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1-MHz ultrasound enhances internal diffusivity in agarose gels
Applied Acoustics 74 (2013) 1117–1121
Contents lists available at SciVerse ScienceDirect
Applied Acoustics journal homepage: www.elsevier.com/locate/apacoust
Technical Note
1-MHz ultrasound enhances internal diffusivity in agarose gels Akira Tsukamoto a, Kei Tanaka b, Tatsuya Kumata b, Kenji Yoshida b, Yoshiaki Watanabe b, Shogo Miyata c, Katsuko S. Furukawa d, Takashi Ushida e,⇑ a
Department of Applied Physics, National Defense Academy of Japan, Yokosuka, Kanagawa 239-8686, Japan Faculty of Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan c Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan d Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan e Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan b
a r t i c l e
i n f o
Article history: Received 3 April 2012 Received in revised form 7 April 2013 Accepted 9 April 2013 Available online 15 May 2013 Keywords: High-frequency ultrasound Internal diffusivity FRAP analysis Agarose gel FITC-dextran Temperature elevation
a b s t r a c t Ultrasound sonification stimulates the release of pharmaceutical compounds from hydrogels. At the surface of hydrogels, cavitation, cavities formed in liquid, activates to stimulate that release under low-frequency ultrasound. Under high-frequency ultrasound, although cavitation activities are highly suppressed, the compounds are still released. Although it remains elusive how high-frequency ultrasound stimulates this release, one hypothesis is that the internal diffusivity is enhanced. In this study, internal diffusivities in agarose gels were estimated with fluorescent recovery after photobleaching (FRAP) analysis. Under 1-MHz ultrasound sonification, internal diffusivity in agarose gels was enhanced. The enhancement of internal diffusivity was larger than that with temperature elevation alone, although temperature elevation was also observed along with the ultrasound sonification. Thus, we found that high-frequency ultrasound sonification enhances internal diffusivity in agarose gels. This enhancement was, at least in part, independent of temperature elevation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Ultrasound sonification stimulates release of drugs from hydrogels [1–6]. Pharmaceutical compounds such as insulin [1,2] and 5fluorouracil [3] have been released in this way. Such release with ultrasound sonification is expected to be employed in new drug delivery systems. With low-frequency ultrasound, activated cavitation interacts with surface of hydrogels to stimulate the release of compounds. Cavitation erodes the surface of hydrogels [4] or accelerates convective flows at the surface [5]. With high-frequency ultrasound, however, those effects are highly suppressed [5]. Instead, temperature elevation of the delivery system seems to stimulate the release [1,3]. However, it remains elusive how high-frequency ultrasound stimulates the release of pharmaceuticals from hydrogels. Internal diffusivity in hydrogels influences the mass transfer of dissolved substances. For example, temperature elevation and glucose concentration stimulate the release of drugs with accelerated mass transfer [7–9]. Because temperature elevation is observed along with the stimulation, internal diffusivity can be a mechanism with which high-frequency ultrasound stimulates release of drugs. However, it is obscure whether ultrasound sonification enhances internal diffusivity in hydrogels. ⇑ Corresponding author. Fax: +81 3 5800 9199. E-mail address: [email protected] (T. Ushida). 0003-682X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apacoust.2013.04.001
Fluorescent recovery after photobleaching, or FRAP, is a fluorescence method that estimates diffusion efficiency without physical contact [10–12]. This analysis does not disturb the ultrasound sonification on hydrogels because the fluorescent lights used for the analysis are exposed optically. In this study, internal diffusivities in hydrogels under ultrasound sonification were estimated with FRAP analysis to understand whether ultrasound sonification enhances internal diffusivity in agarose gels. 2. Materials and methods 2.1. Agarose gel samples Agarose gel samples containing FITC-dextran were prepared as follows. First, agarose powder (3% (w/v); Agarose ME, Wako, Japan) and FITC-dextran (0.1 mg/ml; 4 kDa, Sigma, MO) were dissolved in TAE buffer (Nippon gene, Japan). Then, the solution was boiled gently for complete dissolution. The boiled solution was cooled gently at room temperature for homogeneous solidification. Solidified agarose gels were cut into agarose gel samples (20 20 5 mm). 2.2. Sonication chamber A sonication chamber was custom made for FRAP analysis under ultrasound sonification at a controlled temperature (Fig. 1). The bottom of the chamber was covered with a cover glass
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(30 30 mm, Matsunami, Japan) so that the fluorescence lights used for FRAP analysis could be transmitted through. The temperature was controlled with temperature-controlled water circulating around the sonication chamber. The circulation water was maintained at 37 °C (RE104, LAUDA, Germany). Agarose gel samples were set at the bottom of the sonication chamber for FRAP analysis (Fig. 1) while the ultrasound transducer was set at the top for ultrasound sonification. Between the agarose gel sample and the ultrasound transducer, TAE buffer containing FITC-dextran (4 kDa; 0.1 mg/ml, Sigma) was placed. Note that the concentration of FITC-dextran in the TAE-buffer is equivalent to that in agarose gel samples.
