Irinotecan Delivery by Microbubble-Assisted ... - ACS Publications

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Irinotecan Delivery by Microbubble-Assisted Ultrasound: In Vitro Validation and a Pilot Preclinical Study J.-M. Escoffre,†,¶,⊥ A. Novell,†,¶ S. Serrière,† T. Lecomte,‡,§ and A. Bouakaz*,† †

UMR Inserm U930, Université François-Rabelais de Tours, PRES Centre-Val de Loire Université, 37044 Tours, France Université François-Rabelais, UMR CNRS 7292, 37032 Tours, France § Service d’Hépato-gastroentérologie et de Cancérologie Digestive, University Hospital CHU, 37044 Tours, France ‡

ABSTRACT: Irinotecan is a powerful anticancer drug with severe systemic side effects that limit its clinical application. Drugtargeted delivery with noninvasive methods is required to enhance the drug concentration locally and to reduce these undesirable events. Microbubble-assisted ultrasound has become a promising method for noninvasive targeted drug delivery. The aim of this study is to evaluate the therapeutic effectiveness of in vitro and in vivo irinotecan delivery based on the combination of ultrasound and microbubbles. In the present study, in vitro results showed that the irinotecan treatment with microbubble-assisted ultrasound induced a significant decrease in cell viability of human glioblastoma cells. Moreover, using subcutaneous glioblastoma xenografts, the in vivo preclinical study in nude mice demonstrated that this therapeutic protocol led to a decrease in tumor growth and perfusion and an increase of tumor necrosis. The conclusions drawn from this study demonstrate the promising potential of this therapeutic approach for the anticancer targeted therapy. KEYWORDS: irinotecan, ultrasound, microbubbles, sonoporation, anticancer therapy, ultrasound imaging

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INTRODUCTION Irinotecan, also named as CPT-11, is a camptothecin analogue used as an anticancer drug. Irinotecan is highly active as a single antineoplastic agent or in combination with other therapeutic agents such as 5-fluorouracil, folinic acid, or cetuximab.1,2 This anticancer drug is currently indicated for the treatment of advanced colorectal cancer3 and, recently, for the treatment of glioblastoma.4,5 Irinotecan is a prodrug of the pharmacologic active metabolite SN-38 and is generated by human carboxylesterases in the liver (hCE1), the gastrointestinal tract (hiCE),6 macrophages present in the tumor,7 and cancer cells such as glioblastoma.8 SN-38 is 100- to 1000-fold more potent than irinotecan.9,10 The mechanism of SN-38 action involves the inhibition of topoisomerase I, a nuclear enzyme that maintains and modulates DNA structures during the cell replication, translation, recombination, and repair.11 SN-38 interferes with the function of topoisomerase I by binding to its active site forming thus stable topoisomerase I/DNA cleavage complexes and preventing religation of the DNA strand.12,13 This inhibition is specifically cytotoxic toward cells in the Sphase and leads to cell death. © 2013 American Chemical Society

The early use of irinotecan in clinical applications revealed that its therapeutic effectiveness is strongly limited by potentially life-threatening toxicities.3 Cellular mechanisms causing irinotecan resistance have been reported as reducing intracellular drug accumulation and alteration in irinotecan metabolism.14 Although treatment of advanced colorectal cancer patients using irinotecan as a single agent has shown response rates of around 30%, these rates can reach 50% when used in combination with other therapeutic molecules.15,16 Several studies have demonstrated a good dose−intensity relationship for irinotecan either as monotherapy or in combination with 5-fluorouracil.17,18 Irinotecan dose escalation is potentially of interest, but it has be evaluated in only phase I and II trials in highly selected patients, and this concept is potentially limited by high risk of severe toxicities such as diarrhea, nausea, vomiting, and neutropenia.19 These toxicities Received: Revised: Accepted: Published: 2667

February 13, 2013 May 14, 2013 May 15, 2013 May 15, 2013 dx.doi.org/10.1021/mp400081b | Mol. Pharmaceutics 2013, 10, 2667−2675

