Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

The overall goal of this procedure is to synthesize core-shell lanthanide-doped upconversion nanocrystals based on a high-temperature co-precipitation approach and to perform biocompatible surface modifications that enable further cellular applications. In the past few years, our lab have developed a very unique lanthanide-doped upconversion of nanocrystals, which indicated very unique optical properties, so which can absorb long-wavelength infrared light and basically convert this light into multicolored emissions at short wavelength range, including UV visible to even the infrared light windows. Such unique nanocrystal structures indicated very promising the properties for biomedical applications.

This method provides a feasible approach for synthesize of core-shell upconversion nanostructures and their biocompatible surface modification technique for light-gate membrane channel protein regulation upon near-infrared light illumination. To begin the procedure, prepare stock solutions of the rare-earth acetate complexes in methanol. Prepare 20 milligrams per milliliter solutions of sodium hydroxide and ammonium fluoride in methanol.

Store the stock solutions at four degrees Celsius. Then, pipette three milliliters of oleic acid and seven milliliters of 1-octadecene into a 50-milliliter, three-neck, round-bottom flask. Add to this, 1.089 milliliters of the yttrium acetate solution, 0.608 milliliters of the ytterbium acetate solution, 83.6 microliters of the thulium acetate solution, and 128.5 microliters of the neodymium acetate solution.

Equip the flask with a stir bar and a thermometer. Heat the flask to 100 degrees Celsius in an oil bath while stirring until the methanol has evaporated. Then, connect the flask to a Schlenk line, and pump down the flask for two to three minutes to remove residual methanol.

Next, put the flask under a nitrogen atmosphere. Heat the reaction mixture at 150 degrees Celsius for one hour while stirring at 700 rpm. Then, allow the mixture to cool to room temperature.

Next, place in a 15-milliliter centrifuge tube two milliliters of the sodium hydroxide stock solution and 2.965 milliliters of the ammonium fluoride stock solution. Tightly cap the tube, and vortex the sodium hydroxide and ammonium fluoride mixture for five seconds. Over the course of five minutes, use a glass pipette to slowly add the sodium hydroxide-ammonium fluoride mixture to the reaction flask while stirring.

Then, heat the reaction mixture to no more than 50 degrees Celsius. Hold the mixture at that temperature for 30 minutes. Then, heat the mixture to 100 degrees Celsius to evaporate the methanol.

Connect the flask to the Schlenk line, and remove residual methanol under vacuum for two to three minutes. Fill the flask with nitrogen gas, and heat the reaction mixture to 290 degrees Celsius at five degrees Celsius per minute. Hold the mixture at or slightly above 290 degrees Celsius for 1.5 hours.

Then, allow the mixture to cool to room temperature while stirring. Transfer the product mixture to a 50-milliliter centrifuge tube. Rinse the residue into the tube with 30 milliliters of ethanol.

Centrifuge the mixture at 4, 000 times g for eight minutes at room temperature. Discard the supernatant, and disperse the solids in 10 milliliters of hexanes by sonication for two minutes. Add 30 milliliters of ethanol to the dispersion, centrifuge the mixture again under the same conditions, and discard the supernatant.

Disperse the solids in five milliliters of hexanes, and store the dispersion at four degrees Celsius. Check the upconverted emission of core-shell UCNs solution upon 808-nanometer laser irradiation in the dark. To begin preparing DBCO-modified UCNs, wash the prepared core-shell nanocrystals in 30 milliliters of ethanol by centrifugation.

Discard the supernatant, and add 10 milliliters of a pH four aqueous solution of hydrochloric acid. Sonicate the mixture for 30 minutes to dissolve the solids. Transfer the mixture to a glass vial, and stir vigorously for two hours.

Then, wash the mixture with four 30-milliliter portions of diethyl ether. Combine the ether fractions, and set aside the washed aqueous layer. Recover trace ligand-free UCNs from the ether layers with 10 milliliters of deionized water, and combine the aqueous layers.

Add 20 milliliters of acetone to the combined aqueous layers, and vortex the mixture for five seconds. Centrifuge the mixture at 35, 000 times g for 10 minutes. Discard the supernatant, and dissolve the precipitate in two milliliters of deionized water to obtain a solution of ligand-free UCNs.

