Transfection Efficiency of TDL Compound in HUVEC Enhanced by Ultrasound-Targeted Microbubble Destruction

Ultrasound in Med. & Biol., Vol. 34, No. 11, pp. 1857–1867, 2008 Copyright © 2008 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/08/$–see front matter

doi:10.1016/j.ultrasmedbio.2008.03.019

● Original Contribution TRANSFECTION EFFICIENCY OF TDL COMPOUND IN HUVEC ENHANCED BY ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION JIAN-LI REN, ZHI-GANG WANG, YONG ZHANG, YUAN-YI ZHENG, XING-SHENG LI, QUN-XIA ZHANG, ZHAO-XIA WANG, and CHUAN-SHAN XU Institute of Ultrasound Imaging, The Second Affiliated Hospital of Chongqing Medical University, Chongqing City, China (Received 30 October 2007; revised 26 January 2008; in final form 6 March 2008)

Abstract—The aim of the present study was to explore the gene transfection efficiency of Tat peptide/plasmid DNA/ liposome (TDL) compound combined with ultrasound-targeted microbubble destruction (UTMD) in human umbilical vein endothelial cell (HUVEC). Tat peptide, plasmid DNA (pIRES2-EGFP-HGF) and Lipofectamine™ 2000 were used to prepare the TDL compound. Microbubbles were prepared using mechanic vibration. The expression of the report gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The viability of HUVEC was measured by MTT assay. mRNA and protein of HGF was analyzed by reverse transcription–polymerase chain reaction and Western Blot. The intensity of green fluorescence and the gene transfection efficiency of TDL compound ⴙ microbubbles ⴙ ultrasound group were higher than those of other groups, and no significantly different viability was found between TDL compound ⴙ microbubbles ⴙ ultrasound group and the other groups. The HGF mRNA and HGF protein of TDL compound ⴙ microbubbles ⴙ ultrasound group were higher than those of other groups. Our finding demonstrated that UTMD could enhance the transfection efficiency of TDL compound without obvious effects on the cell viability of HUVEC, suggesting that the combination of UTMD and TDL compound might be a useful tool for the gene therapy of ischemic heart disease. (E-mail: [email protected]) © 2008 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound-targeted microbubble destruction, Cell-permeable peptides, TDL compound, Gene transfection, Ischemic heart disease.

Shim et al. 2006; Yamaguchi et al. 2005). Hepatocyte growth factor, a recently characterized growth factor with a disulfide-linked heterodimer structure, is originally identified in the plasma of rats after partial hepatectomy and its receptor, c-Met, is a transmembrane tyrosine kinase protooncogene. Hepatocyte growth factor participates in mitogenesis (Rubin et al. 1991), motogenesis (Stoker et al. 1987), morphogenesis (Montesano et al. 1991) and angiogenesis (Bussolino et al. 1992). Recent studies have shown that HGF is a cardioprotective factor through angiogenesis, anti-apoptosis, antioxidative stress and antifibrosis (Ahmet et al. 2002; Kitta et al. 2001; Taniyama et al. 2002), suggesting HGF could be a promising therapeutic gene for treating ischemic heart disease (Jayasankar et al. 2005; Yamaguchi et al. 2005). Gene therapy requires an efficient and safe carrier for delivering therapeutic genes to targeted cells. There are two kinds of gene delivery vector, viz., viral vector and nonviral vector (Miyazaki et al. 2006; Saras-

INTRODUCTION Ischemic heart disease is a major health problem worldwide. Although great efforts have been made in its treatment, no ideal treatment is available. Exploring novel therapeutic strategies can improve the clinical outcomes. Gene therapy shows considerable promise as a new modality for treating ischemic heart disease. Many genes have been applied for gene therapy in ischemic heart disease, including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), hypoxia-inducible factor-1alpha (HIF-1␣) and angiopoietin-1 (Ang-1) (Bougioukas et al. 2007; Hao et al. 2007; Iwakura et al. 2003; Kido et al. 2005; Losordo and Dimmeler 2004;

Address correspondence to: Dr. Chuan-Shan Xu, Institute of Ultrasound Imaging, Second Affiliated Hospital, Chongqing Medical University, No.76, Lin Jiang Road, YuZhong District, Chongqing City, 400010, China. E-mail: [email protected] 1857

1858

Ultrasound in Medicine and Biology

Volume 34, Number 11, 2008

Fig. 1. Fluorescent microscopic images of pIRES2-EGFP-HGF expression in HUVEC (⫻200). Cells were transfected with all treated factors. Cells were cultured for 24 h and the reporter gene expression was observed using fluorescent microscopy. C indicates blanked control group; D indicates plasmid DNA; T indicates TDL compound; M indicates microbubbles; U indicates ultrasound. (a-c) C group; (d-f) T group; (g-i) T⫹M group; (j-l) T⫹U group; (m-o) T⫹M⫹U group; (p-r) D⫹M⫹U group. Bright field images (a,d,g,j,m,p); fluorescence images (b,e,h,k,n,q); overlay image (c,f,i,l,o,r).

