Name: anticancer metal complexes: synthesis and cytotoxicity evaluation by the mtt assay
Uploaded: 2013-11-10T21:00:00
Description: Watch this Scientific Journal Video about Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay at JoVE.com

资金从欧洲研究委员会收到了欧盟第七框架计划（FP7/2007-2013）下/ ERC赠款协定没有[239603]

<ol><li>步骤2（2.1-2.6），如果一个手套箱不可用，跳到另一种;准备的V（V）配合物（ 图2）; 18行为步骤1.1-1.4的手套箱。 <ol><li>溶解0.42的配位体的毫摩尔的 H 2升的无水THF中，并将其添加到VO的等效量的（O I镨）在THF中的搅拌溶液3。 </li><li>在室温下15分钟搅拌反应混合物，并除去真空下挥发物。 </li><li>加冷正己烷并在真空下将其删除。暗紫色粉末应以定量收率得到?...

<ol>
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Y., Ashenhurst, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+Titanium(IV)+Complexes:+Renaissance.">Cytotoxic Titanium(IV) Complexes: Renaissance.</a> European Journal of Inorganic Chemistry. , 2203-2218 (2009).</li><li>Buettner, K. M., Valentine, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinorganic+Chemistry+of+Titanium.">Bioinorganic Chemistry of Titanium.</a> Chemical Reviews. 112, 1863-1881 (2012).</li><li>Meker, S., Margulis-Goshen, K., Weiss, E., Magdassi, S., Tshuva, E. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+and+Hydrolysis+of+trans-Ti(IV)+Complexes+of+Salen+Ligands:+Structure-Activity+Relationship+Studies.">Cytotoxicity and Hydrolysis of trans-Ti(IV) Complexes of Salen Ligands: Structure-Activity Relationship Studies.</a> Inorganic Chemistry. 51, 1796-1804 (2012).</li><li>Tshuva, E. Y., Peri, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+cytotoxic+titanium(IV)+complexes;+Insights+on+the+enigmatic+involvement+of+hydrolysis.">Modern cytotoxic titanium(IV) complexes; Insights on the enigmatic involvement of hydrolysis.</a> Coordination Chemistry Reviews. 253, 2098-2115 (2009).</li><li>Shavit, M., Peri, D., Manna, C. M., Alexander, J. S., Tshuva, E. 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A., Fakhree, M. A. A., Omidi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Toxicity+of+Nanogenomedicine+in+MCF-7+Cell+Line:+MTT+assay.">Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay.</a> Journal of Visualized Experiments. (26), e1191 (2009).</li><li>Gonzalez, R. J., Tarloff, J. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+drug+screening+with+panels+of+human+tumor+cell+lines+using+a+microculture+tetrazolium+assay.">Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.</a> Cancer Research. 48, 589-601 (1988).</li><li>Weyermann, J., Lochmann, D., Zimmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+note+on+the+use+of+cytotoxicity+assays.">A practical note on the use of cytotoxicity assays.</a> International Journal of Pharmaceutics. 288, 369-376 (2005).</li><li>Chmura, A. 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在这个手稿中描述的方法结合化学合成与生物细胞活力检测。与关于活细胞的任何生物的方法，这是至关重要的，在层流罩的工作，并保持在无菌条件，包括使用无菌有机溶剂。另外，制备的水解不稳定的金属为基础的药物，并存储应进行惰性条件下，对​​其中的手套箱或舒伦克线技术应该被利用。 该技术的选择惰性的环境下工作，主要是依赖于手套箱的可用性。如果有?...

