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Ultrasonics Sonochemistry 18 (2011) 1165–1171 Contents lists available at ScienceDirect Effect of local dual frequency sonication on drug distribution from polymericnanomicelles Hadi Hasanzadeh a,1, Manijhe Mokhtari-Dizaji a,⇑, S. Zahra Bathaie b, Zuhair M. Hassan c a Department of Medical Physics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iranb Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iranc Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran To overcome the side effects caused by systemic administration of doxorubicin, nanosized polymeric Received 17 January 2011 micelles were used in combination with dual frequency ultrasonic irradiation. These micelles release Received in revised form 12 March 2011 the drug due to acoustic cavitation, which is enhanced in dual frequency ultrasonic fields. To form the Accepted 17 March 2011 drug-loaded micelles, Pluronic P-105 copolymer was used, and doxorubicin was physically loaded into Available online 1 April 2011 stabilized micelles with an average size of 14 nm. In this study, adult female Balb/C mice were trans-planted with spontaneous breast adenocarcinoma tumors and were injected with a dose of 1.3 mg/kg doxorubicin in one of three forms: free doxorubicin, micellar doxorubicin without sonication and micel- lar doxorubicin with sonication. To increase cavitation yield, the tumor region was sonicated for 2.5 min at simultaneous frequencies of 3 MHz (ISATA = 2 W/cm2) and 28 kHz (ISATA = 0.04 W/cm2). The animals were sacrificed 24 h after injection, and their tumor, heart, spleen, liver, kidneys and plasma were sepa- Breast adenocarcinoma rated and homogenized. The drug content in the tissues was determined using tissue fluorimetry(350 nm excitation and 560 nm emission), and standard drug dose curves were obtained for each tissue.
The results show that in the group that received micellar doxorubicin with sonication, the drug concen-tration in the tumor tissue was significantly higher than in the free doxorubicin injection group (8.69times) and the micellar doxorubicin without sonication group (2.60 times). The drug concentration inother tissues was significantly lower in the micellar doxorubicin with sonication group relative to the freedoxorubicin (3.35 times) and the micellar drug without sonication (2.48 times) groups (p < 0.05). We con-clude that dual frequency sonication improves drug release from micelles and increases the drug uptakeby tumors due to sonoporation. The proposed drug delivery system creates an improved treatment capa-bility while reducing systemic side effects caused by drug uptake in other tissues.
Ó 2011 Elsevier B.V. All rights reserved.
desired site of action and increasing its uptake using sonication,the side effects are minimized and the therapeutic efficiency is in- The main mechanisms of biological action for ultrasound in- creased. Various nanosized carriers such as liposomes [16], poly- clude the generation of thermal energy, sonoporation, the meric micelles [17,18] and core–shell nanoparticles [15,19–21] enhancement of local microjets due to inertial cavitation (which have been reported as vehicles for passive targeting. Polymeric mi- are further enhanced by multifrequency sonication [1–3] and the celles [22] and core–shell nanoparticles [19,23] have received con- enhanced permeability of blood capillaries [4–10]. All of these siderable attention due to their self-assembly characteristics in an mechanisms could potentially be used to enhance drug uptake lo- aqueous solution. These properties offer the possibility for a un- cally. Doxorubicin is of great importance in the treatment of leuke- ique biodistribution of drugs to target solid tumors [18], and it mia and solid tumors such as in breast and ovarian cancer and has been reported that doxorubicin-conjugated block copolymer sarcoma pulmonary metastasis, but its clinical use is hampered micelles were effective in the treatment of solid tumors due to pro- by its myelotoxicity and its cumulative cardiotoxicity when longed circulation in the blood [17,24]. Generally, it has been re- administered systemically [11–15]. By targeting this drug to the ported that anticancer agents incorporated in polymeric micelleshave an enhanced blood circulation time (EPR: enhanced penetra-tion and retention effect) and a suppressive effect on the growth of ⇑ Corresponding author. Tel.: +98 21 82883893; fax: +98 21 88006544.
solid tumors [18,21–24]. Many polymeric micelles are convenient E-mail address: [email protected] (M. Mokhtari-Dizaji).
to use because they can conveniently escape the reticuloendothe- 1 Present address: Assistant Professor in Medical Physics, Department of Medical lial system (RES) and undergo renal extraction because of their Physics, Semnan University of Medical Sciences, Semnan, Iran.