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limit dosing of irinotecan in patients with poor health status.20 To overcome these limitations, the development of more efficient and targeted delivery methods is required to increase the local concentration of irinotecan at the desired site while minimizing side effects to healthy tissues. Ultrasound-mediated drug delivery shows great promise in improving the therapeutic effectiveness of a chemotherapeutic agent by increasing the local deposition and reducing the systemic side effects.21 The combination of high frequency ultrasound (1−10 MHz) and ultrasound contrast agents (i.e., consisting of gas microbubbles) is known to enhance the extravasation and the intracellular delivery of genes and drugs into the cells and tissues.22,23 The exposure of microbubbles to low acoustic pressures (i.e., mechanical indices less than 0.2) causes alternatively the shrinking and the expansion of microbubbles during the respective phases of the compression and the rarefaction of the ultrasound wave. These microbubble’s oscillations may induce intense liquid flow around the microbubbles, so-called microstreaming.24,25 At even high acoustic pressures (i.e., mechanical indices higher than 0.2), the microbubbles undergo large oscillations, which lead to violent collapse and likely their destruction. In this regime, the microbubble disruption might be accompanied by the generation of shock waves in the liquid medium close to the microbubbles.26 In the case of an asymmetrical collapse, jet formation may occur when collapsing microbubble is located near the cell membrane or endothelial barrier.27 As described in the available literature, the acoustic phenomena including microstreaming, shock waves, and microjets can transiently enhance the permeability of tumor vasculature and cell membrane through the generation of membrane nanopores and/or the stimulation of paracellular and transcellular pathways.28,29 Choijmants et al. first reported the synergetic effects of ultrasound for irinotecan delivery.30 However, this investigation focused mainly on the intracellular delivery of irinotecan using ultrasound alone (i.e., without microbubbles). Although it was reported that the microbubble-assisted ultrasound increased the therapeutic effectiveness of anticancer drugs such as doxorubicin31−33 compared to ultrasound alone, this approach has not been yet explored to enhance the irinotecan delivery and by that its bioavailability into the tumor. The aim of our study is to investigate whether the successive administration of irinotecan and microbubbles in combination with ultrasound application is an efficient strategy for targeted drug delivery. Hence, we investigated the in vitro and in vivo therapeutic potential of the combination of irinotecan and microbubble-assisted ultrasound on human glioblastoma model. The in vitro therapeutic effectiveness of this method was monitored by an MTT assay. Then, its in vivo effects on the tumor growth and perfusion were assessed by ultrasound imaging and histological analysis. This methodology addresses the following research: (i) Does the combination of irinotecan and microbubbleassisted ultrasound improve the glioblastoma cell death in comparison to irinotecan alone? (ii) Does the combination of irinotecan and microbubble-assisted ultrasound induce tumor regression? The outline of this manuscript is as follows: the experimental methodology is described in the next section (Experimental Section). Then, results from in vitro and in vivo irinotecan delivery are successively presented. Finally, these results are discussed before the main conclusions on the present study are drawn.

