Bubble Liposomes and Ultrasound Exposure ... - ACS Publications

Jan 16, 2014 - ... gene; a phosphorodiamidate morpholino oligomer (PMO) designed to skip this mutated exon in the mRNA induces dystrophin expression...
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Bubble Liposomes and Ultrasound Exposure Improve Localized Morpholino Oligomer Delivery into the Skeletal Muscles of Dystrophic mdx Mice Yoichi Negishi,*,†,‡ Yuko Ishii,†,‡ Hitomi Shiono,†,‡ Saki Akiyama,† Shoko Sekine,† Takuo Kojima,† Sayaka Mayama,† Taiki Kikuchi,† Nobuhito Hamano,† Yoko Endo-Takahashi,† Ryo Suzuki,§ Kazuo Maruyama,§ and Yukihiko Aramaki† †

Department of Drug Delivery and Molecular Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan § Laboratory of Drug and Gene Delivery Research, Faculty of Pharma-Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan S Supporting Information *

ABSTRACT: Duchenne muscular dystrophy (DMD) is a genetic disorder that is caused by mutations in the DMD gene that lead to an absence of functional protein. The mdx dystrophic mouse contains a nonsense mutation in exon 23 of the dystrophin gene; a phosphorodiamidate morpholino oligomer (PMO) designed to skip this mutated exon in the mRNA induces dystrophin expression. However, an efficient PMO delivery method is needed to improve treatment strategies for DMD. We previously developed polyethylene glycol (PEG)-modified liposomes (Bubble liposomes) that entrap ultrasound contrast gas and demonstrated that the combination of Bubble liposomes with ultrasound exposure is an effective gene delivery tool in vitro and in vivo. In this study, to evaluate the ability of Bubble liposomes as a PMO delivery tool, we tested the potency of the Bubble liposomes combined with ultrasound exposure to boost the delivery of PMO and increase the skipping of the mutated exon in the mdx mouse. The results indicated that the combination of Bubble liposomes and ultrasound exposure increased the uptake of the PMO targeting a nonsense mutation in exon 23 of the dystrophin gene and consequently increased the PMO-mediated exon-skipping efficiency compared with PMO injection alone, leading to significantly enhanced dystrophin expression. This increased efficiency indicated the potential of the combination of Bubble liposomes with ultrasound exposure to enhance PMO delivery for treating DMD. Thus, this ultrasound-mediated Bubble liposome technique may provide an effective, noninvasive, nonviral method for PMO therapy for DMD muscle as well as for other muscular dystrophies. KEYWORDS: Bubble liposomes, ultrasound, delivery of an antisense phosphorodiamidate morpholino oligomer (PMO), muscular dystrophy, exon-skipping therapy independent clinical trials.10−12 In particular, PMOs that exclude exon 23 from dystrophin transcripts have yielded superior results in the mdx mouse model of muscular dystrophy compared to treatment with 2′-O methyl phosphorothioate antisense oligonucleotide.8,13 PMOs are characterized as charge-neutral molecules in which the ribose or deoxyribose rings of DNA/RNA are replaced by morpholine rings, and the phosphodiester linkages are replaced by uncharged phosphorodiamidate linkages. PMOs exhibit superior stability and safety compared with other antisense oligonucleotide chemical compounds; however, the delivery efficacy of the charge-neutral PMO

1. INTRODUCTION Duchenne muscular dystrophy (DMD) is the most common serious form of muscular dystrophy and is associated with nonsense or frame-shift mutations in the dystrophin gene, which lead to this lethal muscle-wasting disorder.1,2 By contrast, mutations that maintain the reading frame and cause internally deleted, partially functional dystrophin are closely related to the milder Becker muscular dystrophy (BMD).3,4 A potential strategy for treating DMD by antisense oligonucleotidemediated exon skipping has been established to induce specific exon skipping of the dystrophin gene and eliminate diseasecausing mutations, leading to the restoration of the reading frame of the gene transcripts.5−9 Recently, 2′-O methyl phosphorothioate RNA and phosphorodiamidate morpholino oligomers (PMOs) have been widely utilized in exon-skipping therapies for DMD and have demonstrated usability in two © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1053

August 11, 2013 December 12, 2013 January 16, 2014 January 16, 2014 dx.doi.org/10.1021/mp4004755 | Mol. Pharmaceutics 2014, 11, 1053−1061