1350-4177/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultsonch.2011.03.018 Author's personal copy
H. Hasanzadeh et al. / Ultrasonics Sonochemistry 18 (2011) 1165–1171 small size, which is approximately 20–100 nm [24–27]. Various recorded with the hydrophone. All of the above conditions were methods to quantify doxorubicin in biological fluids have been re- applied in a continuous wave mode and for a sonication duration viewed [28]. Reversed-phase liquid chromatography coupled with of 3 min. Each recorded signal comprised of 32768 data points col- fluorescence detection is the method of choice to assay of doxoru- lected with a sampling rate of 10 MHz, which includes the primary bicin [29–31], although alternative schemes such as electrochemi- frequency and the pressure waves coming from the oscillating cal detection [32], UV spectrophotometric [33] detection and mass walls of the cavities. To extract the frequency contents, signals spectrometry [34] have also been used. However, fluorescence were analyzed in MATLAB software version 7.0.1 (Mathworks, detection is often the most appropriate detection method for doxo- USA) using an FFT function with a hamming window. Background rubicin considering its simplicity of use, selectivity and sensitivity noise was measured by placing the hydrophone in the usual mea- surement conditions and setting the intensity of the unit to zero; In this study, polymeric micelles were prepared using Pluron- this background was subtracted from the signal amplitude in each ic P-105, which is a triblock copolymer consisting of blocks of measurement condition. The subharmonic peak in the FFT of the PPO (Poly Propylene Oxide) and PEO (Poly Ethylene Oxide) in measured pressure signal was recorded. Each experimental condi- the form PEO37–PPO56–PEO37. After loading doxorubicin into tion was repeated 5 times.
these micelles and checking the drug release upon sonicationin vitro, their anti-tumor effect was tested in vivo. Ultrasound was used to enhance the intracellular drug uptake from micelles.
Using a spectrophotometric method, the drug content in several Doxorubicin was obtained from Pharmacia (Italy). Pluronic P- tissues was quantified.
105 was provided by the BASF Corp. (Mount Olive, NJ, USA). N-N-Diethylacrylamide (NNDEA) was obtained from Polysciences (War-rington, USA). N,N0-Bis(acryloyl)cystamine (BAC) was obtained 2. Materials and methods from Fluka (Sigma–Aldrich, UK), and benzoyl peroxide (BP) wasobtained from Merck (Merck KGaA, Darmstadt, Germany).
2.1. Animal model 2.4. Preparation of micelles Female inbred Balb/C (6–8 weeks) mice were purchased from the breeding colony at the Pasteur Institute of Iran (Tehran, Iran).
Polymeric micelles were prepared using Pluronic P-105, which Mice were housed under standard conditions (constant tempera- is a triblock copolymer consisting of blocks of poly (propylene ture, humidity and 12 h dark–light cycles) and had access to food oxide) (PPO) and poly (ethylene oxide) (PEO) in the form PEO37– and water ad libitum. The tumor model was a syngenic of murine PPO56–PEO37. A solution of NaCl and 10 wt.% Pluronic P-105 in dis- spontaneous breast adenocarcinoma, which was chopped into tilled water was added to a round-bottom balloon, which was stir- fresh pieces of about 2–3 mm diameter and then transplanted into red for 20 min while immersed in a water bath under a nitrogen the flank region of the mice. Tumors reached a diameter of about purge at a temperature of 65 °C. A mixture of BAC, BP and NNDEA 7–9 mm in 7–10 days, at which point they were ready to be used (26:1:55 weight ratio) was added to the above solution, and it was in experiments. All animal experiments and protocols were evalu- allowed to stir at this temperature under a nitrogen purge for 3.5 h.
ated and approved by the Animal and Ethics Review Committee of After turning the nitrogen off, the mixture was allowed to poly- the Tarbiat Modares University (Tehran, Iran).
merize for 19.5 h. The size distribution of the micelles was mea-sured by dynamic light scattering (DLS) (Malvern Instruments 2.2. Ultrasonic setup and sonication conditions Ltd., Malvern, UK).
