nanoparticles
Acoustic cavitation in the presence of gold nanoparticles with the appropriate amount and size has been introduced as an approach to improve the therapeutic effects on tumors
35
Declaration of interests
■ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Neda Attaran Department of Medical Nanotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran Tel/Fax: +98-214-486-5154 Email: [email protected]
Ahmad Shanei Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Hadi Akbari-Zadeh Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Mohammad Reza Salamat Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Milad Baradaran-Ghahfarokhi Department of Medical Radiation Engineering, Faculty of Advanced Sciences & Technologies, Isfahan University, Isfahan, Iran November 10, 2019
36
S0041-624X(19)30720-6 https://doi.org/10.1016/j.ultras.2019.106061 ULTRAS 106061
To appear in:
Ultrasonics
Received Date: Revised Date: Accepted Date:
4 August 2018 10 November 2019 29 November 2019
Please cite this article as: A. Shanei, H. Akbari-Zadeh, N. Attaran, M. Reza Salamat, M. Baradaran-Ghahfarokhi, Effect of Targeted Gold Nanoparticles Size on Acoustic Cavitation: An in vitro Study on Melanoma Cells, Ultrasonics (2019), doi: https://doi.org/10.1016/j.ultras.2019.106061
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© 2019 Published by Elsevier B.V.
Effect of Targeted Gold Nanoparticles Size on Acoustic Cavitation: An in vitro Study on Melanoma Cells Ahmad Shanei1, Hadi Akbari-Zadeh1*, Neda Attaran2*, Mohammad Reza Salamat 1, Milad Baradaran-Ghahfarokhi3 1
2
Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Department of Medical Nanotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran
3
Department of Medical Radiation Engineering, Faculty of Advanced Sciences & Technologies, Isfahan University, Isfahan, Iran
*
Corresponding authors: Tel/Fax: +98-214-486-5154. E-mail addresses: [email protected] (N. Attaran),
[email protected] (H. Akbari-Zadeh).
Abstract When a liquid is irradiated with high intensities of ultrasound irradiation, acoustic cavitation occurs. Since cavitation can be fatal to cells, it is utilized to destroy cancer tumors. Considering cavitation onset and bubbles collapse, the required ultrasonic intensity threshold can be significantly decreased in the presence of nanoparticles in a liquid. The effects of gold nanoparticles size on acoustic cavitation were investigated in this in vitro study. For this purpose, ultrasonic waves were used at intensities of 0.5, 1 and 2 W/cm2 and frequency of 1 MHz in the presence of F-Cys-GNPs with 15, 23 and 79 nm sizes and different concentrations (0.2, 1 and 5 µg/ml) in order to determine their effects on the viability of melanoma cells. This was performed at different incubation times 12, 24 and 36 h. The viability of melanoma cells decreased at higher concentrations and sizes of F-Cys-GNPs. The lowest viability of melanoma cells was seen in those containing 79, 23, and 15 nm F-Cys-GNPs. This finding could be explained from the concept that the nucleation sites on the surface of GNPs increase with an increase in size of GNPs, which
1
results in an increase in the number of cavitation bubbles. Acoustic cavitation in the presence of gold nanoparticles can be used as a way for improving therapeutic effects on the tumors.
Highlights
Using gold nanoparticles as a nucleation site to decrease the threshold intensity of the cavitation
Nucleation sites on the surface of gold nanoparticles increase with the increased size of nanoparticles
Acoustic cavitation in the presence of gold nanoparticles with the appropriate amount and size has been introduced as an approach to improve the therapeutic effects on tumors
Keywords Gold nanoparticle; Acoustic cavitation; Melanoma cells; Cancer cells targeting
1. Introduction In recent years, different applications of therapeutic ultrasound as an efficient way of tumor treatment have been successfully developed. This development is based on ultrasound waves’ interactions with tissues, which creates biological effects [1]. Acoustic cavitation is considered one of the most important bio effects of ultrasound, which is characterized by the formation, oscillation and collapse of bubbles in media irradiated with ultrasound waves [2–5]. In this regard, two types of cavitations exist; namely, stable cavitation and inertial cavitation. In the former, the bubbles oscillate around an equilibrium radius without collapsing, which happens during a considerable number of acoustic cycles. The latter case occurs at high ultrasound intensities and can be fatal to cancerous cells [2,6]. Currently, increasing ultrasound efficacy for therapeutic applications via reducing the threshold intensity for the occurrence of inertial cavitation and providing targeted treatment for the selected tissue are the main concerns of researchers [7].