2.3. FRAP analysis In FRAP analysis, the diffusion efficiency at optical focal regions is estimated by the time required for fluorescence recovery after photobleaching. The speed of fluorescence recovery depends on the influx of fluorescence into the optical focal region from which fluorescence has been photobleached. The influx of fluorescence is further dependent on diffusion efficiency near the optical focal region. First, the optical focal regions used for FRAP analysis were set at the internal areas of agarose gel samples. The optical focal regions were set so that they were at least 1 mm away from the gel surface. If the optical focal regions were too close to the gel surface, the internal diffusivity would be overestimated because free water out of the gel has higher diffusivity. Furthermore, optical focal regions were set at center areas of the gels. In those center areas, 10 10 mm at the center, the pressure amplitude of the ultrasound sonification was stable. The pressure amplitude of ultrasound sonification is described in detail below. For photobleaching, intense excitation light was irradiated on agarose gel samples for 30 s (Fig. 2A). After photobleaching, the time courses of fluorescence recovery were obtained by measuring the fluorescence intensity every 10 s (Fig. 2B). Those time courses were obtained with weaker excitation light compared with that for photobleaching to avoid unnecessary photobleaching. The obtained time courses of fluorescence recovery were outlined to calculate the fluorescence recovery times (T1/2), or the time required for half of the original fluorescence intensity to be recovered (Fig. 2C). T1/2 instead of actual diffusivities were obtained for estimating diffusivities in this study because fluorescence images with an epi-fluorescence microscopy are distorted by out-of-focus light when samples are thick (more than 50 lm) [16]. In thin samples (less than 50 lm), fluorescence recovery speeds are calculated from fluorescence recovery curves those are obtained with original fluorescence intensities and fluorescence intensities just after photobleaching [16]. Thus, although precise calculation of actual
Transducer
Sonication chamber Circulating water
Power amplifier
TAE buffer with FITC-dextran
Function generator
Agarose gel sample Objective lens
Cover glass
Fig. 1. The sonication chamber used for ultrasound sonification and FRAP analysis. Ultrasound was irradiated from the transducer toward agarose gel samples at the bottom.
diffusivity is difficult in this study, diffusivities were estimated with T1/2 with original fluorescence intensities and fluorescence intensities just after photobleaching. Optical setups used for photobleaching and for obtaining time courses of fluorescence recovery were as follows. Excitation light, including that for photobleaching and that for measuring fluorescence recovery, was generated with a xenon lamp (Olympus, UXL-75XB). The excitation light passed through an excitation filter (485 nm, Omega, VT) and the objective lens (40, LUCPlanFL, Olympus, Japan) before it finally irradiated the agarose gel samples. ND filters were inserted between the excitation filter and the objective lens to obtain the time courses of fluorescence recovery. The emitted fluorescence from the agarose gel samples was collected by the objective lens and filtered with an emission filter (530 nm, Omega). The fluorescence was obtained with a 3CCD camera (Hamamatsu Photonics, Japan) for quantification. In order to understand the mechanism of internal diffusivity under ultrasound sonification, FRAP analysis was done for three spots in each agarose gel sample: first, without ultrasound sonification; second, with ultrasound sonification; and third, again without ultrasound sonification. Those three FRAP analyses were done with different optical focal regions to avoid artifacts due to photobleached fluorescence. Such three-region FRAP analyses were done on three independent agarose gel samples. In order to understand the internal diffusivity under elevated temperature, FRAP analysis were done with five different optical focal regions in a single agarose gel sample. At each temperature, five independent optical focal regions were used for independent FRAP analysis. Those FRAP analysis were done after the temperature in the agarose gel sample was confirmed to be stable. The temperature measurement is described in detail in the following section. 2.4. Temperature measurement in agarose gel samples The temperatures in agarose gel samples were measured by tracking fluorescence emission [13]. Compared with temperature measurement by thermometers, this method can realize temperature measurement without interfering with the ultrasound sonification. Furthermore, this method is more sensitive to temperature at the optical focal regions where the FRAP analyses were done. Temperatures at those optical focal regions are difficult to measure by thermometers because they are difficult to access. The temperature-sensitivity of fluorescence emission at the optical focal regions is discussed in detail in Section 3. 2.5. Ultrasound sonification Agarose gel samples were irradiated with high-frequency ultrasound with a planar ceramic ultrasound transducer (1 MHz, 30 30 mm). Air pockets attached to the surface of the ultrasound transducer were entirely removed to avoid scattering of the ultrasound transmission. The distance between the ultrasound transducer and agarose gel sample was 30 mm. In order to eliminate backscatter ultrasound from the ultrasound transducer, a sound buffer (Shinetsu-kagaku, Japan) was attached to the transducer. The ultrasound signal was generated by a function generator (WF1966, NF Corporation, Japan) and amplified by a power amplifier (A150, ENI, NY) before it was transmitted to the ultrasound transducer. The pressure amplitude of ultrasound sonification was set to be 200 kPa (Peak-negative-pressure). The pressure amplitude of agarose gel samples was measured with a needle hydrophone (Dr. Müller Instruments, Germany; less than 0.5 mm in sensitive diameter) (Fig. 3A). When the pressure amplitude was measured with the hydrophone, the cover glass and agarose gel samples were removed to position the hydrophone
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(A)
Agarose gel sample
(B)
Objective lens
Objective lens
Xenon lamp
(C)
Agarose gel sample ND filter
CCD camera
Xenon lamp
Fluorescence recovery
Photobleaching
I original Fluorescence intensity I half
I photobleached
Time T1/2
Fig. 2. FRAP analysis. (A) Intense excitation light was irradiated on an agarose gel sample for photobleaching. (B) Fluorescence recoveries were monitored to obtain time courses by measuring fluorescence intensities every 10 s. (C) The time course of fluorescence recovery was outlined to calculate the fluorescence recovery times (T1/2), i.e., the times required for fluorescence to recover its intensity after photobleaching (Iphotobleached) to half (Ihalf) of the original fluorescence intensity (Ioriginal).
