Synthesis and Evaluation of Clinically Translatable Targeted Microbubbles Using a Microfluidic Device for In Vivo Ultrasound Molecular Imaging.
Synthesis and Evaluation of Clinically Translatable Targeted Microbubbles Using a Microfluidic Device for In Vivo Ultrasound Molecular Imaging.
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使用微流体装置的体内超声分子成像的临床转换靶向微泡的合成和评估。
DOI：
10.3390/ijms24109048
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发表时间：
2023-05-20
影响因子：
5.6
通讯作者：
中科院分区：
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本研究的主要目的是使用微流体装置合成用工程蛋白质配体功能化的对比微泡 (MB)，以体内靶向乳腺癌特异性血管 B7-H3 受体，以进行超声诊断成像。我们使用针对人/小鼠 B7-H3 受体选择的高亲和力亲和体 (ABY) 来工程化靶向 MB (TMB)。我们向该 ABY 配体引入了 C 端半胱氨酸残基，以促进与 DSPE-PEG-2K-马来酰亚胺 (M. Wt = 2.9416 kDa) 磷脂的位点特异性缀合，用于 MB 制剂。我们优化了生物共轭反应条件，并将其应用于使用 DSPE-PEG-ABY 和 DPPC 脂质体（5:95 摩尔%）的 TMB 微流体合成。通过流室测定在表达人 B7-H3 (MS1B7-H3) 的 MS1 内皮细胞中体外测试 TMB 与 B7-H3 (MBB7-H3) 的结合亲和力，并通过免疫染色在转基因小鼠模型 (FVB/N-Tg (MMTV-PyMT)634Mul/J) 的乳腺肿瘤中测试 TMB 与 B7-H3 (MBB7-H3) 的结合亲和力，在血管内皮细胞中表达小鼠 B7-H3分析。我们成功地优化了使用微流体系统生成 TMB 所需的条件。合成的MB对MS1细胞表现出更高的亲和力，该细胞被设计为表达更高水平的hB7-H3，并且在活体动物中注射TMB后，在小鼠肿瘤组织的内皮细胞中也表现出更高的亲和力。与野生型对照细胞（MS1WT；36.2 ± 7.5/FOV）相比，MBB7-H3 与 MS1B7-H3 细胞结合的平均数量（平均值±标准差）估计为每视野 (FOV) 354.4 ± 52.3。非靶向 MB 对两种细胞均未表现出任何选择性结合亲和力（MS1B7-H3 为 37.7 ± 7.8/FOV，MS1WT 细胞为 28.3 ± 6.7/FOV）。经离体免疫荧光分析验证，荧光标记的 MBB7-H3 在体内全身注射后共定位于肿瘤血管，表达 B7-H3 受体。我们通过微流控装置成功合成了一种新型MBB7-H3，这使我们能够按需生产用于临床应用的TMB。这种临床可翻译的MBB7-H3在体外和体内均表现出与表达B7-H3的血管内皮细胞显着的结合亲和力，这显示了其作为人类应用的分子超声造影剂的临床转化潜力。
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 (MBB7-H3) was tested in vitro in MS1 endothelial cells expressing human B7-H3 (MS1B7-H3) 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 MBB7-H3 binding to MS1B7-H3 cells was estimated to be 354.4 ± 52.3 per field of view (FOV) compared to wild-type control cells (MS1WT; 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 MS1B7-H3 and 28.3 ± 6.7/FOV for MS1WT cells). The fluorescently labeled MBB7-H3 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 MBB7-H3 via microfluidic device, which allows us to produce on demand TMBs for clinical applications. This clinically translatable MBB7-H3 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.
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影响因子：
6.7
作者：
Anderson CR;Rychak JJ;Backer M;Backer J;Ley K;Klibanov AL
通讯作者：
Klibanov AL
影响因子：
4.7
作者：
Bam, Rakesh;Laffey, Makenna;Wilson, Katheryne E.
通讯作者：
Wilson, Katheryne E.
影响因子：
5.6
作者：
Calabro, Luana;Sigalotti, Luca;Maio, Michele
通讯作者：
Maio, Michele
影响因子：
4.7
作者：
Wright, Thaiesha A.;Rahman, Monica Sharfin;Bennett, Camaryn;Johnson, Madolynn R.;Fischesser, Henry;Ram, Natasha;Tyler, Amoni;Page, Richard C.;Konkolewicz, Dominik
通讯作者：
Konkolewicz, Dominik
DOI：
10.1007/978-1-0716-1617-8_15
发表时间：
2021
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
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作者：
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