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Pharmacy and Life Science Committee on the Care and Use of Laboratory Animals. C57BL/10ScSnmdx mice (mdx)38 carrying a nonsense mutation in exon 23 of the dystrophin gene39 were used for each PMO delivery experiment. Normal C57BL/6 mice were used as a positive control for reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. 2.3. Preparation of Bubble Liposomes. The Bubble liposomes were prepared using previously described methods.35,36 Briefly, PEG liposomes composed of 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) (NOF Corporation, Tokyo, Japan) and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-polyethyleneglycol (DSPE-PEG 2000 -OMe) (NOF Corporation, Tokyo, Japan) in a molar ratio of 94 to 2, 6, or 10 were prepared via a reverse-phase evaporation method. In brief, all reagents were dissolved in 1:1 (v/v) chloroform/diisopropyl ether. Phosphate-buffered saline was added to the lipid solution, and the mixture was sonicated, followed by evaporation at 47 °C. The organic solvent was completely removed, and the size of the liposomes was adjusted to less than 200 nm using extruding equipment and a sizing filter (pore size: 200 nm) (Nuclepore Track-Etch Membrane, Whatman plc, U.K.). The lipid concentration was measured using a Phospholipid C test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The Bubble liposomes were prepared from liposomes and perfluoropropane gas (Takachio Chemical Ind. Co. Ltd., Tokyo, Japan). First, 2 mL sterilized vials containing 0.8 mL of liposome suspension (lipid concentration: 1 mg/mL) were filled with perfluoropropane gas, capped, and then pressurized with an additional 3 mL of perfluoropropane gas. The vial was placed in a bath-type sonicator (42 kHz, 100 W) (BRANSONIC 2510j-DTH, Branson Ultrasonics Co., Danbury, CT, USA) for 5 min to form the Bubble liposomes. The zeta potential and mean size of the Bubble liposomes were determined using the lightscattering method with a zeta potential/particle sizer (Nicomp 380ZLS, Santa Barbara, CA). 2.4. Ultrasound Imaging of the Bubble Liposomes. For analysis of the destruction efficiency of each type of Bubble liposome (DPPC:DSPE-PEG2000-OMe = 98:2, DPPC:DSPEPEG2000-OMe = 94:6, or DPPC:DSPE-PEG2000-OMe = 90:10), the Bubble liposomes (100 μL of lipid concentration: 1 mg/mL) diluted with PBS (7 mL) were dispensed into 6-well plates. B-mode recordings were acquired using a high-frequency ultrasound imaging system (NP60R-UBM, 105 NEPA GENE, Co., Ltd., Chiba, Japan). After ultrasound exposure (frequency, 1 MHz; duty, 50%; intensity, 2 W/cm2; time, 60 s) using an ultrasound generator (Sonitron 2000, NEPA GENE, Co., Ltd., Chiba, Japan), ultrasonographic images were captured for the various types of Bubble liposomes. Each echo signal after ultrasound imaging was analyzed using ImageJ software. The data (n = 3) are shown as relative mean intensities. The mean intensities of the echo signals at the ROIs (regions of interest) (n = 3) are also shown. 2.5. In Vivo Gene Delivery into the Skeletal Muscles of Mice with Bubble Liposomes and Ultrasound Exposure. In vivo gene delivery into the skeletal muscles of mice with Bubble liposomes and ultrasound exposure was performed using previously described methods.37 Briefly, ICR mice (5 weeks old, male) were anesthetized with pentobarbital. A 40 μL suspension of pDNA (10 μg) and Bubble liposomes (30 μg) was injected into the tibialis muscles of the ICR mice. Ultrasound exposure (frequency, 1 MHz; duty, 50%; intensity, 2 W/cm2; time,

