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. Author manuscript; available in PMC: 2015 Feb 19.
Published in final edited form as: Ultrason Sonochem. 2012 Jul 5;20(1):171–179. doi: 10.1016/j.ultsonch.2012.06.015

Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

Shufang Chang a,*,#, Juan Guo a,#, Jiangchuan Sun a,#, Shenyin Zhu c, Yu Yan a, Yi Zhu a, Min Li a, Zhigang Wang b, Ronald X Xu d
PMCID: PMC4332827  NIHMSID: NIHMS648324  PMID: 22841613

Abstract

Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid micro-bubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin– biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm2) for 30 s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90 ± 6.19)%. The expression of wtp53 mRNA after transfection was (97.08 ± 12.18)%. The cell apoptosis rate after gene therapy was (39.67 ± 5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells.

Keywords: Ovarian cancer, Ultrasound, Microbubbles, Gene transfection, LHRHa peptide

1. Introduction

Ovarian cancer is one of the most fatal gynecologic malignancies primarily localized in the peritoneal cavity [1]. Many women with ovarian cancer present with the advanced disease at the time of diagnosis since no effective screening method is available. Standard therapeutic options, such as cytoreductive surgery followed by adjuvant chemotherapy, have poor long-term outcomes for the treatment of ovarian cancer. Therefore, new therapeutic strategies, such as gene therapy, were explored in recent years [2]. Gene therapy inhibits the expression of oncogenes or replaces the activated tumor suppressor genes with their wild-type copies. Among various gene therapy options for ovarian cancer, targeting p53 tumor suppressor gene may provide a clinically promising treatment because a mutation in p53 gene presents in nearly 60% of the ad vanced ovarian cancers [3]. Gene therapy can be delivered to cancer cells by either viral or non-viral methods. Viral vectors have been used in preclinical studies and phase I/II clinical trials for delivering wild-type p53 (wtp53) gene to ovarian cancer cells with high transfection efficiency [4–6]. However, they pose a potential risk of insertional mutagenesis and interference responses [7,8]. Non-viral gene delivery systems are relatively safe and easier to apply, but they suffer from low transfection efficiency and transient gene expression [9,10].

Ultrasound-targeted microbubble destruction (UTMD) is a promising technique for non-viral delivery of gene therapy. UTMD is able to stimulate the permeabilization of cell membranes and increase the regional uptake of plasmid DNA [11–13]. However, UTMD typically uses non-targeted microbubbles that are readily aggregated in liver or spleen, leading to a low concentration at the disease site. Therefore, developing tumor-targeting strategies may potentially improve the gene delivery efficiency.

We synthesized tumor-targeting microbubbles for UTMD medicated delivery of gene therapy to ovarian cancer cells. Luteinizing Hormone–Releasing Hormone (LHRH) was used as a targeting moiety since it is expressed in 70% of ovarian cancer cell lines but not expressed in most of the healthy human organs [14]. In this study, lipid microbubbles were conjugated with a LHRH analog (LHRHa) to target human ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFPN1-wtp53 plasmid. Ultrasound pulses applied to the microbubbles enabled UTMD mediated delivery of the wtp53 gene to the cancer cells. Microbubble binding and gene transfection efficiencies were characterized by bright field and laser scanning confocal fluorescence microscopic imaging. The expression level of wtp53 mRNA after gene therapy was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Cell apoptosis and cell cycle were analyzed by flow cytometry. Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene therapy efficiency in ovarian cancer cells. To the best of the authors’ knowledge, using targeted microbubbles for UTMD mediated delivery of wtp53 gene to ovarian cancer cells has not been reported elsewhere.

2. Materials and methods

2.1. 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). SKOV3 cells (LHRH receptor negative) were obtained from School of Life and Health Sciences, Chongqing Medical University (Chongqing, 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 streptomycin. Exponentially growing cells were used for all the experiments.

