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Year : 2020  |  Volume : 16  |  Issue : 6  |  Page : 1250-1257

Comparison of the effects of ultrasound in a repetitive mode and acoustically active lipospheres in the presence of doxorubicin on breast adenocarcinoma

1 Department of Medical Physics and Physiology, School of Medical Sciences, Arak University of Medical Science, Arak, Iran
2 Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
3 Department of Medical Physics, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
4 Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
5 Department of Pathology, Faculty of Veterinary Medicine, Tehran University of Medical Sciences, Tehran, Iran

Date of Submission01-Aug-2016
Date of Decision06-Dec-2019
Date of Acceptance21-Jan-2020
Date of Web Publication18-Dec-2020

Correspondence Address:
Parviz Abdolmaleki
Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, P. O. Box, 14115-154, Tehran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_839_16

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 > Abstract 

Purpose: We want to compare the synergistic effect of low-intensity, dual-frequency (dual) ultrasound (US), applied in a repetitive sonication mode, and acoustically active lipospheres (AALs) containing doxorubicin (DOX) in a murine model (Balb/C).
Subjects and Methods: The tumor-bearing mice were divided into nine groups, namely two untreated groups (control and sham), and seven experimental groups, including treated with dual-frequency US (150 kHzcontinuous + 1MHzpulse), triple exposure (3×30min) dual-frequency US, DOX (2 mg/kg intravenous), DOX in combination with single exposure (30 min) to dual-frequency US (drug + dual), DOX in combination with triple (3 × 30 min) exposure to dual-frequency US (drug + dual [REP]), AALs containing the drug-loaded (AAL), and a group receiving AAL in combination with single exposure (30 min) dual-frequency US (AAL + dual), respectively.
Results: The effectiveness of DOX on tumor growth was enhanced by a factor of three when combined with the triple exposures of dual US (drug + dual [REP]). This combination protocol further increased the times needed for each tumor to 2 and 7 times its initial volume, respectively by 94% and 36% compared to the drug group. During the 30 days, following the treatment of tumors, the relative volume of tumors in AAL group was 118% less than that of the drug group. The survival rate of the groups treated with drug and AAL + dual was increased by 78.7% and 167% compared with sham, respectively.
Conclusion: Although as a short treatment, a major improvement in treatment was observed by (drug + dual [REP]) compared with other treatments, the AAL + dual treatment compared with (drug + dual [REP]) showed an increase in the survival rates, hence more preferable over long periods.

Keywords: Acoustically active lipospheres, breast adenocarcinoma, doxorubicin, dual-frequency ultrasound, repetition exposure

How to cite this article:
Soleimani H, Abdolmaleki P, Mokhtari-Dizaji M, Toliat T, Tavasoly A. Comparison of the effects of ultrasound in a repetitive mode and acoustically active lipospheres in the presence of doxorubicin on breast adenocarcinoma. J Can Res Ther 2020;16:1250-7

How to cite this URL:
Soleimani H, Abdolmaleki P, Mokhtari-Dizaji M, Toliat T, Tavasoly A. Comparison of the effects of ultrasound in a repetitive mode and acoustically active lipospheres in the presence of doxorubicin on breast adenocarcinoma. J Can Res Ther [serial online] 2020 [cited 2021 Dec 4];16:1250-7. Available from: https://www.cancerjournal.net/text.asp?2020/16/6/1250/303899

 > Introduction Top

The toxicity of doxorubicin (DOX) presents a major dose-limiting factor, hence the fact that any means of reducing the required effective dose, meaning an increase in the effectiveness of the drug, is highly desirable.[1] The application of an extracorporeal source of focused ultrasound (US) energy could provide a potential means of inducing coagulative necrosis in targeted tissues without damaging the overlying and surrounding vital structures.[2],[3] US has been widely used to increase the permeability of different biological barriers, such as cell walls;[2],[4],[5] moreover, its coadministration has been reported to enhance the activity of cancer chemotherapeutic drugs.[6],[7],[8],[9],[10],[11] Low-intensity US (intensity < 5.0 W/cm2) can suppress cell proliferation and clone formation, improve the effects of anticancer chemicals, and deactivate cells through indirect mechanisms.[12],[13],[14] In addition to these studies, much research has been done to find new strategies for localized drug delivery, using engineered delivery vehicles and US energy.[15],[16],[17] Methods using US and acoustically active lipospheres (AAL) include small gas bubbles surrounded by thick oil shells, enclosed by an outer most lipid layer,[18] which might be used to carry bioactive substances to vascular endothelium and cell membrane.[19],[20] When AALs carry the drug, they become dually effective drug delivery vehicles, being both the carrier and activator ultrasonic drug delivery vehicles.