inside the sonication chamber. The tip of the hydrophone was set at the position where agarose gel samples were set. Pressure amplitudes were measured in the plane to understand pressure distributions (14 14 mm; Fig. 3B). The planar pressure distribution showed that the center region, approximately 10 10 mm, was relatively stable in pressure. The real pressure amplitude that agarose gel samples experience could be larger than the amplitude measured with the hydrophone, 200 kPa, because ultrasound reflects at the cover glass. The pressure amplitude that agarose gel samples experience could be up to twice the amplitude measured with the hydrophone. In some experiments, the temperatures of the agarose gel samples were varied in order to investigate the effects of temperature elevation on the internal diffusivity of agarose gel samples. 2.6. Statistical analysis Data are expressed as the means ± SEMs (standard errors of the mean). Differences between groups were calculated by Mann– Whitney test. Statistical significance is shown with the symbol ‘‘’’ when p is below 0.05. The symbol ‘‘NS’’ is used to indicate non-significant cases as needed. 3. Results 3.1. Ultrasound sonification enhanced internal diffusivity In order to understand whether ultrasound sonification enhances internal diffusivity in hydrogels, internal diffusivities in agarose gels were estimated with FRAP analysis. In single agarose
gel samples, FRAP analysis was done three times at three different spots. Among the three FRAP analyses, the first and third were done with ultrasound sonification and the second FRAP analysis without ultrasound sonification. Fluorescence recoveries after photobleaching in FRAP analysis with ultrasound sonification were more rapid than those without ultrasound sonification (Fig. 4A). Fluorescence recovery times (T1/2) were calculated from the obtained time courses of fluorescence. As a result, T1/2 with ultrasound sonification was less than that without ultrasound sonification (Fig. 4B). This result indicates that ultrasound sonification enhanced internal diffusivity in agarose gels. 3.2. Ultrasound sonification elevated temperature in agarose gels In some fluorescent dye, fluorescence emission is sensitive to temperature elevation [13]. Thus, we attempted to measure temperatures at focal regions with fluorescence emission. In order to understand whether FITC-dextran in agarose gel samples could be used as a temperature indicator, fluorescence emission from FITC-dextran at focal regions was measured at different temperatures with heated circulation water. As a result, the fluorescence emission decreased according to temperature elevation (Fig. 5). This result indicated that alteration of fluorescence emission from FITC-dextran could be used as a temperature indicator at focal regions. In order to understand whether ultrasound sonification elevates temperature in agarose gel samples, temperatures at focal regions were measured with fluorescence emission. After a 20-min ultrasound sonification, fluorescence emission at focal regions decreased 5.1 ± 0.9% (mean ± SEM, n = 3 agarose gel samples). This decrease was analogous to the decrease observed in an agarose
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(A)
(B) + 25 %
Water bath
Hydrophone - 25 %
Preamplifier Oscilloscope
Fig. 3. Sound field measurement. (A) Pressure amplitude at the region where agarose gel samples were set was measured with a hydrophone without cover glass and agarose gel samples. (B) Pressure amplitudes were measured in a plane (14 14 mm). Measured pressure amplitude was normalized to the maximum pressure amplitude measured in the plane (0 dB).
(A)
at 37 °C without ultrasound sonification). Comparing this T1/2 decrease with that under ultrasound sonification, T1/2 decreased to 0.56 ± 0.01 (normalized to T1/2 at 37 °C without ultrasound sonification). Because temperature elevations with circulation water heated to 52 °C (4.8 ± 0.2%) and that with ultrasound sonification (5.1 ± 0.9%) were analogous, these results indicated that ultrasound sonification further enhanced internal diffusivity, and that ultrasound sonification can enhance internal diffusivity independent of temperature elevation.
4. Discussion
T1/2 (sec)
(B)
N.S.
200 150
*
100 50 0 1st FRAP (w/o US)
2nd FRAP (w/ US)
3rd FRAP (w/o US)
Fig. 4. Fluorescence recoveries in FRAP analysis under ultrasound sonification. (A) Among three FRAP analyses, the first and third (1st FRAP and 3rd FRAP) were done with ultrasound sonification; the second FRAP analysis (2nd FRAP) was done without ultrasound sonification. (B) Fluorescence recovery times (T1/2) were calculated with the obtained time courses of fluorescence.
