Abstract Microbubble formulations have a long history for enhancement of ultrasound (US) imaging and recently also for therapeutic applications. Previously, a series of freeze-dried bubble formulations based on the lipids DSPC and DSPG were developed. Here, we have attempted to scale-up the production process for future more extensive studies. Bubbles were prepared by homogenization of a lipid dispersion in a perfluoropropane atmosphere in a medium size (300–500 mL) homogenizer and then freeze-dried for better storage stability. In total, 300 freeze-dried vials were prepared. The properties of the bubbles were similar to those previously prepared on a lab scale with the difference that they were slightly larger and also had a better stability. The re-entrapped gas concentration after re-constituted freeze-dried bubbles was 9.4 µL/µmol lipid. The re-entrapped rate was 72.3% of fresh bubble before freeze-drying (13.0 µL/µmol lipid). The half-life of US imaging signal of the re-constituted freeze-dried bubbles in water in vitro was shorter than that of the fresh bubbles (2.7 min vs. 3.3 min). A leak of Evans Blue, that binds to albumin, from mouse ear blood vessel was observed after combination of bubble and US irradiation of 1 MHz for 1 min. As a result of bubble vibration by US irradiation, vascular endothelial cell bond opened and Evans Blue leaked. Toxicity of bubble was tested in rats. No toxicity was found after a single injection in the dose range tested. No serious toxicity was seen after repeated injections (one daily injection during 15 days).

中文翻译：

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用于超声成像和治疗的冷冻干燥脂质稳定微泡制剂的放大生产、表征和毒性

摘要 微泡制剂在增强超声 (US) 成像方面有着悠久的历史，最近也用于治疗应用。此前，已经开发了一系列基于脂质 DSPC 和 DSPG 的冻干气泡配方。在这里，我们试图扩大生产过程以供未来更广泛的研究。通过在中等大小 (300–500 mL) 均质器中在全氟丙烷气氛中均质脂质分散体来制备气泡，然后冷冻干燥以获得更好的储存稳定性。总共制备了 300 个冻干小瓶。气泡的性质与之前在实验室规模上制备的相似，不同之处在于它们略大并且稳定性更好。重新构成冻干气泡后重新捕获的气体浓度为 9.4 µL/µmol 脂质。冷冻干燥前新鲜气泡的重新捕获率为 72.3%（13.0 µL/µmol 脂质）。体外水中重组冻干气泡的 US 成像信号的半衰期短于新鲜气泡的半衰期（2.7 分钟 vs. 3.3 分钟）。结合气泡和 1 MHz 的 US 照射 1 分钟后，观察到与白蛋白结合的伊文思蓝从小鼠耳血管泄漏。由于美国辐射引起的气泡振动，血管内皮细胞键打开，伊文思蓝泄漏。在大鼠中测试了气泡的毒性。在测试的剂量范围内单次注射后未发现毒性。重复注射后未见严重毒性（15 天内每天注射一次）。体外水中重组冻干气泡的 US 成像信号的半衰期短于新鲜气泡的半衰期（2.7 分钟 vs. 3.3 分钟）。结合气泡和 1 MHz 的 US 照射 1 分钟后，观察到与白蛋白结合的伊文思蓝从小鼠耳血管泄漏。由于美国辐射引起的气泡振动，血管内皮细胞键打开，伊文思蓝泄漏。在大鼠中测试了气泡的毒性。在测试的剂量范围内单次注射后未发现毒性。重复注射后未见严重毒性（15 天内每天注射一次）。体外水中重组冻干气泡的 US 成像信号的半衰期短于新鲜气泡的半衰期（2.7 分钟 vs. 3.3 分钟）。结合气泡和 1 MHz 的 US 照射 1 分钟后，观察到与白蛋白结合的伊文思蓝从小鼠耳血管泄漏。由于美国辐射引起的气泡振动，血管内皮细胞键打开，伊文思蓝泄漏。在大鼠中测试了气泡的毒性。在测试的剂量范围内单次注射后未发现毒性。重复注射后未见严重毒性（15 天内每天注射一次）。在气泡和 US 照射 1 MHz 1 分钟后观察小鼠耳血管。由于美国辐射引起的气泡振动，血管内皮细胞键打开，伊文思蓝泄漏。在大鼠中测试了气泡的毒性。在测试的剂量范围内单次注射后未发现毒性。重复注射后未见严重毒性（15 天内每天注射一次）。在气泡和 US 照射 1 MHz 1 分钟后观察小鼠耳血管。由于美国辐射引起的气泡振动，血管内皮细胞键打开，伊文思蓝泄漏。在大鼠中测试了气泡的毒性。在测试的剂量范围内单次注射后未发现毒性。重复注射后未见严重毒性（15 天内每天注射一次）。