Two ultrasonic systems were used in a cubic Perspex water tank 2.5. Loading doxorubicin into micelles (25  20  20 cm3) in an orthogonal geometry. The first systemwas designed in our lab (Tarbiat Modares University, Ultrasound To load doxorubicin into injectable micelles, it was necessary to Lab, Tehran, Iran) in collaboration with the Pars Nahand Engineer- find the optimal drug loading into micelles through a standard ing company (Pardis Technology Park, Tehran, Iran) to operate in curve, which was measured using a UV spectrophotometer (Shima- the low kHz range with a center frequency of 27.7 kHz (nominal dzu, Model RF-1500, Japan) at 350 nm.
frequency of 28 kHz), a bandwidth of 421 Hz and a probe diameter To obtain the curve, several known concentrations (0–0.12 mg of 60 mm. The second system was a 3-MHz therapeutic unit of doxorubicin in a 1 ml solution of micelles) were prepared, and (SM3670, Shrewsbury Medical Ltd., Shropshire, UK) with a 30- their fluorescence emissions were read at 350 nm. It is notable that mm-diameter probe and 5-cm2 effective radiation area (ERA).
at higher concentrations, the fluorescent emission decreases due to Probes were held fixed in the tank wall through a pair of circular self-quenching of the doxorubicin. Fluorescence intensity (counts) holes in such a way that the central beam axis for each probe versus doxorubicin concentration (mg/ml) yielded a straight line of was perpendicular to the other. Acoustic calibration for the power positive slope for increasing concentrations. The results show that and intensity of the devices was carried out in degassed water in there is a significant correlation between the fluorescence intensity the tank using a radiation force balance (Shrewsbury Medical Co., and the doxorubicin concentration (R2 = 0.96, correlation is signif- Shropshire, UK, ±10%) for the therapeutic unit and the hydrophone icant at 0.01). A linear regression analysis was applied between the method in the cubic chamber for our unit (PA124, Precision Acous- fluorescence intensity and the doxorubicin concentration and the tics Ltd., Dorchester, Dorset, UK; calibration range: 10 kHz–3 MHz results are shown in Fig. 1.
with a sensor diameter of 25 mm). Different single frequency son- To examine drug release in vitro, a custom exposure chamber ication conditions including 28 kHz (0.02 and 0.04 W/cm2) and was constructed according to the spectrophotometric characteris- 3 MHz (1 and 2 W/cm2) were demonstrated (Hasanzadeh et al., tics of doxorubicin obtained above (kex = 350 nm, kem = 560 nm); 2010). In addition, a dual frequency combination (28 kHz + 3 MHz) this chamber recorded the change in light emission due to doxoru- at the above-mentioned intensities was studied. All reported bicin release under sonication (Fig. 2). A digital camera (Sony, DSC experimental intensity values consist of the spatial average/tem- P-93, Japan) with a multi-band filter (Alexa FluorÒ 350/488/594, poral average (ISATA). In each sonication condition, subharmonic USA) was used to record images (BMP images with dimensions signal amplitudes at 14 kHz and 1.5 MHz in the water tank were of 640  480 pixels), and a background was recorded without son-

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H. Hasanzadeh et al. / Ultrasonics Sonochemistry 18 (2011) 1165–1171 the complex. The complex was stored at 4 °C, and its stability wasmeasured for one month.
Intensity (Count) = 4590.5Concentration+ 52.82 2.7. Biodistribution and in vivo study Because the goal of the current study was to work below the le- vel of hyperthermia, it was necessary to measure the temperature rise due to sonication in vivo. Three female Balb/C mice were se-lected and placed into the sonication condition after being anes- escence intensity (Count) 100
thetized. The temperatures of the tumors and their surroundingenvironment were monitored every second during sonication with a portable digital thermometer containing a dual thermocouple in- put. The tissue temperature was monitored invasively using K-type Doxorubicin concentration (mg/ml)
wire thermocouples (TP-01, Lutron Electronic Enterprise Co., Tai-wan, 200–1372 °C, 0.1 mm thickness, ±0.1 °C). Each of these mic- Fig. 1. Micellar drug loading standard curve.