Article

EXPERIMENTAL SECTION

Chemicals. Irinotecan in HCl salt (Camptosar, Pfizer France) was a generous gift from Dr. Tournamille (CHRU ToursCRC Henry S. Kaplan, Tours, France). Contrast agents (QA 3411, Bracco Research Geneva, Switzerland) were used for the contrast-enhanced ultrasound imaging and drug delivery using microbubble-assisted ultrasound. These contrast agents are microbubbles consisting of a gaseous core of nitrogen and perfluorobutane mixture surrounded by PEGylated phospholipid shell. The median diameter in volume ranges from 2.3 to 2.6 μm.31 Cell Culture. Human glioblastoma astrocytoma cells (U-87 MG) were derived from a malignant glioma (European Collection of Cell Cultures, Salisbury, UK). These cells are capable of developing a malignant glioblastoma tumor in nude mice. Cells were grown as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM High W/GlutaMAX-I; Life Technologies, Saint-Aubin, France) supplemented with 10% heatinactivated fetal calf serum (FCS; Life Technologies, SaintAubin, France). The cells were routinely subcultured every 4 days and incubated at 37 °C in humidified atmosphere with a 5% CO2 incubator. Ultrasound Setup. Ultrasound waves were generated by a single-element custom-made transducer with a center frequency of 1 MHz. The transducer had a diameter of 15 mm and was naturally focused at 30 mm. It was driven with an electrical signal generated by arbitrary waveform generator (Agilent, Santa Clara, CA) and then amplified by a power amplifier (ADECE, Artannes sur Indre, France). The peak negative pressure of the acoustic wave was measured in a separate setup using a calibrated PVDF needle hydrophone (diameter 0.2 mm; Precision Acoustics Ltd., Dorschester, UK) at the natural focal distance of the transducer. In Vitro Irinotecan Delivery. As previously described,31 U87 MG cells were trypsinized, washed once, and resuspended in OptiMEM High W/GlutaMAX-I (Life Technologies, SaintAubin, France) supplemented with 1% FCS. During the procedure, the cell suspension was maintained in a water-bath at 37 °C (Grant Instruments Ltd., Cambridge, UK). The cell suspension (5 × 105 cells in 1.5 mL) was placed in the polystyrene cuvette (45 mm height, 10 mm inside diameter, 12 mm outside diameter; Fischer Scientific SAS, Illkirch, France), and 2.2 μL of gas microbubbles was added just before ultrasound application. Thus, a microbubble-to-cell ratio of 5 was achieved, and a range of irinotecan concentrations (0.05− 500 μg/mL) was assessed. The center of the plastic cuvette was positioned at the focal distance of the transducer in a deionized water tank at 37 °C. The cell suspension was kept uniform through a gentle magnetic stirring during ultrasound application. Subsequently, the cell suspension was exposed to 1 MHz sinusoidal ultrasound waves with a pulse repetition period of 100 μs, 40 cycles per pulse, and for 30 s (i.e., optimal acoustic parameters for gene and drug delivery). The applied acoustic pressure (i.e., peak negative pressure) was selected at 400 kPa.34,35 After ultrasound application, 500 μL of cells were cultured in 24 well cell culture plates (Corning Life Science BV, Amsterdam, The Netherlands) and incubated at 37 °C in a humidified atmosphere with a 5% CO2 incubator. Four hours later, 1 mL of OptiMEM High W/GlutaMAX-I supplemented with 10% FCS was added to each well and incubated at 37 °C in humidified atmosphere with a 5% CO2 incubator for 24 h. 2668

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injection, a first set of nonlinear contrast images at low transmitted power was performed. Video clips of 36 s were recorded at 10 frames/s to study the tumor perfusion. After image acquisition, quantitative analysis of the tumor perfusion was carried out using VevoCQ software (VisualSonics Inc., Toronto, Canada). Motion artifacts were corrected thanks to this software, and a region of interest (ROI) corresponding to the tumor was manually defined. Then, a time−intensity curve for the ROI was computed, and the peak enhancement (PE), which corresponds to relative blood volume, was determined.39,40 Histopathological Analysis. Tumors were removed and fixed in the formol−acetic acid solution. Then, histological samples were embedded in paraffin, cut at approximately 4 μm, and prepared using conventional hematoxylin/eosin protocol (Novaxia, Saint-Laurent Nouan, France). An expert clinical pathologist reviewed the stained tissue sections (Le Net Pathology Consulting, Amboise, France). The tissue sections were examined by light microscopy on a Leica Diaplan microscope. The size of the neoplastic nodule on the slide was measured with a ruler. The percentage area of necrosis and the mitotic rate (average number of mitotic figures per high power field) was calculated on 10 randomly selected fields. Blood Biochemistry Tests. To evaluate the acute toxicity of the therapeutic protocol, biochemical serum tests were used. Liver toxicity was evaluated by measuring blood aspartate aminotransferase (AST) and alanine aminotransferase (ALT). On day 39, each mouse in the control and treated groups was subjected to retro-orbital blood sampling. Blood samples were centrifuged (1200 g, 5 min) to obtain serum to measure AST and ALT levels (University Hospital CHRU Bretonneau, Tours, France). Statistical Analysis. Descriptive statistics was performed using StatPlus:mac (version 5.8.3.8 2001−2009 Analyst Soft Inc.). Statistical analysis was executed using the nonparametric Mann−Whitney U test (significance was defined as p < 0.05) or linear correlation Pearson test (perfect positive correlation was defined as r close to 1).