gel sample which was heated with heated circulation water at 52 °C (4.8 ± 0.2% (mean ± SEM, n = 3 focal regions in an agarose gel sample)). These results indicated that ultrasound sonification elevates temperature in agarose gel samples. 3.3. Ultrasound sonification can enhance internal diffusivity independent of temperature elevation Temperature elevation enhances internal diffusivity in hydrogels [7–9]. In order to understand whether temperature elevation alone enhanced internal diffusivity under ultrasound sonification, internal diffusivities were estimated under temperature elevation without ultrasound sonification. When an agarose gel sample was heated with circulation water, T1/2 decreased according to the temperature elevation (Fig. 6). When circulation water was heated to 52 °C, T1/2 decreased to 0.71 ± 0.03 (normalized to T1/2
We found that high-frequency ultrasound sonification enhances internal diffusivity in agarose gels. This enhancement was, at least in part, independent of temperature elevation. This temperature elevation can originate from ultrasound absorption and heat generation at the ultrasound transducer [14]. In this study, such temperature elevations were attempted to be suppressed with temperature-controlled circulation water around the sonication chamber. However, the temperature elevation was not effectively suppressed. Thus, instead of suppressing temperature elevation, internal diffusivities were also estimated with temperature elevation alone in order to understand whether the enhancement of internal diffusivity under ultrasound sonification is dependent on the temperature elevation. Alteration in fluorescence emission was used as a temperature indicator in this study. In general, fluorescence emission is sensitive to temperature elevation [13]. As well as other fluorescence, FITC-dextran was sensitive to temperature alteration (Fig. 5). In this study, this alteration in fluorescence emission was also measured under ultrasound sonification. There was a concern that ultrasound sonification itself might alter fluorescence emission without a temperature increase. However, alterations in fluorescence emission under ultrasound sonification were negligible because no rapid alteration in emission was observed when ultrasound sonification was halted. Thus, fluorescence emission from FITC-dextran was supposed to be sensitive mainly to temperature alteration in this study. Physical mechanisms by which high-frequency ultrasound sonification enhanced internal diffusivity remain obscure in this study. Although high-frequency ultrasound less activates cavitation compared with low-frequency ultrasound [5], high-frequency ultrasound sonification could activate cavitation to induce cavitation oscillation, micro-jets and micro-streaming. Our results do not exclude the possibility that high-frequency ultrasound sonification stimulated the release of pharmaceuticals with those activations
A. Tsukamoto et al. / Applied Acoustics 74 (2013) 1117–1121
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5
Fluorescence decrease at focal regions (%)
Gel 1_cooling 4
Gel 1_heating
3
Gel 2 Gel 3
2 1 0 -1 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
Temperature in circulation water (C) Fig. 5. Dependence of fluorescence emission of FITC-dextran on temperature elevation and decrease.
In conclusion, 1-MHz continuous ultrasound sonification enhanced internal diffusivity in agarose gels. This enhancement was, at least in part, independent of temperature elevation. Acknowledgements The authors are grateful to Prof. Mami Matsukawa for considerable discussions. This work was supported in part by a grant from the Translational Systems Biology and Medicine Initiative (TSBMI) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from Nanobio Integration, University of Tokyo, Japan. Fig. 6. T1/2 obtained under temperature elevation (circle dots) and under ultrasound sonification (triangle dot). T1/2 values are normalized to the T1/2 measured at 37 °C without ultrasound sonification.
of cavitation in previous studies. In this study, however, the fluorescence molecule used for FRAP analysis was present both inside and outside of the agarose gel samples. The observed enhancement was independent of modulation in gel surface because the fluorescence dye used for FRAP analysis was present both inside and outside of agarose gel samples at same concentration. Thus, surface modulation should have little influence on FRAP analysis. Acoustic streaming is another typical non-thermal effect observed under ultrasound sonification. This phenomenon occurs due to the pressure gradient generated by absorption of ultrasound during propagation. It is obscure whether this phenomenon could be induced in agarose gels because this phenomenon is often observed in free liquid with abundant volume. If ultrasound attenuation due to dissipation and/or absorption is enough to induce the pressure gradient in free water in agarose gels, that phenomenon could be realized. Releasing pharmaceuticals by enhancing internal diffusivity with high-frequency ultrasound without cavitation dynamics would be ideal for drug delivery systems. Although cavitation dynamics are powerful and have the power to induce beneficial effects, they can also harm surrounding native tissues. Internal diffusivity promotion could be a safer system, along with novel approaches such as controlling surface barriers on the hydrogel surface with ultrasound sonification [2]. By optimizing conditions for ultrasound sonification, such as frequency, pressure amplitude and pulse duration, such enhancement of internal diffusivity could prove to be as powerful as cavitation dynamics. Especially, pressure amplitude should be further reduced because the pressure amplitude of agarose gel samples in this study could be high enough to induce cavitation dynamics due to ultrasound reflection [15].
