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Jul 26, 2015 - Targeted Microbubbles for Ultrasound Mediated Short Hairpin RNA Plasmid Transfection to Inhibit Survivin Gene Expression and Induce ...
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Targeted Microbubbles for Ultrasound Mediated Short Hairpin RNA Plasmid Transfection to Inhibit Survivin Gene Expression and Induce Apoptosis of Ovarian Cancer A2780/DDP Cells Yong Zhang,†,§,# Shufang Chang,*,† Jiangchuan Sun,†,# Shenyin Zhu,∥,# Caixiu Pu,† Yaowei Li,† Yi Zhu,† Zhigang Wang,‡ and Ronald X. Xu†,⊥ †

Department of Obstetrics and Gynecology, Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China § National Engineering Research Center of Ultrasound Medicine, Chongqing 400010,China ∥ Department of Pharmacy, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China ⊥ Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio 43210, United States ‡

ABSTRACT: Nonviral gene transfer by ultrasound-targeted microbubble destruction (UTMD) is an promising technique for RNA interference (RNAi) therapy. Targeting silence survivin gene may provide an important therapeutic option for patients with ovarian cancer. However, UTMD mediated RNAi therapy typically uses nontargeted microbubbles with suboptimal gene transfection efficiency. In this work, a LHRHa targeted microbubble agent and recombinant expression plasmid of shRNA targeting survivin gene (pshRNA survivin) were constructed for UTMD mediated pshRNA survivin therapy in ovarian cancer A2780/DDP cells that express LHRH receptors. The targeted microbubbles (TMBs) mixed with the pshRNA survivin were added to cultured ovarian cancer cells followed by ultrasound exposure (1 MHz, 0.5 W/cm2) for 30 s. After transfection for 48 h, the expression of survivin mRNA and protein were (0.36 ± 0.036) and (0.05 ± 0.02), respectively. The cell proliferation inhibitory rates at 24, 48, and 72 h after treatment are (42.08 ± 3.20)%, (54.60 ± 1.02)%, and (74.25 ± 2.14)%, respectively, and the apoptosis rate was (28.99 ± 2.70)%. The expression of apoptosis related protein caspase-9 and caspase-3 were (0.95 ± 0.09) and (2.6 ± 0.21). In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher RNAi efficiency and higher cell apoptosis rate and cell proliferation inhibitory rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance RNAi efficiency in ovarian cancer cells. This novel method for RNA interference represents a powerful, promising no viral technology that can be used in the tumor gene therapy and research. KEYWORDS: ovarian cancer, ultrasound, microbubbles, survivin, RNA interference, apoptosis

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INTRODUCTION

therapeutic strategies, such as RNA interference (RNAi), have been explored for effective treatment at the early stage.5,6 RNA interference (RNAi) is a naturally occurring process of sequence-specific post-transcriptional gene silencing. It is an

Ovarian cancer is the third most prevalent gynecologic cancer with the highest tumor-related mortality.1−3 Cytoreductive surgery followed by adjuvant chemotherapy is the standard treatment for ovarian cancer. Despite many efforts in clinical surgery and chemotherapy, the 5-year survival rate of ovarian cancer has not changed significantly over the last decades, partially owing to that fact that many patients have developed the advanced disease at the time of diagnosis.4 Therefore, novel © XXXX American Chemical Society

Received: December 13, 2014 Revised: April 5, 2015 Accepted: July 26, 2015

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streptomycin. Exponentially growing cells were used for the whole experiment. Construction of pshRNA-Survivin. The pshRNA-survivin was constructed by Institute of Viral Hepatitis at Chongqing Medical University. It contains a 56-nucleotide long specific survivin sequence, i.e., 5′-CTAGAAAAAAAAGCATTCGTC CGGTTGC CAGTACTC GCA ACC GGA CGA ATG CTT TC-3′. Preparation of Nontargeting Lipid Microbubbles (NMBs). NMBs were prepared following a modified process as described previously.25 Briefly, five milligrams of 1,2dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, Avanti Polar Lipids Inc., Alabaster, AL, USA), 0.5 mg of 1,2distearoyl-sn-glycero-3-phosphatidyl-ethanolamine (DSPE,Avanti), and 1 mg of 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) were dissolved in a 1.5 mL vial containing 50 μL of 100% glycerine and 450 μL of phosphate buffered saline (PBS). The vial was incubated in a 40 °C water bath for 30 min. After that, the container was degassed, refilled with perfluorobutane gas (Research institute of Physical and Chemical Engineering of Nuclear Industry, Tianjing, China), and mechanically vibrated at 4 kHz for 45 s in a dental amalgamator (YJT Medical Apparatuses and Instruments, Shanghai, China). The generated NMBs were washed with PBS and centrifuged at 800 rpm for 5 min. The supernatant was collected and the NMB concentration was estimated by a bright field microscope and sterilized by cobalt 60 (60Co) irradiation. The size distribution of the NMBs was determined by a 3000SSA Zatasizer (Malvern Instruments Inc., Westborough, MA). Preparation of Targeted Lipid Microbubbles (TMBs). LHRHa-targeted lipid MBs (TMBs) were fabricated by a modified emulsification process as described previously.25 The process consists of three consecutive steps of biotinylating MBs (BMBs), avidinylating BMBs (BSMBs), and conjugating BSMBs with biotinylated LHRHa peptide. The experimental details for each step are described below. BMBs were prepared by replacing 1,2-distearoyl-sn-glycero-3-phosphatidyl-ethanolamine with 1,2-distearoyl-sn-glycero-3-phosphatidyl-ethanolamine-N-[biotinyl(polyethylene glycol) 2000] (Avanti) in the NMBs recipe. Then those biotinylated MBs (BMBs) were washed with PBS solution three times in a bucket rotor centrifuge at 1000 rpm for 3 min to remove excess unincorporated lipids from the MBs. After that, 50 μg of streptavidin (SA, Beijing Biosynthesis Biotechnology Co., Ltd., China) per 108 MBs was then added to the washed MB dispersion. After incubation at 4 °C for 20 min, the MBs were washed three times at the same centrifugation conditions to remove the unreacted streptavidin and obtain BSMBs. After that, the BSMBs were incubated at 4 °C with 50 μg of biotinylated LHRHa peptides (with amino acid sequence: pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NH2, synthesized by Beijing SciLight Biotechnology Co. Ltd., Beijing, China) per 108 MBs for another 20 min. Free ligands were removed through washing with PBS. The concentration of the TMBs and NMBs were estimated by a bright field microscope and sterilized by cobalt 60 (60Co) irradiation. Targeted Binding of TMBs in Vitro. The binding affinity of the TMBs was tested on the LHRH receptor positive ovarian cancer cells. A2780/DDP cells (1 × 105cells/well) were incubated with the TMBs (1 × 106/mL) and the NMBs (1 × 106/mL), respectively, in Costar cell culture clusters at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. Considering the buoyancy of the MBs, the cell culture clusters were placed

