Doxorubicin Delivery into Tumor Cells by Stable Cavitation without

Article pubs.acs.org/molecularpharmaceutics

Doxorubicin Delivery into Tumor Cells by Stable Cavitation without Contrast Agents Kamel Chettab,*,†,‡,§,∥ Jean-Louis Mestas,⊥ Maxime Lafond,⊥ Djamel Eddine Saadna,†,‡,§ Cyril Lafon,⊥ and Charles Dumontet†,‡,§,∥ †

Université de Lyon, Université de Lyon 1, 69000 Lyon, France INSERM U1052, Centre de Recherche en Cancérologie de Lyon, 69008 Lyon, France § CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon, 69008 Lyon, France ∥ Hospices Civils de Lyon, Pierre Bénite, France ⊥ Université Lyon, Université Lyon 1, INSERM, LabTAU, F-69003 Lyon, France ‡

ABSTRACT: Doxorubicin, alone or in combination with other anticancer agents, is one of the most widely used chemotherapeutic agents and is administered in a wide range of cancers. However, the use of doxorubicin is limited due to its potential serious adverse reactions. Previous studies have established the ability of high intensity focused ultrasound (HIFU) in combination with various contrast agents to increase intracellular doxorubicin delivery in a targeted and noninvasive manner. In this study, we developed a new sonoporation device generating and monitoring acoustic cavitation bubbles without any addition of contrast agents. The device was used to potentiate the delivery of active doxorubicin into both adherent and suspended cell lines. Combining doxorubicin with ultrasound resulted in a significant enhancement of doxorubicin intracellular delivery and a decrease in cell viability at 48 and 72 h, in comparison to doxorubicin alone. More importantly and unlike previous investigations, our procedure does not require the addition of contrast agents to generate acoustic cavitation and to achieve high levels of doxorubicin delivery. The successful translation of this approach for an in vivo application may allow a significant reduction in the dosage and the adverse effects of doxorubicin therapy in patients. KEYWORDS: doxorubicin, cavitation, cell death

1. INTRODUCTION Doxorubicin (adriamycin) is an anthracycline antibiotic used for the treatment of many different tumor types, including bladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple myeloma, and Hodgkin’s lymphoma. There are many proposed mechanisms by which doxorubicin may induce cell death, including DNA synthesis inhibition, DNA alkylation, and free radical generation. Doxorubicin is known to bind to nuclear DNA and thus inhibit topoisomerase II, which may be the main mechanism of action.1−3 The use and therapeutic benefits of doxorubicin are limited by the potential for severe myocardial damage and poor distribution in solid tumors.4,5 Several efforts to develop novel anthracyclines with more potent activity and/or less cardiac toxicity has resulted in more than 2000 analogues. However, anthracyclines, especially doxorubicin alone or in combination with others anticancer agents, remain one of the most widely used chemotherapeutic compounds since their National Cancer Institute approval in 1974.6−8 To overcome the cardiotoxicity associated with conventional free doxorubicin therapy, many strategies have been applied including the coadministration of cardioprotective © XXXX American Chemical Society

agents and the use of liposomal and encapsulation technologies.9,10 To increase the short circulation half-life of liposome drugs by preventing uptake by the reticuloendothelial system, a pegylated liposomal formulation of doxorubicin such as Caelyx has been developed.11,12 However, the pegylation may reduce the accumulation of liposomal drugs in the tumor tissue13 and present a high affinity for the skin, causing handfoot syndrome in approximately 50% of patients dosed at 50 mg/m2. To overcome these limitations and the adverse events on healthy cells, the development of more efficient, safe and targeted delivery technology is necessary to reduce the therapeutic dose and represents an important issue for cancer therapy. To be effective, anticancer drugs must achieve two goals. First, the drug must penetrate in the tumor tissue specifically and efficiently. Second, the clinical dose should be low but sufficient to have a therapeutic effect while avoiding adverse effects. Received: September 30, 2016 Revised: January 9, 2017 Accepted: January 9, 2017

A

DOI: 10.1021/acs.molpharmaceut.6b00880 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 1. Experimental setup. A PC and LabVIEW software controlled the generation of an electrical signal through two PXI cards (6602 and 5412). This signal is amplified then applied to the couple of ceramics connected in parallel. The cavitation noise inside the tube was listened by a PCD (passive cavitation detector or hydrophone). The cavitation signal was preamplified then acquired by the acquisition card (PXI5620) and saved on the computer for post treatment.

