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Effect of gold nanoparticle size on acoustic cavitation using chemical dosimetry method
Accepted Manuscript Effect of gold nanoparticle size on acoustic cavitation using chemical dosimetry method Ahmad Shanei, Mohammad Mahdi Shanei PII: DOI: Reference:
S1350-4177(16)30152-3 http://dx.doi.org/10.1016/j.ultsonch.2016.05.010 ULTSON 3220
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
28 April 2015 6 May 2016 6 May 2016
Please cite this article as: A. Shanei, M.M. Shanei, Effect of gold nanoparticle size on acoustic cavitation using chemical dosimetry method, Ultrasonics Sonochemistry (2016), doi: http://dx.doi.org/10.1016/j.ultsonch. 2016.05.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of gold nanoparticle size on acoustic cavitation using chemical dosimetry method
Ahmad Shaneia*, Mohammad Mahdi Shaneib a
Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences,
Darvaze Shiraz Blvd., Isfahan, Iran. b
Nanomaterials group, Department of Materials Engineering, Tarbiat Modares University,
Tehran, Iran.
*Corresponding Author: E-mail address: [email protected]
Tel: +98 31 37929080 Fax: +98 31 36688597
ABSTRACT When a liquid is irradiated with high intensities of ultrasound irradiation, acoustic cavitation occurs. Acoustic cavitation generates free radicals from the breakdown of water and other molecules. Cavitation can be fatal to cells and is utilized to destroy cancer tumors. The existence of particles in liquid provides nucleation sites for cavitation bubbles and leads to decrease the ultrasonic intensity threshold needed for cavitation onset. In the present investigation, the effect of gold nanoparticles with appropriate amount and size on the acoustic cavitation activity has been shown by determining hydroxyl radicals in terephthalic acid solutions containing 15, 20, 28 and 35 nm gold nanoparticles sizes by using 1 MHz low level ultrasound. The effect of sonication intensity in hydroxyl radical production was considered. The recorded fluorescence signal in terephthalic acid solutions containing gold nanoparticles was considerably higher than the terephthalic acid solutions without gold nanoparticles at different intensities of ultrasound irradiation. Also, the results showed that the recorded fluorescence signal intensity in terephthalic acid solution containing finer size of gold nanoparticles was lower than the terephthalic acid solutions containing larger size of gold nanoparticles. Acoustic cavitation in the presence of gold nanoparticles can be used as a way for improving therapeutic effects on the tumors.
Keywords: Acoustic cavitation; gold nanoparticle; chemical dosimetry; terephthalic acid
1. Introduction The cavitation phenomenon is the basis of a variety of mechanical and chemical processes induced in liquids by ultrasound [1]. Acoustic cavitation is characterized by the formation, oscillation and collapse of bubbles in media irradiated with ultrasound [2]. Cavitation can be occurred as stable and transient modes. In stable mode, the bubbles oscillate around an equilibrium radius during a considerable number of acoustic cycles. In transient cavitation, bubbles grow rapidly and expand up to several times of their original size, and violently collapse during a single acoustic compression cycle [2]. In fact, very high shear stresses and shock waves are produced during the collapse. Moreover, high pressure and temperature at the collapse region can produce free radicals. Transient cavitation can be fatal to cells, and is utilized to destroy tumors [3]. There are a number of methods for determining and quantifying cavitation [4]. Sazgarnia et al have studied the cavitation potential via two methods of sonoluminescence detection and terephthalic acid (TA) chemical dosimetry at therapeutic intensities of ultrasound [5]. In this research, TA solution was also utilized as a chemical dosimeter to quantify the free hydroxyl radicals generated by collapse of the transient cavities resulting from low intensity ultrasound. This dosimetry is based on the fluorometric method, which is very sensitive to hydroxyl radical measurement. Acoustic cavitation generates free radicals from the breakdown of water and other molecules. When water is sonicated, OH radicals are formed on thermolysis of H2O. The initial step in the decomposition of water is the production of hydroxyl and hydrogen radicals. Simplified equations for production of free radicals by collapse of cavitation in water solutions are previously described by Sazgarnia and Shanei [6]. Such chemical products also may be used to measure cavitation activity. It has been shown that terephthalic acid (TA) [benzene-1, 4-dicarboxylic acid] is suitable for detecting and quantifying free hydroxyl radicals generated by the collapse of cavitation bubbles in ultrasound irradiations.
During this process, TA solution as a dosimetric solution reacts with a hydroxyl radical generated through water sonolysis. Therefore, 2-hydroxyterephthalic acid is produced that can be detected using fluorescence spectroscopy with an excitation and emission wavelengths of 310 and 425 nm, respectively [7, 8]. Cavitation phenomenon could be facilitated by cavitation nuclei such as gas trapped in solid particles in the medium or in crevices in the walls of a vessel containing the irradiated liquid [9]. On the basis of a few reports, the existence of a particle in a liquid provides a nucleation site for cavitation bubble because of its surface roughness leads to decreased threshold intensity of the cavitation, and is also responsible for increasing the quantity of bubbles when the liquid is irradiated by ultrasound [10,11]. Gold nanoparticles (GNPs) have been characterized as novel nanomaterials for use in cancer therapy because of their special optical properties [12,13]. Their low toxicity, good uptake by mammalian cells, and antiangiogenetic properties make GNPs highly attractive for medical applications [14]. The aim of this investigation was to evaluate the effects of GNPs with appropriate amount and size on acoustic cavitation using chemical dosimetry method.