important biological process for modulating gene expression. It has been recognized as a potential therapeutic tool for silencing genes linked to various diseases including cancer.7−9 Despite high therapeutic potentials of siRNAs, their clinical dissemination was hurdled by several critical limitations in drug delivery. First, interfering RNAs have relatively small molecular weights. They are subject to rapid degradation and urinary excretion, resulting in a short plasma half-life.10 Second, it is challenging to deliver intact siRNA into the cytoplasm of the target cells for high transfection efficiency.11 Ultrasound-targeted microbubble destruction (UTMD) technology is a promising approach for no-viral gene delivery.12,13 UTMD is able to produce transient pores in cell membranes, stimulate cell membrane permeabilization, and significantly increase the gene transfection efficiency.14,15 Recently, UTMD has been used for siRNA delivery with the enhanced gene silence efficiency.16−19 However, UTMD typically uses nontargeted microbubbles that are readily aggregate in the reticuloendothelial system (RES), leading to a low concentration at the disease site. Therefore, it is advantageous to deliver siRNA by tumor-targeted microbubbles. Cell specific ligands such as peptide and antibodies have been deployed to modify microbubbles for targeted diagnosis and therapy.20−22 In the previous study, we have synthesized an ovarian cancer targeting microbubbles by conjugating LHRHa on the surface of the lipid microbubbles.23−25 These tumortargeting microbubbles have been used for ultrasound mediated wide-type p53 gene transfection to ovarian cancer A2780/DDP cells with the significantly improved efficiency.25 Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is involved in drug resistance and apoptosis inhibition of cells.26−30 It provides an attractive target for anticancer therapies since it selectively expresses in embryonic tissues and most types of tumors but not in normal adult tissues. Several studies have demonstrated that ultrasoundmediated silencing of survivin using shRNA plasmid microbubbles without targeting is able to induce cell apoptosis and enhance antitumor efficacy.31−33 In this study, the LHRHadecorated microbubbles were mixed with pshRNA-survivin for UTMD-mediated targeted delivery of shRNA-survivin to the cancer cells. The resultant silencing of the survivin expression was evaluated by real-time quantitative polymerase chain reaction (RQ-PCR). Cell apoptosis was analyzed by flow cytometry and Hoechst 33258 staining. The expressions of survivin protein, apoptosis related protein caspase-3, and caspase-9 were evaluated by Western blot. Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene silence efficiency in ovarian cancer cells. To the best of the authors’ knowledge, using LHRHa targeted microbubbles for UTMD mediated delivery of pshRNA-survivin to ovarian cancer cells has not been reported elsewhere.

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EXPERIMENTAL SECTION Cell Lines and Cell Cultures. Human ovarian cancer A2780/DDP cells (LHRH receptor positive) were a generous gift from Professor Zehua Wang at Wuhan Union Hospital (Wuhan, China). The cells were maintained in a HyClone RPMI 1640 medium (Fisher Scientific, Shanghai, China) at 37 °C in a humidified 5% CO2 atmosphere, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL B