Cell Viability. The cell viability was evaluated using a methylthiazolyldiphenyltetrazolium bromide (MTT) colorimetric assay. Twenty-four hours after treatment, the cell medium was replaced with a 0.5 mg/mL MTT solution (Life Technologies, Saint-Aubin, France), and the cells were incubated at 37 °C in humidified atmosphere with a 5% CO2 incubator for 1.5 h. Afterward, the MTT solution was substituted by pure dimethyl sulfoxide solution (Sigma-Aldrich, St. Louis, MO), and the cells were incubated for 10 min under gentle agitation (i.e., 20 rpm) at room temperature. The optical density (OD) was then measured at 570 nm (OD570) to determine the amount of formed formazan crystals and at 690 nm (OD690) as a reference. The cell viability was calculated as:36 cell viability =

(OD570 x − OD690 x) (OD570control − OD690 control)

× 100

In Vivo Irinotecan Delivery. All procedures were performed according to the ethical guidelines and were approved by the Animal Care and Regional Committee for Ethics in Animal Experiments, Val-de-Loire (No. 2011-09-2). Male Swiss nu/nu mice were purchased from Charles River (L’Arbresle, France). They were maintained at constant room temperature with 12 h light cycle in a ventilated isolation cages. The mice were 6 weeks old at the beginning of the experiments, weighing 25−30 g. Under gaseous anesthesia (Aerrane, Baxter, Deerfield, IL), U-87 MG cells (3 × 106 cells/mouse in 100 μL PBS) were subcutaneously injected in the two flanks of each mouse. A total of 15 mice were divided into three experimental groups: (1) control group (i.e., w/o treatment), (2) irinotecan group (i.e., injection of irinotecan on its own), and (3) irinotecan+sonoporation (i.e., injection of irinotecan followed by sonoporation). Under gaseous anesthesia, tumor treatment was initiated when the whole tumor was perfused and reached a volume of 100 mm3. It consisted of a direct intravenous (i.v.) administration of irinotecan via penil vein (20 mg/kg b.w.) followed 1 h later by a direct i.v. bolus injection of gas microbubbles (70 μL). This delay is required to reach the pharmacological peak of SN-38 in the blood.37 The tumor was covered with ultrasound transmission gel and exposed to 1 MHz sinusoidal ultrasound waves with a pulse repetition period of 100 μs, 40 cycles per pulse (40% duty cycle), at peaknegative pressure of 400 kPa during 3 min. The treatment was performed on days 28, 32, and 36 of the tumor growth. Anatomical Ultrasound Imaging. A comparative study between caliper and ultrasound imaging for the measurement of tumor dimensions was performed on ten untreated tumors. Tumor dimensions were determined using digital vernier caliper and ultrasound imaging (Vevo 2100 System, VisualSonics Inc., Toronto, Canada) at 21 MHz (MS-250 probe) and for a mechanical index of 0.43. Ultrasound B-scans were used to image the subcutaneous tumors and to measure their lengths and their widths. Then, the tumor volume was calculated using the formula:38 tumor volume =

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RESULTS In Vitro Irinotecan Delivery. The cell viability was assessed by the MTT assay 24 h after irinotecan delivery with or without microbubble-assisted ultrasound, and the results are shown in Figure 1. The exposure of U-87 MG to microbubble-assisted ultrasound at 400 kPa without irinotecan induced a slight decrease in the cell viability, with no significant difference in comparison to the control condition (90 ± 5% vs 100 ± 3%). As shown in Figure 1, when the glioblastoma cells were only treated with low concentrations of irinotecan alone (0.05−5 μg/mL), the cell viability was not significantly different from the control condition without irinotecan (*p > 0.05). At irinotecan concentrations of 50 and 500 μg/mL, the cell viability significantly decreased compared to low concentrations of irinotecan (***p < 0.001) and reached 79 ± 2% and 40 ± 1%, respectively. Cells treated with low concentrations (0.05 to 5 μg/mL) of irinotecan and insonified with ultrasound at 400 kPa in the presence of microbubbles showed a significant decrease in their viability in comparison to the treatments with irinotecan alone (***p < 0.001) or microbubble-assisted ultrasound alone (***p < 0.001). The combination of ultrasound and microbubbles and irinotecan with higher concentrations (50 and 500

length × width2 2

Contrast-Enhanced Ultrasound Imaging (CEUS). CEUS was performed on the 31st, 35th, and 39th days of the tumor growth. A bolus injection of 70 μL of gas microbubbles was injected via the penil vein. Immediately after contrast agent 2669



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Figure 1. In vitro irinotecan delivery using microbubble-assisted ultrasound. U-87 MG cells were incubated with a range of irinotecan alone at a range of concentrations (0.05−500 μg/mL) or combined with ultrasound sequences at 400 kPa for 30 s and microbubbles. Twenty-four hours after treatment, cell viability was measured by an MTT assay. Data expressed as mean ± SEM were calculated from five independent experiments.

