ultrasound - triggered bubble liposome destruction ( sonoporation ) has been proposed as a safe nonviral means of gene therapy that can target many different cells or tissues .   in the field of cardiovascular medicine , this method may have significant potential for the introduction of therapeutic genes directly into vascular cells or cardiomyocytes [ 1 , 2 ] . sonoporation can only be clinically effective if the dose - effect relationship between the amount of bubble liposome and transfection efficiency is first established . however , few reports have already examined this dose - effect relationship and the safety of the procedure . transfection efficiency in sonoporation depends on various conditions including type of microbubble , mode of ultrasound , frequency of ultrasound , intensity of acoustic pressure , concentration of microbubble , dose of dna , duration of insonification , incubation time of cell with dna , repeat count of insonification , type of targeted cell , and other physicochemical conditions like temperature and carbon dioxide concentration , . greenleaf et al . reported that ultrasound acoustic pressure , dna concentration , and repeat count of insonification correlated with transfection rate . then , chen et al . showed that transfection rate reached plateau when dna concentration was increased . greenleaf et al . also showed that transfection rate peaked and fell off according to the change in liposome concentration . they thought it might be derived from cellular toxicity of large amount of liposome . li et al . reported that cell viability decreased along with the increase in microbubble concentration . guo et al . demonstrated that cell viability decreased with the increase in duration of insonification . suzuki et al . and li et al . showed that cell viability decreased with the increase in ultrasound acoustic pressure [ 8 , 9 ] . on the basis of those previous findings , we planned to examine the effects of amount of plasmid dna , liposome concentration , duration of insonification , repeat count of insonification , and time of incubation with liposome , cell , and dna on transfection rate , which was measured by means of hgf protein release into culture medium . briefly , apical halves of cardiac ventricles from 1-day - old wistar rats were separated , minced , and dispersed with 0.1% collagenase type ii ( worthington biochemical corp . , myocytes were segregated from nonmyocytes using a discontinuous percoll gradient ( sigma chemical co. , inc . , st . after centrifugation , the upper layer consisted of a mixed population of nonmyocyte cell types and the lower layer consisted almost exclusively of cardiac myocytes . after the myocytes had been incubated twice on uncoated 10-cm culture dishes for 30 minutes to remove any remaining nonmyocytes , the nonattached viable cells were plated on gelatin - coated 24-well culture plates or 10-cm culture dishes and then cultured in dmem ( life technologies , grand island , ny ) supplemented with 10% fcs ( life technologies , grand island , ny ) .   after 24-hour incubation in dmem with fcs , the culture medium was changed to serum - free dmem , and all experiments were performed 24 hours later . this purification procedure has well been established [ 11 , 12 ] , and > 95% of the cells obtained by this method were cardiomyocytes . preparation of rat hepatocyte growth factor ( hgf ) expression plasmid dna was described previously . briefly , rat hgf cdna cloned by polymerase chain reaction was inserted into the unique xho i site between the cytomegalovirus immediate - early enhancer - chicken -actin hybrid promoter and rabbit -globin poly a site of the pcaggs expression plasmid . the resulting plasmid , pcaggs - hgf , was grown in escherichia coli dh5 ( figure 1(a ) ) . the plasmid was purified with a quiagen plasmid dna kit and dissolved in te buffer . the purified plasmid dna was stored at 20c and diluted to the required concentration with distilled water immediately before use . liposome microbubble , shu 508a , consists of palmitic acid and galactose and provides echogenic micron - sized air bubbles when suspended in water . the diameter of bubbles ranges from 2 to 8  m , and 97% are smaller than 6  m . these air bubbles are stabilized by palmitic acid , which forms a molecular film that lowers the surface tension of the aqueous vehicle . the shu 508a bubbles are nontoxic , have a neutral ph , are biodegradable , and are made from a physiologically occurring substance . the physiochemical properties of shu 508a bubbles are typical of a saccharide solution . before performing the experiments for dose - effect relationships using liposome sonoporation , we needed to determine the most appropriate ultrasound mode for the sonoporation procedure for efficient transfection . we tested four modes of ultrasound : pulsed wave doppler , color flow doppler , continuous wave doppler , and harmonic power doppler , which are available with standard echocardiographic machinery in a clinical laboratory . we performed a simple transfection experiment at the same acoustic pressure of 0.5  w / cm for each ultrasound mode , using a single condition with 60  g hgf plasmid dna , 1  10  particles / ml of shu 508a liposome , 30  sec insonification , 15  min of dna incubation , and 3 repetitions of insonification . after confluence had been reached in a 35  mm petri dish , the medium was changed to fresh defined serum - free medium . pulsed wave doppler , color flow doppler , and continuous wave doppler were insonified from psk-25at acoustic transducer with toshiba ssa-380a ( toshiba medical systems ) , and harmonic power doppler was insonified from s3 transducer with sonos 5500 ( phillips medical systems ) . continuous - wave doppler ultrasound was the most efficacious and was used for subsequent experiments . the medium in 35  mm petri dishes containing the cardiomyocytes was changed to fresh defined serum - free medium from dmem+10% fcs . rat hgf plasmid dna was diluted with distilled water , and a volume corresponding to 60 , 120 , or 180  g was added to each of the 20 petri dishes per dna dose . cells on each dish were then treated with continuous - wave doppler ultrasound ( frequency of 2.5  mhz and acoustic intensity of 0.5  w / cm from a psk-25at acoustic transducer with toshiba ssa-380a ultrasound system ) with shu 508a liposome ( 1  10  particles / ml ) for acoustic exposure time of 30 or 60 seconds at room temperature ( figure 1(b ) ) . in a separate series of experiments , we tested four liposome concentrations ( 0 , 1  10 , 1  10 or 1  10  particles / ml ) , three insonification repetitions ( 1 insonification only , 3 or 5 insonifications for 30 seconds ) , and three dna incubation times ( 15 , 60 or 120  min ) . after the incubation , the culture medium was changed to normal dmem+10% fcs and the cells were cultured for 72 hours .   