Next, dissolve 200 milligrams of polyacrylic acid in 20 milliliters of deionized water by sonication for 20 minutes. Add to this the solution of ligand-free UCNs while stirring vigorously. Adjust the mixture pH to 7.4 with a one-molar solution of sodium hydroxide.

Sonicate the mixture for 30 minutes, and stir the mixture for 24 hours at room temperature. Then, centrifuge the mixture at 35, 000 times g for 10 minutes. Wash the solids by centrifugation in 10 milliliters of deionized water four times.

Disperse the washed solids in eight milliliters of deionized water to obtain a 10 milligram per milliliter solution of polymer-modified UCNs. Centrifuge one milligram of the polymer-modified UCNs at 35, 000 times g for 10 minutes. Store the remaining solution at four degrees Celsius.

Wash the solids by centrifugation in one-milliliter portions of dry DMF three times. Then, dissolve the precipitate in 200 microliters of dry DMF. Add to this solution 12.2 milligrams of HOBT, 14 milligrams of EDC, five milligrams of DBCO-amine, and 16 microliters of DIPEA.

Stir the mixture at room temperature for 24 hours. Then, centrifuge the mixture for 10 minutes at 35, 000 times g. Wash the solids four times by centrifugation in one-milliliter portions of DMSO.

Disperse the DBCO-modified UCNs in 0.2 milliliters of DMSO, and store the dispersion at four degrees Celsius. First, seed one times 10 the fifth HEK293 cells in a 12-well plate, and incubate the cells at 37 degrees Celsius for 24 hours. Then, in a microcentrifuge tube, combine one microgram of the chosen plasmid with two microliters of P3000 transfection agent in 100 microliters of MEM.

In another microcentrifuge tube, combine 1.5 microliters of the transfection reagent with 100 microliters of MEM. Incubate both mixtures at room temperature for 10 minutes. Then, combine the mixtures, and add 400 microliters of low-serum MEM.

Next, wash the incubated cells twice with one-milliliter portions of serum-free DMEM. Distribute the 600-microliter transfection mixture into the wells of the 12-well plate. Incubate the cells at 37 degrees Celsius for four hours.

Then, remove the medium, and wash the cells twice with one-milliliter portions of DMEM. Distribute among the wells one milliliter of a tetra-acetylated N-azidoacetyl-mannosamine solution in DMEM. Incubate the cells at 37 degrees Celsius for two days.

Then, wash, trypsinize, and re-culture the cells in a confocal dish overnight. Next, replace the medium with one milliliter of fresh DMEM containing two microliters of a 50-milligram per milliliter solution of DBCO-UCNs. Incubate the cells at 37 degrees Celsius for two hours, and wash the cells twice with DMEM.

Turn off the light of the fume hood and add the appropriate diene buffer solutions to the cells for calcium imaging, and incubate the cells for 30 minutes in the dark. Wash the cells with serum-free DMEM. Irradiate the cells with 808-nanometer, near-infrared light delivering 0.8 watts per square centimeter.

Alternate five minutes of irradiation with five-minute breaks until the cells have been irradiated for 20 minutes. Image the cells with a confocal microscope. Transmission electron microscopy of the core and core-shell UCNs showed spherical structures with a shell thickness of about five nanometers.

High-resolution TEM showed d-spacing consistent with highly crystallized nanostructures. Following surface modification, the UCNs were easily dispersed into buffer solution. Dynamic light scattering indicated that the hydrodynamic diameter of DBCO-UCNs in aqueous solution is about 96 nanometers.

The zeta potentials of the PAA and DBCO-modified UCNs indicate negatively charged surfaces, which is consistent with solubility and stability in aqueous buffer solutions. Upconverted emission tests showed that PAA and DBCO-modified UCNs had the same emission patterns as the base core-shell UCNs when irradiated with 808-nanometer light. Azide-tagged HEK293 cells expressing light-gated channel proteins were used to test the bioapplications of DBCO-UCNs.

Localization of the UCNs at the azide tags was attributed to the DBCO groups being stained with an azide-containing dye. When these cells were exposed to near-IR light, the DBCO-UCNs facilitated the flow of calcium ions across the cell membrane, as shown by confocal imaging and flow cytometry with a fluorescent calcium indicator. After watching this video, you should have a good understanding of how to prepare the core-shell upconversion nanocrystals with biocompatible surface modification and their further bio applications to activate the light-gated ion channel in living cells by following this procedure.