一种生物工程螯合蛋白 B 敏感气体囊泡纳米系统，可随超声波生物显微镜图像灰度级强度的增加而响应。

IF 2.4 3区医学 Q2 ACOUSTICS

Ultrasound in Medicine and Biology Pub Date : 2024-10-10 DOI:10.1016/j.ultrasmedbio.2024.09.015

Felipe Vianna Garrute , Ana Beatriz F. Pacheco , George J. Lu , João Carlos Machado

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引用次数: 0

摘要

目的：本研究旨在促进改良气囊（GV）与凝血酶 B（CTSB）蛋白酶的相互作用，并通过超声波（US）分析其背向散射信号：方法：我们修改了编码 GvpC 的基因序列，使其包含 CTSB 分裂，并在大肠杆菌重组系统中表达了该蛋白。纯化后的蛋白质被加入到去除了原始 GvpC 的 GVs 制剂（ΔGV）中，形成了修饰 GV（GV*）。用 Western 印迹测试比较了开始（T0）和与 CTSB 反应 24 小时（T24）后带有 GvpC 和工程化 GvpC 的 GVs。21 MHz US B-模式和非线性对比模式（总功率为 5%）对具有 GVwt、ΔGV（剥离 GV）、GV* 和 CTSB + GV* 样品的 US 模型进行成像。此外，21 MHz US B-mode还对具有肿瘤细胞系胞外部分（TCEF）和TCEF + GV*样本的US模型进行了成像。100%的全 US 功率用于折叠 GV 结构：在 Western 印迹上，我们检测到 GV* 与 CTSB 培养 24 小时后，工程 GvpC 的水平与 T0 时的浓度相比有所下降，这表明 CTSB 分解了工程 GvpC。确定了模型横截面图像上的感兴趣区，与 PBS（对照组）、GVwt、ΔGV 和 GV* 相比，B 模式图像平均灰度级强度显著增加（p < 0.05）。与含有 GVwt、ΔGV 和 GV* 的样品相比，CTSB + GV* 的非线性模式图像灰度级强度分别增加了 11.79、7.86 和 14.75 分贝。与 TCEF 培养的 GV 制剂和 TCEF + GV* 样品相比，TCEF + GVwt 的信号增加了 81%：美国后向散射信号强度的增加表明，GVs 是一种潜在的蛋白酶活性生物传感器，可能有助于检测富含蛋白酶的组织区域。

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A Bioengineered Cathepsin B-sensitive Gas Vesicle Nanosystem That Responds With Increased Gray-level Intensity of Ultrasound Biomicroscopic Images

Objective

This work aimed to promote the interaction of a modified gas vesicle (GV) with cathepsin B (CTSB) protease and analysed their backscattered signal by ultrasound (US).

Methods

We modified the sequence of the gene coding for GvpC to contain a CTSB cleavage and expressed the protein in an Escherichia coli recombinant system. The protein was purified and added to GVs preparations in which the original GvpC was removed (ΔGV), constituting the modified GV (GV*). Western blot testing was used to compare GVs with GvpC and engineered GvpC at starting (T0) and after 24 h (T24) reacting with CTSB. A 21 MHz US B-mode and non-linear contrast mode (5% total power) imaged US phantoms having samples of GVwt, ΔGV (stripped GV), GV* and CTSB + GV*. Also, a 21 MHz US B-mode imaged US phantoms having a tumour cell line extracellular fraction (TCEF) and the TCEF + GV* sample. A 100% total US power was applied to collapse the GV structure.

Results

On Western blotting, we detected a decrease in engineered GvpC levels 24 h after the incubation of GV* with CTSB, compared with the concentration at T0, suggesting that CTSB cleaved the engineered GvpC. Regions-of-interest over image of phantom cross-sections were determined and the B-mode image mean grey-level intensity resulted in a significant (p < 0.05) increase comparing CTSB + GV* with PBS (control), GVwt, ΔGV and GV*. Non-linear mode image grey-level intensity from CTSB + GV* increased by 11.79, 7.86 and 14.75 dB from samples containing GVwt, ΔGV and GV*, respectively. GV preparations incubated with TCEF and the TCEF + GV* sample showed an increase of 81% in signal compared with TCEF + GVwt.

Conclusion

The increased US backscattered signal intensity suggests GVs as a potential biosensor for protease activity, possibly aiding the detection of protease-rich tissue regions.

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来源期刊

Ultrasound in Medicine and Biology 医学-核医学

CiteScore

6.20

自引率

6.90%

发文量

325

审稿时长

70 days

期刊介绍： Ultrasound in Medicine and Biology is the official journal of the World Federation for Ultrasound in Medicine and Biology. The journal publishes original contributions that demonstrate a novel application of an existing ultrasound technology in clinical diagnostic, interventional and therapeutic applications, new and improved clinical techniques, the physics, engineering and technology of ultrasound in medicine and biology, and the interactions between ultrasound and biological systems, including bioeffects. Papers that simply utilize standard diagnostic ultrasound as a measuring tool will be considered out of scope. Extended critical reviews of subjects of contemporary interest in the field are also published, in addition to occasional editorial articles, clinical and technical notes, book reviews, letters to the editor and a calendar of forthcoming meetings. It is the aim of the journal fully to meet the information and publication requirements of the clinicians, scientists, engineers and other professionals who constitute the biomedical ultrasonic community.