in a separate set of experiments , we examined the effect of culture period on the amount of dna product that is hgf protein by discontinuing culture at 24 , 48 , and 72 hours and measuring the amount of rat hgf protein in the medium . the total amount of protein content in the cultured cells was measured and used to correct the hgf level in each dish . we measured rat hgf protein using an eia kit ( institute of immunology co. , ltd . , tokyo , japan )   and protein content of cultured cells using a modified lowry protein assay kit ( pierce biotechnology , rockford ) . to determine the safety of sonoporation , in a separate experiment , cultured cells were exposed to 0.1% trypan blue for 5  min just after ultrasound insonification . this allowed assessment of sarcolemmal membrane damage and was performed for each concentration of liposome , each insonification time , and each number of repetitions of insonification . the number of stained and unstained cells in the dishes was counted and used to calculate the percentage of intact cells . the degree of cellular injury caused by sonoporation was determined by examining the insonified cells by scanning electron microscopy ( hitachi s-4800 ) .   immediately after ultrasound insonification in the presence of liposome , the cardiomyocytes were fixed with phosphate - buffered 2.5% glutaraldehyde for 4 hours , followed by postfixation with 1% osmium tetroxide for 1 hour , and then were conventionally prepared for scanning electron microscopy . comparisons of parameters from experimental groups were performed with unpaired t tests and resulting p - values were corrected according to the bonferroni method . in analyses , the concentration of hgf protein in the culture medium increased as the culture period after ultrasonic transfection was extended . the transfection consisted of three 30-sec insonifications a 15-min incubation with hgf dna ( 60  g ) and liposome ( 1  10  particle / ml ) ( figure 3(a ) ) . after 72 hours of culture , hgf protein concentration in the culture medium was measured and corrected using the protein content of the cultured cells . hgf protein concentration in the culture medium was 0.54  0.049  ng / ml / mg and was highest when 60  g of dna was administered with a liposome concentration of 1  10  particles / ml , a15-min incubation , and three 30-sec insonification .   although the nominal mean values of hgf protein after transfection of 120 and 180  g dna were lower than those after transfection of 60  g , the differences were not statistically significant ( figure 3(b ) ) . hgf protein concentration in the culture medium was 0.56  0.053  ng / ml / mg and was highest when the incubation time was 15  min with a liposome concentration of 1  10 particles / ml , 60  g dna , and three 30-sec insonification .   although the mean values of hgf protein after transfection for 60 and 120  min were lower than those after 15  min incubation , the differences were not statistically significant ( figure 3(c ) ) . hgf protein concentration in the culture medium was 0.59  0.052  ng / ml / mg and was highest when the insonification period was 30  sec with 60  g dna , a liposome concentration of 1  10  particles / ml , and 15-min incubation . there was no significant difference in hgf production in cells insonified for 30 and 60  min ( figure 3(d ) ) . hgf protein concentration in the culture medium was 0.53  0.053  ng / ml / mg and was nominally highest when the liposome concentration was 1  10  particles / ml and insonification consisted of three 30-sec ultrasound exposures , though it was statistically similar to that obtained with 1  10  particles / ml .   at a higher liposome concentration of 1  10  particles / ml , hgf protein concentration decreased ( figure 3(e ) ) . hgf protein concentration in the culture medium was 0.54  0.053  ng / ml / mg and was highest when three 30-sec insonifications were given , with a liposome concentration of 1  10  particles / ml and 60  mg dna . this protein production was statistically higher than in cells given one or five insonifications ( figure 3(f ) ) . the percentage of dead cells was 14.7  0.9% and was higher in the cells given five 30-sec insonifications at a liposome concentration of 1  10  particles / ml ( figure 4(a ) ) . the percentage of dead cells increased with increasing concentrations of liposome ( figure 4(b ) ) . the dead cell count was 24.8  2.9% and was highest when the liposome concentration was 1  10  particles / ml and three 30-sec insonifications were used . the percentage of dead cells increased as the number of insonification repetitions increased ( figure 4(c ) ) . the dead cell count was 14.7  0.9 % and was highest when five repetitions of the insonification step were given , with a liposome concentration of 1  10  particles / ml . no particular changes were evident on the surfaces of untreated control cultured cardiomyocytes when viewed with the scanning electron microscope at low and high magnification ( figures 5(a ) and 5(b ) ) . after sonoporation with a low concentration of liposome ( figure 5(c ) ) and with a high concentration of liposome ( figure 5(d ) ) , microdimples or pores were observed on the surfaces of the cultured cardiomyocytes . considerable efforts have been made to develop methods that will allow effective and safe introduction of vectors into cells for gene therapy .   