is dependent upon passive diffusion for cell entry, resulting in decreased effectiveness.14,15 To enhance the therapeutic efficiency, cell-penetrating peptides (CPPs) have been conjugated to PMOs that target dystrophin exons in mdx mice. The delivery of these PMOs has resulted in significant restoration of dystrophin expression at lower dosages compared with unmodified PMOs.16,17 However, these highly positive-charged peptides are accompanied by higher toxicity, with an LD50 near 100 mg/kg, making their clinical application difficult.17 Thus, although PMO delivery is anticipated to be a feasible, safe approach for clinically treating DMD, delivery efficiency must be improved to achieve therapeutic effects in DMD patients. Among nonviral physical delivery methods,18−21 ultrasound exposure enhances the efficiency of drug or gene delivery into tissues and cells, a technique known as sonoporation.19 It is believed that this effect creates transient pores in the cell membrane by ultrasound cavitation activity, enabling the transport of extracellular molecules into viable cells.20,22−24 Furthermore, a combination of microbubbles, contrast agents for medical ultrasound imaging, and ultrasound exposure has enabled a tissue-specific gene delivery after ultrasound-induced cavitation while reducing cellular damage. This combination produces transient changes in the permeability of the cell membrane and enables the site-specific intracellular delivery of macromolecules like dextran, pDNA, peptides, and siRNA both in vitro and in vivo.25−28 However, microbubbles vary with regard to their size, stability, and targeting function. Liposomes, which are useful carriers of drugs, antigens, and genes, can easily be prepared in a variety of sizes and modified to add a targeting function.29−34 Therefore, we hypothesized that polyethylene glycol (PEG)-modified liposomes containing an ultrasound imaging gas could be novel gene delivery carriers. To address the variable properties of microbubbles, we developed novel liposomal bubbles, “Bubble liposomes”, to deliver genes in vitro and in vivo.35,36 In addition, we demonstrated that the combinations of Bubble liposomes with ultrasound exposure could achieve the efficient intramuscular delivery of basic fibroblast growth factor (bFGF)-expressing plasmid into skeletal muscle in a hindlimb ischemia model, resulting in the improvement of disease symptoms.37 However, whether Bubble liposomes will be a feasible and effective tool for the clinical use of PMO therapy remains unclear. In this study, we assessed the feasibility and effectiveness of Bubble liposomes in combination with ultrasound exposure for PMO delivery in dystrophic mdx mice. The results indicated that the combination of Bubble liposomes and ultrasound exposure increased the uptake of a PMO targeting the mutated mouse dystrophin exon 23 and consequently increased the efficiency of PMO-mediated exon skipping as compared with PMO injection alone, leading to significantly enhanced dystrophin expression. This increased efficiency demonstrates the potential of Bubble liposomes in combination with ultrasound exposure to enhance PMO delivery and to reduce the PMO dosage for treating DMD.

2. MATERIALS AND METHODS 2.1. Antisense Oligonucleotides. The PMO M23D(+7-18) (5′-GGCCAAACCTCGGCTTACCTGAAAT-3′) was purchased from Gene Tools (Philomath, OR, USA). The sequences were designed to anneal to the last 7 bases of exon 23 and the first 18 bases of intron 23.9 2.2. Animals. The use of animals and relevant experimental procedures were approved by the Tokyo University of 1054

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were examined by electrophoresis in 2% agarose gels. The intensity of the bands of the PCR products amplified from the treated muscles in the mdx mice was measured by the ImageJ software (National Institutes of Health, Bethesda, MD). The exon 23skipping efficiency in tibialis anterior muscle was calculated from the following formula: [(the intensity of the skipped band)/(the intensity of the skipped band + the intensity of the unskipped band)]. 2.8. Immunohistochemistry. Serial sections were cut from the treated tibialis anterior and control muscles. The sections were stained with the NCL-DYS2 monoclonal antibody (Novocastra Laboratories), which reacts strongly with the C-terminal region of dystrophin. Alexa Fluor 546 (Invitrogen) was used as a secondary antibody. The sections were evaluated by fluorescence microscopy (Axiovert 200 M, Carl Zeiss, or KEYENCE, BZ8100). For dystrophin-positive fiber counting, the maximum number of dystrophin-positive fibers in one section was counted using a fluorescence microscope. The muscle fibers were defined as dystrophinpositive if more than two-thirds of a single fiber exhibited continuous stainining.40 2.9. Evans Blue Dye Uptake. The Evans blue dye uptake study was performed as previously reported.41 Briefly, mdx mice treated with PMO were exercised at 12 m/min for 30 min by a

