Name: Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay (Video) | JoVE Journal
Uploaded: 2013-11-10T21:00:00.000+00:00
Duration: 11 min 14 s
Description: Learn to synthesize air-sensitive titanium and vanadium anticancer agents and evaluate their cytotoxicity using the MTT assay on human cancer cell lines. This method enables precise IC50 determination for metallo drug development.

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Funding was received from the European Research Council under the European Community&#39;s Seventh Framework Programme (FP7/2007-2013) / ERC Grant agreement no&#160;[239603]

<ol>
	<li>Preparation of the V (V) complex (Figure 2);18 conduct steps 1.1-1.4 in a glove-box; if a glove-box is not available, skip to the alternative steps 2 (2.1-2.6).
	<ol>
		<li>Dissolve 0.42 mmol of the ligands H2L in dry THF and add it to a stirring solution of equivalent amounts of VO(OiPr)3 in THF.</li>
		<li>Stir the reaction mixture at room temperature for 15 min, and remove the volatiles under vacuum.</li>
		<li>Add cold hexane and remove it under vacuum. A dark purple powder should be obtained in a quantitative yield.</li>
		<li>Weigh 8 mg of the obtained compound in an Eppendorf vial. Skip to step 3.</li>
	</ol>
	</li>
	<li>Prepare the V (V) complex using a Schlenk line.
	<ol>
		<li>Dissolve 0.42 mmol of the ligand H2L in dry THF in a dry Schlenk flask with a septum cap, attach to a Schlenk line and apply alternating vacuum/inert gas (N2 or Ar) environments; it is recommended to apply three such rounds and wait 2-5 min on the vacuum stages.</li>
		<li>Add dry THF via syringe through the septum cap.</li>
		<li>Add equivalent amounts of VO(OiPr)3 in a THF solution, which had been prepared similarly by hooking a proper dry Schlenk flask to the line, applying an inert environment&#160;and adding the reagents through a syringe from the vanadium solution to that of the ligand.</li>
		<li>Stir the reaction mixture at room temperature for 15 min&#160;and remove the volatiles under vacuum; if there is a concern of unstable products, perform Schlenk type distillation of the volatiles under inert atmosphere; use inert gas flow to attach the required glassware that had been predried.</li>
		<li>Add cold hexane and remove it under vacuum. A dark purple powder should be obtained in a quantitative yield.</li>
		<li>Weigh 8 mg of the obtained compound in an Eppendorf vial.</li>
	</ol>
	</li>
	<li>Preparation of the Ti (IV) complex (Figure 2);28 conduct steps 3.1-3.3 in a glove-box; if a glove-box is not available, adjust the alternative steps discussed for the vanadium analogue (step&#160;2) that employ a Schlenk line instead.
	<ol>
		<li>Dissolve 0.35 mmol of the ligand H2L in dry THF and add it to a stirring solution of equivalent amounts of Ti(OiPr)4 in THF.</li>
		<li>Stir the reaction mixture at room temperature for 2 hr. Remove the volatiles under vacuum to give a yellow product in a quantitative yield.</li>
		<li>Weigh 8 mg of the obtained compound in Eppendorf vial.</li>
	</ol>
	</li>
	<li>Preparation of HT-29 cells 96-well plate
	<ol>
		<li>Culture HT-29 cells in a 75 cm2 flask with RPMI-1640 medium that contains 1% penicillin/streptomycin antibiotics, 1% L-glutamine, and 10% fetal bovine serum (FBS), at 37 &#176;C with 5% CO2.</li>
		<li>Remove the medium when the HT-29 cells reach maximal confluency (90-100%, every 3-4 days without subculture). Wash with 1 ml of trypsin (0.25%)/EDTA (0.05%) solution and remove it.</li>
		<li>Add 1 ml of trypsin (0.25%)/EDTA (0.05%) solution and incubate for 5 min.</li>
		<li>Detach the HT-29 cells from the flask and add 10 ml of the medium to disable the trypsin activity. Transfer 5 ml of the cells mixture to a tube.</li>
		<li>Use pipette to transfer a few drops of the cells mixture into counter chamber. The counter chamber includes 5 x&#160;5 squares.</li>