2. Materials and Methods 2.1. Synthesis and characterization of gold nanoparticles The 15, 20, 28 and 35 nm GNPs were synthesized according to the method described by Frens [15]. Basically, 50 mL of 0.01% HAuCl4 solution (by weight, Exp: 0.01 g in 100 mL water) was heated to boiling while stirring in a 100 mL beaker. Then, 350 µL of 1% (by weight) trisodium citrate solution was quickly added to the auric solution. The solution changed color within several minutes depending on sizes of the nanoparticles. The color change is slower for larger nanoparticles than for small nanoparticles.
The amount of citrate solution determines the size of nanoparticles. The approximate amount of citrate for 15, 20, 28 and 35 nm GNPs is 390, 370, 350 and 340 µL, respectively. Transmission Electron Microscopy (TEM) showed that, the GNPs shape made in this way typically was nearly spherical with an average diameter of 15±3 nm, 20±3 nm, 28±4 nm
and 35±5 nm. 2.2. Ultrasound generator system Ultrasound irradiation was provided a therapeutic ultrasound unit (215X; coproduct of Novin Medical Engineering Co, Tehran, Iran; and EMS Co, Reading, Berkshire, England) in continuous and pulsed modes at a frequency of 1 MHz with an intensity of 0- 2 W/cm2 (ISATA). The frequency range of therapeutic ultrasound was 1-3 MHz. 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 spatial averaged-temporal averaged in our experiments. An ultrasound transducer with a surface area of 7.0 cm2 was horizontally submerged in the bottom of a glass container filled with degassed water. 2.3. Preparation of terephthalic acid solution The dosimetry solution of TA was prepared according to the standard protocols: containing TA (2 mmol/L, Aldrich) in almost 800 ml deionized water and then treated with 5 ml NaOH (1 M). The solution was stirred for about one hour [7]. The solution was kept in a cool and dark place (4 °C) to prevent photochemical reactions. A cylindrical chamber was constructed from PVC in order to sonicate the TA solution (5 cm3). Chamber’s floor was made from a thin acoustically transparent parafilm layer. 2.4. Calibration curve in fluorescence measurement Initially 2-hydroxyterephthalic acid was synthesized and a stock solution of the standard 2 hydroxyterephthalic acid (HTA) was prepared by reaction of bromoterephthalic acid with sodium hydroxide [16]. Volumes of solution of 500 cm3 were made up with water. The concentration of
the solution with respect to HTA was 2×103 mol/L. The stock solution was diluted initially to a concentration of 2×105 mol/L and then using this solution further dilution of known concentration was made. The fluorescence of each solution was measured using spectrofluorometer with an excitation and emission wavelengths of 310 and 425 nm, respectively. Fluorescence intensity versus HTA concentration was plotted and given a straight line of positive slope for concentration from 1×106 to 1×105 mol/L. 2.5. Experimental protocol of chemical dosimetry The chamber containing TA solution was located in the container filled with degassed water in the near field of probe at 5 mm away from the surface of the probe. To perform experiments under progressive-wave conditions and acoustic reflection free, the inner surfaces of the container was covered by foam [7]. Sonication time of TA solution was 20 min. The measurements were performed on TA solutions containing 15, 20, 28 and 35 nm GNPs sizes and the amount of GNPs was 20-80 mg with step of 20 mg. After ultrasound exposure at different intensities (0.5, 1 and 2 W/cm2) in continuous mode on each solution, its fluorescence signal intensity was recorded using a spectrofluorometer (FP-6200, Jasco, Japan) with an excitation and emission wavelengths of 310 and 425 nm, respectively. The irradiated solutions were kept in a dark place through the experiment and fluorometric assessments and were measured within 2-4 hours after sonication. Before ultrasound irradiation, the fluorescence signal of solutions was also measured. Each experiment was repeated three times on the new samples. 2.6. Data analysis A statistical analysis was performed using SPSS version 16 statistical software. According to the Kolmogorov-Smirnov normality test, the data distribution was normal. Consequently, the fluorescence signal intensity in different solutions and ultrasonic intensities was also compared using one way analysis of variance. Data are presented as Mean ± SD. P<0.05 was considered significant.
3. Results
Hydroxyl radical production was measured in different TA solutions: TA solutions containing 15, 20, 28 and 35 nm GNPs sizes and TA solution without GNPs in the field of 1 MHz ultrasound waves at 0.5, 1 and 2W/cm2 intensities by the TA dosimetry method (excitation wavelength = 310 nm, emission peak wavelength = 420 nm, emission and excitation band width = 5 nm). An example of the fluorescence emission spectrum in TA solutions containing 15, 20, 28 and 35 nm sizes GNPs and with 60 mg amount following 1 MHz ultrasonic irradiation with 2 W/cm2 intensity in continuous mode is presented in Fig.1. Fig. 1 As shown in Fig.1, the highest fluorescence signals were recorded in the TA solutions containing 35, 28, 20 and 15 nm GNPs, respectively. Fig.2 shows the particle-amount dependence of the fluorescence signal intensity in the TA solutions with GNPs to that of a solution without GNPs with 2 W/cm2 intensity in continuous mode. For this case, the size of GNPs was fixed to 35 nm. Fig. 2 As can be seen in Fig.2, the recorded fluorescence signal intensity increased at higher amount of GNPs up to 60 mg. When the added amount of GNPs increased more than 60 mg, however, the recorded fluorescence signal intensity became lower. On the basis of our results, a significant difference in the recorded fluorescence signal intensity between TA solutions containing 35 nm GNPs in the different amounts of GNPs and the TA 2
solution without GNPs with 2 W/cm intensity was observed (P < 0.05). Fig. 3 shows the particle-size dependence of the fluorescence signal intensity in the TA solutions with GNPs to that of a solution without GNPs with 2 W/cm2 intensity in continuous mode. In this case, the amount of GNPs was fixed to 60 mg as the highest ratio of the fluorescence signal intensity was obtained with this amount as observed in the previous case of GNP dependence.