60 s) was immediately applied at the injection site. A Sonitron 2000 (NEPA GENE, CO., LTD.) was used as an ultrasound generator. Several days after the injection, the mice were euthanized and sacrificed. The ultrasound-exposed areas of the tibialis muscles were collected and homogenized. The cell lysates and tissue homogenates were prepared in lysis buffer (0.1 M TrisHCl (pH 7.8), 0.1% Triton X-100, and 2 mM EDTA). Luciferase activity was measured using a luciferase assay system (Promega, Madison, WI) and a luminometer (LB96 V, Belthold Japan Co. Ltd., Tokyo, Japan). The activity is indicated by relative light units (RLU) per mg of protein. The pcDNA3-Luc plasmid, derived from pGL3-basic (Promega, Madison, WI), was used as an expression vector encoding the firefly luciferase gene under the control of a cytomegalovirus promoter. 2.6. In Vivo PMO Delivery into the Skeletal Muscles of mdx Mice Treated with Bubble Liposomes and Ultrasound Exposure. Mdx mice (5−6 weeks old, male) were anesthetized with pentobarbital throughout each procedure. A 40-μL suspension of the PMO (5 μg) and Bubble liposomes (30 μg) was injected into the tibialis muscle of mdx mice, and ultrasound exposure (frequency, 1 MHz; duty, 50%; intensity, 2 W/cm2; time, 60 s) was immediately applied at the injection site. A Sonitron 2000 (NEPA GENE, Co, Ltd. Chiba, Japan) was used as the ultrasound generator. Two weeks after the injection, the mice were euthanized, and the tibialis muscle in the ultrasound-exposed area was collected and embedded in an OCT compound and immediately frozen at −80 °C. The specimens were analyzed by RT-PCR and immunohistochemistry. To determine the uptake of PMO into the tibialis anterior muscles of mdx mice, a fluorescein-labeled PMO purchased from Gene Tools (Philomath, OR, USA) was delivered into the tibialis anterior muscles of mdx mice by the intramuscular injection with or without Bubble liposomes and ultrasound (frequency, 1 MHz; duty, 50%; intensity, 2 W/cm2; time, 60 s). Immediately after the treatment, each muscle was collected, embedded in an OCT compound, and immediately frozen at −80 °C. The sections were evaluated by fluorescence microscopy (Axiovert 200 M, Carl Zeiss, or KEYENCE, BZ8100). The nuclei were stained with DAPI. For dystrophin-positive fiber counting, the maximum number of dystrophin-positive fibers in one section was counted using a fluorescence microscope. 2.7. RNA Preparation and RT-PCR Analysis. RNA preparation and RT-PCR analysis were performed according to previously described methods.6 Briefly, total RNA was extracted from sections cut from frozen tissue blocks using RNAiso Plus (Takara Bio Inc., Japan) according to the manufacturer’s instructions. Complementary DNA was synthesized from 1 μg total RNA in a 20 μL reaction using Prime Script Reverse Transcriptase (Takara Bio Inc., Japan). RT-PCR was first performed with 100 ng total RNA as the starting material for 30 cycles of amplification with ExTaq (Takara Bio Inc., Japan). Forward and reverse primers amplifying from exons 20 and 26, respectively (Ex20Fo, 5′-CAGAATTCTGCCAATTGCTGAG- 3′; Ex26Ro, 5′-TTCTTCAGCTTGTGTCATCC-3′), were used at an annealing temperature of 55 °C with an extension time of 2 min at 72 °C. A 1 μL aliquot of the RT-PCR product was used as the template in a 50 μL secondary nested PCR with ExTaq (Takara Bio Inc., Japan) and inner primers (Ex20Fi, 5′-CCCAGTCTACCACCCTATCAGAGC-3′; Ex26Ri, 5′-CCTGCCTTTAAGGCTTCCTT-3′). The secondary PCR was performed for 25 cycles under cycling conditions identical to the primary amplification. The products

Figure 1. Exon-skipping therapy with morpholino oligonucleotides delivered by Bubble liposomes and ultrasound exposure in mdx mice. The mdx mouse carries a nonsense point mutation in exon 23 of the dystrophin gene and lacks dystrophin expression in all of its muscles. Specifically designed morpholino oligonucleotides (PMO) are able to skip exon 23 in mdx mice and produce a partly functional dystrophin protein.9 A mixture of the PMO and Bubble liposomes in solution was intramuscularly injected into the tibialis anterior muscles of mdx mice. Ultrasound exposure was immediately applied at the injection site as described previously.37 The combination of Bubble liposomes and ultrasound exposure enabled site-specific PMO delivery after ultrasound-induced cavitation, while reducing cellular damage. The delivery mechanism is likely a combination of transient changes in the permeability of the cell membrane and focused ultrasound-specific intracellular delivery of PMO in the mdx muscles. PMO: phosphorodiamidate morpholino oligomer. US: ultrasound. 1055