The aim of this study was to compare the synergistic effects of US and the AALs + DOX on adenocarcinoma tumor growth regarding breast cancer in animal models of Balb/c; this research further aimed at evaluating and comparing the effectiveness of the two kinds of treatments. Therefore, this study includes two main sections, the first of which determines the effectiveness of drug in drug + US group (DOX in combination with single (30 min) or triple (3×30min) exposure to dual-frequency US) compared to drug group. The second section investigates the synergistic effect of US and AAL + drug on the same condition. Changing the therapeutic agents can conduce to reducing the dosage and side effects of the dangerous and sometimes deadly, anticancer DOX.

 > Subjects and Methods Top

Doxorubicin hydrochloride (DOX) was obtained from EBEWE Pharma Ges. m.b.H.Nfg.KG, A-4866 Unterach, Austria, and LIPOID S PC 3 was purchased from Lipoid Company. Pluronic F-68, Coconut oil from Cocos nucifera, cholesterin (95%), Brij R-98, ketamine, and xylazine were purchased from Sigma-Aldrich. Perfluoro-compound FC-72 was obtained from organic U. S. A company.

Female Balb/C strain mice, aged 4–5 weeks, were purchased from Pasteur Institute, Tehran, Iran. Throughout the experiments, they were handled under standard conditions concerning transportation, humidity, temperature, water, and feeding. After 1 week, the mice were transplanted (homograft) as described in the following section. It was used 4T1 breast cancer cell line to establish tumor-bearing female Balb/c mice. The breast tumor cell line was obtained from Pasteur Institute (Tehran, Iran) and was cultured in Roswell Park Memorial Institute - 1640 supplemented with 10% fetal bovine serum (Gibco, USA), 2 mM L-glutamine and 1% penicillin–streptomycin (Gibco, USA). Following the preparation, 1 × 106 4T1 cells were injected subcutaneously into the flank regions of female Balb/c mice.[21],[22],[23] After the inoculated tumor volume reached 200–300 mm3 in the Immunology Department of Tarbiat Modares University, Tehran, Iran. The source animal was anesthetized according to other studies.[24],[25] The tumor was then extracted under sterile conditions, cut into small pieces (2–3 mm3 each), and washed with phosphate-buffered saline solution. The receptor mouse was simultaneously anesthetized through the same method. Flank skin near the breast was then opened where a piece of the extracted tumor (free of necrotic or fat tissues) was transplanted under sterile conditions. After 1 week of tumor implantation, skin hair of the tumor area was removed by hair remover cream; next, the tumor volume was measured by a digital caliper (ks-43, ±1 mm, canon, Germany), and calculated as follows: Tumor volume (mm3) = (length × width2)/2.[26] The tumor was measured (each measurement was repeated 3 times) once every 2–3 days until its diameter was about 8–9 mm. The animals with sufficiently large tumors were isolated and prepared for further experiments. The initial tumor volume before the treatment was 350 ± 50 mm3, and the weight was 19 ± 2.5 g. All experimental procedures were performed in accordance with the protocols approved by the Universities Federation for Animal Welfare.[27]

Animals in the groups receiving US were treated by 30 min of sonication after DOX injection, with the time determined based on the results of previous studies.[24],[25],[28],[29] Based on other studies, a single dose of DOX (2 mg/kg) was chosen for our experiments.[30],[31],[32] The animal was once again anesthetized and carefully positioned between the two US probes. Afterward, the tumors were measured with calipers (using the same method described above) once every 3 days for 30 days, with each measurement repeated 3 times. The collected data were then used to calculate tumor growth parameters, and the relative volume growth (%) was calculated using the following formula:[24],[27]

where V and V0 are the volume of a tumor on the day after treatment and the initial volume, respectively.

The tumor growth inhibition (TGI) percentage was estimated using the following formula:[27]

Where Vt,day and Vc,day are the relative volume of the tumor on the same day in the treatment and control (or sham) groups, respectively.

The survival period for each mouse was also measured and expressed in days in the results section. Mice that died of bleeding or were sent to the pathology (to verify the correct tumor type and additional histopathological studies) were not included in the data set.

