The overall goal of this protocol is to prepare conductive polymer nanoparticles that exhibit photothermal properties, and evaluate their cytotoxicity. Conductive polymer nanoparticles are ideal agents for photothermal therapy because they absorb light in the near infrared range and convert it to heat to kill cancer cells. The main advantage of polymeric phototherapy agents is that they can be tailored to tune their absorption properties by a compatibility and tumor targeting ability to enable effective non-invasive localized cancer therapy.
The polymers we are using for photothermal therapy can also be used for alternative energy applications, sensors and electrochromics. Individuals new to this method may struggle because the reaction is sensitive to moisture. A good understanding of airless synthesis techniques is essential.
Visual demonstration of cell culture assays is critical as sterile techniques are difficult to master. All multi-role play assays require accuracy and consistency to achieve proper results. To a three-neck, round-bottom flask secure a septum, a condenser fitted with an inlet control adapter, and a gas outlet control adapter.
Connect the gas outlet adapter to a bubbler. Next, connect the inlet adapter to an argon Shlenk line with thick-walled PVC tubing. Begin the flow of argon into the reaction flask, and let the gas flow for several minutes.
After turning on the vacuum, use a Bunsen burner to flame dry the reaction, and then purge the apparatus with argon. Repeat this twice more to achieve an airless environment. Use a syringe to add one point zero seven grams of EDOT to the apparatus through the septum.
Add 20 milliliters of Tetrahydrofuran into the flask and stir. Prepare a dry ice acetone bath in a Low-form Dewar flask, and chill the EDOT flask for 15 minutes at minus 78 degrees Celsius. Then drop wise at 11 millimoles of n-butyllithium in hexanes while maintaining a temperature of minus 78 degrees Celsius.
After complete addition, stir for an additional hour. Extreme caution must be taken when handling organolithium reagents. These solutions are exceptionally hazardous, and should only be handled by someone who has been extensively trained in their use.
Remove the dry ice acetone bath. And, immediately begin a drop wise addition of 14 point 13 milliliters of a one Molar Zinc Chloride solution, stirring for one hour at room temperature. Next, add four millimoles of one four Dialkoxy two five Dibromobenzene, and 80 micromoles of palladium tetrakis triphenylphosphine to the reaction.
Begin re-fluxing the reaction at 75 degrees Celsius with an oil bath. Each day, use a syringe to take a small sample of the reaction mixture. Precipitate the sample into two milliliters of one Molar hydrochloric acid, and use two milliliters of dichloromethane to extract the material.
Spot the extract on a silica thin layer chromatography, or TLC plate, alongside pre-made solutions of purified EDOT and the corresponding one four Dialkoxy two five Dibromobenzene. Use a 60 to 40 solution of ethyl acetate and hexanes to develop the plate in a glass chamber. Once the reaction is complete, in three to five days, cool the reaction to room temperature.
Add 10 milliliters of one Molar hydrochloric acid followed by 20 milliliters of dichloromethane to quench the reaction. Transfer the reaction work-up to a separatory funnel, and isolate the organic layer. Wash the organics with deionized water until the water washes are no longer acidic.
Next, dry the organics over 15 grams of magnesium sulfate. After filtering off the solid, remove the solvent using a rotary evaporator to yield a yellow orange solid. In a glass vile, prepare one milliliter of two percent weight by volume PSS-co-MA in water, and add a small stir bar.
Prepare a 16 milligram per milliliter solution of monomer in chloroform and transfer 100 microliters of it into a microcentrifuged tube. Dissolve 30 milligrams of dodecylbenzene sulfonic acid in the microcentrifuge tube, and then use an automatic vortex mixer for 30 to 60 minutes to homogenize the solution. Next, add the homogenized organic phase in 10 microliter aliquot to the two percent PSS-co-MA solution every 60 seconds.
After complete addition, dilute the solution with two milliliters of water and remove the stir bar. Place the emulsion in an ice bath, and then use a probe to sonicate emlusion with two 10 second intervals at an amplitude of 30 percent. Remove the vial from the ice bath, and replace the stir bar to continue mixing the emulsion.
Next, add three point eight microliters of a one hundred milligrams per milliliter aqueous solution of iron chloride to the monomer emulsion, and stir for one hour to yield polymer nanoparticles stabilized with PSS-co-MA. Transfer the nanoparticles suspension into a seven milliliter centrifuge tubes, and centrifuge the suspension at 75, 600 times G for three minutes. Finally, recover the supernatant, and dialyze in water for 24 hours using 100 kilodalton molecular weight cut-off dialysis tubing.
Culture SCOV three ovarian cancer cells in DMEMs supplemented with 10 percent fetal bovine serum at 37 degrees Celsius and five percent carbon dioxide in T 75 flasks. After dissociating and counting cells using standard protocols, feed cells at a density of 5, 000 cells per well in a 96 well plate. Incubate the cells for 24 hours at 37 degrees Celsius and five percent carbon dioxide.
Immediately before use, delude the nanoparticles suspension into full growth medium at a concentration of one milligram per milliliter. Filter the nanoparticles suspension through a sterile zero point two micrometer filter. Then dilute the suspension to the desired exposure concentrations with full growth medium supplemented with one percent penicillin streptomycin.
Gently remove the medium from each well and replace with 100 microliters of the nanoparticles suspension at various exposure concentrations. After incubating the cells, carefully remove the solution, and then replace the medium with 100 microliters of sterile filtered zero point five milligrams per milliliter MTT solution. Incubate the cells for two to four hours in the incubator.
Then examine the cells under a microscope to check for formation of Formazan crystals. Carefully remove the MTT solution and replace it with 100 microliters of dimethyl sulfoxide. Place the plate on a shaker and mix for several minutes.
Finally, measure the absorbents of each well at 590 nanometers and 700 nanometers. These values correspond to the peak absorbents of Formazan product and a baseline respectively. The resulting monomers can be characterized by hydrogen and carbon 13 NMR spectroscopy.
Negishi coupling reactions attach the phenyl ring to the EDOT causing the phenyl proton peak to shift from seven point one PPM to seven point eight PPM. The theyenel proton also shifts up field to six point five PPM. The four protons on the ethylenedioxy bridge carbons split into two sets of multiplets at four point three PPM.
The carbon 13 NMR spectrum exhibits peaks at one 70, one 45, one 40 and one 13 for the thyenel carbons, and one 50, one 20, and one 12 for the phenylene carbons. The cital compatibility of polymer nanoparticles is determined using an MTT cell viability assay. Within the nanaparticle concentration range of zero point two three to 56 micrograms per milliliter, then nanoparticles do not decrease cell viability to less than 90 percent of the control.
Typically a reduction in cell viability of less than 20 percent is consisdered acceptable. Once mastered, it takes about a week to synthesize the Monomer, another week to purify it, and less than a day to prepare and purify the nanoparticles. The citotoxicity study can be completed in two and a half days from cell seeding to the conclusion of the assay.
While attempting in vitro cell studies, it is critical to follow proper aseptic techniques. Additional studies such as live dead assays, microscopy studies, and in vivo biodistribution, and photothermal ovulation studies can be performed with the nanoparticles prepared through these protocols to better understand their potential as photothermal agents for cancer therapy.
