Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

The overall goal of the following experiment is to develop a next generation photosensitizer for photodynamic therapy or PDT application in cancer therapy. This is achieved by adapting a precipitation method of fabrication to make water dispersable conducting polymer nanoparticles. As a second step, the nanoparticles are incubated with cancer cells in vitro, followed by treatment with light to generate reactive oxygen species or ROS, which are the active radicals that partake in PDT.

Next treatment effectiveness is evaluated by quantitative assays and qualitative imaging to determine live versus TED fractions of the cell population. The results show that aggressive cancers such as ovarian cancer, modeled here by OAR three abundantly take up the nanoparticle photosensitizer reported in this work and that the PDT treatment is highly effective in vitro. In that case, the PDT effectiveness correlates directly with the amount of nanoparticles taken up by the different cell lines.

The main advantage of this technique over the existing methods like chemotherapy, radiotherapy, and surgery, is that the treatment can be targeted specific towards disease site to reduce the side effects The active material used shows no in vitro cytotoxicity and further development will make the nanomaterial system more specific towards tDCS site. Begin this procedure with culturing of cell lines and preparation of the M-E-H-P-P-V stock solution as described in the text protocol. Add 50 microliters of the undiluted M-E-H-P-P-V stock solution into three milliliters of TETRAHEDRAN or THF.

Label this solution as diluted M-E-H-P-P-V stock solution transfer to a one centimeter quartz Q vet and measure the absorbance at 495 nanometers by ultraviolet visible spectroscopy. If the absorbance of the diluted M-E-H-P-P-V stock solution is higher than 0.17, dilute the undiluted M-E-H-P-P-V stock solution by adding more TH F1 milliliter at a time until the measured absorbance at 495 nanometers is in the range of 0.13 to 0.17. Next, prepare the nanoparticles by precipitation method by transferring one milliliter of a blended M-E-H-P-P-V-P-C-B-M solution into a one milliliter syringe with a needle attached to it.Rapidly.

Inject one milliliter of the blended M-E-H-P-P-V-P-C-V-M solution into four milliliters of deionized water stirring at 1200 RPM stops during immediately after injection. Use these nanoparticles without further processing to perform PDT on cells incubated with nanoparticles. First label a set of 96 well plates as shown in the text protocol.

Call then harvest the cells from culture flasks by removing media and washing the cells twice with docos phosphate buffered saline or DPBS followed by incubation of the cells with 0.05%tryin for 10 minutes at two milliliters of delcos modified eagle medium supplemented with 10%fetal bovine serum to the resulting cell solution. Mix the solution properly to separate cell clusters into singlets. Take 100 microliters of the cell suspension and add to 900 microliters of DMEM supplemented with 10%FBS mix well and place 10 microliters of this suspension onto a hemo cytometer.

Count the cells using the hemo cytometer and adjust the concentration of the cell suspension to 50, 000 cells per milliliter. Then seed the 96 well plate by adding 50 microliters of the cell solution into the wells, thus seeding 2, 500 cells per well. Next, prepare different nanoparticle concentrations by adding 2100 and 180 microliters of the prepared nanoparticle suspension to DMEM to obtain a final volume of two milliliters.

After 24 hours, wash the wells with one X-D-P-B-S and at 50 microliters of increasing concentrations of nanoparticles into the wells. Each cell line has triplicates for each concentration of nanoparticles. 24 hours after edition of the nanoparticles.

Wash the cells with one X-D-P-B-S and add 50 microliters of Hank's balanced salt solution or HBSS to each well, to irradiate the cells warm up the lamp of the solar simulator for 15 minutes. Place a UV filter in front of the lamp to filter out UV light. Calibrate the lamp with a reference solar cell by adjusting the lamp power to obtain 0.5 sun intensity at the surface of the 96 well plate here.

That condition was achieved with 218 watts of power supplied to the lamp. Place the 96 well plate under the lamp with the lid open and irradiate the cells for 60 minutes, which results in a light dosage of 180 joules per square centimeter following a radiation. Replace the HBSS with 50 microliters of DMEM, supplemented with 10%FBS and incubate the plates for the respective time periods after photodynamic therapy.

After each time period, measure the cell viability by adding 10 microliters of MTT to the well. Incubate the plate for four hours fores on crystals to form. Then add 50 microliters of the solubilization solution into the wells and incubate the plate for six hours.

To dissolve the fores on crystals. Measure the cell viability by recording the absorbance at 570 nanometers with a microplate reader to measure the intrinsic cytotoxicity of the nanoparticles. Read the 96 well plate without applying PDT to it to measure the uptake of nanoparticles.

Detection of ROS formed after PDT and detection of apoptosis necrosis after PDT First turn on the lamps of the microscope and the laser. 30 minutes before imaging. Put the Petri dish containing the fixed cells on the stage of the microscope.

Collect the fluorescence from nanoparticles. RROS detecting reagent propidium died. An XIN five fitsi and DPI overlay the phase contrast images and the fluorescence images in image J.Software representative results after incubation of nanoparticles with different cell lines are shown here.

Ocar three cell line shows the highest uptake followed by a 5 49 and MDA MB 2 31. TE 71 does not show any fluorescence of nanoparticles. The cell viability measurements on these cell lines when kept in the dark are shown by the bar graphs here.

There is almost no cell death up to 96 hours in any of the cell lines. The ROS formation detected by ROS detecting reagent is shown here for of car three cell line. In this control sample.

There is no green fluorescence as it has no nanoparticles and no light dose. However, when both the nanoparticle and light doses are given to the cells immediately after PDT, the bright green fluorescence from R os detecting reagent can be seen. The cell viability after DT is shown here by the bar graph for various nanoparticle doses.

The results of the live dead double staining experiment are shown here in which the of car three cell line undergoes necrosis at highlight dose, whereas at low light doses, the cells undergo apoptotic death. After watching this video, you should have a good understanding of how to fabricate composite conducting polymer nanoparticles for the application of photodynamic therapy based disease treatments.