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Figure 2. Uptake of fluorescein-labeled the PMO into tibialis anterior muscles of mdx mice after intramuscular delivery of the PMO with Bubble liposomes and ultrasound exposure. Fluorescein-labeled PMO was delivered into tibialis anterior muscles of mdx mice by intramuscular injection of PMO with or without Bubble liposomes and ultrasound exposure (frequency, 1 MHz; duty, 50%; intensity, 2 W/cm2; time, 60 s). The PMO uptake into the treated muscles was evaluated by fluorescent microscopy. Sectioned tibialis anterior muscle in mdx mice treated with PMO (a, b, c), and in mdx mice with PMO, Bubble liposomes, and ultrasound exposure (d, e, f). Nuclei are stained with DAPI. Each high-magnification image is indicated by an open square. The red arrows indicate the spaces between muscle fibers with peripheral or centrally located nuclei. Quantitative evaluation of the total fluorescein-positive fibers in the tibialis anterior muscles (g). Scale bar = 200 μm. PMO: phosphorodiamidate morpholino oligomer. BLs: Bubble liposomes. US: ultrasound.

3. RESULTS AND DISCUSION Previously, we reported intramuscular delivery using Bubble liposomes with ultrasound exposure of plasmid DNA expressing the luciferase gene. We also demonstrated that angiogenic gene delivery by Bubble liposomes and ultrasound exposure into the skeletal muscle in a hindlimb ischemia model resulted in an improvement in disease symptoms.37 In this previous experiment, Bubble liposomes containing 6 mol % PEG were used; however, the preparation of Bubble liposomes with varying DPPC/PEG2000 ratios might further improve the gene delivery efficacy in the combination of Bubble liposomes and ultrasound exposure. Therefore, we prepared three types of Bubble liposomes with varying DPPC/PEG2000 ratios and compared their size, destruction efficiency upon ultrasound exposure, and delivery efficiency. The size of the Bubble liposomes containing 6 or 10 mol % PEG ranged from 400 to 500 nm. Bubble liposomes containing 2 mol % PEG had a broader size range (548.9 ± 898.6 nm) (Table S1 in the Supporting Information). These results suggest that the size of the Bubble liposomes increases if the PEG concentration is lowered. However, when comparing ultrasonographic images of the Bubble liposomes with varying DPPC/PEG2000 ratios, there was no difference. When the Bubble liposomes were subjected to a previously determined level of ultrasound exposure,37

running system on a treadmill (model MK-680C, MUROMACHI KIKAI Co., Ltd., Tokyo, Japan). An electric shock bar grid at the end of the tread delivered a mild shock to the mice if they stopped running. A solution of 5 μg/μL Evans blue dye (Wako Pure Chemical Industries, Ltd.) in saline was injected via the tail vein 30 min after the exercise. The injection volume for each mouse was determined by the weight of the animal: 50 μL/10 g body weight. The mice were sacrificed 24 h after the Evans blue dye injection, and the tibialis anterior and gastrocnemius muscles were dissected. Their muscles were incubated in formamide (5 μL/mg body weight) at 55 °C overnight, and the supernatants were quantitated in a spectrophotometer at 620 nm as previously reported.42 For the microscopic evaluation of the dye uptake, the treated muscles were collected, frozen, sectioned, counterstained with DAPI, and evaluated by fluorescence microscopy (Axiovert 200 M, Carl Zeiss, or KEYENCE, BZ8100). 2.10. Creatine Kinase Measurement. Blood was collected through a small cut in the end of the tail 30 min after the exercise. Creatine kinase levels were analyzed by a clinical pathology laboratory (Oriental Yeast Co., Ltd., Tokyo, Japan). 2.11. Statistical Analyses. All data are shown as the mean ± SD (n = 3 to 6). The data were considered to be statistically significant when P < 0.05. A t test was used to calculate the statistical significance. 1056

