Canary Center for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine, Palo Alto, CA 94304, USA.
Int J Mol Sci. 2023 May 20;24(10):9048. doi: 10.3390/ijms24109048.
The main aim of this study is to synthesize contrast microbubbles (MB) functionalized with engineered protein ligands using a microfluidic device to target breast cancer specific vascular B7-H3 receptor in vivo for diagnostic ultrasound imaging. We used a high-affinity affibody (ABY) selected against human/mouse B7-H3 receptor for engineering targeted MBs (TMBs). We introduced a C-terminal cysteine residue to this ABY ligand for facilitating site-specific conjugation to DSPE-PEG-2K-maleimide (M. Wt = 2.9416 kDa) phospholipid for MB formulation. We optimized the reaction conditions of bioconjugations and applied it for microfluidic based synthesis of TMBs using DSPE-PEG-ABY and DPPC liposomes (5:95 mole %). The binding affinity of TMBs to B7-H3 (MB) was tested in vitro in MS1 endothelial cells expressing human B7-H3 (MS1) by flow chamber assay, and by ex vivo in the mammary tumors of a transgenic mouse model (FVB/N-Tg (MMTV-PyMT)634Mul/J), expressing murine B7-H3 in the vascular endothelial cells by immunostaining analyses. We successfully optimized the conditions needed for generating TMBs using a microfluidic system. The synthesized MBs showed higher affinity to MS1 cells engineered to express higher level of hB7-H3, and in the endothelial cells of mouse tumor tissue upon injecting TMBs in a live animal. The average number (mean ± SD) of MB binding to MS1 cells was estimated to be 354.4 ± 52.3 per field of view (FOV) compared to wild-type control cells (MS1; 36.2 ± 7.5/FOV). The non-targeted MBs did not show any selective binding affinity to both the cells (37.7 ± 7.8/FOV for MS1 and 28.3 ± 6.7/FOV for MS1 cells). The fluorescently labeled MB upon systemic injection in vivo co-localized to tumor vessels, expressing B7-H3 receptor, as validated by ex vivo immunofluorescence analyses. We have successfully synthesized a novel MB via microfluidic device, which allows us to produce on demand TMBs for clinical applications. This clinically translatable MB showed significant binding affinity to vascular endothelial cells expressing B7-H3 both in vitro and in vivo, which shows its potential for clinical translation as a molecular ultrasound contrast agent for human applications.
这项研究的主要目的是使用微流控装置合成与工程蛋白配体结合的对比微泡（MB），以在体内靶向乳腺癌特异性血管 B7-H3 受体进行诊断超声成像。我们使用针对人/鼠 B7-H3 受体的高亲和力亲和体（ABY）来工程靶向 MB（TMB）。我们在该 ABY 配体中引入了一个 C 末端半胱氨酸残基，以促进与 DSPE-PEG-2K-马来酰亚胺（MWt = 2.9416 kDa）磷脂的定点缀合，用于 MB 配方。我们优化了生物偶联的反应条件，并将其应用于使用 DSPE-PEG-ABY 和 DPPC 脂质体（5:95 摩尔%）的基于微流控的 TMB 合成。通过流式细胞仪分析，在表达人 B7-H3 的 MS1 内皮细胞（MS1）中以及通过免疫染色分析，在表达鼠 B7-H3 的血管内皮细胞的转基因小鼠模型（FVB/N-Tg（MMTV-PyMT）634Mul/J）的乳腺肿瘤中，体外测试了 TMB 与 B7-H3（MB）的结合亲和力。我们成功优化了使用微流控系统生成 TMB 所需的条件。合成的 MB 显示出与表达更高水平 hB7-H3 的 MS1 细胞以及在活体动物中注射 TMB 后在小鼠肿瘤组织的内皮细胞更高的亲和力。估计结合到 MS1 细胞的 MB 的平均数量（平均值±SD）为 354.4±52.3 个/视场（FOV），而野生型对照细胞（MS1；36.2±7.5/FOV）。非靶向 MB 对两种细胞均没有表现出任何选择性结合亲和力（MS1 为 37.7±7.8/FOV，MS1 细胞为 28.3±6.7/FOV）。体内系统注射后，荧光标记的 MB 与表达 B7-H3 受体的肿瘤血管共定位，通过体外免疫荧光分析得到验证。我们已经成功地通过微流控装置合成了一种新型 MB，这使得我们能够按需生产用于临床应用的 TMB。这种具有临床转化潜力的 MB 在体内外均显示出与表达 B7-H3 的血管内皮细胞的显著结合亲和力，这表明它有潜力作为人类应用的分子超声造影剂进行临床转化。