above their critical radii and thus collapse violently: this is the inertial cavitation regimen. Collapsing bubbles emit a broadband noise that can be used to estimate the inertial cavitation activity.30 Depending on the regimen, ultrasonic cavitation induces various mechanical effects in the medium, leading to transient or irreversible cell damage.21,31−33 Microstreaming is defined as the creation of micrometric flow around stably oscillating bubbles. In a biological context, the high induced shear stress and surface divergence can lead to the stretching and the opening of biological structures.31 Also, cell membranes can be disrupted.34,35 As a result of inertial cavitation events, the violent stress inflicted to cells can result in membrane permeabilization.36 The aim of our study is to investigate the ability of stable cavitation without contrast agents to potentiate doxorubicin delivery into adherent breast cancer cell line MDA-MB231 and the CCRF-CEM, a human chronic myelogenous leukemia cell line growing in suspension. For this we developed a device including cavitation monitoring software, which allows enhancement of doxorubicin uptake from the surrounding medium into cells. The successful translation of this strategy for an in vivo application could allow significant reduction in the dosage and in the adverse effects of doxorubicin therapy.

Recently, the use of high frequency ultrasound (US) combined with various microbubbles as ultrasound contrast agents was explored to enhance the intracellular delivery of drugs in vivo and in vitro.14−18 Microbubble-assisted ultrasound exposures are commonly assumed to cause acoustic cavitation,19−21 which results in the creation of transient pores in the cell membrane allowing transfer of nucleic acids, antibodies, and drugs.15,22−25 Different types of microbubbles (Vevo Micromarker, BR14, SonoVue, and Optison) were used in several studies implying an application of various US parameters depending on the microbubbles and ultrasound device used. Studies using the combination of ultrasound and microbubbles to enhance drugs delivery into cells apply a lower acoustic pressure than in our study to induce formation of cavitation while avoiding violent collapse that can cause cell death.15,26 Recently, Kovacs et al.27 demonstrated that microbubble-enhanced focused ultrasound disrupted locally and reversibly the blood−brain barrier and potentiated doxorubicin diffusion from the microvasculature into brain tissue, which resulted in a significant increase of survival and in a slower disease progression in two syngeneic mouse models of glioblastoma multiforme. The combination of ultrasound and microbubbles is relatively complex to optimize, resulting in time- and moneyconsuming experiments. Consequently, the results obtained are difficult to apply to other US devices and targets and particularly to in vivo applications. The use of cavitation without contrast agents, referred to as unseeded cavitation, is expected to broaden the range of cavitation applications. For this, the ultrasonic system has to allow the initiation of the phenomenon of cavitation and its maintenance over time. Three different kinds of cavitation regimens should be distinguished. The first one is stable cavitation, during which the gas bubbles present in the water grow by rectified diffusion because of the negative pressure created by the acoustic wave and oscillate within the pressure field. The stable bubble cloud reflects a part of the ultrasound beam, with a characteristic acoustic signature: the linear oscillations of stable bubbles result in the emission of the subharmonic of half the excitation frequency.28,29 When the ultrasonic power increases the movement of the bubbles becomes nonlinear, with the presence of reversible collapse associated with the raising of noise in addition to the F/2 line that correspond to a transitional phase of the cavitation phenomena. With higher power, bubbles grow

2. MATERIALS AND METHODS 2.1. Cell Lines and Culture. The breast cancer cell line MDA-MB231 (ATCC: HTB-26) and CCRF-CEM (ATCC: CCL-119), a human T lymphoblastoid nonadherent cell line, were purchased directly from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were authenticated by ATCC by generating human short tandem repeat profiles by simultaneously amplifying multiple STR loci and amelogenin (for gender determination) using the Promega PowerPlex Systems. MDA-MB231 and CCRF-CEM cell lines were grown in Dulbecco’s modified Eagle medium (DMEM) and RPMI 1640 medium, respectively. Both media were supplemented with 10% heat inactivated fetal calf serum (FCS), 200 UI/mL of penicillin, and 200 μg/mL of streptomycin. The cells were maintained at 37 °C with 5% CO2. All reagents were purchased from Invitrogen (Carlsbad, CA, USA). Using MycoAlert kit (Mycoplasma Detection Kit, Lonza) all cell lines were tested mycoplasma-negative. B


Article


Figure 2. Cavitation signal spectrum generated during sonication.