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h after treatment and subjected to Western blotting to determine the efficiency in inhibiting survivin protein expression. Western blotting analysis was performed following standard methods. Briefly, cell lysates were resolved under reducing conditions at a concentration of 50 μg of protein of each sample per lane, proteins were isolated by SDS-PAGE. PVDF membranes were incubated overnight with primary antibody (1:2500 dilution, Sigma, America). Immunodetection with a secondary peroxidase-conjugated antibody (1:2500 dilution, Sigma, America) and chemiluminescence was performed according to the manufacturer’s protocol (Beyotime Biotech, China). To confirm equal protein loading per lane, the nitrocellulose membranes were subsequently reprobed with a 1:5000 dilution of an anti-β-actin antibody (Abcam, U.K.) and developed as described above. Band optical density (OD) was analyzed using a Labworks 4.6 UVP-image capture and analysis software package. The analysis results were expressed in the format of mean ± standard deviation (SD) as the ratio percentage of the protein of interest OD versus the β-actin OD. Cell Viability Assay. After the exposure of the ultrasound pulses, the cells were seeded in a 24-well plate, incubated for 24, 48, and 72 h and washed in PBS for three times. The number of viable cells in each treatment group relative to PBS control was assessed using MTT. Data were collected for three replicates each and used to calculate the respective means and the standard deviations. The percentage inhibition was calculated using the formula: inhibition of cell proliferation rate (%) = [(1 − experimental OD value)/control OD value] × 100%. Detection of Apoptosis by Flow Cytometer. Apoptosis was quantified by a combined staining of Annexin V and propidium iodide (PI) using Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer’s instructions. Propidium iodide (PI) at a concentration of 0.5 μg/mL was added to the cell suspension immediately before flow cytometry analysis (BD Biosciences, USA). The percentage apoptosis rate was calculated using the following formula: apoptosis rate (%) = [(number of early apoptosis cells + mature apoptosis cells)/ number of total cells] × 100%. Single Annexin positive cells were defined as early apoptosis cells, both Annexin and PI positive cells were defined as mature apoptosis cells. Detection of Apoptosis by Hoechst 33258 Fluorescent Staining. After the exposure of the ultrasound pulses, the cells were seeded in a 6-well plate, incubated for 48 h and washed in PBS for three times. The cells were stained in Hoechst 33258 (4 g/mL, Beyotime Institute of Biotechnology, China) for 30 min at 37 °C, fixed for 10 min in 4% paraformaldehyde (Sigma, St. Louis, MO, USA), and then observed under fluorescence microscopy. Apoptotic cells were defined with the changes of nuclear morphology. Normal nuclei showed diffusely and homogeneously low-intensity fluorescent, apoptotic nuclei that were hyperchromatic and compact at condensed or granular state. Transmission Electron Microscopy Observation. The ultrastructure of control group and pshRNA-TMBs + US group cells were observed by transmission electron microscopy. In brief, 48 h after treatment, the A2780/DDP cells were washed with PBS, trypsinized, pelleted by centrifuge (12000 r/min) at 4 °C for 15 min, and fixed with 2.5% glutaraldehyde. The cells were prepared for transmission electron microscopy with an electron microscope according to standard protocols and

upside down to maximize the cell−MB interaction. After a 30 min static exposure, the cell culture clusters were turned over, and the MBs bound to the cells was examined by an inverted bright field microscope. pshRNA-Survivin Delivery by Microbubble-Assisted Ultrasound. MBs-pshRNA-survivin complex was prepared by the flowing process. TMBs or NMBs were suspended in PBS at a concentration of 1× 108/mL. Ten microliters of TMB or NMB suspension was mixed with 100 μL of pshRNA-survivin (10 μg/mL) at 4 °C for 30 min to obtain the MBs/pshRNAsurvivin mixture. A2780/DDP cells (1 × 105 cells/well) were seeded in 96-well plates 24 h to allow cell adhesion. After that, the cells were equally divided into the following seven treatment groups: (a) applying PBS only (i.e., “negative control”); (b) applying pshRNA-survivin only (i.e., “pshRNA only”); (c) applying pshRNA-survivin-NMBs (i.e., “pshRNANMBs”); (d) applying pshRNA-survivin -TMBs (i.e., “pshRNA-TMBs”); (e) applying pshRNA-survivin followed by ultrasound destruction (i.e., “pshRNA+US”); (f) applying pshRNA-NMBs followed by ultrasound destruction (i.e., “pshRNA-NMBs+US”); (g) applying pshRNA-survivin-TMBs followed by ultrasound destruction (i.e., “pshRNA-TMBs + US”). An equivalent amount of the MBs/pshRNA-survivin mixture at a dose of 1.6 μg pshRNA-survivin was applied to each treatment group. Considering the buoyancy of the MBs, the cell culture clusters were placed upside down to maximize the cell−MB interaction. After a 30 min static exposure, the clusters were turned over and ready for ultrasound exposure; the free MBs that did not bind to the cells were not removed from the cell culture. For treatment groups (e), (f), and g), a 1 MHz piezoelectric ceramic transducer (model CGZZ, Ultrasonographic Image Research Institute, Chongqing Medical University, Chongqing, China) was immersed 2 mm above the cell suspension within the cell culture medium. Ultrasound pulses with an averaged intensity of 0.5 W/cm2 were applied to the medium for 30 s. Analysis of Survivin Silence Efficiency by Real-Time Quantitative Polymerase Chain Reaction (RQ-PCR). Cells were harvested 48 h after treatment and subjected to RQ-PCR to determine the efficiency in survivin silence. Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA with a reverse transcription kit (Promega, Madison, WI). The resultant cDNA was amplified using a specific primer pair for human survivin: forward 5′-ATAGTCGACATGGGTGCCCCGACGTTG-3′, reverse 5′-CTCGGATCCTCAATCCATGGCAGCCAG-3′. β-actin was used as an internal standard and its mRNA was amplified with primers: forward 5′CTGAGAGGGAAATCGTGCGT-3′, reverse 5′-CCACAGGATTCCATACCCAAGA-3′. Set up solutions for a test of the Applied Biosystems SYBR Green PCR kit on the BioRad iCycler iQ Real-Time Detection System are PCR mix 12.5 μL, Primer mix 5 μL, double-distilled water 5 μL, cDNA 2.5 μL. The cycling program was performed as follows: 50 °C, 2 min; 95 °C, 2 min; 95 °C, 15 s; 58 °C, 20 s; 72 °C, 45 s; 72 °C, 10 min. The ratio of survivin mRNA expression relative to β-actin mRNA expression was calculated. Furthermore, the relative survivin mRNA expression was normalized against that derived from the control siRNA-transfected group. The level of mRNA expression of targeted gene was 2−ΔΔCt (ΔCt = Ct targeted gene − Ctβ‑actin, ΔΔCt = ΔCtexperiment − ΔCtcontrol). Analysis of Survivin, Caspase-9, and Caspase-3 Expression by Western Blotting. Cells were harvested 48 C