rothermometers had two wire thermocouples placed in theanimal's body. The temperature changes (°C) at different depths ication. The frequency and intensity were selected previously using in mice versus duration of sonication under dual frequency sonica- a subharmonic analysis method to enhance the acoustic cavitation tion conditions was evaluated for different depths relative to the at 28 kHz and 3 MHz [35].
skin in the center of the tumor (close to the ultrasonic probes) to Each sonication protocol was run 5 times, and the RMSE (root about 4 cm below the skin until animal posterior layer in 1 cm mean square error) values of the images were used to compare be- steps. All of the thermocouples provided inputs to the control unit, which contained a microprocessor connected to a computer via anRS-232 port. The temperatures were recorded every second (as .doc files) using thermometer software (Multilogger Thermometer CHY502A, Taiwan), which was connected to a PC, and the temper- ature rise under sonication conditions was recorded every 30 s for20 min. The water temperature was controlled using a digital ther- In the above equation, x i and x0 are signal amplitudes of the mometer and an electrical heater and was fixed at 32 °C. According images in each group and the background images, respectively. Fi- to our temperature measurements, a 2.5 min sonication time was nally, the average of the RMSEs of the image components was used deemed suitable for the next phase of our in vivo study. We have to compare the ability of different sonication conditions to release evaluated a control group without sonication but we did not have the drug from the micelles.
any temperature changes. Therefore, that obtained results are due Doxorubicin was stirred into the micellar solution to load it into to sonication rather than a vasculature lesion provoked by probe the micelles; to separate the free drug from the encapsulated drug, insert in tumor mass.
the solution was filled into a dialysis bag (30 mm diameter, 5 kDa To study the drug distribution in animals, 9 female Balb/C mice cut off, BioGene, Mashhad, Iran) and dialyzed against water for 2 h.
with tumor diameters of about 7–9 mm were randomly divided The correct time was determined by sampling from the dialysis bag into 3 groups as follows: IV injection of free doxorubicin, IV injec- at different times until the drug fluorescence no longer changed.
tion of micellar doxorubicin and IV injection of micellar doxorubi- According to the standard curve of the drug in micelles, the opti- cin with sonication under the optimum conditions mentioned mum drug loading into micelles was determined. All experiments above. After drug injection via the tail vein at a dose of 1.3 mg/kg were performed in triplicate.
(the amount of drug injected was 26 lg for each animal in differentgroups), animals were anesthetized and placed in a special cage inthe vicinity of the probes in the ultrasonic field; in only the sonica- 2.6. Stability studies of the micellar drug tion group, the tumor region was exposed for 2.5 min to the se-lected sonication protocol, which was determined by the To investigate the stability of the micellar drug, the fluorescence subharmonic amplitude analysis (Fig. 3).
amplitude of the micellar drug at 350 nm was measured over time, The concentration of total doxorubicin in plasma is maximum and the amplitude of this emission from each day was normalized at 2 min following injection and decreased bi-exponentially to to day zero (the time of drug encapsulation) to find the stability of underdetectable levels after 24 h [15,28,33]. Therefore animalswere exposed 2 min following injection. Based on previous study,the distribution of doxorubicin into tumors was maximum 24 hfollowing injection. The levels of doxorubicin in the tumors in-creased until 24 h [28]. Therefore, after 24 h, the animals were sac-rificed, blood was collected using a heparinated syringe from theheart, and tissues (heart, liver, spleen, kidney and tumor) were dis-sected and lyophilized. The blood was centrifuged (1500 rpm for10 min), and the plasma was separated and kept for later analysis.
Tissue extracts were obtained according to available protocols [36].
The doxorubicin concentration in the tissues and plasma wasdetermined by a fluorescence assay using a UV spectrophotometer(Shimadzu, Model RF-1500, Japan) at 350 nm. Standard curves foreach tissue were established by adding known amounts of doxoru- Fig. 2. Setup for the measurement of doxorubicin release in vitro. Elements include bicin to tissue extracts of control mice to obtain various concentra- the 3-MHz probe, the 28-kHz probe, the filter on which the camera was mounted, tions; these curves were used later to obtain the drug the light source that is placed at the bottom and the drug chamber that was placedin the water box on the light source.
concentration in different tissues from the experimental groups.