2
The collapse of these bubbles produces high temperature and pressure (5,000 K and 800 Pa). This great energy causes pyrolysis reaction that ultimately leads to the production of reactive oxygen species (ROS) [8,9], which can cause cell death by damage to DNA, protein, lipids, and mitochondria [10]. On the basis of a few reports, considering cavitation onset and bubbles collapse, the required ultrasonic intensity threshold can be significantly decreased in the presence of particles and dissolved gases in a liquid. This may provide nucleation sites for cavitation bubbles and is also responsible for increasing the quantity of bubbles when the liquid is irradiated by ultrasound [11,12]. Chen et al. stated that in the presence of the micro particles, cavitation increases due to an increase in the number of cavities in the liquid [13]. In 2005, Tuziuti et al. showed that the alumina particles increase the output of sono chemical reaction in an aqueous KI solution though it was mainly related to the amount and size of the particles [14]. They also found a relation between the acoustic cavitation and the oxidation reaction index of a KI solution during ultrasound irradiation [14]. On the other hand, in the past few years, usage of gold nanoparticles (GNPs) for biological and nano medical applications has been dramatically increased due to their excellent biocompatibility, inertness, stability, lack of cytotoxic effects, ability to be functionalized with various biochemical molecules, and strong spectroscopic capabilities [15–17]. Moreover, the size of GNPs can easily be controlled during the synthesis by adjusting the ratio gold salt to reducing agents in the chemical reaction [18]. Furthermore, in vivo studies have indicated that GNPs have the ability to be distributed in different organs and tissues within the body, and they can even penetrate subcellular organelles [19]. When GNPs are accumulated at the tumor site, a variety of methods can be used
for diagnosis and treatments of cancer cells [20,21]. In this study, the combination of ultrasound waves with a cavitation enhancing agent (GNPs) was studied as a means of increasing the efficacy of cancer treatment. Various approaches, such as folic acid targeting have been suggested for targeting cancer cells. Folic acid is water-soluble and exists in both cancerous and healthy cells due to the folic acid-receptor. It is one of the Bcomplex group vitamins and is essential for proper cell function, especially with an important 3
role in the DNA nucleotide synthesis and cell division. Therefore, the cells need it to continue their life, however, cancer cells require more folic acid due to their high growth rates [22]. It has been suggested that this receptor makes an appropriate targeting agent due to its particular characteristics such as relatively overexpression level in cancerous tissues, low expression in normal tissues, and its possible conjugation GNPs [23]. The uptake of nanoparticles in cancer cells depends on a variety of factors such as size, shape, and concentration [24]. The conjugation of folic acid to nanoparticles was performed for the same uptake of GNPs in cells at different sizes. Shanei et al. evaluated the effect of GNPs’ size on acoustic cavitation using chemical dosimetry method [25]. They showed that the nucleation sites on the surface of particles increase with the increased size of particles, which results in an increase in the number of cavitation bubbles [25]. In this study, the effect of cavitation due to the change in the GNPs’ size on the viability of melanoma cells was evaluated. For this purpose, GNPs with different sizes (13, 23 and 79 nm) and concentrations (0.2, 1 and 5 µg/ml) in combination with 1 MHz ultrasound (including 0.5, 1 and 2 W/cm2 for 3 min) at three different post-treatments (12, 24 and 36 h) times were considered.
2. Materials and Methods 2.1 Materials and characterization techniques All the chemical compounds used in this research were purchased from Merck (Germany), Fluka (Switzerland) and Sigma Aldrich (USA). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4.3H2O, 99.5% purity) and sodium citrate dihydrate (C6H5Na3O7.2H2O) were purchased from Merck. Folic acid (FA) was obtained from Sigma Aldrich, USA, and deionized water was also used in the experiments.
4
UV–visible spectra were recorded by using a SPEKOL 2000 (Analytik Jena, UK) spectrophotometer. Transmission electron microscopy (TEM) was performed using a Zeiss EM 900 and it was used to investigate morphology and size of GNPs. The surface charge and hydrodynamic size were determined by a ZEN 3600 nanosizer (Malvern, UK) and Dynamic Light Scattering method (DLS), respectively.
2.2. Synthesis of gold nanoparticles in different sizes 2.2.1. Synthesis of <20 nm gold nanospheres 13 nm GNPs were synthesized by the citrate reduction of HAuCl4 [20,26,27]. The glassware was cleaned in aqua regia (3 parts HCl, 1 part HNO3), rinsed with H2O, and then oven-dried prior to use. A mixture containing 5 mL of HAuCl4 (0.2%, w/w) and 90 mL of water was prepared and refluxed under stirring. Next, 5 mL of sodium citrate trihydrate solution (1%, w/w) was quickly added, and after the color changed from pale yellow to deep red, the solution was refluxed for an additional 15 min. This process allowed the solution to cool down to room temperature. The nanoparticles made in this way typically have an average size around 13 nm.
2.2.2. Synthesis of 20 to 100 nm gold nanospheres Various sizes of nanoparticles, from 20 to 100 nm, were synthesized according to the method described by Frens [28]. For this step, 50 mL of 0.01% HAuCl4 solution was heated to boiling temperature while stirring in a 100 mL round bottom flask. Then, a few hundred µL of 1% of trisodium citrate solution was quickly added to HAuCl4 solution so that the color was changed from yellow to red or purple color within several minutes, depending on the sizes of the nanoparticles. The amount of citrate solution determines the size of the nanoparticles [29]. The 350 µL and 230 µL of 1% of trisodium citrate solution obtain GNPs with around 23 nm and 79 nm in diameters, respectively.
