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 Inhibitors,research,lifescience,medical and the safety of the procedure [3]. 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 Inhibitors,research,lifescience,medical cell, and other physicochemical conditions like temperature and carbon dioxide concentration, Inhibitors,research,lifescience,medical [3]. Greenleaf et al. reported that ultrasound acoustic pressure, DNA concentration, and repeat count of insonification correlated with transfection rate [4]. Teupe et al. demonstrated that duration of insonification did not affect transfection rate [5]. Then, Chen et al. showed that transfection rate reached plateau when DNA concentration was increased [6]. Greenleaf et Inhibitors,research,lifescience,medical al. also showed that transfection rate peaked and fell off according to the change in liposome concentration [4]. 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 [1]. Guo et al. demonstrated that cell viability decreased with the increase in duration of insonification [7]. Suzuki et al. and Li et al. showed that cell viability decreased with the increase in ultrasound acoustic selleck chemicals llc pressure [8, 9]. On the basis of those previous findings, we planned Inhibitors,research,lifescience,medical to examine the effects of amount of plasmid DNA, liposome concentration, duration of insonification, repeat count of insonification, ADP ribosylation factor and time of incubation with liposome, cell, and DNA on transfection rate, which was measured by means of HGF protein release into culture medium. 2. Materials and Methods 2.1. Cell Culture Primary cultures of neonatal ventricular myocytes were prepared as described previously [10]. 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., Freehold, NJ). Myocytes were segregated from nonmyocytes using a discontinuous Percoll gradient (Sigma Chemical Co., Inc., St. Louis, MO). After centrifugation, the upper layer consisted of a mixed population of nonmyocyte cell types and the lower layer consisted almost exclusively of cardiac myocytes.