2.2. Chemicals. Doxorubicin hydrochloride (2 mg/mL) was purchased from Accord Health Care France, SAS. 2.3. Ultrasonic Setup. The ultrasonic setup is composed of two piezo-ceramic focused transducers of 50 mm diameter, 50 mm curvature, and F = 1.1 MHz frequency. These are separated by an angle of 90° and placed in order to match their respective focal points. An in-house hydrophone is placed in the water tank to record acoustic cavitation emissions from the focal area (Figure 1). The excitatory signal is generated by a waveform generator (PXI 5412 National Instruments, Austin, TX) and amplified with a 400 W power amplifier (1040LA, E&I Ltd., Rochester, NY). This signal is a burst of 660 sinusoidal cycles with frequency of 1.1 MHz and a repetition frequency of 250 Hz. Thus, the duty cycle (DC) is 15%. The voltage amplitude on the transducers couple is set at 175 Vpp, which leads to an acoustic peak negative pressure of 3.2 MPa at the focal point (in the Eppendorf tube; 6.4 MPa without the tube; measurement with an optical hydrophone FOPH2000 (RP Acoustics, Leutenbach, Germany)). The focal volume is about 2 × 2 × 2 mm3 corresponding to half the peak pressure in linear regimen. This amplitude has been adjusted to maximize the occurrence of the half-harmonic frequency component, characteristic of oscillating bubbles, with lower occurrence of broadband noise (inertial cavitation). To characterize this regimen, we are interested in the knowledge of two specific parameters established from the frequency spectrum of the cavitation signal. The first is the amplitude of the F/2 line emergence (IF1/2 (dB), Figure 2), which is characteristic of the presence of cavitation29 and in particular the stable cavitation in the absence of inertial cavitation.26 The second one is the average of frequency spectrum CI (cavitation index in dB) that characterizes the inertial cavitation level (gray area in Figure 2). IF1/2 is also present in inertial regimen and tends to disappear flooded in the noise of the strong inertial cavitation regimen. To favor the presence of oscillating bubbles the choice of the ultrasonic power applied is high enough to induce formation of bubbles, but sufficiently gentle to avoid violent collapse. To analyze the cavitation signal, 214 points have been acquired on each burst (600 μs length) with a frequency of 32 MHz. On each spectrum the F/2 level (IF1/2) and the cavitation index (CI) were saved for post-treatment (Figure 2). To complete the device parameters, we studied the sonolysis and the hydroxyl radical OH• production in a PBS + 2 mM terephthalic acid (TA) medium (Table 1) to evaluate the cavitation regimen for the acoustic parameter chosen for in vitro

Table 1. Ultrasound Parameters and Radical Hydroxyl Production by Sonolysisa sonolysis (N = 6)

CCRF-CEM cell treatment (N = 22)

5.50 ± 0.75 7.59 ± 3.65 0.029 ± 0.023 174.8 3.2

1.78 ± 1.52 12.27 ± 2.75 x 174.8 3.2

1.1

1.1

3

3

parameters ⟨Cl⟩ (dB) ⟨IF1/2⟩(dB) HTA concentration (μM) ⟨voltage⟩ (Vpp) peak negative pressure (MPa) ultrasound frequency: F (MHz) mechanical index: PNP(MPa) MI = F(MHz)

a

The peak negative pressures were estimated from measurements in 2 mL Eppendorf tubes filled of degassed water. For sonolysis, 60 s sonication of 2 mM terephthalate acid solution (650 μL in 2 mL Eppendorf tube) was realized (n = 6). ⟨CI⟩ and ⟨IF1/2⟩ were the mean of post treatment values coming from the sonolysis treatment analysis or from the sonication of CEM samples (with or without doxorubicin).

treatment. The OH• dosage was made by the fluorescence of the hydroxyterephthalic acid produced by the reaction of OH• and TA.37 2.4. Doxorubicin Delivery by Ultrasound. Forty-eight hours prior to sonication, CCRF-CEM cells were seeded at 0.2 to 0.4 × 106 cells/mL, in order to achieve 0.5−0.8 × 106 cells/ mL on the day of ultrasound exposure. MDA-MB231 cells were plated at 2 × 106 cells per 75 cm2, and the aim was to have cells at approximately 70% confluence 48 h later. Prior to sonication cell lines were harvested and centrifuged (i.e., 5 min, 300g) then washed twice with Opti-MEM reduced-serum medium, and 650 μL of cell suspensions (2 × 106 cells/mL in Opti-MEM) were placed in a 2 mL Eppendorf tube and doxorubicin was added at 2 μM and 0.1 μM to MDA-MB231 and CCRF-CEM cell suspensions, respectively. After ultrasound application according to the parameters indicated in Table 1, 300 μL of cell suspension was immediately removed and used for doxorubicin fluorescence quantification using a fluorescence-activated cell sorter (FACS) (LSR II, BD Biosciences). The remaining cells were placed in complete medium in a 6-well cell culture plate and incubated at 37 °C in humidified atmosphere with 5% CO2 incubator for 48−72 h until quantification of cell viability. 2.5. Doxorubicin Uptake Analysis. Immediately after doxorubicin addition and ultrasound exposure, cells were C