2.3. Conjugation of Cysteamine to Folic Acid
5
First, the folic acid N-hydroxysuccinimide active ester (FA-NHS) was synthesized by the previously reported method, as follows [20]: A solution of anhydrous dimethyl sulfoxide and triethylamine was stirred for 10 min. Then, 0.25 g folic acid was gradually added to this mixture and continuously stirred in the dark overnight. Next, folic acid was mixed with 0.1 g dicyclohexylcarbodiimide and 0.1 g Nhydroxysuccinimide, and the mixture was stirred for further 24 h. The by-product, dicyclohexylurea (DCU), was removed by filtration. DMSO and triethylamine were evaporated under vacuum. Second, for the preparation of cysteamine-folic acid conjugate, vacuum dried FA-NHS was dissolved into the mixture of DMSO and trimethylamine. 0.1 g cysteamine was then added to the mixture and stirred overnight. The resulting yellow solid cysteamine-folic acid conjugate was obtained by filtration and then with twice washing by ethylether.
2.4. Preparation of GNPs with Cysteamine-Folic Acid Conjugate The adhesion of nanoparticles into the organic molecules has been studied on several occasions. In this work, GNPs were conjugated with folic acid via cysteamine as a thiol linker. The cysteamine-folic acid was bound to the GNPs’ surface by a single coupling reaction to produce cysteamine-folic acid conjugated GNPs (F-Cys-GNPs). Conjugation of cysteamine-folic acid on the GNPs surface was carried out as follows [20]: The dried cysteamine-folic acid conjugate was gradually added to the solution of the prepared GNPs and the mixture was stirred for 4 h at room temperature in order to obtain cysteaminefolic acid conjugated GNPs (F-Cys-GNPs). Finally, the prepared F-Cys-GNPs were purified by centrifugation and double re-precipitation from distilled water. The steps for the procedure of preparing F-Cys-GNPs were shown in Figure 1 according to which, in the first step, the conjugation of cysteamine to folic Acid is carried out in the presence of dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), and in the second step, the cysteamine-folic acid compound is conjugated on the freshly prepared GNPs surface to obtain F-CysGNPs.
6
Figure 1. Preparation of GNPs with Cysteamine-Folic Acid Conjugate: First step: the conjugation of cysteamine to folic Acid in the presence of DCC and NHS; Second step: the conjugation of cysteamine-folic acid on GNPs surface
2.5. Characterization techniques 2.5.1. Optical studies of particles Ultraviolet–visible (UV–Vis) spectroscopy is one of the important characterization techniques to study the optical properties of GNPs and GNPs conjugated with cysteamine-folic acid (F-Cys-GNPs). These spectra were recorded by using a SPEKOL 2000 (Analytik Jena, UK) spectrophotometer in a quartz cuvette with 1.0 cm path length. Fourier transform infrared spectroscopy is used to study functional groups on the surface of materials using the discrete energy levels for vibrations of atoms in these groups. Therefore, it was used to verify the conjugation of folic acid to GNPs. FTIR spectra of F-Cys-GNPs and FA were recorded by using Avatar 370 FT-IR Therma Nicolet spectrometer.
2.5.2.
Morphology and size studies of particles
Transmission electron microscopy (TEM) was performed using a Zeiss EM 900 and it was used to investigate the morphology and size of GNPs and its conjugation with cysteamine-folic acid. The samples 7
for TEM measurements were prepared by placing a droplet of the colloidal solution onto a carbon-coated copper grid and allowing it to dry in the air naturally. Based on the TEM images, the size distributions of the final product were determined by counting at least 300 particles. The surface charge and hydrodynamic size were determined by a ZEN 3600 nanosizer (Malvern, UK) and Dynamic Light Scattering method (DLS), respectively.
2.6. Cell culture Melanoma cancer cells were purchased from Iran Cell Bank of Pasteur Institute (Tehran, Iran). The cells were cultured in 25 cm2 culture flasks in Dulbecco’s modified Eagle’s medium (DMEM Gibco Laboratories, CergyPontoise, France) supplemented with 10 % fetal bovine serum (FBS GibcoLaboratories, CergyPontoise, France), 2 mM glutamine, 100 U penicillin per ml, and 100 mg streptomycin per ml (Gibco Laboratories, CergyPontoise, France). They were grown in a humidified cell incubator at 37°C under 5% CO2 atmosphere and 95% air. Melanoma cells separated from the flask surface when the cells filled 70% of the flask using Trypsin-EDATA and seeded in the plates after counting. Before applying treatment protocols, they were allowed to adhere and grow overnight in cell culture medium.
