This protocol provides a reference on construction and characterization of photo responsive drug-dye systems, especially the setup of light radiation. This technique shows the advantage of simple fabrication, high drug loading capacity, and photo controlability. This technique can be used to treat colorectal tumor with the help of optical fiber to deliver lights for activating the drug release at the tumor site.
Begin by weighing 10 milligrams of the boron dipyrromethene chlorambucil or BC prodrug and dissolve it in one milliliter of dimethyl sulfoxide, or DMSO, in a 1.5-milliliter micro tube. Cover the BC solution with foil then prepare 0.4 milligrams per milliliter IR783 in filtered deionized water and transfer 300 microliters in a 1.5-milliliter micro tube. Place this micro tube on a vortex mixer at 1500 RPM.
Next with the end of the 20 microliters pipette tip touching the inner wall of the micro tube, add 20 microliters of the BC solution to the IR783 solution over 10 seconds at a constant rate. Place the micro tube on the vortex mixer for 30 seconds to obtain the IR783/BC nanoparticles. Then place the nanoparticle solution on a rack fully covered with foil.
Centrifuge the resulting IR783/BC nanoparticle solution for 10 minutes at 2000 G and four degrees Celsius to remove aggregates. Collect the supernatant, leaving approximately 20 microliters in the tube to avoid disturbing the pellet before discarding it. After centrifuging the supernatant two times for 30 minutes at 30, 000 G and 4 degrees Celsius, collect the nanoparticle precipitate from both centrifications.
Resuspend the nanoparticles in 300 microliters of PBS. Quantify the content of IR783 and BC by high-performance liquid chromatography, or HPLC, using the elution method. Calculate the prodrug encapsulation efficiency, or EE percent, and loading capacity or, LC percent.
To measure the average size of the IR783/BC nanoparticles with a dynamic light scattering, or DLS, instrument. Add 200 microliters of IR783/BC nanoparticle solution in a cuvette and insert the cuvette in the holder for measurement. Set the measurement type as size and the measurement temperature as 25 degrees Celsius.
Perform three measurements with a duration of 20 seconds for each measurement. To measure the surface charge of the IR783/BC nanoparticles with the DLS instrument, dilute 25 microliters of IR783/BC nanoparticle solution with 725 microliters of deionized water in a 1.5-milliliter micro tube. Add the solution into a zeta potential test cuvette.
Place the cuvette in the sample groove. Cap the sample groove. Next, set the measurement type as zeta potential and the temperature as 25 degrees Celsius.
Perform 10 measurements. Once done, prepare the samples for transmission electron microscopy, or TEM, imaging by adding 10 microliters of IR783/BC nanoparticle solution on a piece of the holey carbon film on a copper grid of 300 mesh and removing seven microliters from the holey carbon film. Leave three microliters of solution on the film overnight for auto evaporation.
Set up an LED lamp at 530 nanometers with an iron stand so that the light directly faces the operating floor. Place an integrating sphere photodiode photometer directly under the LED lamp. Turn on the LED lamp and open the cap of the photometer.
Record the irradiance. Set the lamp parameters using the associated software and adjust the input current in the milliampere to set the irradiance as 50 milliwatts per square centimeter. Dilute the IR783/BC nanoparticle solution with deionized water to 50 micromolar, based on BC concentration.
Add 200 micromolar IR783/BC nanoparticle solution into a 1.5-milliliter micro tube. Place the tube on a foam block having a groove fitting the size of the micro tube and at the same height as the photometer. Open the cap of the tube.
Switch on the LED lamp and irradiate the nanoparticle solution for 1, 2, 3, 5, 7, and 10 minutes. After light irradiation, quantify BC consumption and Cb release by HPLC and calculate the remaining BC and Cb release. IR783/BC nanoparticles were successfully fabricated in this study using a flash precipitation method.
The synthesized nanoparticles were present as a purple solution while the aqueous solution of IR783 was blue. The IR783/BC nanoparticles exhibited an average size of 87.22 nanometers with a polydispersity index, or PDI, of 0.089;demonstrating a narrow size distribution. The surface charge was approximately minus 29.8 millivolts, indicating the negatively charged sulfonate groups of IR783.
The nanoparticle size was maintained at 85 nanometers for at least 48 hours after fabrication, while its PDI remained less than 0.2. No significant change was observed in the size distribution at 0, 24 and 48 hours after fabrication. Aggregates and fragments were observed after lighter irradiation.
Size and distribution changes were observed after three and five minutes of light irradiation. Prodrug BC was photo cleaved in 10 minutes. Meanwhile, chlorambucil was released with a recovery efficiency of around 22%within the same period.
The IR783/BC nanoparticles displayed significant cytotoxicity on human colorectal tumor cells HCT 116 under light irradiation at 530 nanometers compared with the non-irradiation group. It is important to touch the inner wall of the micro tube tightly with the end of the peptides and place the micro tube on the vortex stably.