Article


Figure 3. Ultrasound enhances doxorubicin uptake in CCRF-CEM cell line in a time-dependent manner. Immediately after sonication for a duration ranging from 15 to 45 s the intracellular doxorubicin uptake (A) and cell mortality due to ultrasound were quantified by FACS following annexin VFITC/PI staining (B). For doxorubicin uptake, the data have been normalized by the median fluorescence intensity of the control group. The ends of the box represent the first and third quartile, the vertical line within the box represents the median, and the whiskers extend to the minimum and maximum values.

described previously in the Material and Methods section. The results presented in Figure 3A indicate that doxorubicin accumulation into CCRF-CEM cells is proportional to the US exposure time. Moreover, our results indicate that a 35 s exposure ensures a good compromise between increased doxorubicin uptake and ultrasound-associated toxicity. As shown in Figure 3B an ultrasonic exposure time of 45 s leads to an increase in cell mortality (+9.1%) compared to cells exposed during 35 s. Therefore, constant parameters of 250 Hz PRF, 15% DC, and 175 Vpp for an exposure time of 35 s were considered to be optimal. These results demonstrate that ultrasound without addition of contrast agents potentiate doxorubicin uptake into cells in a time-dependent manner. 3.2. Enhancement of Doxorubicin Uptake by Ultrasound. In order to determine the effect of ultrasound on doxorubicin uptake, the adherent cell line MDA-MB231 and nonadherent cell line CCRF-CEM were exposed to 250 Hz PRF, 15% DC, and 175 Vpp for 35 s. Immediately after the ultrasound exposure, quantitative determination of doxorubicin uptake was evaluated by flow cytometry as described in Materials and Methods. The autofluorescence of doxorubicin was used directly to quantify cellular uptake. The results presented in Figure 4A,B confirm a significant enhancement of intracellular doxorubicin delivery by ultrasound in comparison with cells incubated with doxorubicin alone (***p < 0.002 in both cell lines). Figure 4C illustrates representative histogram plots showing the enhancement of intracellular doxorubicin uptake into MDA-MB231 cells after US exposure. A nonparametric Wilcoxon test was used to study the reproducibility between four independent experiments with at least three technical replicates per experiment. Statistical analysis showed no significant difference in doxorubicin uptake providing evidence that support the reproducibility of the developed device and the reliability of the developed cavitation monitoring software. 3.3. Enhancement of Doxorubicin-Induced Cell Death by Ultrasound. We have previously demonstrated that exposure to inertial cavitation does not induce changes that could alter the cytotoxicity of doxorubicin.38 As the stable cavitation regimen used here results in more gentle effects, we can assume that our present acoustic parameters did not induce changes in doxorubicin cytotoxicity. Sonications were performed at a cell concentration of 2 × 106 cells/mL using appropriate ultrasound parameters as described above. Cell

harvested by centrifugation (i.e., 5 min, 300g) then washed twice in phosphate buffered saline (PBS). Since doxorubicin itself is fluorescent, it was used directly to measure cellular uptake without additional markers, fluorescence intensity being proportional to the amount of doxorubicin internalized. The cells were resuspended in 200 μL of PBS prior to FACS analysis. Doxorubicin red fluorescence quantification was recorded by flow cytometry (FACS) and analyzed using BD FACSDiva software. During FACS analysis the gate was drawn to include all cells and 10,000 events were analyzed. 2.6. Analysis of Cell Viability. Forty eight or seventy-two hours after treatment, the evaluation of doxorubicin-induced reduction of cell viability in cells exposed to ultrasound alone and to doxorubicin with or without ultrasound exposition, was evaluated by annexin V-FITC/propidium iodide (PI) staining using flow cytometry. Briefly, suspension or trypsinized adherent cells including cells present in the culture medium were harvested, washed twice in PBS, and stained with an annexin V-FITC/PI apoptosis kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. During FACS analysis the gate is drawn to include all cells and exclude debris. Results were expressed as a percentage of annexin V-FITC/PI positive cells using BD FACSDiva software. In each experiment cells exposed to the same ultrasound parameters were used to evaluate the direct toxic effect of US. 2.7. Statistical Analysis. Results are representative of four independent experiments with at least three technical replicates per experiment. A statistical analysis using the Wilcoxon test was performed on each test series to determine which groups were statistically different with the statistical analysis software JMP 12.1.0 (SAS Institute, Cary, NC, USA) and a two-tailed significance level of 0.05.