Preparation of acoustically active lipospheres

AALs were prepared according to the method described by Fang et al.[33] Phosphatidylcholine (2.8%, w/v in the final product), cholesterol (1.2% in the final product), and co-emulsifier (0.8% in the final product) were dissolved in an appropriate volume of chloroform–methanol (2:1). The organic solvent was evaporated in a rotary evaporator at 40°C–45°C, 80 RPM to obtain a thin film; solvent traces were removed by maintaining the lipid film under a vacuum for 24 h. The film was hydrated through double-distilled water using a probe-type sonicator (ultrasonic processor Hielscher up 400s [400 watts, 24 kHz], Germany) for 15 min in ice bath. After that, coconut oil (1.8% in the final product) and perfluorocarbon (16% in the final product) were added to the system, followed by high-shear homogenization (Heidolph DIAX 100 KB 30/100W [Germany]) for 15 min; the solution was once again sonicated for 15 min in ice bath.

Droplet size and zeta potential

The mean particle size (z-average) and zeta potential of the AALs were measured by a laser scattering method (Malvern Nano ZS_ 90, Worcestershire, UK). The formulations were diluted 100-fold with double-distilled water prior to the measurement. The evaluations were repeated three times.

Doxorubicin encapsulation in acoustically active lipospheres

AALs were once again prepared according to the methods listed above. However, 10 ml of distilled water was added to doxorubicin hydrochloride (2 mg/ml) for loading; the drug was in the dark for 24 h at 4°C, after which, coconut oil and Pluronic F-68 were added under the same conditions.

High-performance liquid chromatographic analysis of doxorubicin

The analyses were carried out using a high-performance liquid chromatographic (HPLC) system. The Knauer liquid chromatography (Smart line; Knauer, Berlin, German) included a double beam UV/Vis detector, Conductivity Detector Alltech 650 with Alltech ERIS 1000 HP Autosuppressor, and a reverse-phase C18 column (H. P. 25 cm × 0.4 cm internal diameter) S5ODS1–8961 (Knauer, Berlin, Germany). The mobile phase consisted of 25% acetonitrile and 75% water in the presence of 0.1% triethylamine; pH was adjusted to 3 with phosphoric acid. The mobile phase was filtered through a 0.45 μm membrane filter (Whatman Mill, Maidstone, Kent, England). The flow rate was 1.0 ml/min, and the column temperature was 30°C. The UV/visible detector was set at 480 nm.

Determine the amount of drug-loaded in acoustically active lipospheres

AALs were centrifuged at 32,000 g for 60 min at 4°C in a Beckman optima MAX ultracentrifuge (Beckman Coulter, USA) to separate the incorporated drug in AALs from the free form of the drug. The supernatants were analyzed by HPLC so as to determine the amount of free form drug. The prepared solution containing encapsulated DOX was then dissolved in appropriate amounts of methanol to release the DOX entrapped in lipospheres. Subsequently, the total amount of DOX in the solution was determined by HPLC at the same wavelength.

In vivo experimental design

The Balb/C female mice were divided into nine groups, each including 10–12 subjects: (1) the control group, receiving no treatments; (2) the sham group, placed in water for 30 min, but receiving no exposure to US; (3) the drug group intravenously administered with 2 mg/kg body weight of DOX alone; (4) receiving combined dual-frequency US 150 kHzcon (0.2 W/cm2) + 1 MHzpl, 80% (2 W/cm2); (5) dual (REP) group, treated only by triple exposures to US dual-frequency 150 kHz con + 1 MHzpl, 80%; (6) drug + dual group, receiving DOX and combined dual-frequency US; (7) the drug + dual (REP) group, receiving DOX and triple exposures of dual-frequency US; (8) AAL group, receiving AALs containing the drug-loaded, and (9) AAL + dual group, receiving AALs containing the drug-loaded and combined dual-frequency US. Groups (4), (5), (6), (7), and (9) received US for 30 min. (5) and (7) protocols were carried out every repeated exposure at approximately 1 week intervals (time interval between repeated exposure was 1 week).

Ultrasound apparatus and intensity measurement

The experimental setup used in this study is shown in the study of Barati.[24] A 150 kHz PZT transducer (T1) with a probe diameter of 30 mm and an effective radiation area (ERA) of 5 cm2 was used (SM3678B, Shrewsbury Medical Co., UK), 0.2 W/cm2, 0.75 × 105Pa. The other source was a 1 MHz PZT transducer (T2) with a 30 mm probe diameter and an ERA of 5 cm2, (Sonoplus 462, Enraf Nonius Co., Netherlands), 2W/cm2, 2.38 × 105Pa. Acoustic calibration for the power and intensity of the device was tested using a hydrophone method in a water-free gas in a cubic chamber (Bruel and Kjaer model 8103, Denmark) and radiation force balance (UPM-DT-10, Netech, USA, ±1 mW). All reported experimental intensities consisted of the spatial average temporal average (SATA). For both systems, we were able to change the intensity (SATA) (W/cm2), sonication mode, and duty cycle at the adjusted sonication duration.