2.7. Ultrasound generator system Ultrasound irradiation was conducted with a therapeutic ultrasound unit (215X; a coproduct of Novin Medical Engineering Co, Tehran, Iran; and EMS Co, Reading, Berkshire, England) in a continuous mode at a frequency of 1 MHz with intensities of 0.5, 1 and 2 W/cm2 for 3 minutes. Acoustic calibration for the power of the device was performed in a degassed water tank using an ultrasound balance power meter (UPM 2000; Netech Corporation, Grand Rapids, MI) with uncertainty of ±1 mW. All quoted intensities were of spatial-temporal average in our experiments. An ultrasound transducer with a surface area of 7.0 cm2 was horizontally submerged at the bottom of a container filled with degassed water and was vertically irradiated to the cells. 8
2.8. Experimental design Various steps of the experiment are as follows: 1- Cell seeding: Melanoma cancer cells were grown in 96-well and 24-well plates. 2- Adding F-Cys-GNPs: The cells were treated with F-Cys-GNPs in different concentrations and sizes. After completion of 12 h incubation time, the supernatant medium of the well was completely discarded and then, the cancerous cells were washed with PBS (three times) to remove the extra nanoparticles. Afterwards, the fresh culture medium was added to them again. 3- Ultrasound irradiation (US): Ultrasound exposure time was set at 3 min for evaluation of different intensities of ultrasound wave. For the 96-well plate, the three adjacent wells of 6.6 mm diameter were simultaneously exposed to ultrasound. However, for the 24-well plate, a single well of 15.6 mm diameter was independently exposed to ultrasound. 4- Cell survival assay: The cell survival was investigated using MTT and trypan blue assay after 12, 24 and 36 h from the interventions. The steps applied to the cells were proportional to the treatment groups. The treatment groups are as follows: (A) Control group: With no treatment; (B) F-Cys-GNPs only: Treated only with F-Cys-GNPs (15, 23, and 79 nm) at different concentrations (0.2, 1 and 5 µg/ml); (C) US only: Treated only with ultrasound wave at 0.5, 1 and 2 W/cm2 for 3 min; (D) F-Cys-GNPs + US: Treated with an ultrasound wave in the presence of 15, 23, and 79 nm F-CysGNPs. Different concentrations of F-Cys-GNPs including 0.2, 1 and 5 µg/ml, and ultrasonic intensities, as well as 0.5, 1 and 2 W/cm2 were used at a frequency of 1 MHz on this treatment group. Table 1 shows the summary of intervention in different treatment groups. To avoid the variability inherent to the test used, all assays were done for three independent experiments.
9
Table. 1: Type of intervention in different treatment groups; the interval between each intervention was 12 h. Interventions
Cell
Cell
Cell
Survival
Survival
Survival
Assay
Assay
Assay
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Cell
Adding F-
US
Seeding
Cys-GNPs
Irradiation
Groups Control
*
F-Cys-GNPs *
*
only Ultrasound only
*
F-Cys-GNPs + *
*
Ultrasound
2.9. Viability tests 2.9.1. MTT Assay The viability of cells was determined through MTT assay, which is a widely used test for cell viability and is based on the reduction of the yellow tetrazolium dye (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT)) to a purple water insoluble formazan in mitochondria of living cells [30–33]. Cells were cultured in a 96-well plate at the concentration of 5,000 cells in each well. After the incubation of cells for 12 h in these wells, they were exposed to different interventions (F-Cys-GNPs and ultrasound waves) as described in experimental group section. The cells were incubated again for three different times (12, 24 and 36 h). Then, after washing the cells with PBS, 90 µL of fresh medium was added to each plate. Next, 10 µL of MTT solution (50 mg/mL in PBS) was added to them and the plates were incubated for 4 h. In the end, the supernatant was removed and 100 µL of DMSO was added to the wells and incubated for 20 min in dark by shaking. The absorbance of the solution was read at 570 nm by using a microplate Elisareader (Bio-Rad, USA) to measure cell viability.
10
The absorbance value for each group was converted into cell viability as follows: the average absorbance value of the control group was taken as 100%; the absorbance of each experimental group was expressed as a percentage of control group value.
(1)
2.9.2. Trypan Blue Exclusion Assay
The trypan blue exclusion assay is one of the common cell survival assays. With this test, living cells can be detected from dead cells by staining. In the living cell, the cell membrane controls the entry of substances into the membrane, but due to lack of this control in the dead cells, trypan blue can penetrate through the membrane and turn them to blue. This assay was done in 24-well plate with 50,000 cells per well.
In this study, the trypan blue assay was done as follows: Initially, the cells were detached from the surface of the 24-well plate; and after centrifugation, trypan blue was added to them. Finally, the counting process of the number of the dead and living cells was performed under a microscope using a hemocytometer.