References [1] Miyazaki S, Yokouchi C, Takada M. External control of drug release – controlled release of insulin from a hydrophilic polymer implant by ultrasound irradiation in diabetic rats. J Pharm Pharmacol 1988;40:716–7. [2] Kwok CS, Mourad PD, Crum LA, Ratner BD. Self-assembled molecular structures as ultrasonically-responsive barrier membranes for pulsatile drug delivery. J Biomed Mater Res 2001;57:151–64. [3] Miyazaki S, Hou WM, Takada M. Controlled drug release by ultrasound irradiation. Chem Pharm Bull 1985;33:428–31. [4] Demanuele A, Kost J, Hill JL, Langer R. An investigation of the effects of ultrasound on degradable polyanhydride matrices. Macromolecules 1992;25:511–5. [5] Lavon I, Kost J. Mass transport enhancement by ultrasound in non-degradable polymeric controlled release systems. J Control Release 1998;54:1–7. [6] Epstein-Barash H, Orbey G, Polat BE, Ewoldt RH, Feshitan J, Langer R, et al. A microcomposite hydrogel for repeated on-demand ultrasound-triggered drug delivery. Biomaterials 2010;31:5208–17. [7] Sershen S, West J. Implantable, polymeric systems for modulated drug delivery. Adv Drug Deliv Rev 2002;54:1225–35. [8] Kikuchi A, Okano T. Pulsatile drug release control using hydrogels. Adv Drug Deliv Rev 2002;54:53–77. [9] Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Adv Drug Deliv Rev 2001;53:321–39. [10] McNally JG. Quantitative FRAP in analysis of molecular binding dynamics in vivo. Methods Cell Biol 2008;85:329–51. [11] Sprague BL, McNally JG. FRAP analysis of binding: proper and fitting. Trends Cell Biol 2005;15:84–91. [12] Wang YX, Shyy JYJ, Chien S. Fluorescence proteins, live-cell imaging, and mechanobiology: seeing is believing. Annu Rev Biomed Eng 2008;10:1–38. [13] Ross D, Gaitan M, Locascio LE. Temperature measurement in microfluidic systems using a temperature-dependent fluorescent dye. Anal Chem 2001;73:4117–23. [14] Chiarelli P, Lanata A, Carbone M. Acoustic waves in hydrogels: a bi-phasic model for ultrasound tissue-mimicking phantom. Mater Sci Eng C 2009;29:899–907. [15] Apfel RE, Holland CK. Gauging the likelihood of cavitation from short-pulse, low-duty cycle diagnostic ultrasound. Ultrasound Med Biol 1991;17:179–85. [16] Berk DA, Yuan F, Leunig M, Jain RK. Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media. Biophys J 1993;65:2428–36.
Contents lists available at SciVerse ScienceDirect
Applied Acoustics journal homepage: www.elsevier.com/locate/apacoust
Technical Note
1-MHz ultrasound enhances internal diffusivity in agarose gels Akira Tsukamoto a, Kei Tanaka b, Tatsuya Kumata b, Kenji Yoshida b, Yoshiaki Watanabe b, Shogo Miyata c, Katsuko S. Furukawa d, Takashi Ushida e,⇑ a
Department of Applied Physics, National Defense Academy of Japan, Yokosuka, Kanagawa 239-8686, Japan Faculty of Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan c Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan d Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan e Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan b
a r t i c l e
i n f o
Article history: Received 3 April 2012 Received in revised form 7 April 2013 Accepted 9 April 2013 Available online 15 May 2013 Keywords: High-frequency ultrasound Internal diffusivity FRAP analysis Agarose gel FITC-dextran Temperature elevation
a b s t r a c t Ultrasound sonification stimulates the release of pharmaceutical compounds from hydrogels. At the surface of hydrogels, cavitation, cavities formed in liquid, activates to stimulate that release under low-frequency ultrasound. Under high-frequency ultrasound, although cavitation activities are highly suppressed, the compounds are still released. Although it remains elusive how high-frequency ultrasound stimulates this release, one hypothesis is that the internal diffusivity is enhanced. In this study, internal diffusivities in agarose gels were estimated with fluorescent recovery after photobleaching (FRAP) analysis. Under 1-MHz ultrasound sonification, internal diffusivity in agarose gels was enhanced. The enhancement of internal diffusivity was larger than that with temperature elevation alone, although temperature elevation was also observed along with the ultrasound sonification. Thus, we found that high-frequency ultrasound sonification enhances internal diffusivity in agarose gels. This enhancement was, at least in part, independent of temperature elevation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Ultrasound sonification stimulates release of drugs from hydrogels [1–6]. Pharmaceutical compounds such as insulin [1,2] and 5fluorouracil [3] have been released in this way. Such release with ultrasound sonification is expected to be employed in new drug delivery systems. With low-frequency ultrasound, activated cavitation interacts with surface of hydrogels to stimulate the release of compounds. Cavitation erodes the surface of hydrogels [4] or accelerates convective flows at the surface [5]. With high-frequency ultrasound, however, those effects are highly suppressed [5]. Instead, temperature elevation of the delivery system seems to stimulate the release [1,3]. However, it remains elusive how high-frequency ultrasound stimulates the release of pharmaceuticals from hydrogels. Internal diffusivity in hydrogels influences the mass transfer of dissolved substances. For example, temperature elevation and glucose concentration stimulate the release of drugs with accelerated mass transfer [7–9]. Because temperature elevation is observed along with the stimulation, internal diffusivity can be a mechanism with which high-frequency ultrasound stimulates release of drugs. However, it is obscure whether ultrasound sonification enhances internal diffusivity in hydrogels. ⇑ Corresponding author. Fax: +81 3 5800 9199. E-mail address: [email protected] (T. Ushida). 0003-682X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apacoust.2013.04.001
Fluorescent recovery after photobleaching, or FRAP, is a fluorescence method that estimates diffusion efficiency without physical contact [10–12]. This analysis does not disturb the ultrasound sonification on hydrogels because the fluorescent lights used for the analysis are exposed optically. In this study, internal diffusivities in hydrogels under ultrasound sonification were estimated with FRAP analysis to understand whether ultrasound sonification enhances internal diffusivity in agarose gels. 2. Materials and methods 2.1. Agarose gel samples Agarose gel samples containing FITC-dextran were prepared as follows. First, agarose powder (3% (w/v); Agarose ME, Wako, Japan) and FITC-dextran (0.1 mg/ml; 4 kDa, Sigma, MO) were dissolved in TAE buffer (Nippon gene, Japan). Then, the solution was boiled gently for complete dissolution. The boiled solution was cooled gently at room temperature for homogeneous solidification. Solidified agarose gels were cut into agarose gel samples (20 20 5 mm). 2.2. Sonication chamber A sonication chamber was custom made for FRAP analysis under ultrasound sonification at a controlled temperature (Fig. 1). The bottom of the chamber was covered with a cover glass
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(30 30 mm, Matsunami, Japan) so that the fluorescence lights used for FRAP analysis could be transmitted through. The temperature was controlled with temperature-controlled water circulating around the sonication chamber. The circulation water was maintained at 37 °C (RE104, LAUDA, Germany). Agarose gel samples were set at the bottom of the sonication chamber for FRAP analysis (Fig. 1) while the ultrasound transducer was set at the top for ultrasound sonification. Between the agarose gel sample and the ultrasound transducer, TAE buffer containing FITC-dextran (4 kDa; 0.1 mg/ml, Sigma) was placed. Note that the concentration of FITC-dextran in the TAE-buffer is equivalent to that in agarose gel samples.