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Figure 1. LHRHa-targeted microbubbles (TMBs) attached to A2780/DDP cells in vitro. Nontargeted lipid microbubbles (NMBs) incubated with A2780/DDP cells (left). LHRHa-targeted lipid microbubbles (TMBs) incubated with A2780/DDP cells (right).

photographed with H7500 transmission electron microscopy (Hitachico Ltd., Japan). Statistical Analysis. The data were obtained from three independent experiments and were presented as mean ± standard deviation (SD) and processed with the statistics software SPSS 13.0. The measurement differences between treatment groups were determined by an analysis of variance (ANOVA) test. The group-wise comparison was carried out by a Tukey’s honestly significant difference post hoc test. A p value of less than 0.05 was considered statistically

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RESULTS Targeted Binding of TMBs in Vitro. The MBs bound to the cells was examined by a bright field microscope. The microscopic images of the A2780/DDP cells after applying TMBs and NMBs. According to the Figure 1, NMBs do not bind with the cells (left), whereas TMBs bind with the cells well (right). Down-Regulation of Survivin mRNA in RQ-PCR. UTMD mediated survivin silence was tested in the A2780/ DDP cells. Figure 2A shows the levels of survivin mRNA expression for seven treatment groups. According to the figure, the expression levels of survivin mRNA were slightly decreased in (b), (c), and (d) groups, and the expression level of survivin mRNA moderately decreased in the (e) and (f) groups, while declining obviously in the (g) group, which showed a 0.58-, 0.44-, and 0.36-fold decrease in the mRNA expression (p < 0.05) (Figure 2A) . The results indicated that UTMD mediated delivery of the mixture of pshRNA-survivin and the TMBs significantly inhibited the expressions of survivin mRNA in A2780/DDP cells. Suppression of Survivin Protein Expression in Western Blotting Analysis. Figure 2B shows the survivin protein expression levels for seven treatment groups obtained by Western blot analysis. There were not obvious changes in expressions of survivin protein for treatment groups (b) and (c) compared with group (a). However, the survivin expression was suppressed in groups (d), (e), (f), and (g). The indexes of survivin/β-actin ratio for treatment groups (a), (b), and (c) are over 1.3, whereas those for treatment groups (d), (e), (f), and (g) are (1.2 ± 0.4), (0.6 ± 0.1), (0.42 ± 0.06), and (0.05 ± 0.02), respectively. Compared with other treatment groups,

Figure 2. Downregulation of survivin mRNA and protein with pshRNA-survivin transfected in A2780/DDP cells. (A) The expression of survivin mRNA determined by quantitative real-time PCR at 48 h after treatment. Data are represented as mean ± SD (n = 3). The survivin mRNA of group (g) was significantly lower than those of the other groups (p < 0.05). Compared with group (g), *p < 0.05; compared with group (a), #p < 0.05. (B) The expression of survivin protein in A2780/DDP cells for different experiment groups detected by Western blot 48 h after treatment. Quantification of band intensity relative to β-actin was shown in columns. Data are represented as mean ± SD (n = 3). The intensity for treatment groups (a), (b), and (c) are over 1.3, whereas those for treatment groups (d), (e), (f), and (g) are (1.2 ± 0.4), (0.6 ± 0.1), (0.42 ± 0.06), and (0.05 ± 0.02), respectively. Compared with other treatment groups, group (g) has the lowest level of survivin protein expression (p < 0.05). Compared with group (g), *p < 0.05; compared with group (a), #p < 0.05.

group (g) has the lowest level of survivin protein expression (p < 0.05) (Figure 2B). The results indicated that UTMD mediated delivery of TMBs-pshRNA-survivin complex significantly inhibited the expressions of survivin protein in A2780/ DDP cells. D

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Molecular Pharmaceutics Inhibiting the Cell Proliferation in MTT Assay. The cell viability was assessed by methyl thiazol tetrazolium (MTT) assay 24, 48, and 72 h after treatment. It was observed that the cell viability was markedly reduced in a time-dependent manner starting with the maximum reduction observed at 72 h after treatment as shown in Figure 3. In the case of groupa (b), (c),