wathi et al. 2007; Xia et al. 2004). Viral vector is the current favorites for intracellular gene delivery, but it has disadvantages such as immunoreactions, toxicity, nonspecificity, high cost, size limits of exogenous DNA and the possibility of random integration of the vector DNA into the host genome (Haider et al. 2008). Nonviral vector has more advantages such as little immunoreactions, lower toxicity, ease and safety in preparation, high gene encapsulation capability and simplicity of use (El-Aneed et al. 2004; Zhang et al. 2007). However, the lower efficiency of nonviral vector needs to be overcome. Cationic liposome has a better membrane affinity and DNA condensation and can enhance the in vitro transfection efficiency. But the toxicity and in vivo low transfection efficiency remain the limitations for cationic

liposome applied to gene therapy (Alton et al. 1999; Madry et al. 2001). In recent years, cell-permeable peptides (CPPs) have been used widely as cellular delivery vectors for their remarkable functions carrying macromolecular substance directly and actively through cellular membrane into plasma or nucleus without cytotoxicity (Astriab-Fisher et al. 2002; Ignatovich et al. 2003; Nakanishi et al. 2003; Rudolph et al. 2003; Tung et al. 2002). Cell-permeable peptides have many advantageous properties such as high affinity of cellular membrane, fast speed of permeating membrane, fast degradation and no destructiveness to the cellular membrane. Transactivating transcriptional activator protein (Tat peptide) from human immunodeficiency virus type 1(HIV-1 Tat), one of most popular CPPs, has membrane translocation

Transfection efficiency of TDL compound enhanced by UTMD ● J.-L. REN et al.

1859

Fig. 1. (Cont’d)

property and noncytotoxicity (Vives et al. 1997). Recent studies showed that Tat peptide/plasmid DNA/liposome (TDL compound) could enhance transfection efficiency of genes and indicated that the combination of liposome and Tat peptide might become a gene carrier (Jianli et al. 2007; Torchilin et al. 2003). However, our previous data demonstrated that the transfection efficiency of TDL compound still needs further enhancement. Ultrasound (US) irradiation can enhance gene transfection to mammalian cells in vitro and in vivo without cell damage. Microbubbles can lower the threshold of US for cavitation, which transiently perforates the cell membrane to allow delivery of gene materials into cells (Haitao et al. 2003; Korpanty et al. 2005; Vancraeynest et al. 2006; Zhigang et al. 2004). Ultrasound-targeted microbubble destruction (UTMD) could significantly increase the gene transfection efficiency (Bekeredjian et al. 2007; Nie et al. 2006). The objective of this study was to explore the possibility that UTMD enhanced the effi-

ciency of TDL compound–mediated Plasmid pIRES2EGFP-HGF transfection and its effects on the viability of human umbilical vein endothelial cell (HUVEC). MATERIALS AND METHODS Tat peptide Tat peptide (H-Tyr-Gly-Arg-Lys-Lys-Arg-ArgGln-Arg-Arg-Arg-OH) was purchased from AnaSpec, Inc. (San Jose, CA, USA). 1 mM Tat peptide in sterile distilled water was prepared and stored at ⫺20°C before use. Plasmid DNA Plasmid pIRES2-EGFP-HGF was constructed in the Institute of Ultrasound Imaging at Chongqing Medical University (Xingsheng et al. 2007). The plasmid was transformed into MAX Efficiency DH5␣ Competent Cells (Invitrogen, Carlsbad, CA, USA) using the heat–

1860

Ultrasound in Medicine and Biology

Volume 34, Number 11, 2008

Fig. 1. (Cont’d)

shock method. The purification of the plasmid was done using a QIAGEN Plasmid Mini Kit (Qiagen, Valencia, CA, USA). DNA was analyzed using OD260:280 ratios, and 1% agarose gel electrophoresis in TRIS-borate buffer and stained with ethidium bromide. In each case, the OD260:280 ratio was between 1.8 and 2, gel electrophoresis showed supercoiled plasmid DNA with no genomic DNA contamination. 0.2 mg/mL pIRES2EGFP-HGF in sterile water was prepared and stored at ⫺20°C. Liposome Lipofectamine™ 2000 (Invitrogen) was purchased and used according to the instruction manual. Preparation of TDL transfection compound Preparation of TDL compound was carried out in 0.15 mol/L NaCl at room temperature. 4 ␮l (1 mM) Tat peptide and 1 ␮g plasmid DNA were diluted in 5 ␮l 0.15