		<li>Count the cells in 5 representative squares using a microscope: the 4 squares in the corners and the one that is in the middle.</li>
		<li>Calculate the estimated amount of cells present. Sum the cells in the 5 representative squares and divide by 20.</li>
		<li>Divide 0.6 by the result of step 4.7. The received number is the amount of cell mixture required per plate. This calculation refers to a plate that contains 0.6 x&#160;106 cells (9,000 cells per well).</li>
		<li>Use pipette to transfer the amount of cell mixture required per plate (as calculated in section 4.8) and add 13.2 ml of the medium (200 &#956;l per well &#215; 66 wells).</li>
		<li>Add the mixture to 96-well plate by 11-channel pipette (200 &#956;l per well, 6 lines, 66 wells in total). One well of each line should remain empty for blank control.</li>
		<li>Incubate the plate at 37 &#176;C with 5% CO2 for 24 hr to allow the cells to attach to the plate.</li>
	</ol>
	</li>
	<li>Insertion of the compounds
	<ol>
		<li>Add 200 &#956;l (or different amount according to the desired concentration range) of dry THF to 8 mg of each compound weighed as described in step 1.4 (or 2.6) or 3.3. Weigh 8 mg of the ligand H2L, too.</li>
		<li>Dilute each compound solution further by adding 60 &#956;l of THF in 9 different Eppendorf vials.</li>
		<li>Transfer with a pipette 60 &#956;l from the mixture in section 5.1 to the first Eppendorf vial, resuspend and transfer 60 &#956;l to the next vial, and so on until 10 different concentrations are obtained (including the parent mixture in section 5.1).</li>
		<li>Dilute 20 &#956;l of each concentration in THF with 180 &#956;l of medium.</li>
		<li>Prepare a control solution with THF only; 20 &#956;l of THF with 180 &#956;l of medium in each measurement.</li>
		<li>Add 10 &#956;l from the resulting solution (including the THF control), to each well that already contains 200 &#956;l of the aforementioned solution of cells in the medium to give final concentrations of the compound of up to 200 mg/L (the concentration range can be changed according to the compound activity).</li>
		<li>You may observe some precipitation at the highest concentration applied depending on the compound solubility.</li>
		<li>Repeat each measurement of the compound 3x (3 lines in the plate of the same compound); each line contains: cells treated with the compound in 10 different concentrations, 1 well containing cells treated with solvent only for control and 1 well without cells for blank control.</li>
		<li>Incubate the loaded plate for 3 days at 37 &#176;C in 5% CO2 atmosphere.</li>
	</ol>
	</li>
	<li>Cytotoxicity measurement using the MTT assay
	<ol>
		<li>Prepare the MTT solution by adding 1 g of MTT powder to a 200 ml solution of RPMI-1640 medium without phenol red. The stock solution can be divided to 15 ml tubes and stored in -20 &#176;C.</li>
		<li>Add 20 &#956;l of MTT solution to each well using 11-channel pipette (except to the blank control wells without the cells from step 4.10), and incubate the plate for additional 3 hr.</li>
		<li>Remove the medium solution from each well.</li>
		<li>Add 200 &#956;l of isopropanol to each well to dissolve formazan. Stir the plate for 0.5 hr until homogeneousness is reached.</li>
		<li>Measure the absorbance at 550 nm for 200 &#956;l of the aforementioned solution by a microplate reader spectrophotometer.</li>
		<li>Repeat each measurement (that includes 3 repetitions, see step 5.8) on 3 different days.</li>
		<li>Calculate the percentage of cell viability by deducting the blank control absorbance value from the measured value, dividing the result by the absorbance of the THF solvent control value and multiplying by 100%.</li>
		<li>Calculate IC50 values using GraphPad Prism software (or equivalent).</li>
		<li>The reported IC50 is the average of all IC50 values collected on at least three different days, and the error value is the standard deviation.</li>
	</ol>
	</li>
</ol>