The animal keeper cage was made up of a polystyrene cylindrical net so that the waves could easily pass through them. The most important feature of this system was the degree of freedom associated with the movement of the cage, which would position the tumor in the flank area, the center of the interference of the two US fields; in this way, the center of the tumors was 1 cm away from each probe.[24]

These devices were equipped with a built-in digital timer. Although the temperature of the solution increases after long sonication periods as employed in the present study (30 min), it is still below the threshold temperature for biological environments (T <40°C–42°C). Tissue temperature was none invasively measured and monitored using K-type wire thermocouples (TP-01, Lutron Electronic Enterprise Co., Taiwan). The total energy density (the total amount of energy delivered per unit area) for a 30-min sonication period was 3960 J/cm2 as follow:

([0.2](w/cm2)+[2](w/cm2)) × [30×60](s)] =3960(J/cm2)Pathology

In order to verify that the correct tumor type was studied and analyzed, tumor tissues were studied histopathologically. All mice in treated and control groups were sacrificed with ether and then necropsied. Tissue samples from the tumor mass of the mammary gland were prepared for microscopic examination. All tissue samples were cut at a thickness of 4 mm and fixed in 10% formalin solution. The tissue sample processing included various stages of dehydration with alcohol, clearing with xylol, and impregnation with melted paraffin. Paraffin-embedded tissue samples were then sectioned in 5 μ thick slices with a microtome and stained with hematoxylin and eosin.[34]

Statistical analysis

Data analysis was done through analysis of variance (ANOVA) followed by least significant difference post hoc for multiple comparison with SPSS V.13 software (SPSS/PC Inc., Chicago, IL, USA). The results are presented as the mean ± standard deviation after verifying a normal distribution and homogeneity variances, ANOVA analysis was done with a significance level of P < 0.05. To reduce the scatter in the data in statistical analysis, the number of subjects in each group was considered to be eight.

 > Results Top

In the present work, the effectiveness of the treatment was assessed by measuring the tumor growth and changing certain US parameters in different studied mouse groups. Since the results obtained for the control group were similar to the sham group in all experiments, the sham results are used as the reference for all the comparisons.

A comparison of the volume changes during the 30 days after sonication and the survival rate of the groups treated is shown, respectively, in [Figure 1] and [Table 1]. Comparative experiments showed that along with the gradual elimination of the drug from the body (from day 12 to day 18), a significant difference was observed between the drug group and drug + dual group (P < 0.05).
Figure 1: Comparison of the survival rate in different groups. Data analysis was done by the analysis of variance followed by a least significant difference. The letters a-d indicate significant differences between the groups

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Table 1: Relative tumor volume data in different groups

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Using the measured volumes and as a comparative measure of tumor growth, we also calculated T2, T5, and T7 times, the times required for each tumor to reach T2, T5, and T7 times its initial volume, respectively. The T2, T5, and T7 values (expressed in days) of the groups are shown in [Table 2].
Table 2: Comparison of T2, T5, and T7 (the time needed for each tumor to reach 2, 5, and 7 times its initial volume, respectively

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The tumor growth inhibition percentage (TGI %) was calculated at different time intervals following the treatment (3, 6, 9… and 30 days after the treatment) [Table 3]. The TGI% for the drug + dual (REP) group compared to sham was 61.35% after 30 days. There were significant differences between the drug + dual (REP) and sham, drug, and dual (REP) groups regarding TGI% (P < 0.005). No significant difference, however, was observed between the drug + dual (REP) and drug + dual groups (P > 0.05). Our findings showed the survival period of the drug + dual (REP), drug + dual, and the dual (REP) groups to increase by 2.21, 2.16, and 1.78 fold compared to the sham group, respectively.
Table 3: Tumor growth inhibition; percentage, for different groups