mol/L NaCl, respectively, with 8:1 ⫹/⫺ charge ratio, then diluted plasmid DNA solution was pipetted to Tat peptide solution and mixed vigorously by pipetting up and down. After the compound was incubated for 30 min at room temperature, 2 ␮l undiluted Lipofectamine™ 2000 was added and again incubated at room temperature for 30 min. Finally 250 ␮l of serum free Dulbecco modified Eagle medium (DMEM) was added. The charge ratio was calculated based on 330 Da per negative charge for DNA and 247 Da per positive charge for Tat peptide (Liu et al. 2005). Preparation of microbubbles 5 mg 1,2-distearoyl-sn-glycero-phosphatidylcholine (DSPC; Sigma, St. Louis, MO, USA), 2 mg 1,2-dipalmitoyl-sn-glycero-3-phosphor-ethanolamine (DPPE; Sigma) and 10% glucose were dissolved in phosphate-buffered saline (PBS) to a final volume of 0.5 mL in 1.5-mL vials. The vials were incubated at 37°C for 30 min. The head-

Transfection efficiency of TDL compound enhanced by UTMD ● J.-L. REN et al.

1861

Fig. 1. (Cont’d)

space of each vial was filled with the perfluoropropane gas, and then the vial was mechanically shaken for 60 s using a dental amalgamator (YJT, medical apparatus and instrument, Shanghai, China). This solution was then diluted with 0.5 mL PBS and sterilized by 60Co irradiation. Cell culture and transfection The design of our study was approved by the Ethics Review Committee. HUVEC were obtained from American Type Culture Collection (ATCC) and grown in DMEM with 10% fetal bovine serum (FBS; Gibco, Bethesda, MD, USA) and 1% penicillin/streptomycin at 37°C with 5% CO2. The cells were seeded in 24-well plates at a density of 0.5 ⫻ 105 per well. At 24 h incubation, cells were transfected after growing to about 60 – 80% confluence. Before transfection, cells were washed twice in PBS. The 24-well plates were assigned randomly into six groups, each group consisting of three

samples: (A) blanked control group; (B) TDL compound group; (C) TDL compound ⫹ microbubbles group (30 ␮l microbubbles were added in 24-well plate, final volume concentration of microbubbles is 10 %); (D) TDL compound ⫹ ultrasound group (continuous insonation was given by ultrasound gene transfection treatment meter [UGT] 1025, (Institute of Ultrasound Imaging of Chongqing Medical University, Chongqing, China) for 10 s with 10 s pause, 1 MHz, 0.75 W/cm2, 60 s totally); (E) TDL compound ⫹ microbubbles ⫹ ultrasound group (microbubbles were added in the same concentration and insonation was performed in the same mode); (F) plasmid DNA ⫹ microbubbles ⫹ ultrasound group (microbubbles were added in the same concentration and insonation was performed in the same mode). Four hours post incubation at 37°C with 5% CO2, compound solution was replaced with 500 ␮l fresh media containing 10% FBS and 1% penicillin/streptomycin. The cells were cultured for another 24 h and the reporter gene enhanced

1862

Ultrasound in Medicine and Biology

Volume 34, Number 11, 2008

Fig. 1. (Cont’d)

green fluorescent protein (EGFP) expression was analyzed. All transfection experiments were performed in triplicate. Cell transfection analysis At 24 h post transfection, the EGFP expression of all groups in HUVEC was observed using fluorescent microscopy. The cells were washed twice in warm, sterile PBS and then treated with 0.25% trypsin for 2 min at 37°C. Cells were collected by centrifugation at 1000 rpm and then were resuspended in 500 ␮l PBS. Transfection efficiency was analyzed using flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA), with the excitation setting at 488 nm. At least 10,000 cells were acquired and the data were analyzed by software CellQuest. MTT assay The influence to cell viability of all treated factors in HUVEC was assessed with the 3-(4, 5-dimthylthia-

zol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. HUVEC were seeded in 96-well plates at the density of 1 ⫻ 104 per well. When the cells grew to about 60 – 80% confluence, old medium was replaced with 200 ␮l DMEM without FBS and penicillin/streptomycin. Then every treated factor was added to each well, respectively, and every sample in triplicate. The cells were grown in a humidified incubator at 37°C with 5% CO2 for 24 h. After 24 h incubation, 20 ␮l of 5 mg/mL MTT solution in PBS was added to each well. After 4 h incubation at 37°C, the supernatant was removed, and the formazan product was washed out by treatment with 150 ␮l dimethyl sulfoxide and measured photometrically in an enzyme-linked immunosorbent assay reader at 570 nm. Untreated cells served as reference and were taken as 100% viability. The percentage of cell viability was calculated using the following equation: cytoactive (%) ⫽ OD of the treatment group/OD of the control group ⫻ 100%.