Figure 2. Correlation curve between anatomical ultrasound imaging and caliper measurements. Under gaseous anesthesia, U-87 MG cells were subcutaneously injected in the flanks of 10 mice. During the tumor growth, the tumor dimensions were measured using caliper and anatomical ultrasound imaging.

induced a significant and additional 2.8-fold decrease in tumor volume in comparison to irinotecan treatment alone, respectively (*p < 0.05). These results suggest that the microbubble-assisted ultrasound potentiate the therapeutic effectiveness of irinotecan. The effect of the irinotecan delivery using microbubbleassisted ultrasound on tumor perfusion was monitored three days after each treatment by contrast-enhanced ultrasound imaging. Figure 3B depicts the time−intensity curves of representative tumor of each experimental group. The irinotecan treatment alone induced around 2.5-fold decrease in tumor perfusion compared to the control group. In addition, as for the tumor volume (Figure 3A), the irinotecan delivery using microbubble-assisted ultasound causes an additional 2.5fold decrease in tumor perfusion in comparison to the irinotecan-treated tumors. The results suggest that irinotecan delivery using microbubble-assisted ultrasound might induce an inhibition of tumor perfusion. To confirm the therapeutic effect of irinotecan delivery using microbubble-assisted ultrasound, histopathological analyses (Figure 4) were carried out at the end of therapeutic protocol (i.e., day 39). The quality of the histological sections, tissue accountability, slide labeling, and tissue placement were good and considered adequate for our study by the pathologist. All tumors showed common histopathological characteristics. Tumors are rounded neoplastic nodules present within the subcutaneous tissue and partially surrounded by thin fibrous capsules. The proliferation was made up of small collections of poorly differentiated neoplastic cells within a fine and delicate vascular stroma. The cells were highly pleomorphic with one or more prominent nuclei. These nuclei were most often eccentrically located in abundant amphophilic cytoplasms. A few multinucleated cells were also observed in all tumors. The main difference in histological characteristics between the three tumor groups lay in the necrosis and mitosis levels and in the infiltration of neutrophils. Indeed, the irinotecan delivery on its own or in combination with microbubbleassisted ultrasound induced a significant 3- and 4.5-fold increase in ischemic necrosis compared with the control group (*p < 0.05), respectively (Figure 4). In addition, the irinotecan delivery using microbubble-assisted ultrasound led to a 35% and 50% decrease in mitosis level in comparison to the irinotecan alone and control groups, respectively (Figure 4). As shown in Figure 4, multifocal infiltrations of neutrophils were

μg/mL) caused approximately 5 and 8-fold decreases in the cell viability compared to the irinotecan treatment alone (*p < 0.001). These results clearly show that the combination of irinotecan with microbubble-assisted ultrasound induced a synergistic effect on the human glioblastoma cell death. For example, to achieve a cell viability of 40%, irinotecan alone needs to be administrated at an extremely high concentration (500 μg/mL), while the same cell viability can be achieved with a concentration of only 0.5 μg/mL of irinotecan when combined with ultrasound and microbubbles. Hence the concentration of irinotecan can be divided by a factor 1000 while achieving the same therapeutic benefit. Finally, our results confirm that ultrasound in association with microbubbles, without the drug, do not induce significant cell death, which remained constant around 10%. In Vivo Irinotecan Delivery. In the present study, ultrasound imaging was chosen to monitor the therapeutic effectiveness of irinotecan delivery by microbubble-assisted ultrasound. Indeed, this imaging modality provided important morphological and functional information.39 Currently, the caliper is the main tool to measure the tumor volume and monitor the therapeutic effectiveness of anticancer treatments of subcutaneous tumor xenografts.41 This is explained by the fact that this method is easy to use and cheap. Our comparative study showed that the anatomical ultrasound imaging measurements were positively correlated to the caliper data (Pearson’s correlation coefficient, 0.99; Figure 2). On the basis of these results, the anatomical ultrasound imaging was chosen to monitor the tumor growth during the therapeutic protocol and hence its therapeutic response to the treatment. Subcutaneous glioblastoma tumors were treated three times, either by i.v. administration of therapeutic dose of irinotecan alone or in combination with microbubble-assisted ultrasound. The therapeutic effectiveness was evaluated by anatomical (Figure 3A) and contrast-enhanced (Figure 3B) ultrasound imaging every four days. No side effect was associated with the therapeutic protocol. The results shown in Figure 3A demonstrate the substantial therapeutic effect observed at the 39th day. Thus, irinotecan treatment on its own led to approximately 2.5-fold decrease in tumor volume compared to the control group (*p < 0.05). Furthermore, the irinotecan delivery in combination with microbubble-assisted ultrasound 2670