2.2. Preparation and characterization of non-targeted lipid microbubbles (NMBs)

1,2-Dipalmito-yl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-gly- cero-3-phosphoethanolamine (DSPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). DPPC(5 mg), DSPE(0.5 mg), DPPA(1 mg), glycerol(50 μl), and phosphate buffered saline (PBS) (450 μl)were mixed in a glass container. The mixture was placed in a 42 °C water bath for 30 min. The mixture 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 five minutes. The supernatant was collected and the NMB concentration was estimated by a bright field microscope. The size distribution of the NMBs was determined by a 3000SSA Zatasizer (Malvern Instruments Inc., Westborough, MA).

2.3. Preparation and characterization of targeted lipid microbubbles (TMBs)

Targeted lipid microbubbles (TMBs) were synthesized by an established protocol consisting of consecutive steps of biotinylating LHRHa peptide, avidinylatng lipid microbubbles, and conjugating avidinylated microbubbles with biotinylated LHRHa peptide. The experimental details for each step are described below.

2.4. Biotinylating LHRHa peptide

We designed a biotinylated LHRHa peptide that is superactive and degradation-resistant by modifying the sequence of a native LHRH peptide. A Gly amino acid was replaced with d-Leu at position 6 in order to obtain a D-Leu6-des-Gly10-Pro9-ehtylamine LHRH analog (LHRHa) [15]. The modified LHRHa peptide was synthesized by Scilight Biotechnology LLC. (Beijing, China) and biotinylated following a protocol established by the company.

The binding affinity of the biotinyated LHRHa peptide was analyzed by an immunocytochemical assay. A2780/DDP cells were inoculated in a Costar cell culture cluster (Sigma Aldrich, Shanghai, China) at 37 °C in a humidified 5% CO2 atmosphere for 24 h. After that, the cells were washed in phosphate buffered saline (PBS) and fixed in 4% formaldehyde for 30 min, washed in PBS again, and washed in 2% bovine serum albumin (BSA) for 30 min to block the non-specific binding. The cells were then incubated with 50 μg biotinylated LHRHa at room temperature for 30 min, washed in PBS, and incubated at 4 °C with a rabbit LHRH polyclonal antibody (CHEMICON International Inc., Shanghai, China) at a ratio of 1:500 for overnight. The incubated cells were then washed in PBS, incubated again at 37 °C with Rhomdamine (TRITC) - labeled Affinipure Goat anti-Rabbit IgG (ZSGB-BIO, Beijing, China) at a ratio of 1:50 for 30 min, and washed in PBS. The binding affinity of the biotinyated LHRHa peptide were imaged by a TEU-2000 inverted fluorescence microscope (Nikon, Shanghai, China). SKOV3 cells were used as a negative control because they do not express the LHRH receptors [7–8]. SKOV3 cells were incubated with the biotinylated LHRHa peptide following the same procedure as that of the A2780/DDP cells. For the blank control, PBS was used in place of LHRHa in order to exclude non-specific cellular interactions.

2.5. Avidinylating lipid microbubbles

Before avidinylation, lipid microbubbles were first biotinylated following the same procedure as that of NMBs except that DSPE was replaced with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl (polyethyleneglycol) (2000)] (i.e., DSPEPEG(2000)–biotin). The biotinylated microbubbles were washed with PBS for three times and centrifuged at 800 rpm for five minutes. The supernatant was collected and incubated with FITC-labeled streptavidin (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) at room temperature with gentle shaking for 30 min. The incubation mixture was washed with PBS for three times to remove free streptavidin and obtain the avidinylated microbubbles labeled with FITC. The microbubbles were diluted with PBS and imaged by a laser scanning confocal fluorescence microscope (Leica Microsystems Ltd., Beijing, China). The avidinylated microbubbles without fluorescence labeling were also synthesized by incubating the biotinylated microbubbles with plain streptavidin.

2.6. Conjugating avidinylated microbubbles with biotinylated LHRHa peptide

One milliliter aqueous suspension of the avidinylated micro-bubbles at a concentration of 1 × 108/ml was mixed with an excessive amount of the biotinylated LHRHa peptide (50 μg) thoroughly and incubated at room temperature with gentle shaking for 30 min. The mixture was kept still for 10 min, the bottom part was discarded, and the residue was washed with PBS for three times in order to remove the free peptide and obtain the targeted microbubbles (TMBs). The size distribution of the TMBs was characterized by a 3000SSA Zetasizer.

