| Objective To optimize the conditions for preparing polymeric microbubbles encapsulating perfluoropentane with high concentration,suitable size and good stabilization and research on influential factors on elimination half life of polymeric acoustic contrast agent,such as cardiac compression,capillary extrusion,ultrasound wave irradiation,temperature,plasma fraction,blood pressure.Methods 1 Polymeric acoustics contrast agent containing perfluoropentane was prepared by single emulsion method.The single-factor test was used to analysis the three preparation conditions,including polymer mass/perfluoropentane volume ratio,hemogenate speed and time were optimized,and the microbubble concentration and size were set as judge standard.Appearance and particle size of the microbubble was observed.To evaluate its stability in vitro,the density of microbubble stored at 4℃ was measured by hemocytometer on the optical microscope in 0,24 hours.2 The acoustic contrast-enhanced experiments were performed in water sac and rabbit kidney using the optimized microbubbles and the arrival time(AT),time to peak(TTP),half time of descending(HT)and peak intensity(PI)were analyzed by TCA software.3 Microbubble diluted in ringer solution were injected into isolated frog heart.We collected and observed microbubble in 0.5,3min comparing with control groups to evaluate the effect of frog heart on microbubble.To validate the influence of capillary extrusion on microbubble,we established rabbit kiney capillary models in vitro with observing microbubbles’ concentration and appearance before or after different times of passing the microcirculation.After renal arteriovenous contrast-enhanced ultrasound,imaging parameters were analyzed by TCA software.4 To investigate the effect of pressure,temperature and plasma fraction,we observed microbubbles’ concentration and appearance in different times(0,5,10,30min),which were under the pressure of 110 mm Hg and no pressure,at 4℃and 39.4℃,in plasma and physiological saline at 39.4℃respectively.5 The influence of ultrasound wave irradiation was evaluate by measuring the echogenicity of polymeric microbubble which were put in ziplock bag in different times at different MI values and different frequencies.Results 1 The preparation conditions were optimized as a polymer mass perfluoropentane volume ratio of 2/1,homogenate speed of 26 000 rpm and homogenate time of 60 sec,which were white suspension,uniformly distributed circular microbubble with mean size of 3.9±1.1μm and a high microbubble concentration of(1.72±0.33)×109/ml.No significant difference was observed in concentration in 24 hours after polymeric microbubble were kept in vitro(P>0.05).2 In water sac,the echo intensity of optimized microbubbles enhanced obviously showing dense fine dot echoes,while immediately decreased to zero right after ultrasonic blasting.The AT,TP,HT and PI were detected as 3.1±0.6 sec,5.2±0.8 sec,4.0±0.7 sec and(4.7±1.1)× 10-5AU,respectively.3 The concentration of polymeric microbubble had no significant difference after passing from frog heart,but deformed microbubbles were seen on the optical microscope.The concentration had significantly difference after passing from microcirculation of rabbit kidney(P<0.05),while the AUC of renal arteriography was 1.23 times as high as renal venography’s(P>0.05).4 Polymeric microbubbles were found to be stable at 39.4℃,in plasma and under applied pressure(110mm Hg)as evidenced by unchanged particle concentration and diameter in 30 minutes(P>0.05).5 The reduction on the intensity of imaging added with the increasing duration of ultrasound beam exposure and MI,decreasing frequency.Conclusion The polymeric microbubbles prepared by optimized conditions were of high concentration,suitable size,good stability in vitro and good contrast-enhanced effect in vivo and in vitro.The influential factors on elimination half life of polymeric acoustics contrast agent were cardiac compression,capillary extrusion and ultrasound wave irradiation.While,temperature,plasma fraction and blood pressure had little effect on half life of polymeric microbubbles. |
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