Viability is also indicated by:
Viability =
× 100
(2)
2.10. Cellular uptake Inductively coupled plasma mass spectrometry (ICP-MS) was used in order to assess the F-Cys-GNPs uptake in melanoma cells. For this purpose, 5 × 105 melanoma cancer cells were cultured in a 6-well plate and they were intervened with the nanoparticles of different sizes. After 12 h incubation time, the noninternalized GNPs were discarded from the wells, and the cells were washed three times using PBS. Afterwards, the cells were detached from the plate bottom using Trypsin-EDTA and the cell suspensions 11
were centrifuged. The residue cells were initially counted with a hemocytometer and then were completely digested by adding a mixture of 37% hydrochloric acid (HCl, 1.5 ml) and 69% nitric acid (HNO3, 0.5 ml) to them. The melanoma cancer cells were sonicated in an ultrasonic bath at 70 °C for 3 h for complete digestion. Finally, the samples were diluted to a volume of 4 mL for cellular uptake analysis.
2.11. Statistical analysis All statistical analyses were done in the SPSS software (version 16.0, Chicago, IL). Mean values and standard deviations were calculated, and statistical significance of the differences between the studied groups was evaluated. All data were analyzed using one-way ANOVA and Tukey test after performing the normality test. A significant level of 0.05 was considered to compare the data.
3. Results and Discussion 3.1. F-Cys-GNPs and GNPs Synthesis and Characterization GNPs were chosen 13 nm in size in order to characterize the synthesized F-Cys-GNPs and verify the conjugation of folic acid to GNPs. UV-Vis spectrometry was utilized to characterize the synthesized FCys-GNPs (Figure 2). It is clear that the F-Cys-GNPs display a typical surface plasmon broadened band at 705 nm, indicating formation of GNPs. The unmodified GNPs show characteristic surface plasmon absorption at 520 nm. As shown in figure 2, this red-shift in the position of the absorption peak indicates that FA has interacted with GNPs. In addition, the absorption peak at 272 nm is likely ascribed to the attached FA moieties [34]. The adsorption of molecules on the surface of GNPs might also perturb the electron density into the metal nanoparticle and change the interaction with the light, a phenomenon which brings change in the SPR band (Figure 2) [28].
12
Figure 2. UV-Vis spectra of GNPs and GNPs conjugated with cysteamine-folic acid
Figure 3 gives the Fourier transform infrared spectra (FTIR) of F-Cys-GNPs. The FA verified the conjugation of folic acid to GNPs (Figure 3). In this figure, the band at 1315 cm-1 corresponds to stretching vibration of -NH2 in folate. C=O stretching band in carboxyl acids was observed at 1627 cm-1 and 1677 cm-1. While the band at 1516 cm-1 belongs to the C=O bond stretching vibration of –CONHgroup. The bands between 3000 and 3500 cm-1 are related to the O-H stretching and NH-stretching vibration bands of folic acid and cysteamine [34]. The absence of absorption band at 2100-2300 cm-1 is because of –SH absorption on the surface of the GNPs. It shows that –SH coordinates with GNPs on the surface of the particles. The band at 2850-2927 cm-1 relates to asymmetric and symmetric C-H stretching vibrations of –CH2.
13
Figure 3. FTIR spectra of F-Cys-GNPs (green line) and FA (blue line)
The hydrodynamic sizes of the GNPs and F-Cys-GNPs measured via DLS were 22.4 nm and 33.8 nm, respectively (Figure 4). Zeta potential measurements were also employed to confirm a good reaction. Figure 5, shows the surface potential of the GNPs (-12.5 mV) and F-Cys-GNPs (-28.5 mV).
(a)
(b)
Figure 4. Hydrodynamic sizes of (a) GNPs and (b) F-Cys-GNPs [35]
14
(a)
(b)
Figure 5. Zeta potential measurements of (a) GNPs and (b) F-Cys-GNPs [35]
The size distribution and morphology of the synthesized GNPs and F-Cys-GNPs were characterized using TEM (Figures 6 and 7). It can be noted that the synthesized GNPs through three various amounts of 1% of trisodium citrate solution have spherical shapes around 15, 23 and 79 nm in diameter. Moreover, the FCys-GNPs obtained from 13 nm GNPs had a spherical shape with a mean diameter of around 15 nm (Figure 6).
15
Figure 6. TEM images and size distribuations of (a) ~13 nm GNPs and (b), its conjugation with cysteamine-folic acid
16
Figure 7. TEM images and size distributions of (a) ~23 nm GNPs and (b) ~79 nm GNPs
3.2. Effect of F-Cys-GNPs on Cell Survival At first, in order to investigate the combined effects of ultrasound irradiation in the presence of F-CysGNPs, it was necessary to evaluate and measure the cytotoxicity level of the nanoparticles on melanoma cancer cells in order to select the appropriate concentrations. The toxicity of the F-Cys-GNPs with 15, 23 and 79 nm sizes at different concentrations and during 12, 24 and 36 h incubation is shown in Figure 8.