however , we still need a breakthrough in the form of a novel vector that will transform cells at high efficiency and with low risk of adverse effects . this is especially true in cardiovascular medicine , where malignant cellular transformation is rare . one of the promising candidates for safe and efficacious gene transfection is a naked plasmid vector that has been modified to have high affinity for cardiovascular tissues but which has no built - in viral components [ 17 , 18 ] . we have developed a method for electroporation of a cytokine gene for treatment of cardiomyopathy . for this reason , we are pursuing the present sonoporation method as a protocol for gene transfection . the hgf protein used in the present study is found in a wide variety of cell types and has multiple biological properties , including mitogenic , motogenic , morphogenic and antiapoptotic activities . several lines of evidence indicate that this molecule has potential for therapeutic use for treatment of heart failure , myocardial infarction , angina , and hypertension [ 2022 ] . hgf may also have enormous therapeutic potential for hepatic and renal disorders , in addition to cardiovascular diseases [ 2326 ] .   in the present study , we showed variations in amount of hgf plasmid dna , liposome concentration , the duration of insonification , and incubation time of the cardiomyocytes with liposome and dna , and their dose relationships with the final amount of hgf protein released from the cultured neonatal cardiomyocytes . we found that specific amounts of liposome and repetitions of insonification were needed for effective protein production from cardiomyocytes .   however , high concentrations of bubble liposome and large numbers of repeat insonifications resulted in decreased cell viability . plasma membrane sonoporation induced by ultrasound and subsequent self - sealing has been reported in previous investigations [ 2729 ] . however , the exact mechanism by which membrane sonoporation causes substance incorporation into the cell is not yet understood . some investigators speculate that the membrane poration results in both transfection efficiency and cellular damage . in the present study , scanning microscopy images revealed some microdimples or pores on the cell surface after sonoporation , which did not exist on the surface of control cardiomyocytes . thus , we speculate that these dimples or pores on the cell surface might be related to transfection efficiency and might be evidence of cellular injury by sonoporation . previous studies of sonoporation of vascular walls revealed that microbubble destruction would cause rupture of microvessels and extravasation [ 3033 ] , which would cancel out some benefits of sonoporation . thus , the poration and self - sealing mechanism needs to be fully investigated and optimized . a sonoporation technique targeting the cardiovascular system has now been developed for gene transfection to myocardium , limb skeletal muscle , and arteries [ 3437 ] . for a variety of target tissues , a number of microbubbles , including liposomes , and a range of ultrasound modes have been developed . the optimal combination of the type of microbubble , ultrasound mode , and target tissue still needs to be resolved [ 3840 ] . however , the principal types of ultrasound used for sonoporation have included pulsed wave doppler or continuous wave doppler with acoustic pressure ranging 0.55  w / cm [ 3437 ] . in the present study , we found that continuous wave doppler at a standard frequency for clinical use , that is , 2.5  mhz and the usual acoustic pressure of 0.5  w / cm , was most effective with our cardiomyocytes . the reason we used one of the standard ultrasound modes with standard settings for clinical use is that we would like to use our sonoporation system eventually in a clinical setting . to avoid the complexity of numerous combinations of experimental conditions , such as amount of dna , concentration of liposome , duration of insonification , repeat count of insonification , length of incubation time , and culture period after gene transfection , we only used several practical combinations for an in vitro experiment for cultured cardiomyocytes . thus , we might have missed other multimodal aspects of dose - effect relationships among these conditions . hgf dna was successfully transferred to cultured cardiomyocytes using sonoporation with a defined liposome concentration and a mode of insonification . a number of trade - offs between transfection efficiency and cellular injury have to be balanced to optimize this sonoporation method .
we transfected naked hgf plasmid dna into cultured cardiomyocytes using a sonoporation method consisting of ultrasound - triggered bubble liposome destruction . 
 we examined the effects on transfection efficiency of three concentrations of bubble liposome ( 1  106 , 
 1  107 , 
 1  108/ml ) , three concentrations of hgf dna ( 60 , 120 , 180  g / ml ) , two insonification times ( 30 , 60  sec ) , and three incubation times ( 15 , 60 , 120  min ) . 
 we found that low concentrations of bubble liposome and low concentrations of dna provided the largest amount of the hgf protein expression by the sonoporated cardiomyocytes . 
 variation of insonification and incubation times did not affect the amount of product . 
 following insonification , cardiomyocytes showed cellular injury , as determined by a dye exclusion test . 
 the extent of injury was most severe with the highest concentration of bubble liposome .   in conclusion , there are some trade - offs between gene transfection efficiency and cellular injury using ultrasound - triggered bubble liposome destruction as a method for gene transfection .