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Figure 3. Effect(s) of in vivo irinotecan delivery using microbubble-assisted ultrasound on tumor volume (A) and perfusion (B) at day 39. Tumor dimensions and perfusion were measured using anatomical and contrast-enhanced ultrasound imaging, respectively, in control, irinotecan treatment on its own, or using microbubble-assisted ultrasound groups. Data expressed as mean ± SEM were calculated from 10 tumors.

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observed in all tumors. However, the tumors treated by irinotecan delivery using microbubble-assisted ultrasound showed a high level of neutrophil infiltrations compared to the irinotecan alone and to the control groups. To evaluate the toxic responses, blood samples and changes in body weight during the study were analyzed. The animals were injected with nontoxic dose of irinotecan (20 mg/kg/4 days) for both groups (irinotecan and irinotecan/MB+US). There were no significant differences in AST and ALT levels between groups (*p > 0.05), and these levels were close to the normal values for mice (AST: 54−298 UI/L; ALT: 17−77 UI/ L). These results indicate that there is no acute toxicity into liver. In addition, there was no significant difference in the body weight increase (3 ± 1 g; *p > 0.05).

DISCUSSION

The present study examined the therapeutic potential of in vitro and in vivo irinotecan delivery using microbubble-assisted ultrasound. First, we validated this approach in vitro showing that irinotecan treatment with microbubble-assisted ultrasound induced a synergistic enhancement in human glioblastoma cell death (Figure 1). In agreement with published data, these results demonstrated indirectly that the observed enhancement in cell death could be ascribed to an increased drug uptake through ultrasound-induced hydrophilic pores29,42 and not due to the application of ultrasound. Indeed, based on the increase or release of marker molecules28,43 and by measuring changes in membrane electrophysiology,44,45 previous studies showed that microbubble-assisted ultrasound induced a transient increase in membrane permeability through the generation of 2671



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Figure 4. Histopathological analysis after irinotecan delivery using microbubble-assisted ultrasound. Representative histological images of subcutaneous glioblastoma tumors stained using conventional hematoxylin/eosin protocol in control and after treatment with irinotecan treatment on its own or in combination with microbubble-assisted ultrasound.

and consequently enhance the drug extravasation and its bioavalability in the tumor interstitial compartment.49 This permeabilization may increase the intracellular delivery of irinotecan in the endothelial cells. Hence, this strategy might potentiate the destruction of tumor vasculature and reduce the nutrient supply of tumors.50 The results of this pilot preclinical study are the basis of preclinical therapeutic protocol, which will require improvements in ultrasound parameters (e.g., acoustic pressure, exposure time) and microbubbles (e.g., dose, type of microbubbles or nanobubbles). Among these improvements, the use of nanobubbles could enhance the intracellular delivery of irinotecan in the tumor cells.51,52 Indeed, current studies indicate that the nanobubbles readily penetrate tumor tissues based on the enhanced permeability and retention effect (i.e., EPR effect) and could be exposed to ultrasound in order to increase the intracellular delivery of the drug. In addition further additional experiments such as drug biodistribution are necessary to optimize the therapeutic effectiveness of irinotecan in subcutaneous and orthotopic glioblastomas or colorectal tumors and to reduce the systemic side effects of this anticancer compound. As mentioned earlier, therapeutic effectiveness of irinotecan is strongly limited by potentially life-threatening toxicities such as diarrhea, nausea, vomiting, and neutropenia. In addition, SN38 is approximately 100- and 1000-fold more potent than irinotecan. Thereby, SN-38 could be more promising drug than irinotecan for anticancer therapy. However, this active compound is poorly soluble in aqueous solutions and in all pharmaceutically acceptable solvents. Therefore, in order to