化疗仍然是在临床上用于各种癌症疾病的治疗采用的主要课程之一，并由此研究大量的，目的是开发新的和改进的抗癌药物世界范围内进行。这样的研究大多开始在化学级，与化合物的设计和制备，接着在体外的细胞毒性生物学评价。细胞活力可以通过各种检测，提供对细胞活性1-2的信息进行评估。 顺铂是铂络合物被广泛用作化学治疗药物，它被认为是一种有效的治疗主要针对睾丸癌和卵巢癌3-4的例子。然而，其狭窄的活动范围和严重的副作用引发的其他烈性过渡金属配合物5-8的研究。其中，钛（IV）和钒（V）配合物表现出较高的活性的有希望的结果，并减少毒性9-16。钛（Ⅳ）络合物是第一顺铂后，由于这些特性，进入临床试验，但是，他们并没有审判，由于配方的困难和水解稳定性。因此，有电流需要开发对这些金属配合物可结合高抗癌活性与耐水性15,17-21改进的衍生物。 中的Ti（IV）和V的制备方法的一个挑战（V）配合物是指将前体试剂的水解不稳定性，因此，在惰性气氛应保持。钛（Ⅳ）和V（Ⅴ）化合物的制备方法为N 2或Ar条件下，在手套箱中，或使用的Schlenk线技术下进行。 用于抗癌活性的评价的一个常见方法是基于MTT（3 - （4,5 - dimethylthiazolyl）-2,5 - 二苯基溴化）测定。此法是由Mosmann的提出在1983年比色可行性分析22。它是非常充分的研究和表征，并评估新细胞毒性化合物的效力时，由于其精确度，快速性，及对多种细胞系被应用能力它被认为是高效的。此活力测定是基于当它暴露于活细胞的MTT分子的颜色变化。的吸光度，这是成正比的存活细胞的数量，并且相比于未处理的对照的测定中，使所测试的化合物的细胞生长抑制的能力的评估。 的MTT比色分析法在96孔板形式23进行。该细胞可能需要预培养在添加了测试药物的前孔中。在预温育时间可以从0-24小时，根据细胞系的属性而变化。细胞通常暴露于药物为24-96小时，这取决于药物的活性。 MTT溶液然后被加入到经处理的细胞，其中所述叫喊超重MTT是由多种线粒体和细胞质酶，操作中存活的细胞（ 图1）24减少到紫色的甲（formazan）。将MTT分子不是由死细胞或红细胞（代谢不活跃的细胞），脾细胞（静息细胞）和伴刀豆球蛋白A刺激淋巴细胞（活化细胞）22减小。经过3-4小时培养用MTT甲臜沉淀。甲臜形成0.5小时培养后开始，但为获得最佳结果，最好是将细胞暴露于MTT至少3小时22。因此，在生长介质中被删除，甲溶解在有机溶剂中，优选异丙醇25，虽然DMSO也可以使用26。消除了介质是因为酚红，这是广泛通用于生长培养基中获得准确的结果，并且沉淀的蛋白质可与吸光度测定25干涉的关键。当经残溶液达到均匀，该溶液的吸光度用酶标仪分光光度计测定。在550nm处的吸光度是成正比的细胞以每孔细胞200-50,000的范围内的数目，并且因此可以检测到非常少量的细胞22。的吸光度表示存活细胞是保持与药物治疗后的金额，并进行比较了未暴露于药物的对照细胞的吸光度。通过适当的软件分析的结果提供了IC 50值（抑制浓度50％）的值及其统计误差的基础上，测定重复几次。 MTT分析是广泛通用的细胞毒性研究中用于筛选新的抗癌化合物，由于它的精度和相对简单。然而，使用MTT测定法，它依赖于酶促反应时，必须考虑各种酶抑制剂可以影响一个MTT的还原ð导致错误的结果27。此外，本MTT实验不提供对药物2的细胞毒性活性的分子机理的任何信息。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biological Industries</td>
			<td>04-007-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane AR</td>
			<td>Gadot</td>
			<td>830122313</td>
			<td>Dried using a solvent drying system</td>
		</tr>
		<tr>
			<td>HT-29 cell line</td>
			<td>ATCC</td>
			<td>HTB-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol AR</td>
			<td>Gadot</td>
			<td>830111370</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Biological Industries</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Sigma-Aldrich</td>
			<td>M5655-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin antibiotics</td>
			<td>Biological Industries</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 with phenol red with L-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI without phenol red</td>
			<td>Biological Industries</td>
			<td>01-103-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran (THF) AR</td>
			<td>Gadot</td>
			<td>830156391</td>
			<td>dried using a solvent drying system</td>
		</tr>
		<tr>
			<td>Ti(OiPr)4</td>
			<td>Sigma-Aldrich</td>
			<td>205273-500ML</td>
			<td>moisture sensitive</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Biological Industries</td>
			<td>03-052-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>VO(OiPr)3</td>
			<td>Sigma-Aldrich</td>
			<td>404926-10G</td>
			<td>moisture sensitive</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>12-channel pipette 30-300 &#956;l</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-channel pipette 5-50 &#956;l</td>
			<td>Finnpipette</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 flask</td>
			<td>NUNC</td>
			<td>156472</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate with lid (flat bottom)</td>
			<td>NUNC</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Binder</td>
			<td>APT.line C150</td>
			<td></td>
		</tr>
		<tr>
			<td>Counter chamber</td>
			<td>Marienfeld-Superior</td>
			<td>650030</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf vial</td>
			<td>KARTELL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glove box</td>
			<td>M. Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>ADS LAMINAIRE</td>
			<td>OPTIMALE 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader spectrophotometer</td>
			<td>Bio-Tek</td>
			<td>El-800</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 20-200 &#956;l</td>
			<td>Finnpipette</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 5-50 &#956;l</td>
			<td>Finnpipette</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