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H. Hasanzadeh et al. / Ultrasonics Sonochemistry 18 (2011) 1165–1171 Fig. 3. Animal sonication setup. A side view of the animal cage in the sonication condition. The 28-kHz probe, 3-MHz probe and thermocouples are shown.
2.8. Statistical analysis Statistical analysis was performed using Microsoft Excel 2003 and SPSS 16 (SPSS/PC Inc., Chicago, IL). The drug content in tissuesis presented as the mean ± SD. To analyze differences between groups, a one way ANOVA was used with a level of significanceof 0.05 (P-value < 0.05).
Fig. 4 shows the results from dynamic light scattering (DSL), which gives a particle size distribution of the synthesized micelles.
As shown in Fig. 4, polymeric micelles had small particle sizes with Time after drug loading (Day)
an average size of 14 nm.
The stability of the encapsulated drug was evaluated over one Fig. 5. Stability of the micellar drug versus time (days).
month. Fig. 5 shows the fluorescence amplitude of the micellardrug at 350 nm. The amplitude of this emission measurement ateach day was normalized to the value from day zero. As shown in Fig. 5, the encapsulated drug is stable for the first 4 days, andabout 90% of the maximum possible stability was maintained over the first ten days.
The results of a subharmonic amplitude measurement at 14 kHz during both single frequency sonication and simultaneous dual- frequency sonication at different intensities are shown in Fig. 6.
For each measurement condition, the amplitude of the processed signal was extracted and recorded after the subtraction of back- ground noise for each frequency component. One of the interesting results of this frequency combination is the synergistic effects in dual frequency sonication using 28 kHz (0.04 W/cm2) and 3 MHz (2 W/cm2) frequencies. The amplitude increase of dual frequency 28 (0.04) kHz+3 (2)MH 28 (0.02) kHz+3 (1)MHz Fig. 6. Signal amplitudes (mV) at 14 kHz (a subharmonic of 28 kHz) of the 28 kHzand 3 MHz sources applied alone and in combination and presented as mean ± SD.
The background has been subtracted from all values. The values in the parenthesesare intensities in W/cm2.
irradiation with respect to single frequency irradiation is due tothe synergistic effect and creates an enhanced cavitation activity Mean intensity (%)
from the combined fields.
It has been previously shown by the authors that the 14 kHz subharmonic amplitude when applying a dual frequency combina- tion of 28 kHz (0.04 W/cm2) and 3 MHz (2 W/cm2) in the continu-ous mode was about 5 times higher than that obtained from the Size distribution of micelles (nm)
algebraic sum of single 28 kHz and 3 MHz irradiation. RMSE values Fig. 4. Result of DLS (dynamic light scattering) on the synthesized micelles.
of the images from dual frequency sonication (28 kHz (0.04 W/ Author's personal copy
H. Hasanzadeh et al. / Ultrasonics Sonochemistry 18 (2011) 1165–1171 Table 1The standard linear regression functions for each tissue given as fluorescence intensity (X: count) versus concentration of doxorubicin (Y: lg/ml).
Linear regression function Correlation of coefficient 28 kHz+3 MHz
regression functions. Fluorescence intensity (counts) versus con-centration of doxorubicin (lg/ml) for each tissue yielded a straight Fig. 7. RMSE values of the images (Mean ± SD) from dual frequency sonication line of positive slope for increasing concentrations. The results (28 kHz (0.04 W/cm2) and 3 MHz (2 W/cm2)).
show that there is a significant correlation between the fluores-cence intensity and the concentration of doxorubicin (R > 0.96, cor- cm2) and 3 MHz (2 W/cm2)) in vitro are shown in Fig. 7. Drug re- relation is significant at 0.01) for each tissue.
lease from the micelles is due to the cavitation activity, which is These curves were later used to obtain the drug concentration enhanced in a dual frequency field; this property is observable in in different tissues in the studied groups. Table 2 shows the results the RMSE values.
