functionalized nanoparticle targeting of ovarian cancer: a technique to facilitate folic acid-conjugated nanoparticle contrast agent uptake by ovarian cancer cells

Functionalized Nanoparticle Targeting of Ovarian Cancer: A Technique to Facilitate Folic Acid-conjugated Nanoparticle Contrast Agent Uptake by Ovarian Cancer Cells

First, mix the folic acid-capped copper sulfide nanoparticles at the proper concentration in fresh RPMI media. Add this nanoparticle solution to each well of the prepared 24-well plate that contains SKOV-3 cells at a concentration of 400 micrograms per milliliter. Incubate at 37 degrees Celsius with 5% carbon dioxide for two hours.
After this, trypsinize the cells with 0.5 milliliters of 0.25% trypsin and EDTA. To neutralize the trypsin, add at least 1 milliliter of folic acid-free RPMI 1640 complete growth media and centrifuge the cells at 123 x g for six minutes. To wash the cells, remove the supernatant.
Resuspend the cells in 2 milliliters of PBS and centrifuge at 123 x g for six minutes. Repeat this wash step twice to remove any unbound nanoparticles.
Then, resuspend the cells with 1 to 2 milliliters of a 2% Tween solution in PBS. Count the cells using a hemocytometer and Trypan Blue. Dilute the cells in a solution of 2% Tween and PBS to the chosen concentration for detection.

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1. Nanoparticle Synthesis and Functionalization
CAUTION: All synthesis should occur in a ventilated chemical fume hood.
<ol>
	<li>Prior to synthesis, filter approximately 300 mL of deionized (DI) water through a 0.2 &#181;m sterile filter.</li>
	<li>Clean a 250 mL glass round bottom flask with a detergent solution and rinse with DI water. Add 0.0134 g of CuCl2&#160;into 100 mL of DI water to create a 1 mM solution.</li>
	<li>Add 0.015 g of folic acid (FA) to the CuCl2&#160;solution and stir for ~5 min using a magnetic stir bar.</li>
	<li>Add Na2S&#183;9H2O (0.024 g in 100 &#181;L DI water) over approximately 10 s to the reaction mixture utilizing a 200 &#181;L pipette. 
	NOTE: Upon addition of the Na2S&#183;9H2O, the solution will change color from a light yellow to a dark brown.</li>
	<li>Cap the reaction and place in an oil bath, set to 90 &#176;C, and continue stirring with a magnetic stir bar. After approximately 15 min, or when the oil bath has reached 85&#8722;90 &#176;C, allow the reaction to proceed for an additional hour. Your mixture should gradually turn to a dark green color. 
	NOTE: Make sure to vent the system while heating the reaction mixture to avoid pressure buildup.</li>
	<li>Remove the reaction vessel from the oil bath and briefly cool at room temperature for approximately 10 to 15 min before transferring to an ice bath.</li>
	<li>Once the reaction mixture has cooled below 20 &#176;C, adjust the pH to 10 utilizing 1M NaOH to dissolve the remaining folic acid into solution.</li>
	<li>Purify the FA-CuS reaction mixture using a 30 kDa centrifugation column. Add solution in 15 mL batches to the column and centrifuge at 3,082 x g for 15 min.</li>
	<li>Once all of the reaction mixture has been concentrated, recombine the concentrated fractions and wash 4x with 15 mL of pH 10 NaOH in the 30 kDa centrifugation column.</li>
	<li>For mass measurements, take 1/3 of the solution (~66 &#181;L) and split into three glass vials. Dry in a vacuum oven overnight at 40 &#176;C under a vacuum of ~27 mmHg.</li>
	<li>Dissolve the other 2/3 of concentrated solution into 250 &#181;L of PBS and store at 4 &#176;C until further use.</li>
	<li>Prior to utilizing the FA-CuS NPs, sonicate them for 30 min in a bath sonicator on a high setting.</li>
</ol>
2. FA-CuS NPs Uptake by Ovarian Cancer Cells
<ol>
	<li>Prior to incubation with FA-CuS NPs, incubate SKOV-3 cells in a T75 flask with 8&#8722;15 mL of folic-acid-free RPMI-1640 media with 10% FBS and 1% penicillin/streptomycin for at least 24 h.</li>
	<li>Seed the cells in 0.5 mL of folic-acid-free RPMI-1640 complete growth media at a density of 0.1 x 106&#160;cells/mL into a 24-well plate.</li>
	<li>The following day, incubate the cells with 400 &#181;g/mL FA-CuS NPS in 0.5 mL of folic-acid-free RPMI-1640 complete growth media for 2 h.</li>
	<li>Following this incubation, trypsinize the cells with 0.5 mL of 0.25% trypsin with EDTA. Add at least 1 mL of folic-acid-free RPMI-1640 complete growth media to neutralize the trypsin, and centrifuge the cells at 123 x&#160;g&#160;for 6 min.</li>
	<li>Remove the supernatant, resuspend the cells in 2 mL of PBS, and centrifuge at 123 x&#160;g&#160;for 6 min. Perform this wash step 2x to remove any unbound NPs.</li>
	<li>Resuspend the cells in 1&#8722;2 mL of PBS with 2% Tween solution.</li>
	<li>Count the cells using a hemocytometer and Trypan Blue. Further, dilute the cells if cell counts are too high. Dilute the cells in PBS with 2% Tween to the chosen concentration for detection.</li>
	<li>The cells are now ready to be analyzed by the PAFC system.</li>
</ol>