2.3. FRAP analysis In FRAP analysis, the diffusion efficiency at optical focal regions is estimated by the time required for fluorescence recovery after photobleaching. The speed of fluorescence recovery depends on the influx of fluorescence into the optical focal region from which fluorescence has been photobleached. The influx of fluorescence is further dependent on diffusion efficiency near the optical focal region. First, the optical focal regions used for FRAP analysis were set at the internal areas of agarose gel samples. The optical focal regions were set so that they were at least 1 mm away from the gel surface. If the optical focal regions were too close to the gel surface, the internal diffusivity would be overestimated because free water out of the gel has higher diffusivity. Furthermore, optical focal regions were set at center areas of the gels. In those center areas, 10 10 mm at the center, the pressure amplitude of the ultrasound sonification was stable. The pressure amplitude of ultrasound sonification is described in detail below. For photobleaching, intense excitation light was irradiated on agarose gel samples for 30 s (Fig. 2A). After photobleaching, the time courses of fluorescence recovery were obtained by measuring the fluorescence intensity every 10 s (Fig. 2B). Those time courses were obtained with weaker excitation light compared with that for photobleaching to avoid unnecessary photobleaching. The obtained time courses of fluorescence recovery were outlined to calculate the fluorescence recovery times (T1/2), or the time required for half of the original fluorescence intensity to be recovered (Fig. 2C). T1/2 instead of actual diffusivities were obtained for estimating diffusivities in this study because fluorescence images with an epi-fluorescence microscopy are distorted by out-of-focus light when samples are thick (more than 50 lm) [16]. In thin samples (less than 50 lm), fluorescence recovery speeds are calculated from fluorescence recovery curves those are obtained with original fluorescence intensities and fluorescence intensities just after photobleaching [16]. Thus, although precise calculation of actual
Transducer
Sonication chamber Circulating water
Power amplifier
TAE buffer with FITC-dextran
Function generator
Agarose gel sample Objective lens
Cover glass
Fig. 1. The sonication chamber used for ultrasound sonification and FRAP analysis. Ultrasound was irradiated from the transducer toward agarose gel samples at the bottom.
diffusivity is difficult in this study, diffusivities were estimated with T1/2 with original fluorescence intensities and fluorescence intensities just after photobleaching. Optical setups used for photobleaching and for obtaining time courses of fluorescence recovery were as follows. Excitation light, including that for photobleaching and that for measuring fluorescence recovery, was generated with a xenon lamp (Olympus, UXL-75XB). The excitation light passed through an excitation filter (485 nm, Omega, VT) and the objective lens (40, LUCPlanFL, Olympus, Japan) before it finally irradiated the agarose gel samples. ND filters were inserted between the excitation filter and the objective lens to obtain the time courses of fluorescence recovery. The emitted fluorescence from the agarose gel samples was collected by the objective lens and filtered with an emission filter (530 nm, Omega). The fluorescence was obtained with a 3CCD camera (Hamamatsu Photonics, Japan) for quantification. In order to understand the mechanism of internal diffusivity under ultrasound sonification, FRAP analysis was done for three spots in each agarose gel sample: first, without ultrasound sonification; second, with ultrasound sonification; and third, again without ultrasound sonification. Those three FRAP analyses were done with different optical focal regions to avoid artifacts due to photobleached fluorescence. Such three-region FRAP analyses were done on three independent agarose gel samples. In order to understand the internal diffusivity under elevated temperature, FRAP analysis were done with five different optical focal regions in a single agarose gel sample. At each temperature, five independent optical focal regions were used for independent FRAP analysis. Those FRAP analysis were done after the temperature in the agarose gel sample was confirmed to be stable. The temperature measurement is described in detail in the following section. 2.4. Temperature measurement in agarose gel samples The temperatures in agarose gel samples were measured by tracking fluorescence emission [13]. Compared with temperature measurement by thermometers, this method can realize temperature measurement without interfering with the ultrasound sonification. Furthermore, this method is more sensitive to temperature at the optical focal regions where the FRAP analyses were done. Temperatures at those optical focal regions are difficult to measure by thermometers because they are difficult to access. The temperature-sensitivity of fluorescence emission at the optical focal regions is discussed in detail in Section 3. 2.5. Ultrasound sonification Agarose gel samples were irradiated with high-frequency ultrasound with a planar ceramic ultrasound transducer (1 MHz, 30 30 mm). Air pockets attached to the surface of the ultrasound transducer were entirely removed to avoid scattering of the ultrasound transmission. The distance between the ultrasound transducer and agarose gel sample was 30 mm. In order to eliminate backscatter ultrasound from the ultrasound transducer, a sound buffer (Shinetsu-kagaku, Japan) was attached to the transducer. The ultrasound signal was generated by a function generator (WF1966, NF Corporation, Japan) and amplified by a power amplifier (A150, ENI, NY) before it was transmitted to the ultrasound transducer. The pressure amplitude of ultrasound sonification was set to be 200 kPa (Peak-negative-pressure). The pressure amplitude of agarose gel samples was measured with a needle hydrophone (Dr. Müller Instruments, Germany; less than 0.5 mm in sensitive diameter) (Fig. 3A). When the pressure amplitude was measured with the hydrophone, the cover glass and agarose gel samples were removed to position the hydrophone
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(A)
Agarose gel sample
(B)
Objective lens
Objective lens
Xenon lamp
(C)
Agarose gel sample ND filter
CCD camera
Xenon lamp
Fluorescence recovery
Photobleaching
I original Fluorescence intensity I half
I photobleached
Time T1/2
Fig. 2. FRAP analysis. (A) Intense excitation light was irradiated on an agarose gel sample for photobleaching. (B) Fluorescence recoveries were monitored to obtain time courses by measuring fluorescence intensities every 10 s. (C) The time course of fluorescence recovery was outlined to calculate the fluorescence recovery times (T1/2), i.e., the times required for fluorescence to recover its intensity after photobleaching (Iphotobleached) to half (Ihalf) of the original fluorescence intensity (Ioriginal).