Fig.3 It can be observed that, at finer size of GNPs, the recorded fluorescence signal intensity is low and increase with the increased size of GNPs. As expected, the cavitation activity increased at higher intensities of the sonication and, in this regard, it is instructive to consider the rate of increase on the amount of fluorescence signal intensity for 1 MHz ultrasound therapy system. For this purpose, by analyzing the TA solutions in the absence and presence of GNPs, the effect of intensity of the sonication on cavitation activity was evaluated. The recorded fluorescence signal intensity in the TA solutions in the absence and presence of GNPs (15, 20, 28, 35 nm) at 0.5, 1 and 2 W/cm2 intensities in continuous mode is shown in Fig. 4. In this case, the amount of GNPs was fixed to 60 mg. Fig. 4
Fig.5 shows that any increase in intensity of the sonication will be associated with an increase in the recorded fluorescence signal intensity in all TA solution. Fig. 5 Statistical comparison of the results showed that the recorded fluorescence signal intensity in the TA solutions containing GNPs (15, 20, 28 and 35 nm) were significantly different in comparison to the TA solution without GNPs at different intensities (0.5, 1 and 2 W/cm2 ) (P < 0.05). On the basis of our results, there was a significant difference in the recorded fluorescence signal intensity between TA solutions containing 20, 28 and 35 nm GNPs and TA solution containing 2
15 nm GNPs at 2 W/cm intensity (P < 0.05).
4. Discussion
In the recent years ultrasonic therapy for tumors has been successfully developed [17]. The bioeffects normally associated with ultrasound exposure are caused by heat, mechanical effects, and acoustic cavitation. Among these special effects, inertial acoustic cavitation is
believed to be the most important one [3]. Acoustic cavitation may be defined as the growth, oscillation, and collapse of small stabilized gas bubbles under the influence of the fluctuating pressure field of an ultrasound wave in a fluid medium [18]. In order to quantify cavitation, certain methods are necessary for determining and quantifying cavitation which are widely applicable methods [18]. Fang et al. showed that the TA dosimetry is suitable for detecting and quantifying free hydroxyl radical as a criterion of cavitation production in medical ultrasound fields [19]. Terephthalate allows one to measure sensitively the formation of OH radicals in aqueous solutions via its fluorescent product 2-hydroxyterephthalate by fluorometry [19]. In this research, TA solution was also utilized as a chemical dosimeter to quantify the free hydroxyl radicals generated by collapse of the transient cavities resulting from low intensity ultrasound. Currently, reducing the ultrasound intensity required for the occurrence of transient cavitation and providing selectivity in target tissue are the main concerns of researchers to increase ultrasound efficacy in the therapeutic procedures. A number of investigators have shown that particle addition has a considerable potential to enhance the yield in the sonochemical reaction [11, 20]. Roy et al. showed that, the measured cavitation thresholds were decreased with increasing dissolved gas content and increasing suspended particle concentration [20]. Chen et al. believed that microparticles increase cavitation erosion by increasing the number of cavities in the suspension. It was found that suspensions containing particles would generate cavitation as opposed to those without particles [8]. Tuziuti et al. showed that the existence of particles in a liquid provides a nucleation site for the cavitation bubble due to its surface roughness, and it leads to decrease in the cavitation threshold responsible for the increase in the quantity of bubbles, when the liquid is irradiated by ultrasound [11]. This suggests that the enhancement in the yield of sonochemical reaction by appropriate particle addition comes from an increase in the number of cavitation bubbles. Thus, Tuziuti et al. study clarifies the fact that particle addition has the potential to enhance the yield of the
sonochemical reactions, leading to an increasing interest in using nanoparticles as sonosensitizers
[11]. In this regard, some studies have shown that the presence of GNPs in a liquid can increase the possibility of cavitation and the bubbles collapse [6, 21]. In a previous study, we investigated the acoustic cavitation potential in TA solutions containing of 8 nm GNPs by the spectrofluorometry method [6]. In this study, the effect of GNPs with appropriate amount and size on the acoustic cavitation activity have been investigated by determining hydroxyl radicals in TA solutions containing 15, 20, 28 and 35 nm GNPs by using 1 MHz low level ultrasound. The amount of GNPs was 20-80 mg with step of 20 mg. Our results showed that, the recorded fluorescence signal intensity from TA solutions containing GNPs (15, 20, 28 and 35 nm) was higher than TA solution without GNPs following irradiation of 0.5, 1 and 2 W/cm2 ultrasound waves in sonication times of 10, 20 and 30 min. There are two approaches to utilize GNPs: Using GNP as a nucleation site to decrease threshold intensity of the cavitation, and also for increasing the quantity of bubbles and their collapse when the liquid is irradiated by ultrasound [15]. Consequently, many more OH radicals are created and the higher level of the fluorescence emission is caused. Therefore, it is predicted that as well as GNPs can increase the cavitation rate in the TA solution containing GNPs. In an in vivo investigation, performed by Sazgarnia et al. the acoustic cavitation in the presence of GNPs presented as a way to improve therapeutic effects on the tumors [22]. Those findings revealed that, ultrasound irradiation alone has an insignificant antitumor effect, which is enhanced by ultrasound irradiation and administration of GNPs [22]. As can be seen in Fig. 2, for 35 nm GNPs size, the recorded fluorescence signal intensity increased at higher amount of GNPs up to 60 mg. This is because, with an increase in the add amount of GNPs, the sum of surface area also increases. Increase in the surface area could
contribute to provide nucleation sites of bubbles active for cavitation. When the added amount of GNPs increased more than 60 mg, however, the recorded fluorescence signal intensity became lower. This is caused by the substantial prevention of ultrasound propagation due to the increase in the number of GNPs. On the basis of our results, the highest fluorescence signals were recorded in the TA solutions containing 35, 28, 20 and 15 nm GNPs respectively. This finding could be explained from the concept that the nucleation sites on the surface of GNPs increase with increase in size of GNPs, which results in an increase in the number of cavitation bubbles. The size dependence in this paper is according to that in Ref 11. It can be observed in Fig. 3 that, at finer size of GNPs, the recorded fluorescence signal intensity is low and increase with the increased size of GNPs. Tuziut et al. explained that the probable reason for the low reaction yield at finer size of particle might be due to that the small particles and the fluid that surround bubbles were in motion together and the particles do not necessarily play a role of wall against bubbles to cause asymmetric collapse, leading to the generation of a large number of bubbles [11].