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<li>Bruijnincx, P. C., Sadler, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+trends+for+metal+complexes+with+anticancer+activity%5BTitle%5D)+AND+%22Current+Opinion+in+Chemical+Biology%22%5BJournal%5D)">New trends for metal complexes with anticancer activity.</a> Current Opinion in Chemical Biology. 12, 197-206 (2008).</li>
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<li>Desoize, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metals+and+metal+compounds+in+cancer+treatment%5BTitle%5D)+AND+%22Anticancer+Research%22%5BJournal%5D)">Metals and metal compounds in cancer treatment.</a> Anticancer Research. 24, 1529-1544 (2004).</li>
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<li>Meléndez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Titanium+complexes+in+cancer+treatment%5BTitle%5D)+AND+%22Critical+Reviews+in+Oncology%2FHematology%22%5BJournal%5D)">Titanium complexes in cancer treatment.</a> Critical Reviews in Oncology/Hematology. 42, 309-315 (2002).</li>
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<li>Djordjevic, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antitumor+activity+of+vanadium+compounds%5BTitle%5D)+AND+%22Metal+ions+in+biological+systems%22%5BJournal%5D)">Antitumor activity of vanadium compounds.</a> Metal ions in biological systems. 31, 595-616 (1995).</li>
<li>Caruso, F., Rossi, M., Pettinari, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anticancer+titanium+agents%5BTitle%5D)+AND+%22Expert+Opinion+on+Therapeutic+Patents%22%5BJournal%5D)">Anticancer titanium agents.</a> Expert Opinion on Therapeutic Patents. 11, 969-979 (2001).</li>
<li>Caruso, F., Rossi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antitumor+titanium+compounds%5BTitle%5D)+AND+%22Mini-Review+in+Medicinal+Chemistry%22%5BJournal%5D)">Antitumor titanium compounds.</a> Mini-Review in Medicinal Chemistry. 4, 49-60 (2004).</li>
<li>Kostova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Titanium+and+vanadium+complexes+as+anticancer+agents%5BTitle%5D)+AND+%22Anti-Cancer+Agents+in+Medicinal+Chemistry%22%5BJournal%5D)">Titanium and vanadium complexes as anticancer agents.</a> Anti-Cancer Agents in Medicinal Chemistry. 9, 827-842 (2009).</li>
<li>Tshuva, E. Y., Ashenhurst, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxic+Titanium%28IV%29+Complexes%3A+Renaissance%5BTitle%5D)+AND+%22European+Journal+of+Inorganic+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Bioinorganic+Chemistry+of+Titanium%5BTitle%5D)+AND+%22Chemical+Reviews%22%5BJournal%5D)">Bioinorganic Chemistry of Titanium.</a> Chemical Reviews. 112, 1863-1881 (2012).</li>
<li>Meker, S., Margulis-Goshen, K., Weiss, E., Magdassi, S., Tshuva, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+antitumor+activity+of+highly+resistant+salan-titanium%28IV%29+complexes+in+nanoparticles%3A+an+identified+active+species%5BTitle%5D)+AND+%22Angewandte+Chemie+International+Edition+in+English%22%5BJournal%5D)">High antitumor activity of highly resistant salan-titanium(IV) complexes in nanoparticles: an identified active species.</a> Angewandte Chemie International Edition in English. 51, 10515-10517 (2012).</li>
<li>Reytman, L., Braitbard, O., Tshuva, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+cytotoxic+vanadium%28V%29+complexes+of+salan+ligands%3B+insights+on+the+role+of+hydrolysis%5BTitle%5D)+AND+%22Dalton+Transactions%22%5BJournal%5D)">Highly cytotoxic vanadium(V) complexes of salan ligands; insights on the role of hydrolysis.</a> Dalton Transactions. 41, 5241-5247 (2012).</li>
<li>Tzubery, A., Tshuva, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxicity+and+Hydrolysis+of+trans-Ti%28IV%29+Complexes+of+Salen+Ligands%3A+Structure-Activity+Relationship+Studies%5BTitle%5D)+AND+%22Inorganic+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Modern+cytotoxic+titanium%28IV%29+complexes%3B+Insights+on+the+enigmatic+involvement+of+hydrolysis%5BTitle%5D)+AND+%22Coordination+Chemistry+Reviews%22%5BJournal%5D)">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. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+cytotoxic+reagents+based+on+non-metallocene+non-diketonato+well-defined+C-2-symmetrical+titanium+complexes+of+tetradentate+bis%28phenolato%29+ligands%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Active cytotoxic reagents based on non-metallocene non-diketonato well-defined C-2-symmetrical titanium complexes of tetradentate bis(phenolato) ligands.</a> Journal of the American Chemical Society. 129, 12098-12099 (2007).</li>
<li>Mosmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+Colorimetric+Assay+for+Cellular+Growth+and+Survival+-+Application+to+Proliferation+and+Cyto-Toxicity+Assays%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">Rapid Colorimetric Assay for Cellular Growth and Survival - Application to Proliferation and Cyto-Toxicity Assays.</a> Journal of Immunological Methods. 65 (83), 55-63 (1983).</li>
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<li>Chmura, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Group+4+complexes+with+aminebisphenolate+ligands+and+their+application+for+the+ring+opening+polymerization+of+cyclic+esters%5BTitle%5D)+AND+%22Macromolecules%22%5BJournal%5D)">Group 4 complexes with aminebisphenolate ligands and their application for the ring opening polymerization of cyclic esters.</a> Macromolecules. 39, 7250-7257 (2006).</li>
<li>Sebaugh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidelines+for+accurate+EC50%2FIC50+estimation%5BTitle%5D)+AND+%22Pharmaceutical+Statistics%22%5BJournal%5D)">Guidelines for accurate EC50/IC50 estimation.</a> Pharmaceutical Statistics. 10, 128-134 (2011).</li>
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</ol>