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Pathological examinations were performed to confirm the obtained information. Histological results from transplanted tumor tissues identified the tumor cell type as papilloma breast carcinoma. Papillary pattern of tumor growth with malignant signs such as pleomorphism and hyperchromatism were seen in the nucleolus of neoplastic cells of the control [Figure 2].
Figure 2: Histological examination of studied tissues. (a) Control, adenocarcinoma of mammary glands. The growth of tumor without any necrotic area is seen. The connective tissue of tumor stroma is little in contrast of severe growth of tumor. Right above a thrombus, embolism of breast cancer in a large vessel in the lung is obvious; right down, two foci of metastatic tumor breast are observed. (b) Treatment with drug alone. A large area of tumor necrosis is seen in the slide. New growth of adenocarcinoma can be observed in the middle of necrotic area. (c) Treatment with drug + dual ultrasound. Breast cancer, a large focus of necrotic cells in the middle of the slide is seen on the right and above, also left and below. The growth of neoplastic cells is seen. (d) Treatment with drug + dual (REP). A large area of necrosis is seen in breast cancer. Papillary growth of tumor cell in the left and above of the slide is observed (H and E, Bar 100 μm). As exemplified in Figure 2 (d), the necrotic area was too large to be observed in one field of low magnification under the light microscope

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In this research, by considering the hydrophilic nature of DOX, it was tried to enter drug into the capsule AALs. Our results show that the success rate was about 37.37%.

The synergistic effect of AAL with the dual US was examined. The analysis of the relative volume changes during the 30 days after sonication is shown in [Table 4]. A considerable difference was observed between AAL + dual and drug groups (P < 0.005).
Table 4: Relative tumor volume data analysis was done by analysis of variance followed by least significant difference

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Total TGI% for 30 days in groups, drug, AAL, and AAL + dual were 8.5%, 26.5%, and 36%, respectively [Figure 3]. Furthermore, the survival rate of the groups treated with drug and AAL + dual was increased by 78.7 and 167% compared with sham, respectively. Test Kaplan–Meier [Figure 4] confirmed the above results.
Figure 3: Tumor growth inhibition (%) results of different groups from day 3–30 after treatment and total tumor growth inhibition during 30 days after treatment in compared to the sham group

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Figure 4: Survival periods of adenocarcinoma tumor-bearing Balb/C mice on treatment. With 2 mg/kg doxorubicin alone, 2 mg/ kg doxorubicin + simultaneous combined dual-frequency orthogonal US (1 MHzpl,80%+150 kHzcon) in 30 min, acoustically active lipospheres + simultaneous combined dual-frequency orthogonal US (1 MHzpl,80%+150 kHzcon) in 30 min and control. Data represent significant differences according to of variance (Kaplan–Mayer) (P ≤ 0.005) between doxorubicin and acoustically active lipospheres + dual-frequency orthogonal US and (P ≤ 0.05) between doxorubicin + dual-frequency orthogonal US and acoustically active lipospheres + dual-frequency orthogonal US

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In addition, [Figure 5]a and [Figure 5]b shows tumor tissue treated with AAL groups (including drug) with or without US radiation and compares cardiac muscle heart tissue (myocardium) between these two treatments [Figure 5]c and [Figure 5]d.
Figure 5: Histopathology of the tumor. (a) corresponds to the acoustically active lipospheres group. Tumor malignant carcinoma, cystic, excessive bleeding can be seen around the tumor. Inflammation and swelling created at least seven focal necrotic of tissue is observed with a magnification of 30 (H and E, Bar 200 μm). (b) In the acoustically active lipospheres + dual (150 kHz con + 1 MHz pl, 80%) group, the tumor has found cystic mode and multiple necrotic foci in throughout tumor are visible (H and E, Bar 100 μm). Comparisons between cardiac muscle heart tissue in animals treated with: acoustically active lipospheres + dual (150 kHz con + 1 MHz pl, 80%) on the left (c) and treated with: acoustically active lipospheres on the right (d). On the left, the heart muscle is healthy (H and E, Bar 500 μm) but it is observed partially necrotic muscle fibers and cardiac repair reaction on the right. There is also a slight swelling and inflammation of myocardial but fibrosis has not formed yet (H and E, Bar 200 μm)

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 > Discussion Top

In the present study, comparing the effect of DOX + dual (REP) US to that of the drug alone, it was shown that the time effectiveness of DOX + dual (REP) US was more than two-fold the time effectiveness of DOX in tumor growth. Combining the drug with triple low-intensity exposure (drug + dual [REP]) can slow the tumor growth and increase T2, T5, and T7 by 93.52%, 43.54%, and 36.03%, respectively, in compared to the drug group. This means that increasing the number of exposures from one to three improves the synergistic effect of dual-frequency US on T2 and T7 by 26.79% and 25.59%, respectively.

