In vivo cell tracking of therapeutic, tumor, and endothelial cells is an emerging field and a promising technique for imaging cardiovascular disease and cancer development. Site-specific labeling of endothelial cells with the MRI contrast agent superparamagnetic iron oxide (SPIO) in the absence of toxic agents is challenging. Therefore, the aim of this in vitro study was to find optimal parameters for efficient and safe SPIO-labeling of endothelial cells using ultrasound-activated CD31-targeted microbubbles for future MRI tracking. Ultrasound at a frequency of 1 MHz (10,000 cycles, repetition rate of 20 Hz) was used for varying applied peak negative pressures (10–160 kPa, i.e. low mechanical index (MI) of 0.01–0.16), treatment durations (0–30 s), time of SPIO addition (-5 min– 15 min with respect to the start of the ultrasound), and incubation time after SPIO addition (5 min– 3 h). Iron specific Prussian Blue staining in combination with calcein-AM based cell viability assays were applied to define the most efficient and safe conditions for SPIO-labeling. Optimal SPIO labeling was observed when the ultrasound parameters were 40 kPa peak negative pressure (MI 0.04), applied for 30 s just before SPIO addition (0 min). Compared to the control, this resulted in an approximate 12 times increase of SPIO uptake in endothelial cells in vitro with 85% cell viability. Therefore, ultrasound-activated targeted ultrasound contrast agents show great potential for effective and safe labeling of endothelial cells with SPIO.

体内对治疗性，肿瘤性和内皮细胞的细胞追踪是新兴领域，也是对心血管疾病和癌症发展成像的有前途的技术。在没有毒剂的情况下，使用MRI造影剂超顺磁性氧化铁（SPIO）对内皮细胞进行位点特异性标记是具有挑战性的。因此，本体外试验的目的这项研究的目的是找到使用超声激活的CD31靶向微泡对内皮细胞进行高效，安全的SPIO标记的最佳参数，以用于将来的MRI跟踪。超声以1 MHz的频率（10,000个周期，重复频率为20 Hz）用于改变施加的峰值负压（10–160 kPa，即低机械指数（MI）为0.01–0.16），治疗时间（0–30） s），添加SPIO的时间（相对于超声开始为-5分钟至15分钟），以及添加SPIO后的孵育时间（5分钟至3小时）。铁特异性普鲁士蓝染色与基于钙黄绿素-AM的细胞生存力测定相结合，用于定义最有效，最安全的SPIO标记条件。当超声参数为40 kPa峰值负压（MI 0.04）时，观察到最佳的SPIO标记，在添加SPIO之前（0分钟）应用了30 s。与对照相比，内皮细胞中SPIO摄取增加了约12倍体外具有85％的细胞活力。因此，超声激活的靶向超声造影剂在利用SPIO进行内皮细胞的有效和安全标记方面显示出巨大潜力。