3. RESULTS 3.1. Ultrasound Enhances Doxorubicin Uptake into CCRF-CEM Cell Line in a Time-Dependent Manner. First of all, constant parameters of 250 Hz PRF, 15% DC, 175 Vpp excitation voltage were determined on the basis of their effect on cell viability of CCRF-CEM cell lines (data not shown). Second, sonication for a duration ranging from 15 to 45 s was tested to evaluate the effect of US exposure time on doxorubicin uptake. Immediately after ultrasound exposure intracellular doxorubicin accumulation was evaluated as D


Article


analyzed 72 h post sonication was 74 ± 5.3% in the presence of 2 μM of doxorubicin alone vs 54.6 ± 12.8% when doxorubicin was combined with ultrasound (***p < 0.0001) (Figure 5B). When cells were exposed to the ultrasound at 250 Hz PRF, 15% DC, and 175 Vpp for 35 s, the cell mortality did not exceed 3.6% and 3.1% in MDA-MB231 and CCRF-CEM cells, respectively, confirming that ultrasound alone did not impact cell viability (Figure 5A,B).

4. DISCUSSION Over the past few years, ultrasonic cavitation has been used as a tool for drug and gene delivery. In numerous studies, investigators have obtained improved transfection efficiency and chemotherapeutic drug delivery using contrast agentassisted ultrasound.15,18,39−41 Unlike previously published studies, which indicated that efficient drug delivery is dependent on the combination of ultrasound and microbubbles,4−18 the results from the present study demonstrate that unseeded cavitation is sufficient to potentiate doxorubicin delivery into adherent and suspension cell lines. Moreover, it has been demonstrated that microbubble parameters such as size, composition, gas, and concentration influence ultrasoundinduced cavitation and consequently its effect on drug delivery.15,39 All these parameters make this combination approach relatively complicated to optimize, and moreover, it is time- and money-consuming. Our study could help to overcome the limiting therapeutic dosage of doxorubicin, by reducing drug dosage and the injury of nontarget tissues and to improve the patient’s quality of life following a doxorubicin treatment. We examined the impact of ultrasound without contrast agents to potentiate doxorubicin delivery into adherent breast cancer cell line MDA-MB231 and CCRF-CEM, a human chronic myelogenous leukemia suspension cell line. First, we developed a user-friendly and cost-effective ultrasound device.42 This device generates a stable cavitation and allows a real-time measure of the cavitation activity. Using constant parameters of 250 Hz PRF, 15% DC, and 175 Vpp, we demonstrated that doxorubicin intracellular delivery depends on the US exposure time (Figure 3A). An exposure time of 35 s was considered to be optimal for MDAMB231 and CCRF-CEM cell lines since no ultrasonicassociated cell death was detected under these conditions (Figures 3B and 5A,B). Four independent experiments allowed

Figure 4. Enhancement of doxorubicin uptake by ultrasound. (A) Adherent cell line MDA-MB231 and (B) nonadherent cell line CCRFCEM were incubated with 2 and 0.1 μM doxorubicin, respectively, and exposed to 250 Hz PRF, 15% DC, and 175 Vpp for 35 s. Immediately following ultrasound exposure quantitative determination of doxorubicin uptake was quantified by FACS. All experiments were performed at least in triplicate and repeated independently four times for each cell line. Statistical analysis using a nonparametric Wilcoxon test was performed and significance was defined as p < 0.05. The meaning of the box and whiskers are explained in the caption of Figure 3. (C) Flow cytometry overlay of doxorubicin fluorescence intensity. MDA-MB-231 cells were incubated with 2 μM doxorubicin (MDAMB231 + Dox) in combination with US (MDAMB231 + Dox + US). MDA-MB231 cells served as control. Immediately after US exposition and extensive washing with PBS, the intracellular fluorescence of doxorubicin was quantified by flow cytometry.

viability was monitored by flow cytometry after annexin VFITC/PI staining. As shown in Figure 5A, 48 h post sonication, the CCRFCEM cell line viability after exposure to 0.1 μM doxorubicin and ultrasound was 49.8 ± 7.1% vs 63.8 ± 4.9% after exposure to doxorubicin only (***p < 0.0001). The same result was obtained with the MDA-MB231 cell line. Indeed, cell viability

Figure 5. Enhancement of doxorubicin-induced cell death by ultrasound. CCRF-CEM (A) and MDA-MB231 (B) cells were incubated with 0.1 and 2 μM doxorubicin alone (DXR), or with ultrasound (DXR + US) at 250 Hz PRF, 15% DC, and 175 Vpp for 35 s. Cells with no treatment (CTRL) or with ultrasound alone at the same parameters indicate above were used as controls. Forty-eight (CCRF-CEM) and 72 h (MDA-MB231) after sonication cell viability was quantified by flow cytometry after annexin V-FITC/PI staining. Statistical analysis using a nonparametric Wilcoxon test was performed, and significance was defined as p < 0.05. The meaning of the box and whiskers are explained in the caption of Figure 3. E