transient membrane nanopores. Thus, the intracellular delivery of small molecules (i.e., ≤ 4 kDa) such as propidium iodide or anticancer drugs is likely governed by passive diffusion through membrane pores with a size ranging from 10 to 150 nm.46,47 The duration of this uptake is naturally dependent on the membrane recovery time, that is, a few seconds to few minutes.29,48 Subsequently, using ultrasound imaging, we have demonstrated that in vivo irinotecan delivery by microbubble-assisted ultrasound led to a significant decrease in tumor growth and perfusion (Figure 3). These data were confirmed by histopathological analysis (Figure 4). Based on these results drawn from this preclinical study, we hypothetize that irinotecan delivery using microbubble-assisted ultrasound may significantly improve the in vivo therapeutic effectiveness of irinotecan for anticancer therapy. This new therapeutic strategy is compatible with current administration of irinotecan-based chemotherapy protocols (i.e., intravenous administration of irinotecan).1,3 For example, in patients with liver metastases from colorectal cancer, a located and controlled ultrasoundtriggered delivery of SN-38 could be able to increase the intratumoral bioavailability of SN-38 and to enhance the tumor response to irinotecan-based chemotherapy. This method could be able to enhance selectivity of antitumoral effect of irinotecan without an increase in systemic toxicity of irinotecan in clinical practice. Moreover, under ultrasound excitation, the microbubble’s cavitation and destruction in tumor microvascular network may promote the transient permeabilization of tumor endothelium 2672



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enhance drug solubility and improve stability of SN-38, nanoparticle systems have been developed and reported.53−55 Nevertheless, most of these nanoparticles required more validation in therapeutic effectiveness and innocuity terms. Alternatively, the encapsulation of irinotecan into nanoparticle systems has also been described.56,57 Among few particle formulations, the Irinophore C is a promising lipid-based formulation of irinotecan.58,59 Indeed, this remarkable formulation mediated an increase in both plasma and tumor levels of the active form of irinotecan as well as maintaining significant plasma levels of SN-38 for extended time periods.56,60 In addition, several studies reported that the Irinophore C is better tolerated than irinotecan and exhibits improved antitumor effectiveness in subcutaneous and orthotopic human glioblastoma and colorectal cancers.56,58,61 This liposomal formulation might be a good candidate for ultrasound-triggered irinotecan delivery. Indeed, the use of microbubble-assisted ultrasound to increase drug release from micro- and nanoparticles and to enhance intracellular delivery of drug has been previously described.32,35,62

