RPMI-1640 medium and fetal bovine serum (FBS) were supplied by Gibco (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-thiazyl)-2,5-diphenyltetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were obtained from Sigma (St. Louis, MO, USA). TRIzol was purchased from Sangon Biological Engineering Technology and Services (Shanghai, China). Polymerase chain reaction (PCR) mixture was from Takara Biotechnology Co., Ltd., (Dalian, China). Lipofectamine™ 2000 reagent was obtained from Invitrogen (Carlsbad, CA, USA).

Microbubbles used in the experiments were made by the Institute of Ultrasound Imaging, The Second Affiliated Hospital of Chongqing Medical University (Chongqing, China). Before use, the microbubbles were washed with phosphate-buffered solution (PBS) three times and sterilized by ⁶⁰Co γ-irradiation. The density of the microbubbles was ∼2.3×10⁹/ml, with a diameter of 2–5 μm. The ultrasonic gene transfection instrument (UGT1025) was able to launch ultrasonic frequencies of 300 kHz at an acoustic intensity of 0.25–2.5 W/cm² and was developed by the Institute of Ultrasound Imaging, The Second Affiliated Hospital of Chongqing Medical University (Chongqing, China).

Recombinant pEGFP-N1-p53 plasmids were purchased from Promega Beijing Biotech Co., Ltd., (Beijing, China) and grown in Escherichia coli (obtained from the Key Laboratory of Infectious Diseases of Chongqing Medical University, Chongqing, China). After abundant amplification, the plasmid was purified using a plasmid extraction kit (Axygen Biosciences, Union City, CA, USA) and was suspended in 2.5 mM Tris-HCl (pH 8.5) at a concentration of 1.0 μg/μl. For preparation of the mixture of plasmid and microbubbles, 4.0 μg of plasmid pEGFP-N1-p53 (4.0 μl) was lightly blended with 200 μl of the microbubble suspension and the mixture was gently incubated for a few minutes at 4°C to help adhesion. Additionally, 4.0 μg pEGFPN1-p53 plasmid (4.0 μl) was lightly blended with 12 μl liposome (Lipofectamine™ 2000, Invitrogen) and then mixed at room temperature for 20 min. This was the mixture of plasmid and liposome used for subsequent experimentation.

Human cervical carcinoma HeLa cell line (KG-042; Biotech Co., Ltd., Nanjing, China) was grown as a monolayer in a 50-ml culture flask and passaged every 2–3 days. Cells were maintained in RPMI-1640 supplemented with 10% FBS and 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. When cells reached subconfluence, they were counted with a hemocytometer (Burker Turk) and seeded in 6-well plates at a concentration of 4×10⁵ cells/well.

Cell transfections were performed under different ultrasonic conditions using 4 or 8 μg of pEGFP-N1-p53 plasmids according to the manufacturer’s instructions. Cells were incubated for 48 h after transfection and then the expression of enhanced green fluorescent protein (EGFP) was observed under a fluorescence microscope. Five horizons were selected randomly for each group. The intensity of EGFP reflected the transfection efficiency of the pEGFP-N1-p53 plasmids.

In the experiments, HeLa cells were divided into five groups as follow: i) control (no treatment); ii) plasmid only; iii) plasmid with ultrasound; iv) plasmid with microbubbles and ultrasound; and v) plasmid with liposome. The ultrasound treatment parameters for the HeLa cells were: continuous wave, 300 kHz, 0.5 W/cm², 30 sec and a 10% concentration of microbubbles.

Cell viability was evaluated by the MTT reduction assay. In brief, HeLa cells were seeded at a density of 5×103 cells/well in 96-well microtiter plates containing 150 μl of RPMI-1640 medium with 10% FBS. After growing to subconfluence, the cells were exposed to various concentrations of microbubbles (1, 5, 10 and 20%), different intensities of ultrasound (0.5, 0.75 and 1 W/cm²), different radiation times (10, 30 and 60 sec) and incubated for 24 h. Then incubation with MTT (5 mg/ml) was carried out in culture medium for 3 h at 37°C. After that, the medium was discarded and the formazan blue, which formed in the cells, was dissolved in 100 μl of DMSO. The absorbance was measured at 490 nm using a Sunrise Remote Microplate Reader (Grodlg, Austria), and then normalized according to the value of the control (untreated cells).