Figure 3. Growth inhibition effect of A2780/DDP cells with different treatments. The proliferation inhibitory rate of cells was determined by MTT 24, 48, and 72 h after treatment. Data are represented as mean ± SD (n = 3). The proliferation inhibitory rate of the pshRNA-survivinNMBs + US group and pshRNA-survivin-TMBs + US group are significantly higher than those of the other groups (p < 0.05). The proliferation inhibitory rate of pshRNA-survivin-TMBs + US group is higher than pshRNA-survivin-NMBs + US group at 24, 48 and 72 h after treatment (p < 0.05). Compared with pshRNA-survivin-TMBs + US group, *p < 0.05.

and (d), the cell inhibiting rate is less than 30% at 72 h after treatment. Other than that, for treatment group (e), the proliferation inhibitory rate at 24, 48, and 72 h after treatment are (19.91 ± 1.61)%, (26.25 ± 1.08)%, and (53.22 ± 3.57)%; for treatment group (f) are (30.97 ± 1.26)%, (43.78 ± 2.41)%, and (59.69 ± 1.57)%; and for treatment group (g) are (42.08 ± 3.20)%, (54.60 ± 1.02)%, and (74.25 ± 2.14)%, respectively. In comparison with other treatment groups, group (g) results in significantly higher proliferation inhibition than other treatment groups (p < 0.05). Those results indicated that UTMD mediated delivery of the TMBs-pshRNA-survivin mixture significantly inhibited the proliferation of A2780/DDP cells. Inducing Apoptosis in a Flow Cytometer Assay, Hoechst 33258 Fluorescent Staining, Western Blotting Analysis, and Transmission Electron Microscopy Observation. Cell apoptosis after UTMD mediated delivery of pshRNA-survivin was evaluated by flow cytometry, as shown in Figure 4A. According to the figure, the apoptosis efficiencies for treatment groups (a)−(g) are (3.56 ± 0.28)%, (6.04 ± 0.43)%, (6.14 ± 0.92)%, (6.80 ± 0.40)%, (7.55 ± 0.82)%, (11.63 ± 1.16)%, and (28.99 ± 2.70)%, respectively. The percentage of apoptotic cells was significantly higher in the treatment group than control group (a). Furthermore, in comparison with other treatment groups, group (g) results in significantly higher apoptosis efficiency (p < 0.05), as indicated by a 8.4-fold increase compared with group (a). Those results show that UTMD mediated delivery of the mixture of pshRNA-survivin and the TMBs significantly increases the cell apoptosis efficiency. Cell apoptosis also detected by Hoechst 33258 fluorescent staining and transmission electron microscopy. Figure 4B(a−g) shows fluorescence microscopic images acquired for seven

Figure 4. Apoptosis efficiency in A2780/DDP cells with different treatments. (A) The proportion of apoptotic cells was determined by flow cytometry 48 h after treatment. Data are represented as mean ± SD (n = 3). The percentage of apoptotic cells was significantly higher in the group (g) (28.99 ± 2.70) as indicated by a 8.4-fold increase compared with group (a) (3.56 ± 0.28). Furthermore, the apoptotic cells in group (g) was greater than those in group (d) (6.80 ± 0.40), group (e) (7.55 ± 0.82), or group (f) (11.63 ± 1.16) (p < 0.05). (B) Nuclear morphology of cells stained with Hoechst-33258 was observed by fluorescence microscopy (×200) at 48 h after different treatment transfection. Data are representative microscopic pictures. (a) Applying PBS only (“control”); (b) applying pshRNA-survivin only (“survivin only”); (c) applying pshRNA-survivin-NMBs (“survivin-NMBs”); (d) applying pshRNA-survivin-TMBs (“survivinTMBs”); (e) applying pshRNA-survivin followed by ultrasound destruction (“survivin+US”); (f) applying pshRNA-survivin-NMBs followed by ultrasound destruction (“survivin-NMBs + US”); (g) applying pshRNA-survivin-TMBs followed by ultrasound destruction (“survivin-TMBs + US”). Treatment group (g) exhibits the strongest blue fluorescence emission, and chromatin condensation could be visualized in many cells (as show in the red arrow). (C) Ultrasound mediated pshRNA-survivin transfection with TMB destruction induces the cell ultrastructural morphology change in A2780/DDP cells. The cell cytoplasm appeared in much of the microfilament and microtubules in the control group (left). However, typical apoptotic cells could be visualized in the pshRNA-survivin-TMBs plus ultrasound group (right). The cells became smaller, and the cytoplasm was concentrated. The chromatin became highly condensed and marginalized (as show in the red arrow). Scale bar is 2 μm.

groups. Figure 4B(a) shows that the cells in the control group (a) exhibited normal nuclear morphology, nuclei fluoresced faint blue, and the color was homogeneous. Treatment groups E

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Figure 5. Expression of caspase-3 and caspase-9 protein in A2780/DDP cells for different experiment groups detected by Western blot 48 h after treatment. Data are represented as mean ± SD (n = 3). (A) Caspase-3 expression was detected by Western blot analysis. Quantification of band intensity relative to β-actin was shown in columns. The intensity in group (g) was significantly higher than those of the other groups (p < 0.05). Compared with group (g), *p < 0.05; compared with group (a), #p < 0.05. (B) Caspase-9 expression was detected by Western blot analysis. Quantification of band intensity relative to β-actin was shown in columns. The intensity in group (g) was significantly higher than those of the other groups (p < 0.05). Compared with group (g), *p < 0.05; compared with group (a), #p < 0.05.