Article


“Investissements d’Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR).

us to confirm the potentiation of doxorubicin delivery in both adherent and suspension cell lines by using ultrasound without any contrast agents. As shown in Figure 4A,B ultrasound significantly enhance intracellular doxorubicin concentration in MDA-MB231 and CCRF-CEM by 45% and 31%, respectively. Significant increase of MDA-MB231 and CCRF-CEM cell death is correlated to an increase of doxorubicin intracellular concentration consecutive to ultrasound exposition (Figure 5A,B). In comparison with previously reported studies, our results demonstrate a significant effect of unseeded cavitation on doxorubicin intracellular uptake and on the potentiation of its effect on cell viability.15−18 Indeed, a study published by Arthur et al.16 suggests that ultrasound alone does not increase doxorubicin uptake by three bladder cancer cell lines. Works published by Escoffre et al.15 in which MDA-231 cell line was used and Lee et al.17 emphasize the need for the combination of ultrasound with contrast agent to potentiate doxorubicin effect on cell viability. In a preclinical model Li et al.43 demonstrated that high cavitation activity resulted in an enhancement of doxorubicin concentration by up to 4.5-fold compared to control, but the ultrasound treated region showed significant hemorrhage area. The use of a hydrophone may help to better control and adjust the level of cavitation and thus to avoid the adverse effects due to ultrasound. The presented study did not explore the dynamics of cell permeabilization and recovery. These characteristics should be studied in order to evaluate the duration during that the cavitation actually potentiates the drug penetration and thus to obtain the maximum from this combination. Subject to further studies on the mechanisms of action of ultrasound, the approach presented in this study offers a remarkable potential of target transfer and transmission of therapeutic drugs while mitigating side effects. Indeed, using ultrasound guidance, ultrasound allows drugs delivery in precise and noninvasive manner without the addition of contrast agent. Nevertheless, several parameters may influence the activity of cavitation (gas content of the medium, medium heterogeneity, temperature). This implies an even higher requirement for an efficient stable cavitation control for a potential in vivo application. This consists in the temporal control of the cavitation regimen and activity level as well as the spatial monitoring. This last point is a key parameter for the reliability of in vivo applications of cavitation.

■

■

REFERENCES

(1) Momparler, R. L.; Karon, M.; Siegel, S. E.; Avila, F. Effect of adriamycin on DNA, RNA, and protein synthesis in cell-free systems and intact cells. Cancer Res. 1976, 36, 2891−5. (2) Fornari, F. A.; Randolph, J. K.; Yalowich, J. C.; Ritke, M. K.; Gewirtz, D. A. Interference by doxorubicin with DNA unwinding in MCF-7 breast tumor cells. Mol. Pharmacol. 1994, 45, 649−56. (3) Gewirtz, D. A. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem. Pharmacol. 1999, 57, 727−741. (4) Outomuro, D.; Grana, D. R.; Azzato, F.; Milei, J. Adriamycininduced myocardial toxicity: New solutions for an old problem? Int. J. Cardiol. 2007, 117 (117), 6−15. (5) Minchinton, A. I.; Tannock, I. F. Drug penetration in solid tumours. Nat. Rev. Cancer 2006, 6, 583−592. (6) Carvalho, C.; Santos, R. X.; Cardoso, S.; Correia, S.; Oliveira, P. J.; Santos, M. S.; Moreira, P. I. Doxorubicin: the good, the bad and the ugly effect. Curr. Med. Chem. 2009, 16, 3267−3285. (7) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev. 2004, 56, 185− 229. (8) Turner, A.; Li, L. C.; Pilli, T.; Qian, L.; Wiley, E. L.; Setty, S.; Christov, K.; Ganesh, L.; Maker, A. V.; Li, P.; Kanteti, P.; Das Gupta, T. K.; Prabhakar, B. S. MADD Knock-Down enhances doxorubicin and TRAIL induced apoptosis in breast cancer cells. PLoS One 2013, 8, e56817. (9) Waterhouse, D. N.; Tardi, P. G.; Mayer, L. D.; Bally, M. B. A comparison of liposomal formulations of doxorubicin with drug administered in free form: Changing toxicity profiles. Drug Saf. 2001, 24, 903−20. (10) Langer, S. Dexrazoxane for the treatment of chemotherapyrelated side effects. Cancer Manage. Res. 2014, 6, 357−363. (11) Hilger, R. A.; Richly, H.; Grubert, M.; Oberhoff, C.; Strumberg, D.; Scheulen, M. E.; Deeber, S. Pharmacokinetics (PK) of a liposomal encapsulated fraction containing doxorubicin and of doxorubicin released from the liposomal capsule after intravenous infusion of Caelyx/Doxorubicinil. Int. J. Clin. Pharmacol. Ther. 2005, 43, 588−9. (12) Twelves, C. J.; Dobbs, N. A.; Aldhous, M.; Harper, P. G.; Rubens, R. D.; Richards, M. A. Comparative pharmacokinetics of doxorubicin given by three different schedules with equal dose intensity in patients with breast cancer. Cancer Chemother Pharmacol. 1991, 28, 302−7. (13) Parr, M. J.; Masin, D.; Cullis, P. R.; Bally, M. B. Accumulation of liposomal lipid and encapsulated doxorubicin in murine Lewis lung carcinoma: the lack of beneficial effects by coating liposomes with poly(ethylene glycol). J. Pharmacol. Exp. Ther. 1997, 280, 1319−1327. (14) Cochran, M. C.; Eisenbrey, J.; Ouma, R. O.; Soulen, M.; Wheatley, M. A. Doxorubicin and paclitaxel loaded microbubbles for ultrasound triggered drug delivery. Int. J. Pharm. 2011, 414, 161−170. (15) Escoffre, J. M.; Piron, J.; Novell, A.; Bouakaz, A. Doxorubicin delivery into tumor cells with ultrasound and microbubbles. Mol. Pharmaceutics 2011, 8, 799−806. (16) Arthur, C.; Flaig, T.; Su, L. J.; Denney, R.; Barnes, F.; Glode, L. M. The effect of ultrasonic irradiation on doxorubicin-induced cytotoxicity in three human bladder cancer cell lines. Ultrasonics 2007, 46, 68−73. (17) Lee, N. G.; Berry, J. L.; Lee, T. C.; Wang, A. T.; Scott Honowitz, S.; Murphree, A. L.; Varshney, N.; Hinton, D. R.; Fawzi, A. A. Sonoporation Enhances Chemotherapeutic Efficacy in Retinoblastoma Cells In Vitro. Invest. Ophthalmol. Visual Sci. 2011, 52, 3868−3873. (18) Lamanauskas, N.; Novell, A.; Escoffre, J. M.; Venslauskas, M.; Satkauskas, S.; Bouakaz, A. Bleomycin delivery into cancer cells in vitro with ultrasound and SonoVue or BR14 microbubbles. J. Drug Target. 2013, 21, 407−14.