As expected, the amount of fluorescence signal intensity for higher intensities was increased. The ultrasound irradiation parameters such as intensity irradiation are effective in hydroxyl radical production and in turn, in cavition production. The amount of hydroxyl radicals
production versus ultrasound intensity show that, with increasing intensity, the hydroxyl radical production is increased. In Fig. 5, it is well known that cavitation activity increases with increasing acoustic intensity and then reaches a peak followed by a decrease with increasing acoustic intensity. With increasing intensity, the hydroxyl radical production and the fluorescence substances are increased. Although fluorescence measurement has high sensitivity, it is inappropriate at high concentration of the fluorescence substance.
5. Conclusion Acoustic cavitation in the presence of GNPs can be used as a way for improving therapeutic effects on the tumors in ultrasonic therapy. 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. TA dosimetry is a suitable method for monitoring the acoustic cavitation effects by measurement of hydroxyl radicals in medical ultrasound ranges.
Acknowledgements This study was performed in Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. Authors would like to thank Novin Medical Engineering Co, Tehran, Iran, for providing the ultrasound unit. In addition, authors are thankful to Head of the Medicinal Chemistry Department, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran for technical support and advice.
References [1] G. Mark, A. Tauber, R. Laupert, H.P. Schuchmann, D. Schulz, A. Mues, C. von Sonntag, OHradical formation by ultrasound in aqueous solution- Part II: Terephthalate and Fricke
dosimetry and the influence of various conditions on the sonolytic yield, Ultrason. Sonochem. 5 (1998) 41-52. [2] H. Tang, C.C. Wang, D. Blankschtein, R. Langer, An investigation of the role of cavitation in low-frequency ultrasound-mediated transdermal drug transport, Pharm. Res., 19 (2002) 11601169. [3] P. Marmottant, S. Hilgenfeldt, Controlled vesicle deformation and lysis by single oscillating bubbles, Nature, 423 (2003) 153-156. [4] G.J. Price, F.A. Duck, M. Holland, T. Berryman, Measurement of radical production as a result of cavitation in medical ultrasound fields, Ultrason. Sonochem, 4 (1997) 165-171. [5] A. Sazgarnia, A. Shanei, H. Eshghi, M. Hassanzadeh- Khayyat, H. Esmaily, M.M. Shanei, Detection of sonoluminescence signals in a gel phantom in the presence of Protoporphyrin IX conjugated to gold nanoparticles, Ultrasonics, 53 (2013) 29-35. [6] A. Sazgarnia, A. Shanei, Evaluation of acoustic cavitation in terephthalic acid solutions containing gold nanoparticles by the spectrofluorometry method. Article ID:376047, Int. J. Photoenergy, (2012). [7] A.H. Barati, M. Mokhtari-Dizaji, H. Mozdarani, S.Z. Bathaei, Z.M. Hassan, Free hydroxyl radical dosimetry by using 1MHz low level ultrasound waves, Iran. J. Radiat. Res., 3 (2006) 163-169. [8] H. Chen, J. Wang, D. Chen, Cavitation damages on solid surfaces in suspensions containing spherical and irregular microparticles, Wear, 266 (2009) 345-348. [9] M. Hodnett, B. Zequiri, A detector for monitoring the onset of cavitation during therapy-level measurements of ultrasonic power, J. phys. Conf. Ser, 1 (2004) 112-117. [10] C.H. Farney, T. Wu, G. Holt, T.W. Murray, R.A. Roy, Nucleating cavitation from laserilluminated nano-particles, Acoust. Res. Lett., 6 (2005) 138-143.
[11] T. Tuziuti, K. Yasui, M. Sivakumar, Y. Iida, N. Miyoshi, Correlation between cavitation noise and yield enhancement of sonochemical reaction by particle addition, J. Phys. Chem., 109 (2005) 4869-4872. [12] X. Huang, P. K. Jain, I. H. El-Sayed, and M. A. El- Sayed, Plasmonic photothermal therapy (PPTT) using gold nanoparticles, Lasers Med. Sci., 23 (2008) 217-228. [13] G. F. Paciotti, L. Myer, D. Weinreich et al., Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery, Drug Deliv., 1 (2004) 169-183. [14] J. C. Y. Kah, M. C. Olivo, C. G. L. Lee, and C. J. R. Sheppard, Molecular contrast of EGFR expression using gold nanoparticles as a reflectance-based imaging probe, Mol. and Cell. Probe, 22 (2008) 14-23. [15] G. Frens, Controlled nucleation for the regulation of the particle size in monodisperse gold suspension, Nat. Phys. Sci., 241 (1973) 20-22. [16] T. J. Mason, J. P. Lorimer, D. M. Bates, Y. Zhao, Dosimetry in sonochemistry: The use of aqueous terephthalate ion as a fluorescence monitor, Ultrason. Sonochem. 1 (1994) S91-S94. [17] I. Rosental, J.Z. Sostaric, P. Riesz, Sonodynamic therapy a review of the synergistic effects of drug and ultrasound, Ultrason. Sonochem., 11 (2004) 349-363. [18] S.B. Barnett, Conclusions and recommendations on thermal and non-thermal mechanisms for biologic effects of ultrasound, Ultrasound Med. Biol., 24 (1998) 41-49. [19] X. Fang, G. Mark, C. von Sonntag, OH radical formation by ultrasound in aqueous solutions, Part I: the chemistry underlying the terephthalate dosimeter, Ultrason. Sonochem., 3 (1996) 57-63. [20] R.A. Roy, S.I. Madanshetty, R.E. Apfel, An acoustic back scattering technique for the detection of transient cavitation produced by microsecond pulses of ultrasound, J. Acoust. Sco. Am., 87 (1991) 2451-2458.