17
18
Figure 8. Melanoma cell viability incubated at the different sizes and concentrations of F-Cys-GNPs for (a) 12, (b) 24 and (c) 36 h
In this study, it was found that the viability of melanoma cells was not affected at the used sizes and 0.2, 1 and 5 µg/ml concentrations of F-Cys-GNPs during the incubation, whereas, under our experimental conditions, F-Cys-GNPs in 10 µg/ml concentration showed detectable cytotoxicity. Until now, there have been a large number of researches concerning GNPs cell cytotoxicity in the living systems. However, there are two conflicting conclusions among the previous investigations. A small group of investigators insisted that GNPs are generally non-toxic while another group of scientists demonstrated the existence of toxicity in their researches [36–39]. The emergence of various opinions is mainly due to the variations in related study factors such as GNPs’ size, shape, surface charge and coating material that can lead to diverse results when determining GNPs’ interactions with biomolecules, cell lines and tissues. According to the published reports, as expected, other factors such as administration routes and doses applied, the time of exposure and examination, assays for assessing cells’ survival, methods for detecting the concentration of GNPs in specific sites and distribution of particles over cells play an important role in corresponding conclusions. Patra et al. have reported that cell type is dependent on response to GNPs [36], while Pan et al. have demonstrated an opposite opinion, indicating that the cytotoxicity of GNPs depends on their size [40]. It has been reported that smaller particle sizes (1 to 2 nm) were found to be highly toxic, while larger sizes were nontoxic [40]. It is necessary to mention that in this study, the 15, 23 and 79 nm nanoparticles were utilized. Chen et al. studied the toxicity of a wide range of injected GNP sizes which were spherical in shape with a diameter of 3 to 100 nm in mice [41]. They found that at the dose they used, the smallest size (less than 5 nm) and the largest size (50 to 100 nm) of GNPs were not toxic. However, they stated that the intermediate size range of 8 to 37 nm had lethal effects on mice. They reported that the systematic toxicity of this size was linked to major organ damage in the liver, spleen, and lungs. While, in the same 19
study, the same size nanoparticles were not toxic on Hela cell line [41]. This study has demonstrated a large inconsistency between the in vitro and in vivo results, and highlights the notion that in vitro experiments may not lead to the same results revealed during in vivo studies. Nevertheless, in vitro studies are a critical and first step for evaluating treatment outcomes of new modalities. Figure 9 shows the internalization value of F-Cys-GNPs in different sizes of them with concentration 5 µg/ml in melanoma cancer cells. These results show that the nanoparticle uptake in 15 and 23 nm does not significantly differ from each other (P> 0.05); however, the absorption of nanoparticles in 79 nm is slightly smaller than that in other two sizes. Considering the small size of the gold particles, it can be anticipated that the cells would internalize the nanoparticle conjugates from the extracellular environment through endocytotic processes. This judgment has been performed on the basis of the reports confirming that the entrance of the GNPs into the cells was carried out by endocytosis [42]. On the other hand, folic acid is one of the most important cells targeting candidates [43]. Cells need it to function properly, while cancer cells require more folic acid due to their high growth rate [22]. The folic acid attached to the GNPs is the best help to absorb in the melanoma cells. The Folate receptor in the cells is the best target for folic acid [44], and F-Cys-GNP can internalize into the cell by absorbing folic acid. In addition, the enhanced penetration and retention effect (EPR effect) stated that molecules with a certain size such as nanoparticles can penetrate the cancerous cells due to cells’ defect like lymphatic drainage. Indeed, this principle predicted that the nanoparticle tendency is more accumulated in cancerous cells than in healthy cells [45].
20
Figure 9. Gold content of cells after 12 h of incubation with different sizes of F-Cys-GNPs
3.3. The Ultrasound Irradiation Effect on Cell Survival in the presence of F-Cys-GNPs with Different Sizes The biological effects of ultrasound can be described among the most effective ways of treating cancers. Combining these waves with some sensitizers can improve these effects. In this study, ultrasonic waves were used at intensities of 0.5, 1 and 2 W/cm2 [46] and frequency of 1 MHz in the presence of F-CysGNPs with 15, 23 and 79 nm sizes to determine their effects on the viability of melanoma cells. This was performed at different incubation times 12, 24 and 36 h. The optimum size and concentration of F-CysGNPs were found to be 79 nm and 5 µg/ml, respectively. Figure 10 shows the percentage viability of the melanoma cell line at incubation times of 12, 24 and 36 h for various experimental groups.
