Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

The overall goal of this procedure is to synthesize immuno targeted magneto plasmonic nano clusters with a strong magnetic moment and a strong near infrared absorbance. This is accomplished by first synthesizing iron oxide core nanoparticles. The second step is to deposit a gold shell onto iron oxide core nanoparticles.

Next, the nano clusters are formed by an oil and water microemulsion approach. The final step is to conjugate monoclonal antibodies to the nano clusters. Ultimately, dark field imaging is used to show the molecular specificity of the immuno targeted magneto plasmonic Nano clusters I approach provides the combination of a simple synthesis nanoparticles with strong magnetic moment without sacrificing the super paramagnetic properties and strong, visible knee infrared absorbents that addresses all major limitations of previous methods, and the procedure will be demonstrated by Grady student, Frank W, who has developed the procedure in my laboratory To make the nanoparticles first set up the LabWare in a fume hood, connect a round bottom flask to a condenser and immerse it in a silicone oil bath on a hot plate.

Monitor the bath with a thermometer that can read above to hundred and 60 degrees Celsius wearing protective garments. Start combining reagents in a metal flask as the reaction temperatures are very high for Pyrex. To make the magnetic nanoparticle suspension combine iron acetylate oleic acid ole lamine one two H, aec, diol, and phenyl ether.

Stir the mixture vigorously and heat it to 250 to 260 degrees Celsius. Do not heat it any higher, or the phenyl ether will boil and the vessel will burst. Maintain the high temperature for an hour under reflux and then let the reaction cool.

Back to room temperature to deposit a gold shell onto magnetic core nanoparticles. Add the following materials to a new round bottom flask, gold acetate oleic acid ole lamine one two. He aec dial and phenyl ether.

Then add five milliliters of the magnetic nanoparticle suspension. Heat this reaction to 180 degrees Celsius and keep it there under reflux for an hour. Then wait for the solution to cool down to room temperature.

To precipitate the hybrid primary nanoparticles, add 50 milliliters of ethanol to the suspension. Then load the suspension into a centrifugation tube and spin it down. Now resuspend the pellet in 25 milliliters of hexane using a sonicate and add back 25 milliliters of ethanol and repeat the centrifugation.

Repeat the wash process three more times. Finally, dry out the pellet of primary hybrid nanoparticles in a vacuum desiccate overnight. Do not proceed until the particles are completely dried out.

To make the hybrid magneto plasmonic nano clusters start by resus suspending the dry primary nanoparticles in a milliliter of hexane. Using a sonicate proceed when no visible precipitate is present. Next dropwise, add the nanoparticle suspension to 10 milliliters of sodium esal sulfate in a 20 milliliter glass capped vial, try to keep the solution in two phases, cap and shake the vial by hand.

Then sonicate the two phase solution for two hours. Position the vial near the center of the bath. The bath will gradually heat up after two hours.

Heat the mixture in an 80 degrees Celsius water bath for 10 minutes. Then let it cool back to room temperature. Now centrifuge the suspension at 100 G for 30 minutes.

Save the supernatant and resuspend the pellet in 0.1 millimolar sodium citrate using 10 minutes of sonication. These nano clusters should be about 180 nanometers in diameter. Next centrifuge, the saved supernatant at 400 G for 30 minutes.

Again, save the supernatant and resuspend the pellet in 0.1 millimolar sodium citrate using 10 minutes of sonication. The nano clusters in this Resus suspension are about 130 nanometers in diameter. Now spin down the saved supernatant at 1500 G for 30 minutes and resuspend the pellet as before for nano clusters with a diameter of about 90 nanometers.

For analysis, transfer 300 microliters of each suspension to individual wells and a 96 well plate and read their UV vis NIR absorption spectra. Also transfer 10 microliters to carbon coated copper grids for TEM imaging to conjugate antibodies to the nano clusters. First, replace the media of the antibody solution with heaps so they can be oxidized.

Transfer 100 microliters of antibodies to a 10 K molecular weight cutoff centrifuge filter and add 3.9 milliliters of four millimolar heaps, pH 7.2. Then spin it down at 3, 250 G at eight degrees Celsius for 20 minutes. Next, add 10 microliters of 100 millimolar sodium par I date to oxidize the antibodies and cover the vial with foil.

Allow the reaction to incubate on a room. Temperature orbital shaker for 30 minutes later. Quench the oxidation reaction by adding half a milliliter of one XPBS.

Now to the oxidized antibodies, add two microliters of the linker solution and set the reaction to shake for an hour at room temperature. After an hour, filter the reaction through another 10 K molecular weight cutoff filter at 3, 250 G for 20 minutes and at eight degrees Celsius. Then transfer the antibody to a new vial and bring the volume up to 100 microliters in one XPBS returning them to their original working concentration ready to conjugate to the nano clusters.

Now collect 100 microliters of nano cluster suspension with an OD of about 1.0. Add one microliter of the modified antibodies and incubate them at room temperature for two hours with mild shaking. After two hours, add 10 microliters of one millimolar five kilodalton thiol.

Peg then incubate the reaction for 15 minutes at room temperature with shaking. Now pellet the antibody conjugated nano clusters at 830 G for three minutes and resuspend the pellet in 100 microliters of 2%weight by volume five. Kilodalton PEG in PBS at pH 7.2 measure the absorbance of the suspension compared to that of the bare nano clusters, there should be a red shift after conjugation.

The nanoparticles can aggregate as noted by a significant shift in the red NIR spectrum. If this happens, increase the concentration of thiol peg to five millimolar, increase the incubation time to 30 minutes and incrementally decrease the centrifugal speed by 200 G until the nanoparticles no longer aggregate. The synthesized iron oxide particles were about five nanometers in diameter.

Following gold shell deposition. Their size increased to about six nanometers. The colloidal color changes from brown for iron oxide nanoparticles to red purple after deposition of the gold shell, and finally to a purple gray color after assembly of the primary particles.

These nano clusters are about 180 nanometers in diameter. The absorbent spectra of various particle sizes show that primary iron oxide core gold shell nanoparticles have a distinctive resonance peak at 530 nanometers. That is not present in bare iron oxide particles.

Also, clusters have much stronger NIR absorbent after conjugating anti EGFR and anti HER two antibodies to the nano clusters. Their specificity was checked against an EGFR positive skin cancer cell line and a HER two positive breast cancer cell line, both types labeled the appropriate cells. In contrast, untargeted pegylated nano clusters labeled neither cell line.

After watching this video, you should have a good understanding of how to synthesize immuno targeting magneto plus mono natal clusters one semester. This technique can be done in hours if it performed properly. This novel non-material can be used in a variety of exciting biomedical applications, including contrast enhancement in mag motive imaging modalities, simultaneous capture and detection of circulating tumor cells, and multimodal molecular imaging combined with photothermal cancer therapy.