[21] A. Sazgarnia, A. Shanei, M.M. Shanei, Monitoring of transient cavitation induced by ultrasound and intense pulsed light in presence of gold nanoparticles, Ultrason. Sonochem., 21 (2014) 268-374. [22] A. Sazgarnia, A. Shanei, A.R. Taheri, N. Tayyebi Meibodi, H. Eshghie, N. Attarane, M.M. Shanei, Therapeutic effects of acoustic cavitation in the presence of gold nanoparticles on a colon tumor model, J. Ultrasound Med, 32 (2013) 475-483.
Fig.1. An example of the fluorescence emission spectrum in TA solutions containing 15, 20, 28 and 35 nm sizes with 60 mg amount GNPs following 1 MHz ultrasonic irradiation with 2 W/cm2 intensity, excitation wavelength of 310 nm, emission and excitation band width of 5 nm
Fig.2. Particle-amount dependence of the fluorescence signal intensity in the TA solutions with GNPs to that of a solution without GNPs at 2 W/cm2 intensity
Fig. 3.Particle-size dependence of the fluorescence signal intensity in the TA solutions with GNPs to that of a solution without GNPs at 2 W/cm2 intensity
Fig. 4. Recorded fluorescence signal intensity in the TA solutions in the absence and presence of GNPs (15, 20, 28, 35 nm) at 0.5, 1 and 2 W/cm2 intensities, amount of GNPs of 60 mg
Fig.5. Recorded fluorescence signal intensity in the different TA solutions at 0.5, 1 and 2 W/cm2 intensities, amount of GNPs of 60 mg
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Highlights
>We executed GNPs as a way for improving cavitation > We examined fluorescence signal in the presence of GNPs in 15, 20, 28 and 35 nm sizes > Fluorescence signal intensity TA solution containing finer size of GNPs was lower than the TA solutions containing larger size of GNPs.
S1350-4177(16)30152-3 http://dx.doi.org/10.1016/j.ultsonch.2016.05.010 ULTSON 3220
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
28 April 2015 6 May 2016 6 May 2016
Please cite this article as: A. Shanei, M.M. Shanei, Effect of gold nanoparticle size on acoustic cavitation using chemical dosimetry method, Ultrasonics Sonochemistry (2016), doi: http://dx.doi.org/10.1016/j.ultsonch. 2016.05.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of gold nanoparticle size on acoustic cavitation using chemical dosimetry method
Ahmad Shaneia*, Mohammad Mahdi Shaneib a
Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences,
Darvaze Shiraz Blvd., Isfahan, Iran. b
Nanomaterials group, Department of Materials Engineering, Tarbiat Modares University,
Tehran, Iran.
*Corresponding Author: E-mail address: [email protected]
Tel: +98 31 37929080 Fax: +98 31 36688597
ABSTRACT When a liquid is irradiated with high intensities of ultrasound irradiation, acoustic cavitation occurs. Acoustic cavitation generates free radicals from the breakdown of water and other molecules. Cavitation can be fatal to cells and is utilized to destroy cancer tumors. The existence of particles in liquid provides nucleation sites for cavitation bubbles and leads to decrease the ultrasonic intensity threshold needed for cavitation onset. In the present investigation, the effect of gold nanoparticles with appropriate amount and size on the acoustic cavitation activity has been shown by determining hydroxyl radicals in terephthalic acid solutions containing 15, 20, 28 and 35 nm gold nanoparticles sizes by using 1 MHz low level ultrasound. The effect of sonication intensity in hydroxyl radical production was considered. The recorded fluorescence signal in terephthalic acid solutions containing gold nanoparticles was considerably higher than the terephthalic acid solutions without gold nanoparticles at different intensities of ultrasound irradiation. Also, the results showed that the recorded fluorescence signal intensity in terephthalic acid solution containing finer size of gold nanoparticles was lower than the terephthalic acid solutions containing larger size of gold nanoparticles. Acoustic cavitation in the presence of gold nanoparticles can be used as a way for improving therapeutic effects on the tumors.