Transfection efficiency of TDL compound enhanced by UTMD ● J.-L. REN et al.

1863

Fig. 1. (Cont’d)

Western blot Proteins were extracted using protein extraction reagent (Kangchen, Shanghai, China), following a protocol provided by the manufacturer. Total protein concentration was determined with the Bradford protein assay (Bradford 1976). Protein samples were mixed in Laemmli loading buffer and boiled for 5 min. Equal protein levels of cell lysates were electrophoresed using a 4% stacking gel and 10% separating gel. The electrophoresed proteins were transferred to a polyvinylidene fluoride membrane (PVDF; Millipore, Bedford, MA, USA) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA). Completion of protein transfer from gels to the membranes was checked by staining the gels with Coomassie Blue R-250. Western blot was performed as described by Towbin et al. (1979). The membranes were washed and blocked in TRIS-buffered saline (1⫻ TBS) with 5.0% nonfat dried milk. The rinsed membranes were incubated overnight with anti-HGF an-

tibody (sc-7949, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution rate. After 2 h incubation with the appropriate species-specific horseradish peroxidase-conjugated secondary antibody (sc-2004, Santa Cruz Biotechnology) at 1:2000 dilution rate, immunoreactive bands were visualized with chemiluminescent substrate (Santa Cruz Biotechnology) according to the manufacturer’s instructions. The protein bands were normalized with ␤-actin, and all blots were quantified with software Quantity One (Bio-Rad). Reverse transcription–polymerase chain reaction mRNA levels were monitored by reverse transcription–polymerase chain reaction (RT-PCR). Total RNA was extracted by phenol– chloroform extraction and ethanol precipitation using TRIZOL (Life Technologies, Gaithersburg, MD, USA). The integrity of RNA was detected by 1.5 % agarose gels containing ethidium bromide. Polymerase chain reaction was performed by

Ultrasound in Medicine and Biology



trasound group was more intense than those of other groups.

*

      &

7

70

78 JURXS

708

Volume 34, Number 11, 2008

'08

Fig. 2. pIRES2-EGFP-HGF transfection efficiency in HUVEC. The percentage of EGFP-positive cells was determined using flow cytometry 24 h post transfection. Data represented as mean ⫾ SD (n ⫽ 3). C indicates blanked control group; D indicates plasmid DNA; T indicates TDL compound; M indicates microbubbles; U indicates ultrasound. *Transfection efficiency differed significantly from other group (p ⬍ 0.05).

two-step RT-PCR kit (AMV, TaKaRa, Tokyo, Japan) using a PCR system (MyCycler, Bio-Rad), following a protocol provided by the manufacturer. Sequences of primers for HGF are: 5⬘-CTAAGACCTGTGGGATGGGC-3⬘ (sense), 5⬘-CTCAAAGATGTCATTGTCCCC-3⬘ (antisense) (383bp) under the following conditions: an initial heating to 94°C for 2 min; then 94°C for 30 s, 55°C for 30 s, 72°C for 45 s for 35 cycles; and then at 72°C for 10 min. The PCR products were affirmed on 1.5% agarose gels containing ethidium bromide. The products were separated alongside a 100-bp DNA molecular weight ladder (Promega, Southampton, UK) for sizing. The bands were normalized with ␤-actin, and all blots were quantified with software Quantity One (BioRad). Statistical analysis Data are expressed as mean ⫾ SD. The analysis of variance (SPSS 14.0 statistical software, SPSS, Inc., Chicago, IL, USA) was used to assess the effects on transfection efficiency, cell viability, protein expression and mRNA expression. A p-value less than 0.05 was considered indicative of a statistically significant difference.

Transfection efficiency Quantitative analysis of gene expression was undertaken to determine the efficiency of gene transfection for all groups. Positive EGFP expression was detected using flow cytometry 24 h post transfection. Transfection efficiency was measured by the percentage of cells that expressed EGFP in the population (Fig. 2). Figure 2 showed that the transfection efficiency of TDL compound, TDL compound ⫹ microbubbles, TDL compound ⫹ ultrasound, TDL compound ⫹ microbubbles ⫹ ultrasound group and plasmid DNA ⫹ microbubbles ⫹ ultrasound group was, respectively, 21.92%, 22.06%, 23.62%, 27.48% and 9.38%. The transfection efficiency of TDL compound ⫹ microbubbles ⫹ ultrasound group was higher than those of other groups. MTT assay Cell viability was one of the important factors to be considered in selecting gene carriers. The influence to cell viability of all treated factors in HUVEC was evaluated by MTT assay (Fig. 3). Figure 3 showed that cell viability of all treated groups had no significant difference from the blanked control group. It was very encouraging that no noticeable cytotoxicity was observed in TDL compound ⫹ microbubbles ⫹ ultrasound group. Western blot analysis Hepatocyte growth factor protein levels were analyzed by Western blot (Fig. 4). Figure 4a showed that the special band of 69 kilodalton (KD) was found in all groups, and the band of TDL compound ⫹ microbubbles ⫹ ultrasound group was more prominent than those of other groups. The faint band that probably represented endogenous HGF was seen in the control group. The

