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Bioengineering

用于实时评估金属有机框架毒理学特征的电池-基底传感

Published: May 26, 2023
doi:
10.3791/65313
, ,
1Department of Chemical and Biomedical Engineering,West Virginia University, 2Department of Anthropology for Energy,West Virginia University, 3Department of Hospitality and Tourism,West Virginia University

Summary

以下研究利用电池-基板阻抗传感（ECIS）评估所选金属有机框架的毒理学特征，这是一种实时，高通量筛选技术。

Abstract

金属-有机骨架（MOFs）是通过金属离子和有机接头在有机溶剂中的配位形成的杂化物。MOFs在生物医学和工业应用中的实施引起了对其安全性的担忧。在此，在暴露于人肺上皮细胞时评估了选定的MOF（沸石咪唑框架）的轮廓。评估平台是一种实时技术（即电池-基板阻抗传感[ECIS]）。本研究确定并讨论了所选MOF对暴露细胞的一些有害影响。此外，这项研究证明了使用实时方法与其他生化测定相比进行全面细胞评估的好处。该研究得出的结论是，观察到的细胞行为变化可能暗示暴露于不同物理化学特征的MOFs以及正在使用的这些框架的剂量时可能引起的毒性。通过了解细胞行为的变化，人们预见了通过专门定制其物理化学特性来改进MOFs用于生物医学应用的安全设计策略的能力。

Introduction

金属-有机骨架（MOF）是通过金属离子和有机接头1,2在有机溶剂中结合而形成的杂化物。由于这种组合的多样性，MOFs具有结构多样性3，可调孔隙率，高热稳定性和高表面积4,5。这些特性使它们在各种应用中具有吸引力的候选者，从气体储存6,7到催化8,9，以及从造影剂10,11到药物输送单元12,13。然而，在此类应用中实施MOF引起了人们对其对用户和环境安全性的担忧。例如，初步研究表明，当细胞暴露于用于MOF合成的金属离子或接头时，细胞功能和生长会发生变化1,14,15。例如，Tamames-Tabar等人证明，ZIF-8 MOF，一种基于Zn的MOF，相对于基于Zr和Fe的MOF，导致人宫颈癌细胞系（HeLa）和小鼠巨噬细胞系（J774）的更多细胞变化。这种效应可能是由于ZIF-8的金属成分（即Zn），它可能在框架崩解和Zn离子释放1时诱导细胞凋亡。同样，Gandara-Loe等人证明，当浓度为10μg/mL或更高时，基于铜的MOF的HKUST-1导致小鼠视网膜母细胞瘤细胞活力的降低最高。这可能是由于在该框架的合成过程中掺入的Cu金属离子，一旦释放，可能会在暴露的细胞中诱导氧化应激15。

此外，分析表明，暴露于具有不同理化特征的MOFs会导致暴露细胞的不同反应。例如，Wagner等人证明，用于永生化人支气管上皮细胞暴露的ZIF-8和MIL-160（一种基于Al的框架）导致细胞反应取决于框架的物理化学性质，即疏水性，大小和结构特征16。作为补充，Chen等人证明，暴露于人正常肝细胞（HL-7702）的浓度为160μg/mL的MIL-100（Fe）导致细胞活力的最大损失，可能是由于该特定框架的金属成分（即Fe17）。

虽然这些研究根据其物理化学特性和暴露浓度对MOFs对细胞系统的有害影响进行分类，从而引发了对框架实施的潜在关注，特别是在生物医学领域，但大多数这些评估都是基于单时间点比色测定。例如，结果表明，当使用（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物）四唑（MTT）和水溶性四唑盐（WST-1）测定时，这些生化试剂在与细胞也暴露于的颗粒相互作用时可能导致假阳性18。四唑盐和中性红试剂在颗粒表面具有很高的吸附或结合亲和力，导致试剂信号干扰19。此外，对于其他类型的测定，例如流式细胞术，以前被证明用于评估暴露于MOFs 20,21的细胞的变化，研究表明，如果要考虑对颗粒的有害影响进行可行的分析，则必须避免主要问题。特别是，必须解决颗粒大小的检测范围，特别是在混合群体中，例如MOF提供的检测范围或细胞变化前用于校准的颗粒参考22。还表明，在细胞标记期间用于此类细胞术测定的染料也可能干扰细胞暴露于23的纳米颗粒。

本研究的目的是使用实时、高通量评估测定来评估暴露于选定 MOF 时细胞行为的变化。实时评估有助于深入了解与曝光窗口相关的时间相关效应16.此外，它们提供了有关细胞 – 底物相互作用，细胞形态和细胞 – 细胞相互作用的变化的信息，以及这些变化如何取决于感兴趣的材料的物理化学性质和暴露时间分别为24,25。

为了证明所提方法的有效性和适用性，使用了人支气管上皮（BEAS-2B）细胞、ZIF-8（沸石咪唑酸盐16的疏水框架）和电池-基底阻抗传感（ECIS）。BEAS-2B细胞代表肺暴露的模型26，以前已用于评估细胞暴露于纳米粘土及其热降解副产物26,27,28时的变化，以及评估纳米材料的毒性，例如单壁碳纳米管（SWCNTs）18。此外，这些细胞已被用作肺上皮功能的模型30多年29。之所以选择ZIF-8，是因为其在催化30中广泛应用，并作为造影剂31用于生物成像和药物递送32，因此在此类应用中具有扩展的肺部暴露潜力。最后，ECIS是一种非侵入性实时技术，以前用于实时评估细胞粘附性、增殖、运动性和形态的变化16,26，这是分析物（材料和药物）与暴露细胞之间各种相互作用的结果16,18,28.ECIS使用交流电（AC）来测量固定在金电极上的电池的阻抗，阻抗变化可以深入了解电池 – 金基板界面处电阻和电容的变化，由细胞 – 细胞相互作用引起的屏障功能以及此类金电极的过电池层覆盖率33,34。使用ECIS允许以非侵入性，实时的方式以纳米级分辨率进行定量测量26,34。