anticancer metal complexes: synthesis and cytotoxicity evaluation by the mtt assay

钛（IV）和钒（V）的配合物是高度有效的抗癌剂。在它们的合成方法的一个挑战是指它们的水解不稳定性，所以它们的制备方法，应在惰性气氛下进行。评价这些复合物的抗癌活性可通过MTT法来实现。 MTT测定是基于酶催还原的MTT分子为甲，当它暴露于活细胞的比色测定法的可行性。减少的结果是在MTT分子的颜色变化。吸光度测量值相对于对照测定下列其与不同浓度的试验化合物，它被转换为所述化合物的抗癌活性，其IC 50值的处理剩余的存活癌细胞的百分比。 MTT法是广泛普遍的，由于其精确性，快速性，和相对简单的细胞毒性研究。 在此，我们预发送一个详细的协议，用于合成气敏感金属类药物和细胞活力的测量，包括制备的细胞板的，该化合物与细胞温育后，使用MTT测定法，并测定IC 50值的可行性测量。

根据既定程序18,28，它们的纯度可以用NMR和元素分析来评估制备的复合物。 从MTT测定接收到的数据进行分析，以评估化合物18,21的细胞毒性。首先，执行从所有其他值减法空白对照吸光度值。第二，在THF溶剂中的控制值被设置为100％生存力，因为没有生长抑制化合物的溶液。根据控制所有其它值被变换成活力的百分比。的浓度应用和计算出的相对存活率百分比...

Watch this Scientific Journal Video about Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay at JoVE.com

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

一种用于合成气敏感的钛和钒的抗癌剂的方法进行说明，随着朝向通过MTT测定人肿瘤细胞系的细胞毒活性进行评价。

抗癌金属配合物的合成与细胞毒性评价MTT法

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Research

JoVE Journal

Chemistry

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

科研

化学

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

酰胺偶联反应为双吡啶基配体及其络合合成，以白金为双核抗癌药物

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

功能化的合成与表征金属 - 有机骨架

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Metal-free Synthesis of Ynones from Acyl Chlorides and Potassium Alkynyltrifluoroborate Salts

Ynones从酰氯和钾Alkynyltrifluoroborate盐无金属合成

Ynones are a valuable functional group and building block in organic synthesis. Ynones serve as a precursor to many important organic functional groups ...

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

一系列钌的合成，表征和反应<em&gt;Ñ</em&gt; -triphos<sup&gt;博士</sup&gt;配合

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

Reverse Microemulsion-mediated Synthesis of Monometallic and Bimetallic Early Transition Metal Carbide and Nitride Nanoparticles

单金属和双金属前过渡金属碳化物和氮化物纳米粒子的反相微乳液介导的合成

A reverse microemulsion is used to encapsulate monometallic or bimetallic early transition metal oxide nanoparticles in microporous silica shells. The ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

Synthesis of a Water-soluble MetalÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œOrganic Complex Array

一个水溶性金属 - 有机络合物阵列合成

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions

Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions

钯<em&gt;ñ</em&gt; - 杂环碳烯配合物：苯并咪唑盐的合成和碳 - 碳键合反应中的催化活性

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from ...

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

钌线粒体钙吸收抑制剂的合成与评价

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

Synthesis and Performance Characterizations of Transition Metal Single Atom Catalyst for Electrochemical CO2 Reduction

Synthesis and Performance Characterizations of Transition Metal Single Atom Catalyst for Electrochemical CO2 Reduction

电化学 CO2还原的过渡金属单原子催化剂的合成及性能表征

This protocol presents both the synthesis method of the Ni single atom catalyst, and the electrochemical testing of its catalytic activity and selectivity ...