 F\WRDFWLYH

WUDQVIHFWLRQHIILFLHQF\

1864

  

RESULTS



Fluorescent microscopy At 24 h post transfection, EGFP expression of all groups in HUVEC was observed using fluorescent microscopy (Fig. 1). Figure 1 showed that no green fluorescence was observed in blanked control group, and green fluorescence was observed in other groups. Green fluorescence of TDL compound ⫹ microbubbles ⫹ ul-

 &

7

70 78 JURXS

708

'08

Fig. 3. The influence to the cytoactive of HUVEC was evaluated by MTT. Data represented as mean ⫾ SD (n ⫽ 3). C indicates blanked control group; D indicates plasmid DNA; T indicates TDL compound; M indicates microbubbles; U indicates ultrasound.

Transfection efficiency of TDL compound enhanced by UTMD ● J.-L. REN et al.

1865

One software (Bio-Rad). Figure 5b showed that the expression of HGF mRNA of TDL compound ⫹ microbubbles ⫹ ultrasound group was significantly higher than those of other groups (p ⬍ 0.05). DISCUSSION The development of gene therapy is still slow because of the safety of viral vector and poor efficiency of nonviral vector. To achieve safe and effective gene delivery, it is necessary to condense the DNA, protect it from degradation by nucleases, achieve cellular internal-

Fig. 4. Western blot for the presence of HGF protein from HUVEC. Cells were transfected with all treated factors. The cells were harvested and the protein extracts were subjected to SDS–polyacrylamide gel electrophoresis and probed by Western blot analysis with antibodies to HGF. (a) Prominent band consistent with HGF was seen in TDL compound ⫹ microbubbles ⫹ ultrasound group, and a faint band was seen in blanked control group. (b) The results represent the mean ⫾ SD for three independent HGF protein sets normalized to ␤-actin. *The expression of HGF protein of TDL compound ⫹ microbubbles ⫹ ultrasound group differed significantly from other groups (p ⬍ 0.05). C indicates blanked control group; T indicates TDL compound; M indicates microbubbles; U indicates ultrasound. ␤-actin was used as an inner reference.

bands were normalized with ␤-actin, and we quantified the expression of HGF protein in all groups with Quantity One software (Bio-Rad). Figure 4b showed that the expression of HGF protein of TDL compound ⫹ microbubbles ⫹ ultrasound group was significantly higher than those of other groups (p ⬍ 0.05). Reverse transcription–polymerase chain reaction analysis Consistent with the results of the Western blot, RT-PCR revealed expression of HGF mRNA in all groups (Fig. 5). Figure 5a showed that the special band of 383bp was found in all groups, and the band of TDL compound ⫹ microbubbles ⫹ ultrasound group was more prominent than those of other groups. The bands were normalized with ␤-actin, and we quantified the expression of HGF mRNA in all groups with Quantity

Fig. 5. RT-PCR analysis of the expression of HGF mRNA in HUVEC. Cells were transfected with all treated factors. RNA was prepared and analyzed by RT-PCR. Prominent band consistent with HGF was seen in TDL compound ⫹ microbubbles ⫹ ultrasound group, and a faint band was seen in blanked control group. (a) The DNA marker 100 bp ladder (Promega) is indicated and sizes of PCR products presented. (b) The results represent the mean ⫾ SD for three independent RNA sets normalized to ␤-actin. *The expression of HGF mRNA of TDL compound ⫹ microbubbles ⫹ ultrasound group differed significantly from other groups (p ⬍ 0.05). C indicates blanked control group; T indicates TDL compound; M indicates microbubbles; U indicates ultrasound. ␤-actin was used as an inner reference.