2.7. Targeted binding of TMBs

The binding affinity of the TMBs was tested on a LHRH polyclonal antibody assay (CHEMICON International, Inc., Shanghai, China) and compared with that of the NMBs. The LHRH polyclonal antibody was mixed with the TMBs and the NMBs respectively, incubated at 4 °C for overnight, and washed with PBS for three times to remove the free antibody. Subsequently, both the TMBs and the NMBs were incubated with TRITC-labeled Affinipure goat Anti-Rabbit IgG at room temperature for 30 min, washed with PBS for three times, and imaged by a laser scanning confocal microscope.

The binding affinity of the TMBs was also tested on the LHRH receptor positive ovarian cancer cells. A2780/DDP cells (5 × 104/ml) were incubated with the TMBs (1 × 105/ml) and the NMBs (1 × 105/ml) respectively in a Costar cell culture cluster at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. The number of the TMBs and the NMBs was counted by a hemacytometer. After an aliquot of the microbubble suspension was diluted to 1:100, 10 μl of the suspension was loaded to the hemacytometer. The number of the microbubbles was counted in the 4 outer squares and the microbubble concentration was calculated by the following formula: microbubble concentration per milliliter = total microbubble count in 4 squares × 2500 × dilution factor. Considering the buoyancy of the microbubbles, the cell culture clusters were placed upside down to maximize the cell- microbubble interaction. After a 30 min of static exposure, the clusters were washed in PBS for three times to remove the unbound microbubbles. The microbubbles bound to the cells were examined by a bright field microscope.

2.8. UTMD mediated gene delivery in vitro

To demonstrate UTMD mediated gene transfection, the wtp53 plasmid, the A2780/DDP cells, and the microbubbles were prepared in advance. The wtp53 plasmid integrated with an enhanced green fluorescent protein vector (pEGFP-N1) by GeneChem Co., Ltd. (Shanghai, China). A2780/DDP cells were incubated at 37 °C in a humidified 5% CO2 atmosphere for 24 h, harvested with trypsin–EDTA (Invitrogen, Shanghai, China), washed by PBS twice, and centrifuged at 1000 rpm for five minutes. The collected cells were re-suspended in medium at a concentration of 1 × 105/ml. The TMBs were suspended in PBS at a concentration of 1 × 108/ml. 10 μl TMB suspension was mixed with 100 μl pEGFP-N1-p53 plasmid (1 μg/μl) at 4 °C for 30 min and washed in PBS for three times to obtain the TMB/plasmid complex. As a negative control, the pEGFP-N1-p53 plasmid was mixed with the NMBs following the same procedure.

UTMD mediated delivery of wtp53 gene was demonstrated in A2780/DDP cells. The cells were equally divided into the following six treatment groups: (a) applying wtp53 only (i.e., “wtp53 only”); (b) applying the mixture of wtp53 and the NMBs without ultra-sound destruction (i.e., “wtp53-NMBs”); (c) applying the mixture of wtp53 and the TMBs without ultrasound destruction (i.e., “wtp53-TMBs”); (d) applying wtp53 followed by ultrasound destruction (i.e., “wtp53 + US”); (e) applying the mixture of wtp53 and the NMBs followed by ultrasound destruction (i.e., “wtp53-NMBs + US”); (f) applying the mixture of wtp53 and the TMBs followed by ultrasound destruction (i.e., “wtp53-TMBs + US”). An equivalent amount of the pEGFP-N1-p53 plasmid was applied to each treatment group. For treatment groups (d)–(f), a 1 MHz piezoelectric ceramic transducer (model CGZZ, Ultrasono-graphic 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. After the exposure of the ultrasound pulses, the cells were seeded in a 24 well plate, incubated for 24 h, and washed in PBS for three times. The report gene transfection was observed by a TEU-2000 inverted fluorescence microscope (Nikon, Shanghai, China). The report gene transfection efficacy was determined by a FACS Vantage SE flow cytometer (BD Biosciences, Shanghai, USA).

