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

Overview

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.

Protocol

1. Nanoparticle Synthesis and Functionalization

CAUTION: All synthesis should occur in a ventilated chemical fume hood.

1. Prior to synthesis, filter approximately 300 mL of deionized (DI) water through a 0.2 µm sterile filter.
2. Clean a 250 mL glass round bottom flask with a detergent solution and rinse with DI water. Add 0.0134 g of CuCl₂ into 100 mL of DI water to create a 1 mM solution.
3. Add 0.015 g of folic acid (FA) to the CuCl₂ solution and stir for ~5 min using a magnetic stir bar.
4. Add Na₂S·9H₂O (0.024 g in 100 µL DI water) over approximately 10 s to the reaction mixture utilizing a 200 µL pipette.
   NOTE: Upon addition of the Na₂S·9H₂O, the solution will change color from a light yellow to a dark brown.
5. Cap the reaction and place in an oil bath, set to 90 °C, and continue stirring with a magnetic stir bar. After approximately 15 min, or when the oil bath has reached 85−90 °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.
6. 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.
7. Once the reaction mixture has cooled below 20 °C, adjust the pH to 10 utilizing 1M NaOH to dissolve the remaining folic acid into solution.
8. 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.
9. 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.
10. For mass measurements, take 1/3 of the solution (~66 µL) and split into three glass vials. Dry in a vacuum oven overnight at 40 °C under a vacuum of ~27 mmHg.
11. Dissolve the other 2/3 of concentrated solution into 250 µL of PBS and store at 4 °C until further use.
12. Prior to utilizing the FA-CuS NPs, sonicate them for 30 min in a bath sonicator on a high setting.

2. FA-CuS NPs Uptake by Ovarian Cancer Cells

1. Prior to incubation with FA-CuS NPs, incubate SKOV-3 cells in a T75 flask with 8−15 mL of folic-acid-free RPMI-1640 media with 10% FBS and 1% penicillin/streptomycin for at least 24 h.
2. Seed the cells in 0.5 mL of folic-acid-free RPMI-1640 complete growth media at a density of 0.1 x 10⁶ cells/mL into a 24-well plate.
3. The following day, incubate the cells with 400 µg/mL FA-CuS NPS in 0.5 mL of folic-acid-free RPMI-1640 complete growth media for 2 h.
4. 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 g for 6 min.
5. Remove the supernatant, resuspend the cells in 2 mL of PBS, and centrifuge at 123 x g for 6 min. Perform this wash step 2x to remove any unbound NPs.
6. Resuspend the cells in 1−2 mL of PBS with 2% Tween solution.
7. 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.
8. The cells are now ready to be analyzed by the PAFC system.

Materials

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