1866

Ultrasound in Medicine and Biology

ization and transfer DNA into the cytoplasm or the cell nucleus without cytotoxicity (Hellgren et al. 2004). Liposome and CPPs can condense the DNA, protect DNA from degradation by nucleases and transfer DNA into the cytoplasm or the cell nucleus (Hyndman et al. 2004). Our previous work showed that TDL compound could enhance the efficiency of gene transfection and optimal Tat/DNA charge ratio was 8:1 in TDL compound (Jianli et al. 2007), so the present study chose 8:1 TDL compound. However, the transfection efficiency of 8:1 TDL compound was only 22.03% and still needed further enhancement. UTMD, as a novel drug delivery carrier, has been extensively applied for site-specific intracellular delivery of macromolecules both in vitro and in vivo (Li et al. 2003; Lindner 2004). The results from the present study showed that UTMD could significantly enhance the gene transfection efficiency of TDL compound. Under fluorescent microscopy, green fluorescence was observed in other groups besides the blanked control group and the intensity of green fluorescence of TDL compound ⫹ microbubbles ⫹ ultrasound group was higher than those of other groups. Flow cytometry showed that the transfection efficiency of TDL compound ⫹ microbubbles ⫹ ultrasound group was 27.48% and higher than those of other groups. MTT showed that the cell viability of TDL compound ⫹ microbubbles ⫹ ultrasound group was not significantly different from those of other groups. As a final component of this study, we assessed the expression of HGF mRNA and HGF protein in HUVEC by RT-PCR and by Western blot respectively. The Western blot analysis (Fig. 4) showed that TDL compound ⫹ microbubbles ⫹ ultrasound group significantly stimulated the expression of HGF protein in HUVEC, and this was confirmed at the HGF mRNA level by RT-PCR analysis as well (Fig. 5). The HGF mRNA and HGF protein of TDL compound ⫹ microbubbles ⫹ ultrasound group were higher than those of other groups, showing that UTMD could upregulate the expressions of the objective gene in the transcriptional and translational levels. Therefore, our findings demonstrated that UTMD can enhance the expression of gene materials mediated by TDL compound in HUVEC, and has no significant influence to cell viability, suggesting that the combination of UTMD and TDL compound might be a safe and efficient gene delivery carrier. The addition of microbubbles increased transfection efficiency of TDL compound when applying ultrasound for gene delivery. There are many advantages to this. First, the microbubbles collapse on the cell membrane, destabilizing its structure and permeability. TDL compound can form a compacted compound by electrostatic interaction and condense the DNA; thus it can easily and actively deliver the DNA into the cells. Second, an ultrasound instrument can be applied conveniently and the index of energy output can

Volume 34, Number 11, 2008

be controlled exactly (Guozhong et al. 2005; Rahim et al. 2006), and ultrasound can be focused on a small domain and adjust the gene transfection and expression in the target tissues. Ultrasound wave that destructed microbubbles in a targeted tissue can improve the target of gene therapy in special time and space (Bekeredjian et al. 2007; Zhigang et al. 2004). Third, local cavitation induced by UTMD can increase permeability of the creature barrier, and therapeutic gene released from microbubbles can enhance the concentration of gene material and increase the expression level of therapeutic gene in target tissue (Suzuki et al. 2007). Therefore, the combination of UTMD and TDL compound provide a safe and efficient approach to deliver therapeutic gene HGF into ischemic heart tissue. In conclusion, the present study manifested that UTMD could significantly enhance the gene transfection efficiency of TDL compound. The combination of UTMD and TDL compound might be a safe and highefficient approach to deliver therapeutic gene HGF into ischemic heart tissue. As the observable difference between in vitro and in vivo, further investigations should concentrate on the efficiency in vivo. Acknowledgements—The authors thank Mr. Xin Li for assistance with flow cytometry and Mr. TingHe Yu for his help. The work was supported by the Key Program of Natural Science Foundation of China (No.30430230) and Hi-Tech Research and Development Program of China (2006 AA02Z4FO).

REFERENCES Ahmet I, Sawa Y, Iwata K, Matsuda H. Gene transfection of hepatocyte growth factor attenuates cardiac remodeling in the canine heart: A novel gene therapy for cardiomyopathy. J Thorac Cardiovasc Surg 2002;124:957–963. Alton EW, Stern M, Farley R, Jaffe A, Chadwick SL, Phillips J, Davies J, Smith SN, Browning J, Davies MG, Hodson ME, Durham SR, Li D, Jeffery PK, Scallan M, Balfour R, Eastman SJ, Cheng SH, Smith AE, Meeker D, Geddes DM. Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: A double-blind placebo-controlled trial. Lancet 1999;353: 947–954. Astriab-Fisher A, Sergueev D, Fisher M, Shaw BR, Juliano RL. Conjugates of antisense oligonucleotides with the Tat and antennapedia cell-penetrating peptides: Effects on cellular uptake, binding to target sequences, and biologic actions. Pharm Res 2002;19:744 – 754. Bekeredjian R, Bohris C, Hansen A, Katus HA, Kuecherer HF, Hardt SE. Impact of microbubbles on shock wave-mediated DNA uptake in cells in vitro. Ultrasound Med Biol 2007;33:743–750. Bekeredjian R, Kuecherer HF, Kroll RD, Katus HA, Hardt SE. Ultrasound-targeted microbubble destruction augments protein delivery into testes. Urology 2007;69:386 –389. Bougioukas I, Didilis V, Ypsilantis P, Giatromanolaki A, Sivridis E, Lialiaris T, Mikroulis D, Simopoulos C, Bougioukas G. Intramyocardial injection of low-dose basic fibroblast growth factor or vascular endothelial growth factor induces angiogenesis in the infarcted rabbit myocardium. Cardiovasc Pathol 2007;16(2):63– 68. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248 –254. Bussolino F, Di Renzo MF, Ziche M, Bocchietto E, Olivero M, Naldini L, Gaudino G, Tamagnone L, Coffer A, Comoglio PM. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 1992;119:629 – 641.