<table><tbody><tr><td>0.025% Trypsin With EDTA&amp;nbsp;&amp;nbsp;</td><td>Corning</td><td>25-053-Cl</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filters&amp;nbsp;&amp;nbsp;</td><td>Millipore</td><td>UFC903024</td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filters&amp;nbsp;&amp;nbsp;</td><td>Millipore</td><td>UFC803024</td><td></td></tr><tr><td>Bright-Line Hematocytometer&amp;nbsp;&amp;nbsp;</td><td>Hausser Scientific</td><td>1492</td><td></td></tr><tr><td>Copper(II) Chloride&amp;nbsp;&amp;nbsp;</td><td>ACROS ORGANICS</td><td>206532500</td><td></td></tr><tr><td>CPX Series Digital Ultrasonic Cleaning Bath&amp;nbsp;&amp;nbsp;</td><td>Fisherbrand</td><td>Model CPX3800</td><td></td></tr><tr><td>Norm-Ject 10 mL Syringes</td><td>HENKE SASS WOLF</td><td>4100-X00V0</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Sigma-Aldrich</td><td>&amp;nbsp;F2442-500ML</td><td></td></tr><tr><td>Folic Acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>&amp;nbsp;F7876-10G</td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>Alfa Aesar</td><td>&amp;nbsp;J62036</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>GIBCO</td><td>&amp;nbsp;15140-122</td><td></td></tr><tr><td>RPMI Midum 1640 (1x) Folic Acid Free&amp;nbsp;</td><td>Gibco</td><td>&amp;nbsp;27016-021</td><td></td></tr><tr><td>SKOV-3 Cells&amp;nbsp;&amp;nbsp;</td><td>ATCC</td><td>HTB-77</td><td></td></tr><tr><td>Sodium Hydroxide Beads&amp;nbsp;</td><td>BDH</td><td>BDH9292-500G</td><td></td></tr><tr><td>Sodium Sulfide Nonahydrate&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>431648-50G</td><td></td></tr><tr><td>Trypan Blue Solution 0.4%&amp;nbsp;&amp;nbsp;</td><td>Amresco</td><td>K940-100ML</td><td></td></tr><tr><td>0.2 &amp;micro;m sterile syringe filter</td><td>VWR</td><td>28145-477</td><td></td></tr><tr><td>Alconox</td><td>Sigma-Aldrich</td><td>242985-1.8KG</td><td>Detergent used for cleaning glassware.</td></tr><tr><td>Tween 20&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P7949-100ML</td><td></td></tr></tbody></table>

Source: Lusk, J. F. et al. <a href="https://www.jove.com/t/60279">Ovarian Cancer Detection Using Photoacoustic Flow Cytometry.</a> J. Vis. Exp. (2020)
In this video, we demonstrate a technique to facilitate the internalization of folic acid-capped copper sulfide nanoparticles (CuS NPs). Folic acid-functionalized CuS NPs have the ability to target folate-receptors which are overexpressed in many ovarian cancer cells.

Source: Lusk, J. F. et al. Ovarian Cancer Detection Using Photoacoustic Flow Cytometry. J. Vis. Exp. (2020)
In this video, we demonstrate a technique to ...

Folate receptors are membrane-associated glycoproteins overexpressed by a majority of ovarian cancer cells. Exploiting these receptors allows for the targeted delivery of diagnostic nanoparticles into the cancer cells.
Begin with a suspension of the desired folic acid-capped nanoparticle contrast agent conjugates. Add this nanoparticle suspension into an adherent culture of ovarian cancer cells. Incubate the culture for the desired time period.
The folic acid functionalized to the nanoparticles helps recognize the folate receptors expressed on the cancer cells, promoting the selective binding of nanoparticle conjugates with the cells. Subsequently, the plasma membrane invaginates, facilitating the cellular uptake and accumulation of nanoparticles.
Harvest the cancer cells. Next, centrifuge to separate the cells from the supernatant containing any unbound nanoparticles. Discard the supernatant and resuspend the nanoparticle-bearing cells in a suitable buffer for downstream applications.

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Functionalized Nanoparticle Targeting of Ovarian Cancer: A Technique to Facilitate Folic Acid-conjugated Nanoparticle Contrast Agent Uptake by Ovarian Cancer Cells

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Functionalized Nanoparticle Targeting of Ovarian Cancer: A Technique to Facilitate Folic Acid-conjugated Nanoparticle Contrast Agent Uptake by Ovarian Cancer Cells