AUTHOR INFORMATION

Corresponding Author

*Address: Centre de Recherche en Cancérologie de Lyon, Equipe Anticorps-Anticancer. INSERM, U1052-CNRS UMR 5286, Faculté Rockefeller, 8 Avenue Rockefeller, 69008 Lyon, France. E-mail: [email protected]. Phone: + 33 4 78 77 72 36. Fax: + 33 4 78 77 70 88. ORCID

Kamel Chettab: 0000-0002-5311-3792 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Dr. Spasevska Ivana for article proofreading. This work was supported by the Lyric grant INCa-DGOS-4664 and the European Project Eurostars E! 6173 named “Oncoson”. It was performed within the framework of the LabEx DevWeCan (ANR-10-LABX-0061) and CeLyA (ANR-10LABX-0060) of Université de Lyon, within the program F


Article


transfection using PEI nanoparticles. J. Controlled Release 2012, 160, 64−71. (42) Chettab, K.; Roux, S.; Mathé, D.; Cros-Perrial, E.; Lafond, M.; Lafon, C.; Dumontet, C.; Mestas, J. L. Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents. PLoS One 2015, 10, e0134247. (43) Li, T.; Wang, Y. N.; Khokhlova, T. D.; D’Andrea, S.; Starr, F.; Chen, H.; McCune, J. S.; Risler, L. J.; Mashadi-Hossein, A.; Hwang, J. H. Pulsed high intensity focused ultrasound (pHIFU) enhances delivery of doxorubicin in a preclinical model of pancreatic cancer. Cancer Res. 2015, 75, 3738−3746.