of tissue spectrofluorimetric measurements from three different Fig. 8 shows the temperature rise due to sonication with com- groups: free doxorubicin, micellar doxorubicin and micellar bined ultrasound irradiation (28 kHz (0.04 W/cm2) + 3 MHz (2 W/ doxorubicin with 28 kHz (0.04 W/cm2) and 3 MHz (2 W/cm2) dual cm2)) in 30 s intervals for 20 min at several depths in the animal frequency sonication in continuous mode. Based on spectrofluori- body. True to expectations, the temperature rise is more serious metric analysis, doxorubicin content (lg) was presented as at a 1 cm depth, which is closer to the ultrasonic probes than in mean ± SD in different tissues.
the other regions. The increase is also seen to some extent at the The drug content in the group that received micellar drugs in 2 cm depth, but the other two depths do not change temperature their tumor tissue was significantly higher (3.34 times) than in significantly. The 1 cm thermocouple was placed into tumor tissue, the group receiving doxorubicin in its free form (p < 0.05). In other and the other three were placed outside the tumor. From this data, non-tumor tissues, the drug content in the micellar group was low- the duration of sonication in future experiments was selected to be er (1.35 times) than in the group that received free doxorubicin 2.5 min; as Fig. 8 illustrates, this length of time causes a tempera- (p < 0.05).
ture rise of no more than 4 °C outside the tumor. The results indi-cate that a 2.5 min sonication causes a temperature rise below the level of hyperthermia (T 6 42 °C). We have evaluated a controlgroup without sonication but we did not have any temperature Ultrasound is a unique tool in the field of drug delivery because it enables improved penetration ability, which allows substances to To study the drug distribution in animal tissues, three forms of reach deep regions in the body, and it is non-ionizing by nature. In doxorubicin were injected into female Balb/C mice that had tumors addition, it may be used to enhance intracellular drug uptake from of about 7–9 mm diameter: free doxorubicin, micellar doxorubicin micelles. The biological action of ultrasound encompasses several and micellar doxorubicin with sonication under optimum condi- mechanisms of action including the generation of thermal energy, tions. The doxorubicin concentration in tissues and in the plasma sonoporation, the enhancement of local microjets due to inertial were determined by a fluorescence assay; the results are shown cavitation (which is enhanced in a multifrequency sonication field in Table 1. In this table, standard curves for each tissue were estab- [1,2]) and the enhancement of the permeability of blood capillaries lished by adding known amounts of doxorubicin to tissue extracts [4–10]. To inhibit an unwanted temperature increase in critical tis- of control mice to obtain standard measurements at different sues above their tolerances, the sonication time was selected as concentrations. The results are shown and are fit with linear 2.5 min. It is also expected that under multifrequency sonicationconditions, enhanced cavitation activity will be observed.
The amount of drug collected from the different groups was 12.64 ± 1.94 lg for the micellar doxorubicin injection group and16.09 ± 1.07 lg for the micellar doxorubicin injection with sonica- tion group. The amount of drug injected was 26 lg for each animal in the different groups. The difference between the amounts of col- lected drug and injected drug might be due to the fact that we did not collect animal urine and we used plasma instead of serum. Fur- thermore, to collect the drug from several tissue extracts, only the filtered supernatant of centrifuged tissue extracts was used to track the drug, but the sediments may also contain some drugs. Fi-nally, the drug content of tissues such as the muscle, lung, gut and brain was not measured. All of these issues may cause a decrease in drug collection.
Time (min)
It is also apparent in Table 2 that sonication of the micellar form of the drug caused a significant increase (2.60 times) in drug up- Fig. 8. The mean ± SD of temperature changes (°C) at different depths in mice (1, 2, take by the tumor and a decrease in drug uptake by other tissues 3 and 4 cm) versus duration of sonication (min) under dual frequency sonicationconditions (3 MHz and 28 kHz).
(from 1.5 times for the spleen to 9.6 times for the heart). It is ex- Author's personal copy
H. Hasanzadeh et al. / Ultrasonics Sonochemistry 18 (2011) 1165–1171 Table 2Doxorubicin content (lg) presented as mean ± SD in different tissues and in different groups studied 24 h after injection.
Micellar doxorubicin Micellar doxorubicin + sonication pected that the accumulation and uptake of the drug from the [2] R. Feng, Y. Zhao, C. Zhu, T.J. Mason, Enhancement of ultrasonic cavitation yield micellar form in the tumor tissue will be higher (8.69 times) than by multi-frequency sonication, Ultrason. Sonochem. 9 (2002) 231–236.
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