The authors declare they have no competing financial interests.

The method described in this manuscript combines chemical synthesis with biological cell viability assay. As with any biological methods concerning viable cells, it is vital to work in a laminar hood, and maintain sterile conditions, including the use of sterile organic solvents. Also, preparation and storage of hydrolytically unstable metal based drugs should be carried out under inert conditions, for which glove box or Schlenk line techniques should be utilized.
The selection of the techniqu...

Chemotherapy is still one of the main courses of treatments employed in the clinic for various cancer diseases, and thus vast amount of research is conducted worldwide with the aim to develop new and improved anticancer drugs. Such studies mostly begin at the chemical level, with the design and preparation of compounds, followed by biological evaluation of the cytotoxic properties in vitro. Cell viability may be assessed by various assays that provide information on cellular activity1-2.
Cisplatin is an example of a platinum complex that is widely used as a chemotherapeutic drug, which is considered an efficient treatment mainly for testicular and ovarian cancers3-4. However, its narrow activity range and severe side effects trigger studies of other potent transition metal complexes5-8. Among others, titanium (IV) and vanadium (V) complexes showed promising results of high activity and reduced toxicity9-16. Ti (IV) complexes were the first to enter clinical trials after cisplatin due to these properties; however, they have failed the trials due to formulation difficulties and hydrolytic instability. There is thus a current need to develop improved derivatives of these metal complexes that may combine high anticancer activity with water resistance15,17-21.
A challenge in the preparation of Ti (IV) and V (V) complexes refers to the hydrolytic instability of the precursor reagents; therefore, inert atmosphere should be maintained. The preparation of Ti (IV) and V (V) compounds is conducted under N2 or Ar conditions in a glove box or using Schlenk line techniques.
One common method for evaluation of the anti-cancer activity is based on the MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) assay. This assay is a colorimetric viability assay that was presented in 1983 by Mosmann22. It is extremely well studied and characterized, and it is considered highly efficient when evaluating the effectiveness of new cytotoxic compounds due to its precision, rapidity, and its capability to be applied on variety of cell lines. This viability assay is based on the color change of the MTT molecule when it is exposed to viable cells. Measurement of the absorbance, which is proportional to the number of viable cells, and comparison to untreated controls, enables assessment of the cell growth inhibition capabilities of the compound tested.
The MTT colorimetric assay is conducted in a 96-well plate format23.&#160;The cells may require preincubation in the wells before the addition of the tested drug. The preincubation times may vary from 0-24 hr according to the cell line properties. Cells are usually exposed to the drug for 24-96 hr depending on the drug activity. MTT solution is then added to the treated cells, where the yellow MTT is reduced to purple formazan by a variety of mitochondrial and cytosolic enzymes that are operational in viable cells (Figure 1)24. The MTT molecule is not reduced by dead cells or red blood cells (metabolically inactive cells), spleen cells (resting cells) and concanavalin A-stimulated lymphocytes (activated cells)22. After 3-4 hr of incubation with MTT the formazan precipitates. The formation of the formazan begins after 0.5 hr of incubation but for optimal results it is best to expose the cells to MTT for at least 3 hr22. Consequently, the growing medium is removed and the formazan is dissolved in an organic solvent, preferably isopropanol25, although DMSO can also be used26. The elimination of the medium is crucial for achieving accurate results since phenol red, which is widely common in growth medium, and precipitating proteins can interfere with the absorbance measurement25. When the formazan solution reaches homogeneous, the absorbance of the solution is measured using a microplate reader spectrophotometer. The absorbance at 550 nm is directly proportional to the number of cells in range of 200-50,000 cells per well, and thus very small amounts of cells can be detected22. The absorbance indicates the amount of viable cells that remained after treatment with the drug, and is compared to the absorbance of control cells that were not exposed to the drug. Analysis of the results by proper software provides the IC50 (inhibition concentration; 50%) values and their statistical errors based on several repetitions of the measurement.
The MTT assay is widely common in cytotoxicity studies for screening new anticancer compounds, due to its accuracy and relative simplicity. However, when using the MTT assay, which is depended on enzymatic reaction, one must consider that various enzyme inhibitors can affect the reduction of MTT and lead to false results27. In addition, the MTT assay does not provide any information on the molecular mechanism of the cytotoxic activity of the drug2.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent/Material&lt;/strong&gt;</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(O&lt;em&gt;i&lt;/em&gt;Pr)&lt;sub&gt;4&lt;/sub&gt;</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(O&lt;em&gt;i&lt;/em&gt;Pr)&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>404926-10G</td><td>moisture sensitive</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>12-channel pipette 30-300 &amp;mu;l</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>12-channel pipette 5-50 &amp;mu;l</td><td>Finnpipette</td><td></td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; 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>CO&lt;sub&gt;2&lt;/sub&gt; 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 &amp;mu;l</td><td>Finnpipette</td><td></td><td></td></tr><tr><td>Pipette 5-50 &amp;mu;l</td><td>Finnpipette</td><td></td><td></td></tr></tbody></table>