(b)−(d) also exhibit faint blue fluorescence emission; only a few chromatin condensations were observed, indicating that direct application of pshRNA-survivin or applying pshRNAsurvivin-loaded microbubbles without ultrasound mediation can induce A2780/DDP cell apoptosis slightly. Treatment groups (e)−(f) exhibit moderate blue fluorescence emission, and some nuclear condensation and morphological changes such as chromatin condensation and fragmentation were observed, indicating that ultrasound mediation may enhance cell apoptosis even without the use of a targeting vector. Treatment group (g) exhibits the highest blue fluorescence emission, and chromatin condensation and fragmentation could be visualized in many cells. Indicating that ultrasound medication of the TMBs-pshRNA-survivin complex yields the highest apoptosis efficiency. Ultrasound medication of the mixture of pshRNA-survivin and the TMBs induced A2780/DDP apoptosis were also observed under transmission electron microscopy. Figure 5C shows representative ultrastructural morphologic changes of A2780/DDP cells 24 h after different treatment. The cell

cytoplasm appeared in much of the microfilament and microtubules in the control group (Figure 4C left). However, typical apoptotic cells could be visualized in the pshRNAsurvivin-TMBs plus ultrasound group. The cells became smaller, the cytoplasm was concentrated, the nucleus became irregular, and the chromatin became highly condensed and marginalized (Figure 4C, right). In addition, we further measured the activity of caspase-3 and caspase-9, the major effector protein of apoptosis. As shown in Figure 5, cells transfected with pshRNA-survivin using ultrasound-targeted TMBs destruction showed more prominent bands of caspase-3 and caspase-9 than other treatment groups. The indexes of caspase-3/β-actin ratio for treatment groups (a), (b), and (c) are less than 0.1, whereas those for treatment groups (d), (e), (f), and (g) are (0.10 ± 0.02), (0.45 ± 0.13), (0.70 ± 0.26), and (0.95 ± 0.09), respectively (Figure 5A). The indexes of caspase-9/β-actin ratio for treatment groups (a), (b), and (c) are less than 0.7, whereas those for treatment groups (d), (e), (f), and (g) are (1.00 ± 0.20), (1.20 ± 0.10), (2.30 ± 0.10), and (2.60 ± 0.21), respectively (Figure F

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Molecular Pharmaceutics

evidenced in our previous study.23−25 Our experiment also showed that US mediation of TMBs/NMBs and pshRNAsurvivin mixture or bold pshRNA yielded the siRNA effect. The survivin mRNA expression was inhibited in comparison with the negative control. However, the survivin protein expressions in the NMBs and pshRNA-survivin mixture group or bold pshRNA-survivin group were not significantly inhibited in comparison with the negative control. The mechanism behind this phenomenon has not been fully understood yet. We believe that it is a complicated process from the transcription to the translation. Further study will be performed to elucidate the mechanism. Several studies have shown that the silence survivin is able to inhibit cell proliferation and induce apoptosis.45−47 In this study, we also investigated whether combination of TMBs and pshRNA with ultrasound mediation could inhibit A2780/DDP cell proliferation and induce apoptosis in vitro. The MTT assay showed that cell proliferation inhibitory rate was significantly increased with combination of TMBs and pshRNA with ultrasound mediation. Flow cytometry analysis and Hoechst 33258 fluorescent staining also revealed that the apoptosis efficiency was significantly higher in cells treated by the mixture of TMBs and pshRNA-survivin followed by ultrasound mediation, as compared with the other treatment groups. Our experiment also showed that bold pshRNA-survivin with ultrasound mediation and pshRNA-survivin/NMBs mixture with ultrasound mediation yielded relatively high inhibitory rates, but the apoptosis effect is not as significant as the inhibitory effect in those groups. The mechanism behind this phenomenon has not been fully understood yet. We believe that the proliferation inhibitory rate and the apoptosis efficiency were correlated with the survivin silence efficiency, indicating that proliferation inhibition and apoptosis induction may contribute to the mechanisms of survivin on A2780/DDP cells. The process of apoptosis can be induced either by the extrinsic pathway, which involves signaling from death receptors at the cell surface, or by the intrinsic mitochondriamediated pathway.48,49 Caspase 3 is the effector for extrinsic apoptosis pathway, and activation of caspase 9 is related to intrinsic mitochondria-mediated apoptosis.50,51 In the present study, we further measured the activity of caspase-9 and caspase-3, two key mediators of cell death. We found that cells transfected with pshRNA-survivin using ultrasound-targeted TMB destruction showed more prominent bands than other treatment groups and that the level of caspase-3 and caspase-9 was correlated with apoptosis efficiency, which indicates that a combination of TMBs and pshRNA with ultrasound mediation induced both caspase3 mediated extrinsic apoptotic pathway and caspase 9 mediated intrinsic apoptotic pathway. In conclusion, our data revealed that, on the basis of the survivin mediated invasion, survivin silence can accomplish a selective apoptosis effect on A2780/DDP cells in vitro. The pshRNA-survivin system mediated by ultrasound in combination with LHRHa-targeted microbubbles may present a novel and attractive approach for ovarian cancer gene therapy. Further in vivo studies will be performed to elucidate the gene silence outcome of the ultrasound mediated MBs/pshRNAsurvivin mixture.