2.9. Expression of wtp53 mRNA after gene transfection

The wtp53 mRNA expressions of the A2780/DDP cells were characterized by reverse transcription-polymerase chain reaction (RT-PCR). The cells were equally divided into the following treatment groups: (1) “PBS”, (2) “wtp53”, (3) “wtp53 + US”, (4) “wtp53-NMBs + US”, (5) “wtp53-NMBs”, (6) “wtp53-TMBs”, and (7) “wtp53-TMBs + US”. For each treatment group, the total RNA was extracted with Trizol reagent (Tiangen Biotech,Co.,Ltd., Beijing, China) and reverse-transcribed into cDNA using a reverse transcription kit (Promega, Madison, WI). The resultant cDNA was amplified using the following primer sequences with a predicted product size of 184 bp: 5′-TGC AGC TGT GGG TTG ATT CC-3′ (forward, wtp53) and 5′-CCA CAC GCA AAT TTC CTT CC-3′ (reverse, wtp53). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Its mRNA was amplified using the following primer sequences with a predicted product size of 310 bp: 5′-TGT TCG TCA TGG GTG TGA ACC-3′ (forward) and 50-AAG GCC ATG CCA GTG AGC TTC-3′ (reverse). The following cycling program was used for RT-PCR: 94 °C for three minutes, 94 °C for 30 s, 56 °C for one minute, 72 °C for 40 s (35 cycles), and 72 °C for 10 min. The obtained RT-PCR products were electrophoresed through 1% agarose gel with ethidium bromide. The intensities of wtp53 and GAPDH were imaged by a digital PCR system (BIORAD, Shanghai, China) and evaluated using a Quantity One software package (BIO-RAD, Shanghai, China). The relative wtp53 expression levels were calculated based on the ratio of wtp53 and GAPDH intensities.

2.10. Cell apoptosis after gene transfection

A2780/DDP cells were harvested with trypsin, washed twice with PBS, centrifuged, and re-suspended at a concentration of 1 × 106/ml in culture medium. The cells were equally divided into the following six treatment groups for apoptosis analysis: (1) applying PBS only (i.e., “negative control”), (2) applying ultrasound only (i.e., “US”), (3) applying NMBs followed by ultrasound destruction (i.e., “NMBs + US”), (4) applying TMBs followed by ultrasound destruction (i.e., “TMBs + US”), (5) applying wtp53 followed by ultrasound destruction (i.e., “wtp53 + US”), (6) applying the mixture of wtp53 and the NMBs followed by ultrasound destruction (i.e., “wtp53-NMBs + US”), and (7) applying the mixture of wtp53 and the TMBs followed by ultrasound destruction (i.e., “wtp53-TMBs + US”). After the treatments, the cells were harvested with trypsin (0.05%)/EDTA (0.02%), washed twice with PBS, and fixed with 70% alcohol at 4 °C for overnight. After that, the cells were incubated at 37 °C in ribonuclease solution (0.25 mg/ml) for 30 min, and stained by propidium iodide (50 μL/ml) on ice and in dark for 30 min. The apoptosis rate of each treatment group was analyzed by flow cytometry.

2.11. Cell cycle analysis

A2780/DDP cells were harvested with trypsin and washed twice with PBS, centrifuged, and re-suspended at a concentration of 1 × 106/ml in culture medium. The cells were equally divided into the following two groups: (1) applying PBS only (i.e., “control”), and (2) applying the mixture of wtp53 and the TMBs followed by ultrasound destruction (i.e., “wtp53-TMBs + US”). 24 h after treatments, the cells were harvested with trypsin (0.05%) /EDTA (0.02%), washed twice with PBS, and fixed with 70% alcohol at 4 °C for overnight, After that, the cells were incubated in 0.25 mg/ml ribonuclease solution at 37 °C for 30 min, followed by staining with 50 μl/ml propidium iodide on ice and in dark for 30 min. For both the control and the treatment groups, the cell cycles were analyzed by flow cytometry.

2.12. Statistical analysis

The experimental results were analyzed using a SPSS 13.0 software package (SPSS, Chicago, IL). The measurements were expressed as mean ± standard deviation. 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 significant.

3. Results

3.1. Synthesis and affinity characterization of TMBs

TMBs were synthesized by conjugating gas-filled lipid micro-bubbles with a LHRHa peptide through a biotin–streptavidin–biotin linkage. The synthesized TMBs have a size distribution of 2527.6 ± 496.4 nm, slightly larger than that of the NMBs (1634.0 ± 621.6 nm). The binding affinities of the TMBs were evaluated by bright field and fluorescence microscopic imaging, as illustrated in Figs. 1–4.