Keywords: Acoustic cavitation; gold nanoparticle; chemical dosimetry; terephthalic acid
1. Introduction The cavitation phenomenon is the basis of a variety of mechanical and chemical processes induced in liquids by ultrasound [1]. Acoustic cavitation is characterized by the formation, oscillation and collapse of bubbles in media irradiated with ultrasound [2]. Cavitation can be occurred as stable and transient modes. In stable mode, the bubbles oscillate around an equilibrium radius during a considerable number of acoustic cycles. In transient cavitation, bubbles grow rapidly and expand up to several times of their original size, and violently collapse during a single acoustic compression cycle [2]. In fact, very high shear stresses and shock waves are produced during the collapse. Moreover, high pressure and temperature at the collapse region can produce free radicals. Transient cavitation can be fatal to cells, and is utilized to destroy tumors [3]. There are a number of methods for determining and quantifying cavitation [4]. Sazgarnia et al have studied the cavitation potential via two methods of sonoluminescence detection and terephthalic acid (TA) chemical dosimetry at therapeutic intensities of ultrasound [5]. In this research, TA solution was also utilized as a chemical dosimeter to quantify the free hydroxyl radicals generated by collapse of the transient cavities resulting from low intensity ultrasound. This dosimetry is based on the fluorometric method, which is very sensitive to hydroxyl radical measurement. Acoustic cavitation generates free radicals from the breakdown of water and other molecules. When water is sonicated, OH radicals are formed on thermolysis of H2O. The initial step in the decomposition of water is the production of hydroxyl and hydrogen radicals. Simplified equations for production of free radicals by collapse of cavitation in water solutions are previously described by Sazgarnia and Shanei [6]. Such chemical products also may be used to measure cavitation activity. It has been shown that terephthalic acid (TA) [benzene-1, 4-dicarboxylic acid] is suitable for detecting and quantifying free hydroxyl radicals generated by the collapse of cavitation bubbles in ultrasound irradiations.
During this process, TA solution as a dosimetric solution reacts with a hydroxyl radical generated through water sonolysis. Therefore, 2-hydroxyterephthalic acid is produced that can be detected using fluorescence spectroscopy with an excitation and emission wavelengths of 310 and 425 nm, respectively [7, 8]. Cavitation phenomenon could be facilitated by cavitation nuclei such as gas trapped in solid particles in the medium or in crevices in the walls of a vessel containing the irradiated liquid [9]. On the basis of a few reports, the existence of a particle in a liquid provides a nucleation site for cavitation bubble because of its surface roughness leads to decreased threshold intensity of the cavitation, and is also responsible for increasing the quantity of bubbles when the liquid is irradiated by ultrasound [10,11]. Gold nanoparticles (GNPs) have been characterized as novel nanomaterials for use in cancer therapy because of their special optical properties [12,13]. Their low toxicity, good uptake by mammalian cells, and antiangiogenetic properties make GNPs highly attractive for medical applications [14]. The aim of this investigation was to evaluate the effects of GNPs with appropriate amount and size on acoustic cavitation using chemical dosimetry method.
2. Materials and Methods 2.1. Synthesis and characterization of gold nanoparticles The 15, 20, 28 and 35 nm GNPs were synthesized according to the method described by Frens [15]. Basically, 50 mL of 0.01% HAuCl4 solution (by weight, Exp: 0.01 g in 100 mL water) was heated to boiling while stirring in a 100 mL beaker. Then, 350 µL of 1% (by weight) trisodium citrate solution was quickly added to the auric solution. The solution changed color within several minutes depending on sizes of the nanoparticles. The color change is slower for larger nanoparticles than for small nanoparticles.
The amount of citrate solution determines the size of nanoparticles. The approximate amount of citrate for 15, 20, 28 and 35 nm GNPs is 390, 370, 350 and 340 µL, respectively. Transmission Electron Microscopy (TEM) showed that, the GNPs shape made in this way typically was nearly spherical with an average diameter of 15±3 nm, 20±3 nm, 28±4 nm
and 35±5 nm. 2.2. Ultrasound generator system Ultrasound irradiation was provided a therapeutic ultrasound unit (215X; coproduct of Novin Medical Engineering Co, Tehran, Iran; and EMS Co, Reading, Berkshire, England) in continuous and pulsed modes at a frequency of 1 MHz with an intensity of 0- 2 W/cm2 (ISATA). The frequency range of therapeutic ultrasound was 1-3 MHz. 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 spatial averaged-temporal averaged in our experiments. An ultrasound transducer with a surface area of 7.0 cm2 was horizontally submerged in the bottom of a glass container filled with degassed water. 2.3. Preparation of terephthalic acid solution The dosimetry solution of TA was prepared according to the standard protocols: containing TA (2 mmol/L, Aldrich) in almost 800 ml deionized water and then treated with 5 ml NaOH (1 M). The solution was stirred for about one hour [7]. The solution was kept in a cool and dark place (4 °C) to prevent photochemical reactions. A cylindrical chamber was constructed from PVC in order to sonicate the TA solution (5 cm3). Chamber’s floor was made from a thin acoustically transparent parafilm layer. 2.4. Calibration curve in fluorescence measurement Initially 2-hydroxyterephthalic acid was synthesized and a stock solution of the standard 2 hydroxyterephthalic acid (HTA) was prepared by reaction of bromoterephthalic acid with sodium hydroxide [16]. Volumes of solution of 500 cm3 were made up with water. The concentration of
the solution with respect to HTA was 2×103 mol/L. The stock solution was diluted initially to a concentration of 2×105 mol/L and then using this solution further dilution of known concentration was made. The fluorescence of each solution was measured using spectrofluorometer with an excitation and emission wavelengths of 310 and 425 nm, respectively. Fluorescence intensity versus HTA concentration was plotted and given a straight line of positive slope for concentration from 1×106 to 1×105 mol/L. 2.5. Experimental protocol of chemical dosimetry The chamber containing TA solution was located in the container filled with degassed water in the near field of probe at 5 mm away from the surface of the probe. To perform experiments under progressive-wave conditions and acoustic reflection free, the inner surfaces of the container was covered by foam [7]. Sonication time of TA solution was 20 min. The measurements were performed on TA solutions containing 15, 20, 28 and 35 nm GNPs sizes and the amount of GNPs was 20-80 mg with step of 20 mg. After ultrasound exposure at different intensities (0.5, 1 and 2 W/cm2) in continuous mode on each solution, its fluorescence signal intensity was recorded using a spectrofluorometer (FP-6200, Jasco, Japan) with an excitation and emission wavelengths of 310 and 425 nm, respectively. The irradiated solutions were kept in a dark place through the experiment and fluorometric assessments and were measured within 2-4 hours after sonication. Before ultrasound irradiation, the fluorescence signal of solutions was also measured. Each experiment was repeated three times on the new samples. 2.6. Data analysis A statistical analysis was performed using SPSS version 16 statistical software. According to the Kolmogorov-Smirnov normality test, the data distribution was normal. Consequently, the fluorescence signal intensity in different solutions and ultrasonic intensities was also compared using one way analysis of variance. Data are presented as Mean ± SD. P<0.05 was considered significant.