5B). Compared with other treatment groups, group (g) has the highest level of caspase-3 and caspase-9 expression (p < 0.05), indicating that ultrasound mediated TMBs-pshRNA-survivin mixture delivery induces A2780/DDP cell apoptosis by down regulating caspase-3 and caspase-9 expression.

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DISCUSSION RNA interference (RNAi) technique is a promising approach for cancer therapy.34 However, cells do not readily take up siRNA. Therefore, clinical applications of siRNA largely depend on the development of delivery systems. In the past decade, numerous transfection methods or delivery devices have been developed and reported.35−37 Even though the viral vectors have high gene silence efficiency, potential risk of insertional mutagenesis and interference response is the major obstacle.38 Nonviral delivery systems are relatively safe and easier to apply, but they suffer from low silence efficiency.39 Therefore, development of a targeted and high efficiency siRNA delivery system is highly desired. Ultrasound-targeted microbubble destruction (UTMD) has recently been used for siRNA delivery both in vitro and in vivo.16,40−42 UTMD is able to stimulate sonoporation, allowing for the transient change in the permeability of the cell membrane and the increase in the site-specific intracellular delivery of biological macromolecules such as DNA and siRNA.43 However, existing microbubbles have some problems with targeting function. We have synthesized ovarian cancer targeted microbubbles for UTMD mediated wtp53 gene transfection to induce A2780/DDP cells apoptosis.25 In this study, we use these targeted microbubbles for UTMD mediated survivin silence to induce ovarian cancer apoptosis. The use of survivin is based on the following underlying rationale: (1) Survivin is upregulated in various malignancies but not expressed in normal adult tissues. It may serve as a diagnostic marker and also as a novel therapeutic target for human cancer. (2) Survivin is a member of the inhibitor of apoptosis protein (IAP) family associated with drug resistance and apoptosis inhibition of ovarian cancer cells.26−30 (3) It has been proven that silencing survivin expression is able to induce the apoptosis of ovarian cancer cells, both in vitro and in vivo.29,44 To demonstrate the UTMD mediated siRNA technique, we incubated the mixture of the TMBs and pshRNA-survivin with A2780/DDP cells for the following reasons: The entrapment efficiency of plasmid on the MBs is relatively low (22.4 ± 4.16%) in our experiment. If we use plasmid loaded microbubbles in the experiment, a large volume of plasmid loaded microbubbles will be need to obtain 1.6 μg of plasmid. After ultrasound pulses were applied to the cell culture, the survivin mRNA and protein were analyzed and compared with the other treatment groups. We found that MBs assisted mediation of pshRNA survivin by ultrasound pulses could inhibit survivin mRNA expression. It was confirmed that ultrasound mediation after combination of TMBs and pshRNA yielded the most prominent effect of gene downregulation, consistent with previous reports.25 Interestingly, TMB assisted pshRNA-survivin delivery with ultrasound mediation also yielded a significantly lower survivin protein expression in comparison with pshRNA-survivin delivery with NMBs or without microbubbles. We believe that the decreased survivin mRNA and protein expression for TMB assisted gene silence is associated with the interaction of ultrasound pulses with the increased number of the TMBs bound with the cancer cells, as

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: Department of Obstetrics and Gynecology, Second Affiliated Hospital of Chongqing Medical University, G