Fig. 1.

Fig. 1

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Bright field and confocal fluorescence microscopic images show targeted binding of biotinylated LHRHa peptide with A2780/DDP cells, while blank and negative controls do not show significant binding. Figures on the left are bright field microscopic images. Figures on the right are the corresponding fluorescence microscopic images. Scale bar = 25 μm. (a1, a2): A2780/DDP cells after incubation with biotinylated LHRHa and TRITC labeled IgG. (b1, b2): A2780/DDP cells after incubation with PBS (blank control); (c1, c2): SKOV3 cells after incubation with biotinylated LHRHa and TRITC labeled IgG (negative control).

Fig. 4.

Fig. 4

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In comparison with non-targeted microbubbles (NMBs), targeted microbubbles (TMBs) bind with A2780/DDP cells well. (a) Microscopic image of NMBs after being incubated with A2780/DDP cells and washed with PBS. (b) Microscopic image of TMBs after being incubated with A2780/DDP cells and washed with PBS.

Fig. 1 verifies that the biotinylated LHRHa peptide binds with the LHRH receptor positive cells. According to Fig. 1a, significant fluorescence emission can be observed on the cell membrane after incubating the A27809/DDP cells with the biotinylated LHRHa peptide and the TRITC labeled IgG. In comparison, neither the A2780/ DDP cells incubated with PBS (Fig. 1b, blank control) nor the SKOV3 cells incubated with the biotinylated LHRHa peptide and the TRITC labeled IgG (Fig. 1c, negative control) have significant fluorescence emission. These results indicate that the binding between the synthesized LHRHa peptide and the A2780/DDP cells is specific.

Fig. 2 verifies the successful biotinylation of lipid microbubbles. Bright field and confocal fluorescence microscopic images were acquired after the biotinylated microbubbles were incubated with FITC-labeled streptavidin. The confocal fluorescence microscopic image in Fig. 2a clearly shows the biotin distribution on the surface of the lipid core–shell structure. The corresponding bright field microscopic image in Fig. 2b indicates that all the microbubbles have been successfully biotinylated.

Fig. 2.

Fig. 2

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Avidinylated microbubbles bind with FITC-labeled streptavidin very well. (a) Confocal fluorescence microscopic image of the streptavidin bound microbubbles. (b) The corresponding bright field microscopic image.

Fig. 3 uses an immunofluorescence technique to verify that the LHRHa conjugated microbubbles (i.e., TMBs) has high binding affinity. Bright field and confocal fluorescence microscopic images were acquired after the TMBs were mixed with LHRH polyclonal antibody and incubated with TRITC-labeled IgG. According to Fig. 3, the TMBs bind with TRITC-labeled IgG with high specificity and affinity. In comparison, the NMBs do not bind with TRITC-labeled IgG (fluorescence image not shown).

Fig. 3.

Fig. 3

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LHRHa conjugated TMBs bind well with TRITC-labeled IgG. (a) Confocal fluorescence microscopic image of the TMBs bound with TRITC-labeled IgG. (b) Corresponding bright field image of the TMBs confirms the microbubble morphology.

Fig. 4 compares the targeted binding capability of the TMBs and the NMBs with A2780/DDP cells. According to Fig. 4a, the NMBs do not bind with the cells, whereas the TMBs bind with the cells very well (Fig. 4b).