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After 2 weeks, the treated muscles were collected and sectioned, and the levels of exon skipping were analyzed by RT-PCR. The full-length transcript was detected as an amplicon of 1093 base pairs, and the in-frame transcript, excluding exon 23, was an 880-base-pair product. The results demonstrated that the exon-skipping efficiency was enhanced by PMO delivery with the combination of Bubble liposomes and ultrasound exposure compared with PMO injection alone, as shown in Figure 3a,b. The dystrophin expression in each treated muscle was further confirmed by immunohistochemistry (Figure 4). The results revealed that the density of dystrophin-positive fibers in the cross-section of the tibialis anterior muscle was increased after treatment with the combination of Bubble liposomes and ultrasound exposure compared with PMO injection alone, as shown in Figure 4a,b. Indeed, the number of dystrophin-expressing fibers was significantly increased by the combination method of Bubble liposomes and ultrasound exposure compared with PMO injection alone (Figure 4c). The enhancement by Bubble liposomes and ultrasound exposure of the exon-skipping efficiency and subsequent dystrophin expression was relatively consistent with the increase in fluorescently labeled PMO uptake in the muscle fibers after the treatment as shown in Figure 2. Although Evans blue dye is a membrane-impermeable dye that binds serum albumin, this dye can easily enter the cell if the plasma membrane of the cell is impaired.43,44 Consequently, the uptake of Evans blue dye by muscle fibers indicates the presence of membrane disruptions in the muscle fibers. To examine the sarcolemmal integrity of the skeletal muscle fibers in the mdx mice after PMO delivery via Bubble liposomes and ultrasound exposure, we injected Evans blue dye and evaluated the uptake of the dyes into the tibialis anterior or gastrocnemius muscles by fluorescent microscopy. As shown in Figure 5a, Evans blue dye positive fibers were reduced in the PMO delivery with Bubble liposomes and ultrasound exposure group compared with the PMO injection alone group. In addition, tibialis anterior muscles in which fibers displayed the dye uptake were extracted, and the dye uptake was measured as described in Materials and Methods.42 PMO delivery via Bubble liposomes and ultrasound exposure decreased the uptake of dye in tibialis anterior muscle compared with PMO injection alone (Figure 5b).Similarly, when the PMO was delivered to another target site, gastrocnemius muscle, by Bubble liposomes and ultrasound exposure, the dye uptake decreased compared with PMO injection alone (data not shown). Membrane disruptions and muscle damage not only enhance the dye uptake but also cause the leakage of cytoplasmic factors, such as creatine kinase.45 Therefore, to assess the serum creatine kinase levels in mdx mice after PMO treatment, we delivered the PMO into the posterior of each muscle (tibialis anterior, quadriceps, gastrocnemius, and biceps femoris) by the combination of Bubble liposomes and ultrasound exposure. As a result, the creatine kinase levels were significantly decreased in the PMO with Bubble liposomes and ultrasound exposure treatment group compared with the PMO injection alone group (data not shown). In addition, we previously reported that treatment with Bubble liposomes and ultrasound exposure does not affect the uptake of dye when specific ultrasound parameters (frequency, 1 MH; duty, 50%; intensity, 2 W/cm2; time, 60 s) are used.37 Thus, these data suggest that PMO delivery via Bubble liposomes and ultrasound exposure results in increased sarcolemmal integrity. In Figure 3, the exon-skipping efficiency improvement was less

Figure 3. Detection of exon 23 skipped dystrophin mRNA by RTPCR. (a) Exon 23 skipping in tibialis anterior muscle was detected by RT-PCR 2 weeks after the intramuscular injection of PMO with or without Bubble liposomes and ultrasound exposure (frequency, 1 MHz; duty, 50%, intensity, 2 W/cm2; time, 60 s). The upper bands (indicated by Exon 22/23/24) correspond to the normal mRNA, and the lower bands (indicated by Exon 22/24) correspond to the mRNA in which exon 23 was skipped. (b) Quantitative analysis by RT-PCR. The exon 23 skipping efficiency in tibialis anterior muscle was calculated using the following formula [(the intensity of the skipped band)/(the intensity of the skipped band + the intensity of the unskipped band)]. The data (n = 3) are shown as the means ± SD. *P < 0.05. PMO: phosphorodiamidate morpholino oligomer. BLs: Bubble liposomes. US: ultrasound.