21
22
Figure 10. Viability percentage of the melanoma cell line at (a) 12, (b) 24 and (c) 36 h incubation times in various experimental groups
There was no significant difference between the group incubated with 5 µg/ml F-Cys-GNP, size of 79 nm, and the control one at incubation times of 12, 24 and 36 h (P >0.5). Statistical comparison of the results showed a significant difference in cell viability between the ultrasound (1 and 2 W/cm2) irradiated groups and the control groups for incubation times of 12, 24 and 36 h (P <0.05), whereas, there was no significant difference in the cell viability between the ultrasound irradiated group at 0.5 W/cm2 intensity and the control for the studied incubation times (P > 0.3). Ultrasound irradiation appears to be an appropriate method for damaging malignant cells due to the intrinsic cellular responses. Indeed, it could generate microbubbles that fluctuate in response to the ultrasonic waves [47] that will collapse under enough intensity to produce the inertial or stable cavitation [48]. The collapse of these bubbles causes the production of ROS [8], which can ultimately cause cell death by damaging the cell organelles [10]. Moreover, significant synergistic effect was seen when ultrasound irradiation was employed in the presence of F-Cys-GNPs (P < 0.02). Statistical analysis showed that there was a significant difference in cell viability between the US irradiated cells in the presence of F-Cys-GNPs and other groups for 12, 24 and 36 h incubation times (P< 0.02). In this regard, some studies have shown that the presence of GNPs in a liquid phase can increase the possibility of cavitation and the bubbles collapse [12]. In a research on the correlation between the decrease in the cavitation threshold and the particles addition, Tuziuti et al. showed that the existence of particle in a liquid provides a nucleation site for cavitation bubble [14]. Sazgarnia and Shanei have evaluated acoustic cavitation in TA solutions containing GNPs by spectrofluorometry method. They reported that the fluorescence signal for TA solution containing GNPs is higher than the TA solution without GNPs in different intensities [12]. This finding could have been related to two types of actions: (1) GNPs acted as cavitation nuclei, that is, the nanoparticles may have
23
acted as the sites for cavitation and increased the cavitation rate; (2) increased collapse of cavities could have been another feasible process. Figure 10 (a, b and c) shows that any increase in incubation time will be associated with a decrease in the cell viability value in the US, and the US in the presence of F-Cys-GNPs groups. The effect of F-Cys-GNPs concentration on the melanoma cell’s viability in the field of 1 MHz ultrasound waves at the intensity of 2 W/cm2 and 36 h incubation time is presented in Figure 11. In this case, the size of F-Cys-GNPs was adjusted in 79 nm.
Figure 11. Effects of F-Cys-GNPs concentration on the melanoma cell’s viability in the field of 1 MHz ultrasound waves at intensity of 2 W/cm2 and 36 h incubation time
As can be seen in Figure 11, the viability of melanoma cells is reduced by increasing the concentration of F-Cys-GNP. On the basis of our results, in the case of 79 nm F-Cys-GNP at 0.2 µg/ml concentration and 2 W/cm2 intensity, the cancerous cell survival has not had a significant difference with ultrasound group
24
(P >0.4). However, increasing concentration (1 and 5 µg/ml) in these combination has provided a significant difference from ultrasound group (P<0.05). Statistical comparison of the combined group results at 2 W/cm2, intensity and different concentrations show a significant difference in all groups containing F-Cys-GNP when compared with each other (P<0.05) (Figure 11). The viability of melanoma cells is reduced by increasing the F-Cys-GNP concentrations. This is mainly due to a fact that, with an increase in the concentration of F-Cys-GNPs, the sum of surface area also increases, which could contribute to providing nucleation sites of active bubbles for cavitation. This cavitation can be fatal to cells and is used to destroy cancerous tumors. Such findings were also confirmed in similar studies using chemical dosimetry method. Shanei et al. investigated the effects of gold nanoparticle size on acoustic cavitation using chemical dosimetry method [25]. They showed that the recorded fluorescence signal intensity increased at higher concentrations of GNPs. On the basis of those results, a significant difference in the recorded fluorescence signal intensity between TA solutions containing 35 nm GNPs in different concentrations of GNPs and TA solution without GNPs with 2 W/cm2 intensity was observed. The effect of the size of GNPs on the viability of melanoma cells in the field of 1 MHz ultrasound waves at the intensity of 2 W/cm2 for 36 h incubation time is presented in Figure 12. In this case, the concentration of F-Cys-GNPs was adjusted on 5 μg/ml as the lowest ratio of the viability of melanoma cells was obtained with this concentration, as observed in the previous case of F-Cys-GNP dependence.
25
Figure12. Effects of F-Cys-GNPs size on the melanoma cell’s viability in the field of 1 MHz ultrasound waves at intensity of 2 W/cm2 and 36 h incubation time
It can be observed that at finer sizes of the F-Cys-GNPs, the viability of melanoma cells is significantly higher and decreases with the increased size of the F-Cys-GNPs. On the basis of our results, the lowest viability of melanoma cells was seen in cells containing 79, 23, and 15 nm F-Cys-GNPs. This finding could be explained from the concept that the nucleation sites on the surface of GNPs increase with an increase in size of GNPs, which results in an increase in the number of cavitation bubbles. Such findings were also confirmed in similar studies using chemical dosimetry method [25]. On the basis of the results obtained by Shanei et al., the recorded fluorescence signal intensity increased at a higher size of GNPs [25]. In this study, to investigate the effect of the ultrasound intensity on cell destruction, comparison of viability of the melanoma cell line in the experimental groups in the field of 1MHz ultrasound waves at intensities of 0.5, 1, and 2 W/cm2 for incubation time 36 h was studied.