Total RNA was extracted from HeLa cells using TRIzol. The quantity of RNA isolates was determined spectrophotometrically using a DNA/RNA Gene-Quant Calculator (Amersham Biosciences, USA). Reverse transcription was performed in 20 μl of reaction mixture containing 2 μg of total RNA, 5.0 units of AMV reverse transcriptase, 50 pmol of oligo(dT) primer, 40 nmol of dNTP mixture, 40 units of RNase inhibitor, 4 μl of 5X RT buffer (Bioer, Hangzhou, China) at 42°C for 1 h and 95°C for 5 min. The following primers for RT-PCR were used: p53 (568 bp), sense 5′-AGCATCTTATCCGAGTGGAAGGAA-3′; antisense 5′-TTATGGCGGGAGGTAGACTGACC-3′. β-actin (386 bp): sense 5′-GATGGTGGGAATGGGTCAGA-3′; antisense 5′-GGAGAGCATAGCCCTCGTAGAT-3′. RT-PCR analysis was performed in 20 μl of reaction mixture containing 1 μl of cDNA reaction mixture, 10 nmol of dNTP mixture, 10 pmol of sense and antisense primers and 2 units of BioReady rTaq polymerase (Bioer, Hangzhou, China). For PCR product analysis, 6 µl of each reaction mixture was electrophoresed on 1.5% agarose gel containing 1% Gold-View^™. The band intensity was analyzed using the Geldoc 2000 system (Bio-Rad, USA) and presented as a percentage of β-actin expression.

HeLa cells growing in 25-ml culture flasks were harvested, washed and fixed with ice cold alcohol (75%) for >24 h. After further being washed twice, cells were incubated with PBS (pH 7.4) containing RNase (5 units) and PI (50 μg/ml) for 15 min at 37°C. Flow cytometry was performed using a FACS Vantage SE flow cytometer.

All data are expressed as the means ± SD. Statistical analyses were performed using the SPSS 13.0 package (SPSS Inc., Chicago, IL, USA). Comparisons between groups were performed by the Student’s t-test and one-way analysis of variance (ANOVA). Statistical significance was accepted at a p-value <0.05.

The viability of HeLa cells under different ultrasonic conditions, as assessed by MTT method, is shown in Table I and Fig. 1A. When ultrasound was conducted with 0.5, 0.7, 1.0 W/cm² and 10 sec, 0.5, 0.75 W/cm² and 30 sec, cell survival was approximately 80%. When ultrasound was 0.5 W/cm² and 60 sec, viability was approximately 70%. As the intensity and time increased simultaneously, cell viability was decreased significantly.

Based on the results of the MTT assay, we chose these ultrasound parameters (30 sec, 0.5 and 0.75 W/cm²; 60 sec, 0.5 W/cm²; 10 sec, 1 W/cm²) as the transfection conditions of the wtp53 gene, for which cell viability was >70%. Cells were incubated for 48 h after transfection and then the expression of EGFP was examined using fluorescence microscopy. As presented in Fig. 2A, when ultrasound was 0.5 W/cm² and 30 sec, expression of EGFP in HeLa cells was highter than that in the other three groups. The result demonstrated that using this acoustic intensity and exposure time, the transfection efficiency of the wtp53 gene was highest.

As shown in the result of the MTT assay (Table II and Fig. 1B), when the microbubble concentration was 20%, and ultrasound parameters were 0.5 W/cm², 30 sec or 0.75 W/ cm², 30 sec, cell survival was <70%. No significant difference was noted between the two groups (p>0.05). When micro-bubble concentration was 1 or 5% and ultrasound parameters were 0.5 W/cm², 30 sec or 0.75 W/cm², 30 sec, cell survival was >80%. That is to say, cell viability was not significantly affected. When the microbubble concentration was 10%, the difference between 0.5 W/cm², 30 sec and 0.75 W/cm², 30 sec was statistically significant (p<0.05). At the same time, we found that when the ultrasound parameters were 0.5 W/cm², 30 sec or 0.75 W/cm², 30 sec, the difference in cell viability was significant for the four concentrations of microbubbles.

Due to the slight effect on cell viability and high transfection efficiency, we chose 0.5 W/cm², 30 sec, continuous wave and a microbubble concentration of 10% as the optimal transfection condition for transfection of the wtp53 gene in subsequent experiments.

Fig. 2B indicates that the two groups, plasmid with microbubbles and ultrasound and plasmid with liposome, showed greater EGFP expression in HeLa cells than that in the other three groups. The transfection rate in the former was higher than that in the latter (p<0.05), while it was higher in the plasmid and liposome group than that in the plasmid only and plasmid and ultrasound groups (p<0.01).

The RT-PCR results (Fig. 3) showed that wtp53 expression was detected in the plasmid with microbubbles and ultrasound group and the plasmid with liposome group, while in the other three groups the expression was not apparent. The wtp53 mRNA level was also increased in the plasmid with microbubbles and ultrasound group compared to the plasmid with liposome group. This demonstrated that the carrier of ultrasound micro-bubbles increased the transfection rate of the wtp53 gene.

To test the role of wtp53 in the cell cycle progression of HeLa cells, the cell cycle distribution was assessed by flow cytometry. As indicated in Table III and Fig. 4, in the plasmid with microbubbles and ultrasound group, 83.33±3.43% of the total number of cells was in the G1 phase of the cell cycle. Compared to the other groups, the number of G1 phase cells was increased significant, while the number of cells in the G2 phase was decreased. This indicated that the wtp53 gene arrested the cell cycle of HeLa cells in the G1 phase.