本研究评估并比较了实时评估MOF诱导的细胞行为变化的简单性和易用性与单点测定评估。这样的研究可以进一步外推，以评估细胞谱，以响应暴露于其他感兴趣的颗粒，从而允许安全的设计颗粒测试并随后帮助实施。此外，这项研究可以补充单点评估的遗传和细胞测定。这可能导致对颗粒对细胞群的有害影响的更明智的分析，并可用于以高通量方式筛选此类颗粒的毒性16,35,36。

Protocol

1. ZIF-8合成 在本例中，使用1：10：100（金属：接头：溶剂）的质量比来合成ZIF-8。为此，测量出六水合硝酸锌，并记录测量结果。利用示例质量比计算接头、2-甲基咪唑和溶剂（即甲醇）所需的量。 将六水合硝酸锌和接头放入两个不同的玻璃小瓶中。将计算量的甲醇量的一半加入六水合硝酸锌中，另一半加入接头中。让每种溶液溶解。 将两种溶液（即分别含有?…

Representative Results

本研究使用常见的 体外 模型细胞系39 （BEAS-2B），旨在证明ECIS评估暴露于实验室合成的MOF后细胞行为变化的可行性和适用性。这些变化评估通过常规比色测定的分析得到补充。 首先评估框架的物理化学特性，以确保所采用方法的可重复性，获得的结果的有效性以及此类结果的相关讨论。例如，ZIF-8的表面形貌分析是通过SEM进行的;图像显示，这些框架?…

Discussion

先前的分析表明，ECIS可用于评估暴露于分析物（即碳纳米管35，药物43或纳米粘土16）的细胞的行为。此外，Stuekle等人使用ECIS评估暴露于纳米粘土及其副产物的BEAS-2B细胞的毒性，发现细胞行为和附着取决于此类材料的物理化学特性42。在此，我们提出确定BEAS-2B细胞响应暴露于MOFs的可能变化，从而有助于旨在区分ZIF-8对 体?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作部分由国家普通医学科学研究所（NIGMS）T32计划（T32 GM133369）和国家科学基金会（NSF 1454230）资助。此外，西弗吉尼亚大学共享研究设施和应用生物物理学的援助和支持也得到了认可。

Materials

 4-[3-(4-idophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1 assay)  Roche 5015944001
0.25% Trypsin-EDTA (1x) Gibco 25255-056
100 mm plates Corning 430167
1300 Series A2 biofume hood Thermo Scientific 323TS
2510 Branson bath sonicator Process Equipment & Supply, Inc.  251OR-DTH
2-methylimidazole, 97% Alfa Aesar 693-98-1
5 mL sterile microtube Argos Technologies T2076S-CA
50 mL  tubes  Falcon 352098
96W10idf well plates Applied Biophysics  96W10idf PET
96-well plates Fisherbrand FB012931
Biorender Biorender N/A
Countess cell counting chamber slides Invitrogen C10283
Countess II FL automated cell counter Life Technologies C0916-186A-0303
Denton Desk V sputter and carbon coater Denton Vacuum N/A
Dimethly sulfoxide  Corning 25-950-CQC
DPBS/Modified Cytiva SH30028.02
Dulbecco's modified Eagle medium Corning 10-014-CV
ECIS-ZΘ Applied Biophysics  ABP 1129
Excel Microsoft Version 2301
Falcon tubes (15 mL) Corning 352196
Fetal bovine serum Gibco 16140-071
FLUOstar OPTIMA plate reader BMG LABTECH 413-2132
GraphPad Prism Software (9.0.0) GraphPad Software, LLC Version 9.0.0
HERAcell 150i CO2 Incubator Thermo Scientific 50116047
Hitachi S-4700 Field emission scanning electron microscope equipped with energy dispersive X-ray  Hitachi High-Technologies Corporation S4700 and EDAX TEAM analysis software
ImageJ software National Institutes of Health N/A
Immortalized human bronchial epithelial cells American Type Culture Collection CRL-9609
Isotemp freezer Fisher Scientific 
Methanol, 99% Fisher Chemical 67-56-1
Parafilm sealing film The Lab Depot HS234526A
Penicillin/Steptomycin Gibco 15140-122
Sorvall Legend X1R Centrifuge  Thermo Scientific 75004220
Sorvall T 6000B DU PONT  T6000B
Trypan blue, 0.4% solution in PBS MP Biomedicals, LLC 1691049
Vacuum Chamber Belart 999320237
Zinc Nitrate Hexahydrate, 98% extra pure Acros Organic 101-96-18-9
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Tags

Metal-organic FrameworksMOFsElectric Cell-substrate Impedance SensingECISHuman Lung Epithelial CellsToxicological ProfileReal-time MonitoringHigh-throughputNanomaterialsSafetyBiomedical Applications
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Rose, O. L., Arnold, M., Dinu, C. Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles. J. Vis. Exp. (195), e65313, doi:10.3791/65313 (2023).
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