26
Figure 13. Cell viability in various experimental groups in the field of 1MHz ultrasound waves at intensities of 0.5, 1, and 2W/cm2 for incubation time of 36 h
Figure 13 shows that the increase in ultrasound intensity decreases the cell viability value in the US, and the US in the presence of F-Cys-GNPs groups. Statistical comparison of the results showed that the viability of melanoma cells in the groups containing 15, 23 and 79 nm F-Cys-GNPs with ultrasound irradiation were significantly different in comparison to that without F-Cys-GNPs at 0.5, 1 and 2 W/cm2 intensities (P < 0.05). Moreover, significant differences are observed in the cell survival with an increase in the size of nanoparticles at special ultrasound intensity (P<0.05). As expected, cell viability for higher intensities was decreased. The ultrasound irradiation parameters such as intensity irradiation were effective in cavitation production and in turn, in the cell viability.
27
Table. 2: Results of cell survival percentage in the presence of F-Cys-GNPs with different concentrations (0.2, 1, 5 µg/ml) and sizes (15, 23, 79 nm) at 2 W/cm2 intensity and 36 h intervention time by using trypan blue assay F-Cys-GNPs with
F-Cys-GNPs with different sizes
different concentrations
15 nm
23 nm
79 nm
0.2 µg/ml
54.11 ± 2.90
50.14 ± 3.14
46.43 ± 4.85
1 µg/ml
47.80 ± 4.27
42.22 ± 4.24
33.61 ± 3.89
5 µg/ml
40.13 ± 4.66
22.49 ± 3.15
18.45 ± 3.50
Another assay was used to evaluate cell survival in order to justify our data. The viability of the cells after radiation was also tested using the trypan blue exclusion method. The results of this assay at 2 W/cm2 intensity and 36 h post-treatment are shown in Table 2. All of the results obtained with both techniques (trypan blue and MTT assays) showed good consistency with each other without any significant differences. Like MTT assay, the results indicated that optimum size and concentration of the F-Cys-GNPs are 79 nm and 5 µg/ml, respectively. In addition, with an increase in size and concentration of nanoparticles, cell survival decreases probably due to an increase in cavitation amount and its effect on the cells. MTT assays measure the cell mitochondrial function as one of the important organelles in the cell and the easiest target for ROS damage. Damage to this organelle can reduce the cellular survival and the consequence of this damage was investigated with the trypan blue assay. Free radicals produced in the presence of endogenous hydrogen peroxide can cause cellular oxidative stress, which is believed to be one of the key mechanisms of cytotoxicity leading to cell death.
Conclusion
28
This study was performed to investigate the effect of different GNPs sizes’ in combination with various US intensities on Melanoma cancer cells’ viability. This combination effect was measured at various nanoparticles concentrations using MTT and trypan blue assays 12, 24 and 36 h after intervention. Our result revealed that increasing the GNPs sizes’ can enhance their therapeutic effect in combination with US irradiation. In addition, sonosensivity effect on Melanoma cancer cells depended on nanoparticles concentration and US intensity. In addition, these results suggest that better therapeutic effects can be taken if the uptake of larger sizes nanoparticles in the cell can be increased and the side effects can be reduced by localizing the treatment to cancerous cells.
Acknowledgements
This manuscript is a part of a research project supported by Isfahan University of Medical Sciences by scientific code 195043. The authors would like to thank from the staff of the Central Laboratory of Isfahan Medical School who has kindly cooperated in this research. Also, we would like to thank the Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran, for the help in the synthesis of nano samples.
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Effect of Targeted Gold Nanoparticles Size on Acoustic Cavitation: An in vitro Study on Melanoma Cells Ahmad Shanei1, Hadi Akbari-Zadeh1*, Neda Attaran2*, Mohammad Reza Salamat 1, Milad Baradaran-Ghahfarokhi3 1
2
Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Department of Medical Nanotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran
3
Department of Medical Radiation Engineering, Faculty of Advanced Sciences & Technologies, Isfahan University, Isfahan, Iran
*
Corresponding authors: Tel/Fax: +98-214-486-5154. E-mail addresses: [email protected] (N. Attaran),
[email protected] (H. Akbari-Zadeh).
Highlights
Using gold nanoparticles as a nucleation site to decrease the threshold intensity of the cavitation
The nucleation sites on the surface of gold nanoparticles increase with the increased size of nanoparticles
Acoustic cavitation in the presence of gold nanoparticles with the appropriate amount and size has been introduced as an approach to improve the therapeutic effects on tumors
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Declaration of interests
■ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Neda Attaran Department of Medical Nanotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran Tel/Fax: +98-214-486-5154 Email: [email protected]
Ahmad Shanei Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Hadi Akbari-Zadeh Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Mohammad Reza Salamat Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Milad Baradaran-Ghahfarokhi Department of Medical Radiation Engineering, Faculty of Advanced Sciences & Technologies, Isfahan University, Isfahan, Iran November 10, 2019
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