Bubble liposomes containing 2 mol % PEG or 6 mol % PEG were efficiently destroyed after the ultrasound exposure, and a small portion (more than 30%) of the Bubble liposomes containing 10 mol % PEG still remained (Figure S1 in the Supporting Information). Furthermore, when we compared the gene delivery efficiency of the Bubble liposomes, we observed that the efficacy of the Bubble liposomes containing 6 mol % PEG was higher than that of the other types of Bubble liposomes (Figure S2 in the Supporting Information). Based on these results, the Bubble liposomes containing 6 mol % PEG were used in subsequent experiments for PMO delivery. We thus investigated whether Bubble liposomes in combination with ultrasound exposure could enhance the uptake of PMO into the muscle fibers of mdx mice as shown in Figure 1. A solution mixture of fluorescently labeled PMO and Bubble liposomes was intramuscularly injected into the tibialis anterior muscles of mdx mice, and ultrasound was immediately applied at the injection site as described previously.37 The mice were sacrificed after the treatment. Tissue sections were analyzed by fluorescent microscopy, which revealed that the relative PMO uptake into the muscle fibers was significantly increased compared to treatment with PMO injection alone as shown in Figure 2. We next examined whether delivering PMOs by a combination of Bubble liposomes and ultrasound exposure could enhance exon skipping by using a PMO targeting exon 23 in the mdx mouse.6,9 A total of 5 μg of PMO with Bubble liposomes was intramuscularly injected into the tibialis anterior muscles of mdx mice, and ultrasound was immediately applied. 1057

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Figure 4. Restoration of dystrophin expression in the tibialis anterior muscles of mdx mice 2 weeks after the intramuscular injection of PMO with or without Bubble liposomes and ultrasound exposure. The dystrophin expression in tibialis anterior muscle was detected by immunohistochemistry 2 weeks after the intramuscular injection of the PMO with or without Bubble liposomes and ultrasound exposure (frequency, 1 MHz; duty, 50%; intensity, 2 W/cm2; time, 60 s). The dystrophin expression was detected by staining with NCL-DYS2 against dystrophin. Scale bar = 200 μm. Tibialis anterior muscle in normal C57BL/6 mice (a), mdx mice (b), mdx mice treated with the PMO (c), mdx mice treated with PMO, Bubble liposomes, and ultrasound exposure (d). Quantitative evaluation of total dystrophin-positive fibers in tibialis anterior muscle (e). Data (n = 6) are shown as the means ± SD. *P < 0.005. PMO: phosphorodiamidate morpholino oligomer. BLs: Bubble liposomes. US: ultrasound. Each higher magnification photograph is indicated by an open square.

exposure enable the direct entry of siRNA into the cytoplasm without involvement of the endosomal pathway.36 Therefore, delivery by Bubble liposomes in combination with ultrasound exposure may enable immediate and direct PMO delivery into the cytoplasm of muscle cells. Because PMO is charge neutral,14,15 it is not easy to complex PMO with a cationic polymer or lipid as a delivery vehicle. Physical methods, such as scrape-loading, syringe-loading, and osmotic-loading, are superior to the cotransfection of PMO with Lipofectin or Lipofectamine for enhancing the delivery of PMO into cells. These procedures cause a temporary disruption of the plasma membrane that enables the direct entry of the PMO into the cytoplasm, bypassing the endosomal pathway.48 Thus, the delivery efficiency might increase due to the appearance of a transient disruption in the cell membrane caused by the spreading of the Bubble liposomes, followed by their eruption upon ultrasound exposure, which is consistent with previous microbubble studies25 Recently, the use of antisense oligonucleotides as well as PMOs for the treatment of DMD has been demonstrated in mouse and dog models of DMD, with no adverse effects.49−51

than 2 times with Bubble liposomes and ultrasound application compared with that of PMO alone. However, the number of dystrophin-positive fibers and suppression of the Evans blue dye uptake improved by nearly 5 to 6 times. Both improvements may be related to the bioeffects of ultrasound and/or ultrasound contrast agents.46,47 Thus, the translational regulation of dystrophin may be directly or indirectly influenced. Further studies will be required to determine the mechanism that leads to the enhancement or maintenance of dystrophin protein expression after PMO delivery by Bubble liposomes and ultrasound exposure. As shown in Figures 2 to 4, the delivery method of Bubble liposomes with ultrasound exposure enhanced PMO uptake into the cytoplasm of muscle fibers, which was followed by the restoration of dystrophin expression in the treated muscles of mdx mice. However, the mechanism of delivery of the PMO by Bubble liposomes with ultrasound exposure remains unclear. Previously, we reported that Bubble liposomes could induce cavitation with a short duration of ultrasound exposure, leading to efficient gene transfer into various type of cells.35 We further demonstrated in vitro that Bubble liposomes and ultrasound 1058