In the previous research, we showed that following the injection of DOX, exposure to US (30 min) at 150 kHzcon potentiates the effect of DOX. Therefore, TGI% (3–21 days) in the drug and drug + 150 kHz groups was 23% and 55%, respectively.[29] With respect to the survival period and TGI %, our results do not reflect any clear difference between the application of a single dose of the drug (DOX) and the application of dual (REP) US, unlike what was observed by the application of 150 kHzcon US in our earlier research.[29] This suggests that the latter treatment increases the survival period in a fashion similar to that caused by a single dose of the drug. Although a clear difference was found between the drug group and drug + dual (REP) groups, no significant difference was seen between the drug + dual (REP) and drug + dual groups. It is possible that with the increase in the number of exposures, a more significant difference is achieved in the survival period, which requires further investigation.

Although several studies have been conducted on restructuring the DOX carriers for higher-targeted and more effective inhibition in tumor cells,[33],[35],[36] none has compared the effectiveness of treatments with AAL as a drug carrier in the presence or absence of dual US.

The investigations showed that US was able to carry biologically active drug and destroy lipospheres through their energy, releasing the drug in the desired location. In this study, the synergistic effect of AAL with dual US was examined, synergistic effects of dual US and AAL were then compared with drug + dual (REP) protocol. Fang and colleagues loaded a lipophilic antioxidant called resveratrol in the AALs. They stated that a nondiluted dose of the drug can be loaded in lipid-coated microbubbles without a deposit. Such kinds of lipospheres are clinically acceptable.[33] In this research, by considering the hydrophilic nature of DOX, it was tried to enter drug into the capsule AALs. In our work, due to the characteristics of the hydrophilic drug, the transpiration and escape velocity of the AALs appeared to be very slow under normal conditions.[32] Our data showed that the efficacy of DOX to day 9 was slightly more than AAL, but due to the features of AALs covering surface, they have longer stability in the circulatory system.

The results suggested a significant difference between these two groups from day 12 to day 24 following the treatment (P < 0.05). It seems that during the gradual withdrawal of the drug-free from body animals, the retention rate drug incorporation into AALs was higher than the free drug. The high frequency produced the force necessary to move the AAL toward the vessel wall and contact it. The frequency pulse then burst and destroyed them.[32],[37] Comparative experiments indicate that the effectiveness of this treatment (AAL) is more impressive with dual US. Moreover, the survival rate in AAL + drug (99.2 ± 20.44 day) group was 35% higher than the drug (66.38 ± 16.01 day) group compared with sham (37.14 ± 7.54 day), corroborating the effectiveness of AAL + drug. Amount of T5 in AAL + dual group to compare with sham, drugs, and drug + dual increased by 61%, 51%, and 14% respectively, indicating the optimal effect of treatment with AAL and US irritation. It is to be noted that the amount of DOX incorporated in AALs was 37.37% in comparison to free DOX. However, there was no difference between AAL + dual and drug + dual groups regarding T2; in the long term, however, (T7) difference was 11%. The survival rate of AAL + dual group, compared with the drug + dual group, showed a 22.4% increase, which is a favorable outcome since the most important factor is “the increase in survival.” Probably, if the amount of injected DOX loading was similar to free DOX, the survival rate in AAL + dual group would show a further increase compared with the drug group. However, the survival rates in the AAL group and AAL + dual group increased by 18.4 and 49.4%, respectively, compared with the drug group, representing a durability effect of the drug incorporated in AAL in the blood circulation compared to free drug and US effect on AAL. Despite the superiority of treatment by drug + dual (REP) over all other treatments, the AAL + dual group, compared to drug + dual (REP), showed an increase of 17.9% in survival rates, which is a sign of superiority over all treatments over long periods.

 > Conclusion Top

The results presented in this study suggest that the number of repetitive exposures of low-intensity US (as a noninvasive tool) might be a major parameter determining the rate of tumor growth suppression in adenocarcinoma in an in vivo model. Moreover, simultaneous treatment with repetitive application of US and the drug is the most efficient in short-term treatments. However, to increase the survival time (long-period effects), the combined treatment of AALs-delivered DOX and US appears to be a highly efficient approach; and according to the amount of injected drug loaded in AAL (37.37%) compared to the free drug could reduce side effects.

According to the present results, it is likely that the combination of these two methods (dual pulse repetition and AAL) will increase the efficacy of treatment. By considering the process of AAL elimination from the circulatory system, this will require further study and experimental research.


The authors gratefully acknowledge the Research Council of Tarbiat Modares University for the financial support.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3], [Table 4]


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