3.2. Gene transfection, mRNA expression, and cell apoptosis

UTMD mediated delivery of wtp53 gene was tested in the A2780/DDP cells. The wtp53 plasmid integrated with pEGFP-N1 was used for fluorescence imaging of gene transfection efficiency. Fig. 5a–f show both bright field and fluorescence microscopic images acquired for six treatment groups. Treatment groups (a)– (c) exhibit negligible fluorescence emission, indicating that direct application of wtp53 or applying wtp53-loaded microbubbles without ultrasound mediation cannot deliver wtp53 gene effectively. Treatment groups (d)–(e) exhibit moderate fluorescence emission, indicating that ultrasound mediation may enhance gene delivery even without the use of a targeting vector. Treatment group (f) exhibits the highest fluorescence emission, indicating that ultrasound medication of the mixture of wtp53 and the TMBs yields the highest gene transfection efficiency. Quantitative analysis of the fluorescence emission intensities for these treatment groups also yielded consistent conclusions. According to Fig. 6, the gene transfection efficiencies for treatment groups (a)–(c) are less than 1%; whereas those for treatment groups (d)–(f) are (7.24 ± 5.15)%, (25.33 ± 4.44)%, and (43.90 ± 6.19)%, respectively. Compared with other treatment groups, group (f) has the highest transfection efficiency and the difference is statistically significant (p < 0.05). This experimental result indicates that UTMD mediated delivery of wtp53 gene loaded TMBs significantly enhances the efficiency for plasmid DNA transfection.

Fig. 5.

Fig. 5

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Bright field and fluorescent microscopic images of the EGFP expression in ovarian cancer A2780/DDP cells after the following treatments: (a1, a2) wtp53 only, (b1, b2) wtp53-NMBs, (c1, c2) wtp53-TMBs, (d1, d2) wtp53 + US, (e1, e2) wtp53- NMBs + US, (f1, f2) wtp53-TMBs + US. Notation “1” represents bright field images, “2” fluorescence images. Scale bar is 50 μm. EGFP expression is observed in the (d), (e) and (f) groups. The strongest fluorescence emission is observed after applying the mixture of wtp53 and TMBs with US mediation.

Fig. 6.

Fig. 6

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Transfection efficiency of the wtp53 gene for different experiment groups detected by flow cytometry 24 h after transfection .Data are represented as mean ± SD(n = 3). The transfection efficiencies of the (d), (e) and (f) groups were significantly higher than those of the other groups. Among these three groups, group (f) has the highest transection efficiency (p < 0.05).

Fig. 7 shows the levels of wtp53 mRNA expression for seven treatment groups. According to the figure, treatment groups (1), (2), (5), and (6) do not express wtp53 mRNA, indicating that wtp53 gene cannot be effectively transfected without ultrasound mediation. Treatment groups (3), (4), and (7) express wtp53 mRNA. The indexes of the wtp53mRNA/GAPDH ratio for these groups are (45.21 ± 7.32)%, (5.06 ± 0.76)%, and (97.08 ± 12.18)%, respectively. Compared with other treatment groups, group (7) has the highest level of wtp53 mRNA expression (p < 0.05), indicating that the ultrasound mediation of the TMBs may significantly increase the level of wtp53 mRNA expression in the A2780/DDP cells.

Fig. 7.

Fig. 7

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RT-PCR analysis of the expression of wtp53 mRNA in A2780/DDP cells after different treatments. wtp53 mRNA expression were analyzed by RT-PCR 24 h after transfection. Cells transfected with EGFP-N1-wtp53 using ultrasound-targeted TMBs destruction showed more prominent bands than other treatment groups. Band #1 indicates negative control group(PBS). Band #2 indicates wtp53 group. Band #3 indicates wtp53 + US group. Band #4 indicates wtp53-NMBs + US group. Band #5 indicates wtp53-NMBs group. Band #6 indicates wtp53-TMBs group. Band #7 indicates wtp53-TMBs + US group. GAPDH was used as an internal reference.

Cell apoptosis after UTMD mediated delivery of wtp53 gene was also evaluated by flow cytometry, as shown in Fig. 8. According to the figure, the apoptosis efficiencies for treatment groups (1)–(7) are (5.87 ± 0.88)%, (8.74 ± 1.31)%, (11.04 ± 1.66)%, (13.47 ± 2.02)%, (14.51 ± 2.71)%, (24.54 ± 3.68)%, and (39.67 ± 5.95)%, respectively. In comparison with other treatment groups, group (7) results in significantly higher apoptosis efficiency (p < 0.05), indicating that UTMD mediated delivery of the mixture of wtp53 and the TMBs significantly increases the cell apoptosis efficiency.

Fig. 8.