inside the sonication chamber. The tip of the hydrophone was set at the position where agarose gel samples were set. Pressure amplitudes were measured in the plane to understand pressure distributions (14 14 mm; Fig. 3B). The planar pressure distribution showed that the center region, approximately 10 10 mm, was relatively stable in pressure. The real pressure amplitude that agarose gel samples experience could be larger than the amplitude measured with the hydrophone, 200 kPa, because ultrasound reflects at the cover glass. The pressure amplitude that agarose gel samples experience could be up to twice the amplitude measured with the hydrophone. In some experiments, the temperatures of the agarose gel samples were varied in order to investigate the effects of temperature elevation on the internal diffusivity of agarose gel samples. 2.6. Statistical analysis Data are expressed as the means ± SEMs (standard errors of the mean). Differences between groups were calculated by Mann– Whitney test. Statistical significance is shown with the symbol ‘‘’’ when p is below 0.05. The symbol ‘‘NS’’ is used to indicate non-significant cases as needed. 3. Results 3.1. Ultrasound sonification enhanced internal diffusivity In order to understand whether ultrasound sonification enhances internal diffusivity in hydrogels, internal diffusivities in agarose gels were estimated with FRAP analysis. In single agarose
gel samples, FRAP analysis was done three times at three different spots. Among the three FRAP analyses, the first and third were done with ultrasound sonification and the second FRAP analysis without ultrasound sonification. Fluorescence recoveries after photobleaching in FRAP analysis with ultrasound sonification were more rapid than those without ultrasound sonification (Fig. 4A). Fluorescence recovery times (T1/2) were calculated from the obtained time courses of fluorescence. As a result, T1/2 with ultrasound sonification was less than that without ultrasound sonification (Fig. 4B). This result indicates that ultrasound sonification enhanced internal diffusivity in agarose gels. 3.2. Ultrasound sonification elevated temperature in agarose gels In some fluorescent dye, fluorescence emission is sensitive to temperature elevation [13]. Thus, we attempted to measure temperatures at focal regions with fluorescence emission. In order to understand whether FITC-dextran in agarose gel samples could be used as a temperature indicator, fluorescence emission from FITC-dextran at focal regions was measured at different temperatures with heated circulation water. As a result, the fluorescence emission decreased according to temperature elevation (Fig. 5). This result indicated that alteration of fluorescence emission from FITC-dextran could be used as a temperature indicator at focal regions. In order to understand whether ultrasound sonification elevates temperature in agarose gel samples, temperatures at focal regions were measured with fluorescence emission. After a 20-min ultrasound sonification, fluorescence emission at focal regions decreased 5.1 ± 0.9% (mean ± SEM, n = 3 agarose gel samples). This decrease was analogous to the decrease observed in an agarose
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(A)
(B) + 25 %
Water bath
Hydrophone - 25 %
Preamplifier Oscilloscope
Fig. 3. Sound field measurement. (A) Pressure amplitude at the region where agarose gel samples were set was measured with a hydrophone without cover glass and agarose gel samples. (B) Pressure amplitudes were measured in a plane (14 14 mm). Measured pressure amplitude was normalized to the maximum pressure amplitude measured in the plane (0 dB).
(A)
at 37 °C without ultrasound sonification). Comparing this T1/2 decrease with that under ultrasound sonification, T1/2 decreased to 0.56 ± 0.01 (normalized to T1/2 at 37 °C without ultrasound sonification). Because temperature elevations with circulation water heated to 52 °C (4.8 ± 0.2%) and that with ultrasound sonification (5.1 ± 0.9%) were analogous, these results indicated that ultrasound sonification further enhanced internal diffusivity, and that ultrasound sonification can enhance internal diffusivity independent of temperature elevation.
4. Discussion
T1/2 (sec)
(B)
N.S.