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

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

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

The complexes were prepared based on established procedures18,28 and their purity may be evaluated by NMR and elemental analysis.
The data received from the MTT assay is analyzed to evaluate the cytotoxicity of the compound18,21. First, subtraction of the blank control absorbance value from all the other values is performed. Second, the THF solvent control value is set to 100% viability, as no growth inhibiting compound was added. All other values are transformed into per...

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

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

The overall goal of this procedure is to prepare anti-cancer metallo drugs and evaluate their cytotoxicity by the MTT assay. This is accomplished by first synthesizing metal-based anti-cancer compounds. The second step is to prepare 96 well plates of cancer cells.Next, the metal-based anti-cancer compounds are inserted into the cancer cells. The final step is to measure the cell viability by the MTT assay, where the reduction of MTT by mitochondrial and cytosolic enzymes to form zen is measured ultimately the anti-cancer activity and half maximal inhibitory concentration. Or I see 50 values of the compounds are determined.Generally individuals new to this method may struggle because it's a multi-step procedure that requires the full attention of the laboratory worker and is placed on strict timeline. Visual demonstration of this method is critical as preparation of cell plate is not straightforward because working with viable cells requires following particular guidelines. Working in a glove box, prepare the vanadium five complex by dissolving 0.42 millimole of the ligand precursor in dry THF.Then add it to a stirring solution of equivalent amounts of vanadium ot tris isop oxide in THF. Stir the reaction mixture at room temperature for 15 minutes before removing the volatiles under vacuum. Next, add a cold hexane and remove it under vacuum.A dark purple powder should be obtained in a quantitative yield. Weigh eight milligrams of the obtained compound in an epi orph vial. To prepare the titanium four complex dissolve 0.35 millimole of the ligand precursor in dry THF, and add it to a stirring solution of equivalent of amounts of titanium tetra isop oxide in THF.Stir the reaction mixture at room temperature for two hours. Then remove the volatiles under vacuum to give a yellow product in a quantitative yield. Weigh eight milligrams of the obtained compound in an eph vial to prepare HT 29 cells in a 96 well plate first culture.The cells in a 75 square centimeter flask as described in the text protocol, remove the medium when the HT 29 cells reach maximal co fluency. After washing the cells in one milliliter of trypsin EDTA solution, add one milliliter of the same solution and incubate for five minutes. Detach the HT 29 cells from the flask and add 10 milliliters of the medium to disable the trypsin activity.Transfer five milliliters of the cell mixture to a tube. Use a pipette to transfer a few drops of the cell mixture into a counter chamber. The counter chamber includes two parts of five by five squares.Count the cells in five representative squares of each part using a microscope. Four squares in the corners and one square in the middle. Calculate the estimated amount of cells present.Sum the cells in the five representative squares of each part. Calculate the average sum of the two parts and divide by 20. Next, divide 0.6 by the quotient.The received number is the amount of cell mixture required per plate. This calculation refers to a plate that contains 9, 000 cells per well. Using a pipette, combine the amount of the cell mixture required per plate with 13.2 milliliters of the medium.Add the mixture to a 96 well plate. Using an 11 channel pipette at 200 microliters per well over six lines containing 66 wells in total. One well of each line should remain empty for the blank control.Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 24 hours to allow the cells to attach to the plate to insert the compounds first, add 200 microliters of dry THF to eight milligrams of the weight compound. Dilute the compound solution further by first adding 60 microliters of THF in nine different einor files. For each solution, use a pipette to transfer 60 microliters from the compound solution to the first einor file.Then resus suspend and transfer 60 microliters to the next vial and so on until 10 different concentrations are obtained. Proceed to dilute 20 microliters of each concentration in THF with 180 microliters of medium. Also, prepare a solvent only control solution by adding 20 microliters of THF to 180 microliters of medium.Next, add 10 microliters from each resulting solution to each well. That already contains 200 microliters of the aforementioned solution of cells in the medium to give final concentrations of the compound of up to 200 milligrams per liter. Each line contains cells treated with the compound in 10 different concentrations.One, control well containing cells treated with solvent only and one well without cells For the blank control, set up the lines to repeat each measurement of the compound three times. Incubate the loaded plate for three days at 37 degrees Celsius in 5%carbon dioxide atmosphere. Prepare the MTT solution as described in the text protocol using an 11 channel pipette.Add 20 microliters of MTT solution to each well except the blank control wells. Incubate the plate for an additional three hours after incubation. Remove the medium solution from each well and add 200 microliters of isopropanol to each well.To dissolve the formin, stir the plate for 30 minutes until homogenous. Measure the absorbance at 550 nanometers for 200 microliters of the homogenous solution by a microplate reader, spectrophotometer repeating each measurement on three different days. Next, calculate the percentage of cell viability by deducting the blank control absorbance value from the measured value, dividing the result by the absorbance of the THF solvent control value and multiplying by 100%Calculate the half maximal inhibitory concentration values using graphing software.The reported IC 50 is the average of all IC 50 values collected on at least three different days, and the error value is the standard deviation. The data received from the MTT assay is analyzed to evaluate the cytotoxicity of the compound. The measurements should be performed on a range of concentrations that will produce a graph displaying an even point distribution at least three points on each plateau and three points to define the slope.Insufficient measurements at high concentrations or low concentrations will impair the nonlinear regression fit of the curve required for determination of IC 50 values, and as a result diminish their accuracy. Relative IC 50 values as determined by the nonlinear regression fit correspond to the concentrations leading to 50%of the cell growth inhibition possible by the compound tested. This is calculated as the middle point between the maximal and minimal cell viability measured, which are not necessarily 100%and 0%respectively.When inspecting the results of the experiments described in the protocol. It is clear that cisplatin as well as the titanium and vanadium complexes are highly active. It is also noticeable that the closer the maximal inhibition value of a given compound is to 100%the closer the relative IC 50 value is to the absolute one when comparing the IC 50 values among the different compounds.The titanium four complex has the highest cytotoxicity and its IC 50 values are lower than those of cisplatin and the Vanadium five complex. However, the free ligand exhibits markedly reduced activity since following addition of the ligand at different concentrations. The cell viability does not drop as significantly as it does for its complexes.This assures that the high anti-cancer activity of the complexes relies on the metal centers While attempting this procedure. It's important to keep sterile conditions at all times. After watching this video, you should have a good understanding of how to evaluate the cytotoxicity of your anti-cancer compounds by the M-T-T-S-A.

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Learn to synthesize air-sensitive titanium and vanadium anticancer agents and evaluate their cytotoxicity using the MTT assay on human cancer cell lines. This method enables precise IC50 determination for metallo drug development.

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

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