DOI: 10.1021/mp500835z Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

(9) Gil-Ranedo, J.; Mendiburu-Elicabe, M.; Garcia-Villanueva, M.; Medina, D.; del Alamo, M.; Izquierdo, M. An off-target nucleostemin RNAi inhibits growth in human glioblastoma-derived cancer stem cells. PLoS One 2011, 6 (12), e28753. (10) Iversen, F.; Yang, C.; Dagnæs-Hansen, F.; Schaffert, D. H.; Kjems, J.; Gao, S. Optimized siRNA-PEG conjugates for extended blood circulation and reduced urine excretion in mice. Theranostics 2013, 3 (3), 201−209. (11) Racz, Z.; Hamar, P. SiRNA technology, the gene therapy of the future? Orv. Hetil. 2008, 149 (4), 153−159. (12) Chen, Z. Y.; Yang, F.; Lin, Y.; Zhang, J. S.; Qiu, R. X.; Jiang, L.; Zhou, X. X.; Yu, J. X. New development and application of ultrasound targeted microbubble destruction in gene therapy and drug delivery. Curr. Gene Ther. 2013, 13 (4), 250−274. (13) Mayer, C. R.; Geis, N. A.; Katus, H. A.; Bekeredjian, R. Ultrasound targeted microbubble destruction for drug and gene delivery. Expert Opin. Drug Delivery 2008, 5 (10), 1121−1138. (14) Fan, Z.; Chen, D.; Deng, C. X. Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles. J. Controlled Release 2013, 170 (3), 401−413. (15) Geis, N. A.; Katus, H. A.; Bekeredjian, R. Microbubbles as a vehicle for gene and drug delivery: current clinical implications and future perspectives. Curr. Pharm. Des. 2012, 18 (15), 2166−2183. (16) Florinas, S.; Nam, H. Y.; Kim, S. W. Enhanced siRNA delivery using a combination of an arginine-grafted bioreducible polymer, ultrasound, and microbubbles in cancer cells. Mol. Pharmaceutics 2013, 10 (5), 2021−2030. (17) Vandenbroucke, R. E.; Lentacker, I.; Demeester, J.; De Smedt, S. C.; Sanders, N. N. Ultrasound assisted siRNA delivery using PEGsiPlex loaded microbubbles. J. Controlled Release 2008, 126 (3), 265− 273. (18) Shi, Q.; Liu, P.; Sun, Y.; Zhang, H.; Du, J.; Li, F.; Du, L.; Duan, Y. siRNA delivery mediated by copolymer nanoparticles, phospholipid stabilized sulphur hexafluoride microbubbles and ultrasound. J. Biomed. Nanotechnol. 2014, 10 (3), 436−444. (19) Florinas, S.; Kim, J.; Nam, K.; Janat-Amsbury, M. M.; Kim, S. W. Ultrasound-assisted siRNA delivery via arginine-grafted bioreducible polymer and microbubbles targeting VEGF for ovarian cancer treatment. J. Controlled Release 2014, 183, 1−8. (20) Geers, B.; De Wever, O.; Demeester, J.; Bracke, M.; De Smedt, S. C.; Lentacker, I. Targeted Liposome-Loaded Microbubbles for CellSpecific Ultrasound-Triggered Drug Delivery. Small 2013, 9 (23), 4027−35. (21) Wang, L.; Li, L.; Guo, Y.; Tong, H.; Fan, X.; Ding, J.; Huang, H. Construction and in vitro/in vivo targeting of PSMA-targeted nanoscale microbubbles in prostate cancer. Prostate 2013, 73 (11), 1147−58. (22) Willmann, J. K.; Paulmurugan, R.; Chen, K.; Gheysens, O.; Rodriguez-Porcel, M.; Lutz, A. M.; Chen, I. Y.; Chen, X.; Gambhir, S. S. US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice. Radiology 2008, 246 (2), 508−518. (23) Liu, H.; Chang, S.; Sun, J.; Zhu, S.; Pu, C.; Zhu, Y.; Wang, Z.; Xu, R. X. Ultrasound-mediated destruction of LHRHa-targeted and paclitaxel-loaded lipid microbubbles induces proliferation inhibition and apoptosis in ovarian cancer cells. Mol. Pharmaceutics 2014, 11 (1), 40−48. (24) Pu, C.; Chang, S.; Sun, J.; Zhu, S.; Liu, H.; Zhu, Y.; Wang, Z.; Xu, R. X. Ultrasound-mediated destruction of LHRHa-targeted and paclitaxel-loaded lipid microbubbles for the treatment of intraperitoneal ovarian cancer xenografts. Mol. Pharmaceutics 2014, 11 (1), 49−58. (25) Chang, S.; Guo, J.; Sun, J.; Zhu, S.; Yan, Y.; Zhu, Y.; Li, M.; Wang, Z.; Xu, R. X. Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells. Ultrason. Sonochem. 2013, 20 (1), 171−179. (26) Ferrandina, G.; Legge, F.; Martinelli, E.; Ranelletti, F. O.; Zannoni, G. F.; Lauriola, L.; Gessi, M.; Gallotta, V.; Scambia, G. Survivin expression in ovarian cancer and its correlation with clinico-

No. 74 Linjiang Road, Yuzhong District, Chongqing 400010, China. Tel: +86 23 6369 3279. E-mail: [email protected]. Author Contributions #

These authors (Y.Z., J.S., and S.Z.) contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Dr. Pan Li (Institute of Ultrasound Imaging, Second Hospital of Chongqing Medical University, and Chongqing, China) for the helpful technical discussion, Dr. Zehua Wang (Department of Obstetrics and Gynecology, Tongji Medical College, Wuhan Union Hospital Huazhong University of Science and Technology, Wuhan, China) for the kind supply of A2780/DDP cells, and Dr. Zhibiao Wang (Director of National Engineering Research Center of Ultrasound Medicine, Chongqing Medical University, Chongqing, China) for the generous support of the experimental facilities. This research was supported by Natural Science Foundation of China (81372799), Bureau of Health Foundation of Chongqing (2010-1-6, 2010-1-39), and Program for Innovation Team Building at Institutions of Higher Education in Chongqing (KJTD201303).

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ABBREVIATIONS USED MBs, microbubbles; US, ultrasound; UTMD, ultrasoundtargeted microbubble destruction; TMBs, LHRH receptor targeted lipid microbubbles; NMBs, nontargeted lipid microbubbles; LHRHa, LHRH analogue; TEM, transmission electron microscopy; PBS, phosphate buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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DOI: 10.1021/mp500835z Mol. Pharmaceutics XXXX, XXX, XXX−XXX