Fig. 8

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Apoptosis efficiency in A2780/DDP cells with different treatments .The percentage of apoptosis cells was determined by flow cytometry 24 h after transfection .Data are represented as mean ± SD(n = 3). The apoptosis efficiencies of the (6) and (7) groups are significantly higher than those of the other groups (p < 0.05). The apoptosis efficiency of group (7) is higher than group (6) (p < 0.05).

With flow cytometry, we are also able to analyze the cell cycle after UTMD mediated gene transfection in A2780/DDP cells. Fig. 9 compares the cell cycles for the control group (“PBS”) and the treatment group (“wtp53-TMBs + US”). According to the figure, the cell proportions of the treatment group in G0/G1, S, and G2/M phases are (62.79 ± 4.65)%, (23.98 ± 2.41)%, and (13.22 ± 2.43)%, respectively. In comparison, those of the control group are (48.97 ± 2.70)%, (34.17 ± 3.67)%, and (16.85 ± 1.31)%, respectively. In comparison with the control group, the treatment group results in a significantly increased cell proportion in the G0/G1 phase (p < 0.05) and a significantly decreased cell proportion in the S phase (p < 0.05), indicating that the UTMD mediated delivery of wtp53-loaded TMBs induces a significant G1 phase arrest and a concomitant reduction in the cell fraction in the S phase.

Fig. 9.

Fig. 9

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Ultrasound mediated wtp53 gene transfection with TMB destruction induces the cell cycle arrest in A2780/DDP cells. Cell cycles were detected by flow cytometry 24 h after transfection for both the control group (PBS) and the treatment group (wtp53-TMBs + US). G0/G1 phase is significantly increased in the TMBs- wtp53 + US group than that in the control group (p < 0.05).

4. Discussion and conclusions

For several decades, gas-filled microbubbles have been used as a hypoechoic contrast agent in clinical ultrasonography [16]. Recent advances in microfabrication and molecular science have enabled novel applications for this traditional technology in the field of multimodal imaging, molecular targeting, and drug delivery. In the arena of multimodal imaging, multiple contrast agents have been encapsulated in lipid, protein, or polymer microbubbles to facilitate simultaneous contrast enhancement in multiple imaging modalities [17,18]. In the arena of disease targeting, the surfaces of microbubbles and nanobubbles have been conjugated with disease-targeting moieties, such as antibodies and peptides, for molecular imaging [19]. Targeted microbubbles with the size around several microns may circulate in blood stream and bind with the over-expressed endothelial biomarkers [20,21]. Targeted nanobubbles or nanoparticles with the size of hundred nanometers or less may penetrate the tumor endothelial barrier and bind with the over-expressed tissue biomarkers [22,23]. In the arena of drug delivery, microbubbles have been used as a biodegradable carrier for targeted delivery, controlled activation, and sustained release of various therapeutic agents [24,25]. Multiple drugs have been encapsulated within the same microbubbles for combinatory treatment and therapeutic synergy [26,27].

More recently, the emergence of gene therapy has further stimulated the microbubble research for the enhancement of gene delivery efficiency [28–31]. The ultrasound-targeted microbubble destruction (UTMD) technique has been developed to facilitate the increased regional uptake of plasmid DNA [11–13]. We also synthesized a tumor-targeting microbubble agent for ultrasound mediated delivery of gene therapy in ovarian cancer cells. Our research strategy is based on the following underlying rationale:

  • (1)

    We targeted LHRH receptors because they are expressed in 70% of the ovarian cancer cell lines but not expressed in most of the healthy human organs [14]. Modified LHRH peptide has been used as a targeting moiety for the delivery of anti-cancer drugs [22,32]. However, to best of our knowledge, no study has been reported for the use of modified LHRH peptide as a microbubbles targeting moiety.

  • (2)

    We demonstrated the targeted binding of the targeted microbubbles in A2780/DDP cells because these are ovarian cancer cells expressing LHRH receptors. SKOV3 cells were used as a negative control because they do not express LHRH receptors. For the purpose of concept approval, our targeted microbubbles were prepared following a three-step procedure: (1) avidinylating microbubbles, (2) biotinylating LHRHa peptide, and (3) conjugating microbubbles with peptide via the avidin–biotin linkage.