200 150
*
100 50 0 1st FRAP (w/o US)
2nd FRAP (w/ US)
3rd FRAP (w/o US)
Fig. 4. Fluorescence recoveries in FRAP analysis under ultrasound sonification. (A) Among three FRAP analyses, the first and third (1st FRAP and 3rd FRAP) were done with ultrasound sonification; the second FRAP analysis (2nd FRAP) was done without ultrasound sonification. (B) Fluorescence recovery times (T1/2) were calculated with the obtained time courses of fluorescence.
gel sample which was heated with heated circulation water at 52 °C (4.8 ± 0.2% (mean ± SEM, n = 3 focal regions in an agarose gel sample)). These results indicated that ultrasound sonification elevates temperature in agarose gel samples. 3.3. Ultrasound sonification can enhance internal diffusivity independent of temperature elevation Temperature elevation enhances internal diffusivity in hydrogels [7–9]. In order to understand whether temperature elevation alone enhanced internal diffusivity under ultrasound sonification, internal diffusivities were estimated under temperature elevation without ultrasound sonification. When an agarose gel sample was heated with circulation water, T1/2 decreased according to the temperature elevation (Fig. 6). When circulation water was heated to 52 °C, T1/2 decreased to 0.71 ± 0.03 (normalized to T1/2
We found that high-frequency ultrasound sonification enhances internal diffusivity in agarose gels. This enhancement was, at least in part, independent of temperature elevation. This temperature elevation can originate from ultrasound absorption and heat generation at the ultrasound transducer [14]. In this study, such temperature elevations were attempted to be suppressed with temperature-controlled circulation water around the sonication chamber. However, the temperature elevation was not effectively suppressed. Thus, instead of suppressing temperature elevation, internal diffusivities were also estimated with temperature elevation alone in order to understand whether the enhancement of internal diffusivity under ultrasound sonification is dependent on the temperature elevation. Alteration in fluorescence emission was used as a temperature indicator in this study. In general, fluorescence emission is sensitive to temperature elevation [13]. As well as other fluorescence, FITC-dextran was sensitive to temperature alteration (Fig. 5). In this study, this alteration in fluorescence emission was also measured under ultrasound sonification. There was a concern that ultrasound sonification itself might alter fluorescence emission without a temperature increase. However, alterations in fluorescence emission under ultrasound sonification were negligible because no rapid alteration in emission was observed when ultrasound sonification was halted. Thus, fluorescence emission from FITC-dextran was supposed to be sensitive mainly to temperature alteration in this study. Physical mechanisms by which high-frequency ultrasound sonification enhanced internal diffusivity remain obscure in this study. Although high-frequency ultrasound less activates cavitation compared with low-frequency ultrasound [5], high-frequency ultrasound sonification could activate cavitation to induce cavitation oscillation, micro-jets and micro-streaming. Our results do not exclude the possibility that high-frequency ultrasound sonification stimulated the release of pharmaceuticals with those activations
A. Tsukamoto et al. / Applied Acoustics 74 (2013) 1117–1121
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5
Fluorescence decrease at focal regions (%)
Gel 1_cooling 4
Gel 1_heating
3
Gel 2 Gel 3
2 1 0 -1 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
Temperature in circulation water (C) Fig. 5. Dependence of fluorescence emission of FITC-dextran on temperature elevation and decrease.
In conclusion, 1-MHz continuous ultrasound sonification enhanced internal diffusivity in agarose gels. This enhancement was, at least in part, independent of temperature elevation. Acknowledgements The authors are grateful to Prof. Mami Matsukawa for considerable discussions. This work was supported in part by a grant from the Translational Systems Biology and Medicine Initiative (TSBMI) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from Nanobio Integration, University of Tokyo, Japan. Fig. 6. T1/2 obtained under temperature elevation (circle dots) and under ultrasound sonification (triangle dot). T1/2 values are normalized to the T1/2 measured at 37 °C without ultrasound sonification.
of cavitation in previous studies. In this study, however, the fluorescence molecule used for FRAP analysis was present both inside and outside of the agarose gel samples. The observed enhancement was independent of modulation in gel surface because the fluorescence dye used for FRAP analysis was present both inside and outside of agarose gel samples at same concentration. Thus, surface modulation should have little influence on FRAP analysis. Acoustic streaming is another typical non-thermal effect observed under ultrasound sonification. This phenomenon occurs due to the pressure gradient generated by absorption of ultrasound during propagation. It is obscure whether this phenomenon could be induced in agarose gels because this phenomenon is often observed in free liquid with abundant volume. If ultrasound attenuation due to dissipation and/or absorption is enough to induce the pressure gradient in free water in agarose gels, that phenomenon could be realized. Releasing pharmaceuticals by enhancing internal diffusivity with high-frequency ultrasound without cavitation dynamics would be ideal for drug delivery systems. Although cavitation dynamics are powerful and have the power to induce beneficial effects, they can also harm surrounding native tissues. Internal diffusivity promotion could be a safer system, along with novel approaches such as controlling surface barriers on the hydrogel surface with ultrasound sonification [2]. By optimizing conditions for ultrasound sonification, such as frequency, pressure amplitude and pulse duration, such enhancement of internal diffusivity could prove to be as powerful as cavitation dynamics. Especially, pressure amplitude should be further reduced because the pressure amplitude of agarose gel samples in this study could be high enough to induce cavitation dynamics due to ultrasound reflection [15].
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