3. Results
Hydroxyl radical production was measured in different TA solutions: TA solutions containing 15, 20, 28 and 35 nm GNPs sizes and TA solution without GNPs in the field of 1 MHz ultrasound waves at 0.5, 1 and 2W/cm2 intensities by the TA dosimetry method (excitation wavelength = 310 nm, emission peak wavelength = 420 nm, emission and excitation band width = 5 nm). An example of the fluorescence emission spectrum in TA solutions containing 15, 20, 28 and 35 nm sizes GNPs and with 60 mg amount following 1 MHz ultrasonic irradiation with 2 W/cm2 intensity in continuous mode is presented in Fig.1. Fig. 1 As shown in Fig.1, the highest fluorescence signals were recorded in the TA solutions containing 35, 28, 20 and 15 nm GNPs, respectively. Fig.2 shows the particle-amount dependence of the fluorescence signal intensity in the TA solutions with GNPs to that of a solution without GNPs with 2 W/cm2 intensity in continuous mode. For this case, the size of GNPs was fixed to 35 nm. Fig. 2 As can be seen in Fig.2, the recorded fluorescence signal intensity increased at higher amount of GNPs up to 60 mg. When the added amount of GNPs increased more than 60 mg, however, the recorded fluorescence signal intensity became lower. On the basis of our results, a significant difference in the recorded fluorescence signal intensity between TA solutions containing 35 nm GNPs in the different amounts of GNPs and the TA 2
solution without GNPs with 2 W/cm intensity was observed (P < 0.05). Fig. 3 shows the particle-size dependence of the fluorescence signal intensity in the TA solutions with GNPs to that of a solution without GNPs with 2 W/cm2 intensity in continuous mode. In this case, the amount of GNPs was fixed to 60 mg as the highest ratio of the fluorescence signal intensity was obtained with this amount as observed in the previous case of GNP dependence.
Fig.3 It can be observed that, at finer size of GNPs, the recorded fluorescence signal intensity is low and increase with the increased size of GNPs. As expected, the cavitation activity increased at higher intensities of the sonication and, in this regard, it is instructive to consider the rate of increase on the amount of fluorescence signal intensity for 1 MHz ultrasound therapy system. For this purpose, by analyzing the TA solutions in the absence and presence of GNPs, the effect of intensity of the sonication on cavitation activity was evaluated. The recorded fluorescence signal intensity in the TA solutions in the absence and presence of GNPs (15, 20, 28, 35 nm) at 0.5, 1 and 2 W/cm2 intensities in continuous mode is shown in Fig. 4. In this case, the amount of GNPs was fixed to 60 mg. Fig. 4
Fig.5 shows that any increase in intensity of the sonication will be associated with an increase in the recorded fluorescence signal intensity in all TA solution. Fig. 5 Statistical comparison of the results showed that the recorded fluorescence signal intensity in the TA solutions containing GNPs (15, 20, 28 and 35 nm) were significantly different in comparison to the TA solution without GNPs at different intensities (0.5, 1 and 2 W/cm2 ) (P < 0.05). On the basis of our results, there was a significant difference in the recorded fluorescence signal intensity between TA solutions containing 20, 28 and 35 nm GNPs and TA solution containing 2
15 nm GNPs at 2 W/cm intensity (P < 0.05).
4. Discussion
In the recent years ultrasonic therapy for tumors has been successfully developed [17]. The bioeffects normally associated with ultrasound exposure are caused by heat, mechanical effects, and acoustic cavitation. Among these special effects, inertial acoustic cavitation is
believed to be the most important one [3]. Acoustic cavitation may be defined as the growth, oscillation, and collapse of small stabilized gas bubbles under the influence of the fluctuating pressure field of an ultrasound wave in a fluid medium [18]. In order to quantify cavitation, certain methods are necessary for determining and quantifying cavitation which are widely applicable methods [18]. Fang et al. showed that the TA dosimetry is suitable for detecting and quantifying free hydroxyl radical as a criterion of cavitation production in medical ultrasound fields [19]. Terephthalate allows one to measure sensitively the formation of OH radicals in aqueous solutions via its fluorescent product 2-hydroxyterephthalate by fluorometry [19]. In this research, TA solution was also utilized as a chemical dosimeter to quantify the free hydroxyl radicals generated by collapse of the transient cavities resulting from low intensity ultrasound. Currently, reducing the ultrasound intensity required for the occurrence of transient cavitation and providing selectivity in target tissue are the main concerns of researchers to increase ultrasound efficacy in the therapeutic procedures. A number of investigators have shown that particle addition has a considerable potential to enhance the yield in the sonochemical reaction [11, 20]. Roy et al. showed that, the measured cavitation thresholds were decreased with increasing dissolved gas content and increasing suspended particle concentration [20]. Chen et al. believed that microparticles increase cavitation erosion by increasing the number of cavities in the suspension. It was found that suspensions containing particles would generate cavitation as opposed to those without particles [8]. Tuziuti et al. showed that the existence of particles in a liquid provides a nucleation site for the cavitation bubble due to its surface roughness, and it leads to decrease in the cavitation threshold responsible for the increase in the quantity of bubbles, when the liquid is irradiated by ultrasound [11]. This suggests that the enhancement in the yield of sonochemical reaction by appropriate particle addition comes from an increase in the number of cavitation bubbles. Thus, Tuziuti et al. study clarifies the fact that particle addition has the potential to enhance the yield of the
sonochemical reactions, leading to an increasing interest in using nanoparticles as sonosensitizers
