The overall goal of this procedure is to investigate the behaviors of curcumin-loaded magnetically and thermally sensitive hydrogels before and during magnetically triggered curcumin release. This method can help answer key questions in the anti-cancer drug field about the encapsulation of hydrophobic anti-cancer drugs such as curcumin for localized delivery to tumor cells. The main advantage of this technique is hydrophobic anti-cancer drugs are encapsulated by a spherical thermal-induced physical-crosslinking between polyethylenime-PNIPAAm and magnetic nanoparticles enabling targeting delivery with high-frequency magnetic fields.
Though this method can provide insight into thermal-induced self-assemblies of PNIPAAm, it can also be applied to other substances such as emulsions, or polymerizations, or nanoparticle preparations. To begin the procedure, place 0.2 grams of PEI, 0.25 grams of polyNIPAM, five milliliters of 9.1 weight percent iron two three oxide nanoparticles dispersed in water, and 20 milliliters of deionized water in a glass vial. Use the same stirring conditions to combine in another vial 0.2 grams of PEI, 0.25 grams of polyNIPAM, five milliliters of 4.76 weight percent aminosilane functionalized iron two three oxide nanoparticles dispersed in water, and 20 milliliters of deionized water.
Next, prepare two batches of 0.8 grams of PEI and 18.2 milliliters of deionized water. Heat both solutions at 70 degrees Celsius, in a water bath, for 30 minutes, while stirring. Then, while still stirring in the water bath, begin sonicating one batch, with a 50-watt probe sonicator.
Using a three milliliter syringe, add this batch to the polyNIPAM and iron oxide suspension drop-wise, at a rate of one milliliter per minute. The addition should be as slow as possible to keep the physical cross-linking minimal. Otherwise, it will need a large magnetic microgel, that is difficult to disperse in water.
Continue sonicating and stirring the suspension at 70 degrees Celsius, for 30 minutes. Then, allow the suspension to cool to room temperature. Turn off the sonicator and remove the suspension from the water bath.
Remove the magnetic stir bar from the suspension. Place a magnet next to the vial and wait for the magnetic polyNIPAM iron oxide microgels to collect at the bottom of the vial. Then, remove the supernatant, and add 25 milliliters of deionized water to the microgels.
Remove the magnet and vortex the mixture to obtain a dispersion of polyNIPAM iron oxide microgels in water. Repeat this process with the polyNIPAM aminosilane functionalized iron oxide suspension and the second batch of the PEI solution, to produce the corresponding functionalized microgel dispersion. In an aluminum foil-wrapped vial, dissolve 100 milligrams of curcumin, and 20 milliliters of HPLC-grade ethanol.
Add two milliliters of the solution to the polyNIPAM aminosilane functionalized iron oxide dispersion. Stir the mixture at 400 RPM, overnight at room temperature. Then, remove the stir bar.
Place a magnet next to the vial, and wait for the curcumin-loaded microgels to collect at the bottom of the vial. Replace the supernatant with 25 milliliters of deionized water, and disperse the microgels by vortexing. Next, to test the magnetically triggered curcumin release, first transfer 10 milliliters of the curcumin-loaded microgel dispersion to a 15 milliliter conical centrifuge tube.
Add two milliliters of deionized water to the tube and vortex to re-disperse the microgels. Place the tube at the center of the coil of a high frequency magnetic field. Apply the magnetic field at 15 kilohertz for 20 minutes.
Every two minutes, transfer 0.5 milliliters of the upper layer of the dispersion to a one milliliter cuvette. Replace each aliquot with 0.5 milliliters of deionized water. Use a thermometer to mix the water into the dispersion and to measure the dispersion temperature.
Measure the absorption of each aliquot at 482 nanometers. Calculate the concentration of released curcumin in each aliquot from a standard calibration curve. Organic inorganic microgels were prepared from polyNIPAM and iron two three oxide nanoparticles.
Microgels prepared with the 3-aminopropyltriethyloxysilane iron oxide particles were found to have a much lower relative iron oxide composition than non-functionalized particles, suggesting that the functionalized particles had cross-linked a larger amount of polyNIPAM than the non-functionalized particles. Aminosilane functionalized microgels, loaded with curcumin, showed increased isolation compared to their precursor microgels, suggesting that the hydrophobic curcumin was at least partially present in the microgel surfaces. Encapsulation of curcumin was confirmed by the appearance of characteristic absorption peaks in the FTIR spectrum of the loaded microgels.
The rate of curcumin release was evaluated with and without exposure to a high frequency magnetic field. The curcumin release rate over a period of 20 minutes was 2.5 times greater in the presence of a high frequency magnetic field. Exposure to the magnetic field increased the bulk solution temperature to approximately 50 degrees Celsius, thus altering the microgel properties to exclude curcumin.
Thermostability tests showed that the microgels remain dispersed in water in temperatures up to 70 degrees Celsius, despite aggregation. After watching this video, you should have a good understanding of how to encapsulation, hydrophobic, and high cancer drugs in polyNIPAM-based magnetic microgels by temperature-induced immersion, and how to perform a rapid targeted delivery by the manipulation of high frequency magnetic fields.