Transfection efficiency of TDL compound enhanced by UTMD ● J.-L. REN et al. El-Aneed A. An overview of current delivery systems in cancer gene therapy. J Control Release 2004;94:1–14. Guozhong W, Shenjiang H, Zhefeng Z. Enhanced reporter gene transfer and expression in cardiac myocytes mediated by ultrasonic destruction of the microbubbles. Chin J Appl Physiol 2005;21:371–375. Haider HK, Elmadbouh I, Jean-Baptiste M, Ashraf M. Nonviral vector gene modification of stem cells for myocardial repair. Mol Med 2008;14:79 – 86. Haitao R, Hong R, Zhigang W. Effects of ultrasonic cavitation on cultured cell membrane in vitro. Chin J Ultrasonogr 2003;12:499 –501. Hao X, Månsson-Broberg A, Grinnemo KH, Siddiqui AJ, Dellgren G, Brodin LA, Sylvén C. Myocardial angiogenesis after plasmid or adenoviral VEGF-A(165) gene transfer in rat myocardial infarction model. Cardiovasc Res 2007;73: 481– 487. Hellgren I, Gorman J, Sylven C. Factors controlling the efficiency of Tat-mediated plasmid DNA transfer. J Drug Target 2004;12:39 – 47. Hyndman L, Lemoine JL, Huang L, Porteous DJ, Boyd AC, Nan X. HIV-1 Tat protein transduction domain peptide facilitates gene transfer in combination with cationic liposomes. J Control Release 2004;99:435– 444. Ignatovich IA, Dizhe EB, Pavlotskaya AV, Akifiev BN, Burov SV, Orlov SV, Perevozchikov AP. Complexes of plasmid DNA with basic domain 47–57 of the HIV-1 Tat protein are transferred to mammalian cells by endocytosis-mediated pathways. J Biol Chem 2003;278:42625– 42636. Iwakura A, Fujita M, Kataoka K, Tambara K, Sakakibara Y, Komeda M, Tabata Y. Intramyocardial sustained delivery of basic fibroblast growth factor improves angiogenesis and ventricular function in a rat infarct model. Heart Vessels 2003;18:93–99. Jayasankar V, Woo YJ, Pirolli TJ, Bish LT, Berry MF, Burdick J, Gardner TJ, Sweeney HL. Induction of angiogenesis and inhibition of apoptosis by hepatocyte growth factor effectively treats postischemic heart failure. J Cardiac Surg 2005;20:93–101. Jianli R, Zhigang W, Xingsheng L, Lin S, Yong Z, Zhaoxia W, Chunjjang Y, Qunxia Z, Chuanshan X. Tat peptide/plasmid DNA/ Liposome (TDL) compound enhances gene transfection in human umbilical vein endothelial cell. Acta Acad Med Milit Tert 2007; 29:2097–2099. Kido M, Du L, Sullivan CC, Li X, Deutsch R, Jamieson SW, Thistlethwaite PA. Hypoxia-inducible factor 1-alpha reduces infarction and attenuates progression of cardiac dysfunction after myocardial infarction in the mouse. J Am Coll Cardiol 2005;46:2116 –124. Kitta K, Day RM, Ikeda T, Suzuki YJ. Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis. Free Radic Biol Med 2001;31:902–910. Korpanty G, Chen S, Shohet RV, Ding J, Yang B, Frenkel PA, Grayburn PA. Targeting of VEGF-mediated angiogenesis to rat myocardium using ultrasonic destruction of microbubbles. Gene Ther 2005;12:1305–1312. Lindner JR. Microbubbles in medical imaging: current applications and future directions. Nat Rev Drug Discov 2004;3:527–532. Li T, Tachibana K, Kuroki M, Kuroki M. Gene transfer with echoenhanced contrast agents: Comparison between Albunex, Optison, and Levovist in mice—Initial results. Radiology 2003;229:423– 428. Liu Z, Li M, Cui D, Fei J. Macro-branched cell-penetrating peptide design for gene delivery. J Control Release 2005;102: 699 –710. Losordo DW, Dimmeler S. Therapeutic angiogenesis, vasculogenesis for ischemic disease. Part I. Angiogenic cytokines. Circulation 2004;109:2487–2491. Madry H, Reszka R, Bohlender J, Wagner J. Efficacy of cationic liposome-mediated gene transfer to mesangial cells in vitro and in vivo. J Mol Med 2001;79:184 –189. Miyazaki M, Obata Y, Abe K, Furusu A, Koji T, Tabata Y, Kohno S. Gene transfer using nonviral delivery systems. Perit Dial Int 2006; 26:633– 640. Montesano R, Matsumoto K, Nakamura T, Orci L. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 1991;67: 901–908. Nakanishi M, Eguchi A, Akuta T, Nagoshi E, Fujita S, Okabe J, Senda T, Hasegawa M. Basic peptides as functional components of nonviral gene transfer vehicles. Curr Protein Pept Sci 2003;4:141–150.