[11]. In this regard, some studies have shown that the presence of GNPs in a liquid can increase the possibility of cavitation and the bubbles collapse [6, 21]. In a previous study, we investigated the acoustic cavitation potential in TA solutions containing of 8 nm GNPs by the spectrofluorometry method [6]. In this study, the effect of GNPs with appropriate amount and size on the acoustic cavitation activity have been investigated by determining hydroxyl radicals in TA solutions containing 15, 20, 28 and 35 nm GNPs by using 1 MHz low level ultrasound. The amount of GNPs was 20-80 mg with step of 20 mg. Our results showed that, the recorded fluorescence signal intensity from TA solutions containing GNPs (15, 20, 28 and 35 nm) was higher than TA solution without GNPs following irradiation of 0.5, 1 and 2 W/cm2 ultrasound waves in sonication times of 10, 20 and 30 min. There are two approaches to utilize GNPs: Using GNP as a nucleation site to decrease threshold intensity of the cavitation, and also for increasing the quantity of bubbles and their collapse when the liquid is irradiated by ultrasound [15]. Consequently, many more OH radicals are created and the higher level of the fluorescence emission is caused. Therefore, it is predicted that as well as GNPs can increase the cavitation rate in the TA solution containing GNPs. In an in vivo investigation, performed by Sazgarnia et al. the acoustic cavitation in the presence of GNPs presented as a way to improve therapeutic effects on the tumors [22]. Those findings revealed that, ultrasound irradiation alone has an insignificant antitumor effect, which is enhanced by ultrasound irradiation and administration of GNPs [22]. As can be seen in Fig. 2, for 35 nm GNPs size, the recorded fluorescence signal intensity increased at higher amount of GNPs up to 60 mg. This is because, with an increase in the add amount of GNPs, the sum of surface area also increases. Increase in the surface area could
contribute to provide nucleation sites of bubbles active for cavitation. When the added amount of GNPs increased more than 60 mg, however, the recorded fluorescence signal intensity became lower. This is caused by the substantial prevention of ultrasound propagation due to the increase in the number of GNPs. On the basis of our results, the highest fluorescence signals were recorded in the TA solutions containing 35, 28, 20 and 15 nm GNPs respectively. This finding could be explained from the concept that the nucleation sites on the surface of GNPs increase with increase in size of GNPs, which results in an increase in the number of cavitation bubbles. The size dependence in this paper is according to that in Ref 11. It can be observed in Fig. 3 that, at finer size of GNPs, the recorded fluorescence signal intensity is low and increase with the increased size of GNPs. Tuziut et al. explained that the probable reason for the low reaction yield at finer size of particle might be due to that the small particles and the fluid that surround bubbles were in motion together and the particles do not necessarily play a role of wall against bubbles to cause asymmetric collapse, leading to the generation of a large number of bubbles [11].
As expected, the amount of fluorescence signal intensity for higher intensities was increased. The ultrasound irradiation parameters such as intensity irradiation are effective in hydroxyl radical production and in turn, in cavition production. The amount of hydroxyl radicals
production versus ultrasound intensity show that, with increasing intensity, the hydroxyl radical production is increased. In Fig. 5, it is well known that cavitation activity increases with increasing acoustic intensity and then reaches a peak followed by a decrease with increasing acoustic intensity. With increasing intensity, the hydroxyl radical production and the fluorescence substances are increased. Although fluorescence measurement has high sensitivity, it is inappropriate at high concentration of the fluorescence substance.
5. Conclusion Acoustic cavitation in the presence of GNPs can be used as a way for improving therapeutic effects on the tumors in ultrasonic therapy. 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. TA dosimetry is a suitable method for monitoring the acoustic cavitation effects by measurement of hydroxyl radicals in medical ultrasound ranges.
Acknowledgements This study was performed in Department of Medical Physics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. Authors would like to thank Novin Medical Engineering Co, Tehran, Iran, for providing the ultrasound unit. In addition, authors are thankful to Head of the Medicinal Chemistry Department, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran for technical support and advice.
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Fig.1. An example of the fluorescence emission spectrum in TA solutions containing 15, 20, 28 and 35 nm sizes with 60 mg amount GNPs following 1 MHz ultrasonic irradiation with 2 W/cm2 intensity, excitation wavelength of 310 nm, emission and excitation band width of 5 nm
Fig.2. Particle-amount dependence of the fluorescence signal intensity in the TA solutions with GNPs to that of a solution without GNPs at 2 W/cm2 intensity
Fig. 3.Particle-size dependence of the fluorescence signal intensity in the TA solutions with GNPs to that of a solution without GNPs at 2 W/cm2 intensity
Fig. 4. Recorded fluorescence signal intensity in the TA solutions in the absence and presence of GNPs (15, 20, 28, 35 nm) at 0.5, 1 and 2 W/cm2 intensities, amount of GNPs of 60 mg
Fig.5. Recorded fluorescence signal intensity in the different TA solutions at 0.5, 1 and 2 W/cm2 intensities, amount of GNPs of 60 mg
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Highlights
>We executed GNPs as a way for improving cavitation > We examined fluorescence signal in the presence of GNPs in 15, 20, 28 and 35 nm sizes > Fluorescence signal intensity TA solution containing finer size of GNPs was lower than the TA solutions containing larger size of GNPs.