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Figure 5. Evans blue dye uptake analyses. The sarcolemmal integrity of the skeletal muscle fibers in the mdx mice after PMO delivery with Bubble liposomes and ultrasound exposure was examined. Evans blue dye uptake into the muscle was evaluated by fluorescent microscopy (a) or the measurement of Evans blue dye uptake (b). (a) Evans blue dye fluorescence photomicrographs of sections from the nontreated tibialis anterior muscles, tibialis anterior muscles treated with PMO alone, and tibialis anterior muscles treated with PMO delivered by Bubble liposomes and ultrasound exposure. The nuclei are stained with DAPI. Scale bar = 200 μm. (b) The columns represent the Evans blue dye content measured at A620 nm after the formamide extraction of nontreated tibialis anterior muscles, tibialis anterior muscles treated with PMO alone, or tibialis anterior muscles treated with PMO delivered by BLs and US. The error bars represent the standard deviation of several tested samples. Data (n = 6) are shown as the means ± SD. *P < 0.005 compared with nontreatment (saline injection alone) or PMO. PMO: phosphorodiamidate morpholino oligomer. BLs: Bubble liposomes. US: ultrasound.

mice can be improved by the combination of Bubble liposomes with ultrasound exposure. Applied as antisense oligonucleotide therapy in a mouse model of DMD muscle, the intramuscular injection of a PMO targeting exon 23 in mdx mice with Bubble liposomes followed by ultrasound exposure enabled the restoration of dystrophin expression followed by the apparent inhibition of Evans blue dye uptake in muscle and reduced creatine kinase levels in the treated mdx mouse. Because the intramuscular injection of PMO alone may be inefficient and restrict its clinical use, this ultrasound-mediated Bubble liposome technique may provide an effective noninvasive, nonviral method for antisense oligonucleotide therapy for DMD muscle as well as for other forms of muscular dystrophy.

Therefore, the strategy of exon skipping in DMD muscle to restore an in-frame, symptomatic, or very mild Becker-like transcript is anticipated to provide a more promising therapeutic option for DMD treatment.52 Human clinical trials with antisense oligonucleotides targeting exon 51 are now underway.10−12 However, to maintain therapeutic efficacy, high and repeated doses of PMO will be required in DMD patients because the cellular uptake of PMO is poor, and PMO is rapidly excreted from the circulation.15 Thus, to establish an ideal and general therapy for patients, a safe and efficient PMO delivery system is required to reduce dosage, frequency, and cost. To address these issues, based on our results, delivering PMOs by a combination of Bubble liposomes and ultrasound exposure may be a feasible method to treat DMD muscles. However, to efficiently treat whole body skeletal muscle in DMD patients, the systemic delivery of PMO will be required because skeletal muscle constitutes approximately 30% of the total body mass. Recently, we have developed plasmid DNAand siRNA-loaded Bubble liposomes that are capable of loading plasmid DNA or siRNA53−55 and systemically delivering plasmid DNA into the ultrasound-focused tissues by increasing vascular permeability. Therefore, it is anticipated that exonskipping-inducing antisense oligonucleotides as well as PMOs can be delivered intravascularly into muscle by the combination of Bubble liposomes with ultrasound exposure, leading to a beneficial whole body muscle treatment for DMD.

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ASSOCIATED CONTENT

S Supporting Information *

Table of particle size and zeta potential of Bubble liposomes. Figure depicting ultrasonographic images of the Bubble liposomes with varying DPPC/PEG2000 ratios. Figure depicting comparison of gene delivery efficacy by Bubble liposomes with varying DPPC/PEG2000 ratios upon ultrasound exposure. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

4. CONCLUSIONS In conclusion, we have demonstrated a novel method for delivering a PMO into skeletal muscle in a DMD model mouse by the combination of Bubble liposomes and ultrasound exposure, suggesting that PMO delivery into the muscle of mdx

Author Contributions ‡

The first three authors contributed equally to this work.

Notes

The authors declare no competing financial interest. 1059

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ACKNOWLEDGMENTS We are grateful to Prof. Katsuro Tachibana (Department of Anatomy, School of Medicine, Fukuoka University) for technical advice regarding the induction of cavitation with ultrasound, to Mr. Masaya Yamane (School of Pharmacy, Tokyo University of Pharmacy and Life Sciences) for excellent technical assistance, and to Mr. Yasuhiko Hayakawa and Mr. Kosho Suzuki (NEPA GENE Co., Ltd.) for technical advice regarding ultrasound exposure. This study was supported by a Grant for Industrial Technology Research (04A05010) from the New Energy and Industrial Technology Development Organization (NEDO) of Japan and a Grant-in-Aid for Scientific Research (B) (23300193) from the Japan Society for the Promotion of Science.

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