(19) Mehier-Humbert, S.; Bettinger, T.; Yan, F.; Guy, R. H. Plasma membrane poration induced by ultrasound exposure: Implication for drug delivery. J. Controlled Release 2005, 104, 213−222. (20) Dalecki, D. Mechanical bioeffects of ultrasound. Annu. Rev. Biomed. Eng. 2004, 6, 229−248. (21) Prentice, P.; Cuschier, A.; Dholakia, K.; Prausnitz, M.; Campbell, P. Membrane disruption by optically controlled microbubble cavitation. Nat. Phys. 2005, 1, 107−110. (22) Reslan, L.; Mestas, J. L.; Herveau, S.; Béra, J. C.; Dumontet, C. Transfection of cells in suspension by ultrasound cavitation. J. Controlled Release 2010, 142, 251−258. (23) Togtema, M.; Pichardo, S.; Jackson, R.; Lambert, P. F.; Curiel, L.; Zehbe, I. Sonoporation Delivery of Monoclonal Antibodies against Human Papillomavirus 16 E6 Restores p53 Expression in Transformed Cervical Keratinocytes. PLoS One 2012, 7, e50730. (24) Suzuki, R.; Namai, E.; Oda, Y.; Nishiie, N.; Otake, S.; Koshima, R.; Hirata, K.; Taira, Y.; Utoguchi, N.; Negishi, Y.; Nakagawa, S.; Maruyama, K. Cancer gene therapy by IL-12 gene delivery using liposomal bubbles and tumoral ultrasound exposure. J. Controlled Release 2010, 142, 245−250. (25) Mestas, J. L.; Lenz, P.; Cathignol, D. Long-lasting stable cavitation. J. Acoust. Soc. Am. 2003 Mar, 113 (3), 1426−30. (26) Watanabe, Y.; Atsuko, A.; Horie, S.; Tomita, N.; Shiro Mori, S.; Morikawa, S.; Matsumura, Y.; Vassaux, G.; Kodama, T. Low-intensity ultrasound and microbubbles enhance the antitumor effect of cisplatin. Cancer Sci. 2014, 99, 2525−2531. (27) Kovacs, Z.; Werner, B.; Rassi, A.; Sass, J. O.; Martin-Fiori, E.; Bernasconi, M. Prolonged survival upon ultrasound-enhanced doxorubicin delivery in two syngenic glioblastoma mouse models. J. Controlled Release 2014, 187, 74−82. (28) Datta, S.; Ammi, A. Y.; Coussios, C. C.; Holland, C. K. Monitoring and simulating stable cavitation during ultrasoundenhanced thrombolysis. J. Acoust. Soc. Am. 2007, 122, 3052−3052. (29) Phelps, A. D.; Leighton, T. G. The subharmonic oscillation and combination-frequency subharmmonic emissions from a reanant bubble: Their properties and generation mechanisms. Acta acust United acust 1997, 83, 59−66. (30) Leighton, T. The acoustic bubble; Academic Press, 1994. (31) Marmottant, S.; Hilgenfeldt, S. Controlled vesicle deformation and lysis by single oscillating bubbles. Nature 2003, 423, 153−156. (32) Wangn, P.; Li, Y.; Wang, X.; Guo, L.; Su, X.; Liu, Q. Membrane damage effect of continuous wave ultrasound on CCRF-CEM human leukemia Cells. J. Ultrasound Med. 2012, 31, 1977−1986. (33) Collis, J.; Manasseh, R.; Liovic, P.; Tho, P.; Ooi, A.; PetkovicDuran, K.; et al. Cavitation microstreaming and stress fields created by microbubbles. Ultrasonics 2010, 50, 273−9. (34) Rooney, J. A. Shear as a mechanism for sonically induced biological effects. J. Acoust. Soc. Am. 1972, 52, 1718−24. (35) Wu, J.; Nyborg, W. L. Ultrasound, cavitation bubbles and their interaction with cells. Adv. Drug Delivery Rev. 2008, 60, 1103−16. (36) Coussios, C. C.; Roy, R. A. Applications of Acoustics and Cavitation to Noninvasive Therapy and Drug Delivery. Annu. Rev. Fluid Mech. 2008, 40, 395−420. (37) Villeneuve, L.; Alberti, L.; Steghens, J. P.; Lancelin, J. M.; Mestas, J. L. Assay of hydroxyl radicals generated by focused ultrasound. Ultrason. Sonochem. 2009, 16, 339−44. (38) Lafond, M.; Mestas, J. L.; Prieur, F.; Chettab, K.; Gracin, S.; Clézardin, P.; Lafon, C. Unseeded inertial cavitation for enhancing the delivery of chemotherapies: A safety study. Ultrasound Med. Biol. 2016, 42, 220−31. (39) Sun, R. R.; Noble, M. L.; Sun, S. S.; Song, S.; Miao, C. H. Development of therapeutic micro-bubbles for enhancing ultrasoundmediated gene delivery. J. Controlled Release 2014, 182, 111−120. (40) Cochran, M.; Wheatley, M. A. In vitro gene delivery with ultrasound-triggered polymer. Ultrasound Med. Biol. 2013, 39, 1102− 1119. (41) Lee, J. L.; Lo, C. W.; Ka, S. M.; Chen, A.; Chen, W. S. Prolonging the expression duration of ultrasound mediated gene G


Doxorubicin Delivery into Tumor Cells by Stable Cavitation without

Recommend Documents