Supplementary file 1_αvβ3-targeted gas vesicles for ultrasound molecular imaging of tumors.docx

PurposeTo achieve ultrasound molecular imaging (UMI) of various tumor types, this study developed a novel integrin-targeted probe, RGD-hGVs, based on gas vesicles (GVs) derived from Halobacterium salinarum NRC-1 (Halo) and cyclic RGDfK peptides. The application potentials of UMI were evaluated in osteosarcoma and melanoma.
Materials and MethodsRGD-hGV acoustic nanoprobes were constructed by conjugating Halo-derived GVs with cyclic RGD peptides. Morphology was characterized by PCM and TEM, and hydrodynamic size and zeta potential were measured. In vitro targeting to bEnd.3, K7M2, and B16-F10 cells was evaluated by flow cytometry and confocal microscopy. Ultrasound imaging was performed in osteosarcoma and melanoma models, followed by immunofluorescence staining for αvβ3 and probe distribution. Biocompatibility was assessed by hemolysis, CCK-8 assays, serum biochemistry, and H&E staining of major organs.
ResultsRGD-hGVs (∼240 nm) demonstrated targeted binding to tumor cells (bEnd.3, K7M2, B16-F10) in vitro and in vivo. They produced stronger, longer-lasting tumor ultrasound signals than non-targeted controls, with immunofluorescence confirming localization to tumor vasculature and cells, and showed no significant toxicity.
ConclusionRGD-hGVs may serve as a novel UMI probe, suitable for diagnosing multiple tumors.

**研究目的** 为实现多种肿瘤类型的超声分子成像（ultrasound molecular imaging，UMI），本研究基于来自盐杆菌NRC-1株（Halobacterium salinarum NRC-1，Halo）的气囊（gas vesicles，GVs）与环状RGDfK肽，开发了一种新型整合素靶向探针RGD-hGVs，并在骨肉瘤与黑色素瘤模型中评估了超声分子成像的应用潜力。

**材料与方法** 通过将盐杆菌来源的气囊与环状RGD肽偶联，构建RGD-hGV声学纳米探针。采用相位对比显微镜（PCM）与透射电子显微镜（Transmission Electron Microscope，TEM）对其形态进行表征，并检测其流体力学粒径与ζ电位。通过流式细胞术与共聚焦显微镜评估其对bEnd.3、K7M2及B16-F10细胞的体外靶向能力。在骨肉瘤与黑色素瘤模型中开展超声成像实验，随后对整合素αvβ3进行免疫荧光染色并观察探针的组织分布。通过溶血实验、CCK-8细胞增殖实验、血清生化检测及主要脏器苏木精-伊红（Hematoxylin-Eosin，H&E）染色评估其生物相容性。

**结果** RGD-hGVs（粒径约240 nm）在体外与体内均能靶向结合肿瘤细胞（bEnd.3、K7M2、B16-F10）；其产生的肿瘤超声信号较非靶向对照组更强且持续时间更久，免疫荧光实验证实其定位于肿瘤血管与肿瘤细胞，且未表现出明显毒性。

**结论** RGD-hGVs可作为一种新型超声分子成像探针，适用于多种肿瘤的诊断。