REFERENCES

(1) Fujita, K.; Sparreboom, A. Pharmacogenetics of irinotecan disposition and toxicity: a review. Curr. Clin. Pharmacol. 2010, 5, 209− 17. (2) Weekes, J.; Ho, Y. H.; Sebesan, S.; Ong, K.; Lam, A. K. Irinotecan and colorectal cancer: the role of p53, VEGF-C and alpha-B-Crystallin expression. Int. J. Colorectal Dis. 2010, 25, 907. (3) Weekes, J.; Lam, A. K.; Sebesan, S.; Ho, Y. H. Irinotecan therapy and molecular targets in colorectal cancer: a systemic review. World J. Gastroenterol. 2009, 15, 3597−602. (4) Vredenburgh, J. J.; Desjardins, A.; Reardon, D. A.; Friedman, H. S. Experience with irinotecan for the treatment of malignant glioma. Neuro Oncol. 2009, 11, 80−91. (5) Jakobsen, J. N.; Hasselbalch, B.; Stockhausen, M. T.; Lassen, U.; Poulsen, H. S. Irinotecan and bevacizumab in recurrent glioblastoma multiforme. Expert Opin. Pharmacother. 2011, 12, 825−33. (6) Hatfield, M. J.; Tsurkan, L.; Garrett, M.; Shaver, T. M.; Hyatt, J. L.; Edwards, C. C.; Hicks, L. D.; Potter, P. M. Organ-specific carboxylesterase profiling identifies the small intestine and kidney as major contributors of activation of the anticancer prodrug CPT-11. Biochem. Pharmacol. 2011, 81, 24−31. (7) Ghosh, S. Cholesteryl ester hydrolase in human monocyte/ macrophage: cloning, sequencing, and expression of full-length cDNA. Physiol. Genom. 2000, 2, 1−8. (8) Wang, W.; Ghandi, A.; Liebes, L.; Louie, S. G.; Hofman, F. M.; Schonthal, A. H.; Chen, T. C. Effective conversion of irinotecan to SN38 after intratumoral drug delivery to an intracranial murine glioma model in vivo. Laboratory investigation. J. Neurosurg. 2011, 114, 689− 94. (9) Sanghani, S. P.; Quinney, S. K.; Fredenburg, T. B.; Sun, Z.; Davis, W. I.; Murry, D. J.; Cummings, O. W.; Seitz, D. E.; Bosron, W. F. Carboxylesterases expressed in human colon tumor tissue and their role in CPT-11 hydrolysis. Clin. Cancer Res. 2003, 9, 4983−91. (10) Mathijssen, R. H.; van Alphen, R. J.; Verweij, J.; Loos, W. J.; Nooter, K.; Stoter, G.; Sparreboom, A. Clinical pharmacokinetics and metabolism of irinotecan (CPT-11). Clin. Cancer Res. 2001, 7, 2182− 94. (11) Ikeguchi, M.; Arai, Y.; Maeta, Y.; Ashida, K.; Katano, K.; Wakatsuki, T. Topoisomerase I expression in tumors as a biological marker for CPT-11 chemosensitivity in patients with colorectal cancer. Surg. Today 2011, 41, 1196−9. (12) Hsiang, Y. H.; Lihou, M. G.; Liu, L. F. Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res. 1989, 49, 5077−82. (13) Goldwasser, F.; Bae, I.; Valenti, M.; Torres, K.; Pommier, Y. Topoisomerase I-related parameters and camptothecin activity in the colon carcinoma cell lines from the National Cancer Institute anticancer screen. Cancer Res. 1995, 55, 2116−21. (14) Candeil, L.; Gourdier, I.; Peyron, D.; Vezzio, N.; Copois, V.; Bibeau, F.; Orsetti, B.; Scheffer, G. L.; Ychou, M.; Khan, Q. A.; Pommier, Y.; Pau, B.; Martineau, P.; Del Rio, M. ABCG2 overexpression in colon cancer cells resistant to SN38 and in irinotecan-treated metastases. Int. J. Cancer 2004, 109, 848−54. (15) Douillard, J. Y.; Cunningham, D.; Roth, A. D.; Navarro, M.; James, R. D.; Karasek, P.; Jandik, P.; Iveson, T.; Carmichael, J.; Alakl, M.; Gruia, G.; Awad, L.; Rougier, P. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet 2000, 355, 1041−7. (16) Saltz, L. B.; Cox, J. V.; Blanke, C.; Rosen, L. S.; Fehrenbacher, L.; Moore, M. J.; Maroun, J. A.; Ackland, S. P.; Locker, P. K.; Pirotta, N.; Elfring, G. L.; Miller, L. L. Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer. Irinotecan Study Group. N. Engl. J. Med. 2000, 343, 905−14. (17) Van Cutsem, E.; Dirix, L.; Van Laethem, J. L.; Van Belle, S.; Borner, M.; Gonzalez Baron, M.; Roth, A.; Morant, R.; Joosens, E.; Gruia, G.; Sibaud, D.; Bleiberg, H. Optimisation of irinotecan dose in the treatment of patients with metastatic colorectal cancer after 5-FU

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CONCLUSIONS In summary, the present work suggested that the irinotecan delivery using microbubble-assisted ultrasound enhanced the in vitro and in vivo therapeutic effectiveness of irinotecan compared with only irinotecan treatment. However, further improvements are still required to potentiate this therapeutic protocol. The coadministration of clinically approved gas microbubbles and anticancer drugs in combination with ultrasound might be a new strategy to improve the efficiency and the safety of conventional chemotherapy treatment and easily translatable to clinic.

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AUTHOR INFORMATION

Corresponding Author

*UMR Inserm U930 Imagerie et Cerveau, Université FrançoisRabelais, CHRU Bretonneau, B1A, 2 bd Tonnellé, 37044 Tours Cedex 9, France. Tel.: +33-0-247479748. Fax: +33-0247479767. E-mail address: [email protected]. Present Address ⊥

Imaging Division, University Medical Center Utrecht, Heidelberglaan 100, P.O. Box 85500, 3508 GA, Utrecht, Netherlands

Author Contributions ¶

J-M.E. and A.N. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge the technical assistance of J.-Y. Tartu (UMR Inserm U930, Tours, France) for the mounting ultrasound setup, Dr. P. Vourc’h for blood sample analysis (University Hospital CHRU Bretonneau, Tours, France), Dr. V. Gouilleux (UMR CNRS 7292, Tours, France) for fruitful discussions, and Bracco Research Geneva for supplying the microbubbles. The Inserm and the European Commission FP7 Program SONODRUGS (NMP4-LA-2008-213706) funded this research study. 2673



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