  • (3)

    We chose to use LHRHa-targeted microbubbles for intraperitoneal delivery of gene therapy. On the one hand, our targeted microbubbles have an averaged diameter of (2527.6 ± 496.4) nm and cannot effectively penetrate the endothelial fenestration in tumor vasculature. Therefore, regional delivery of the microbubbles is necessary. On the other hand, intraperitoneal gene therapy may present an effective treatment for ovarian cancer because 90% of ovarian cancers are confined to the peritoneum, even at relapse [33]. This closed compartment permits uncomplicated intraperitoneal delivery of concentrated plasmid and targeted-microbubbles for effective transfection without the need to penetrate the leaky tumor vasculature.

In this study, we fabricated the targeted microbubbles (TMBs) by conjugating a modified LHRHa peptide with lipid microbubbles via avidin–biotin interactions. The binding affinity tests showed that the TMBs bound well with the LHRH receptor positive A2780/DDP cells, whereas the non-targeted microbubbles (NMBs) did not bind with the cells. Therefore, the interaction between the TMBs and the A2780/DDP cells is specific. To demonstrate the UTMD mediated gene delivery technique, we incubated the mixture of the TMBs and the wpt53 plasmid with the A2780/DDP cells. After ultrasound pulses were applied to the cell culture, the gene transfection efficiency, the wtp53 mRNA expression, and the cell apoptosis were analyzed and compared with the other treatment groups. Our experiment showed that TMB assisted gene delivery with ultrasound mediation was able to achieve a gene transfection efficiency of around 43.9%, which was significantly higher than that of the other treatment groups. This result is consistent with previous reports [31,34–36]. Interestingly, TMB assisted gene delivery with ultrasound mediation also yielded a significantly higher reporter plasmid expression in comparison with gene delivery with NMBs or without microbubbles. The mechanism behind this phenomenon has not been fully studied yet. We believe that the increased mRNA expression for TMB assisted gene delivery is associated with the interaction of ultrasound pulses with the increased number of the TMBs bound with the cancer cells, as evidenced by the bright field microscopic images in Fig. 4. First of all, the application of the ultrasound pulses may introduce the sonoporation effect for the increased gene delivery efficiency [37]. Second, applying ultrasound pulses to microbubbles may cause energy burst and cavitation for enhanced drug delivery efficiency [38,39]. Finally, microbubbles with exposure to ultrasound pulses elicit a Ca2+ influx, which leads to activation of BKCa channels and a subsequent, local hyperpolarization of the cell membrane [40]. This local hyperpolarization of the cell membrane may facilitate uptake of macromolecules through endocytosis and macropinocytosis. Thus, the microbubbles may help to increase transfection efficiency over the use of ultrasound alone, a phenomenon that we have observed in our experiments.

Several studies showed that the transfection of wtp53 might be mediated by stimulating the cell apoptosis [41–43]. In the present study, flow cytometry revealed that the apoptosis efficiency was significantly higher in cells treated by the mixture of TMBs and pEGFP-N1-wtp53 followed by ultrasound mediation, as compared with the other treatment groups. The apoptosis efficiency was correlated with the wtp53 transfection efficiency, indicating that apoptosis may be one way of explaining the mechanisms of wtp53 on A2780/DDP cells. Cell cycle analysis also revealed that bursting the LHRHa-targeted microbubbles with ultrasound to deliver wtp53 plasmid was able to induce G0/G1 phase arrest of A2780/DDP cells at 24 h of treatment, accompanied by a reduction in the cell fraction in S phase.

In summary, we synthesized the targeted microbubbles for ultrasound mediated delivery of wtp53 gene to ovarian cancer cells. Our study revealed that, on the basis of the p53-mediated invasion, wtp53 can accomplish a selective apoptosis effect on A2780/DDP cells in vitro. The wtp53 gene system mediated by ultrasound in combination with LHRHa-targeted microbubbles may present a novel and attractive approach for ovarian cancer gene therapy.

Acknowledgements

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 (30801228), Natural Science Foundation of Chongqing (CSTC, 2008BB5405), Bureau of Health Foundation of Chongqing (07-2-098), National High Technology Research and Development Program of China (2006AA02Z4FO) and National Institute of Health in United States (CA159077).

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