1867

Nie F, Xu HX, Tang Q, Lu MD. Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells. World J Gastroenterol 2006;12:7508 –7513. Rahim A, Taylor SL, Bush NL, ter Haar GR, Bamber JC, Porter CD. Physical parameters affecting ultrasound/microbubble-mediated gene delivery efficiency in vitro. Ultrasound Med Biol 2006;32: 1269 –1279. Rubin JS, Chan AM, Bottaro DP, Burgess WH, Taylor WG, Cech AC, Hirschfield DW, Wong J, Miki T, Finch PW. A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor. Proc Natl Acad Sci U S A 1991;88:415– 419. Rudolph C, Plank C, Lausier J, Schillinger U, Muller RH, Rosenecker J. Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells. J Biol Chem 2003;278:11411–11418. Saraswathi TR, Kavitha B, Vijayashree Priyadharsini J. Gene therapy for oral squamous cell carcinoma: an overview. Indian J Dent Res 2007;18:120 –123. Shim WS, Li W, Zhang L, Li S, Ong HC, Song IC, Bapna A, Ge R, Lim YT, Chuah SC, Sim EK, Wong P. Angiopoietin-1 promotes functional neovascularization that relieves ischemia by improving regional reperfusion in a swine chronic myocardial ischemia model. J Biomed Sci 2006;13:579 –591. Stoker M, Gherardi E, Perryman M, Gray J. Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature 1987; 327:239 –242. Suzuki R, Takizawa T, Negishi Y, Hagisawa K, Tanaka K, Sawamura K, Utoguchi N, Nishioka T, Maruyama K. Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound. J Control Release 2007;117:130 –136. Taniyama Y, Morishita R, Aoki M, Hiraoka K, Yamasaki K, Hashiya N, Matsumoto K, Nakamura T, Kaneda Y, Ogihara T. Angiogenesis and antifibrotic action by hepatocyte growth factor in cardiomyopathy. Hypertension 2002;40:47–53. Torchilin VP, Levchenko TS, Rammohan R, Volodina N, Papahadjopoulos-Sternberg B, D’Souza GG. Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome-DNA complexes. Proc Natl Acad Sci U S A 2003;100:1972–1977. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76:4350 – 4354. Tung CH, Mueller S, Weissleder R. Novel branching membrane translocational peptide as gene delivery vector. Bioorg Med Chem 2002;10:3609 –3614. Vancraeynest D, Havaux X, Pouleur AC, Pasquet A, Gerber B, Beauloye C, Rafter P, Bertrand L, Vanoverschelde JL. Myocardial delivery of colloid nanoparticles using ultrasound-targeted microbubble destruction. Eur Heart J 2006;27:237–245. Vives E, Brodin P, Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 1997;272:16010 – 16017. Xia D, Zhang MM, Yan LN. Recent advances in liver-directed gene transfer vectors. Hepatobiliary Pancreat Dis Int 2004;3:332–336. Xingsheng L, Zhigang W, Lin S, Jianl R, Chunjiang Y, Yuanyi Z, Haitao R, Pan L. Construction and expression of enhanced green fluorescent protein and HGF co-expression vector. J Chongqing Medical University 2007;32:470 – 473. Yamaguchi T, Sawa Y, Miyamoto Y, Takahashi T, Jau CC, Ahmet I, Nakamura T, Matsuda H. Therapeutic angiogenesis induced by injecting hepatocyte growth factor in ischemic canine hearts. Surg Today 2005;35:855– 860. Zhang S, Zhao B, Jiang H, Wang B, Ma B, Cationic lipids and polymers mediated vectors for delivery of siRNA. J Control Release 2007;123:1–10. Zhigang W, Zhiyu L, Haitao R, Hong R, Qunxia Z, Ailong H, Qi L, Chunjing Z, Hailin T, Lin G, Mingli P, Shiyu P. Ultrasoundmediated microbubble destruction enhances VEGF gene delivery to the infarcted myocardium in rats. Clin Imaging 2004;28:395–398.