This method employs topical application of nanoparticles to image microscopic tumors, especially in malignancies that do not yet have access to the vasculature, such as the metastasis of ovarian cancer. This technique is ratiometric, imaging targeted and non-targeted nanoparticles in a single scan to diminish false signals and reveal the true extent of the tumor. These nanoparticles can be administered by intraperitoneal or intravenous injection for high precision imaging of ovarian cancer and many other tumor types.
For gold nanostar core synthesis place a conical flask containing 800 milliliters of freshly prepared ascorbic acid solution on a magnetic stir plate at four degrees Celsius and induce a steady vortex. Quickly add eight milliliters of freshly prepared tetrachloroauric acid solution to the vortex. Within seconds nanostars will form and the solution will assume a dark blue color.
Pour the nanostar suspension into 50-milliliter conical tubes for centrifugation and aspirate all but the last 200 microliters of supernatant from each tube at the end of the spin. Use a micropipette to resuspend the nanoparticles in each tube before pooling all of the nanoparticles in a single dialysis cassette. Then dialyze the nanoparticles for at least three days against two liters of deionized water changing the water daily.
For silica shell formation, first add 10 milliliters of isopropanol, 500 microliters of TEOS, 200 microliters of deionized water, and 60 microliters of dye to a 50-milliliter conical tube. And add three milliliters of ethanol, and 200 microliters of ammonium hydroxide to a 15-milliliter tube. Next, sonicate the dialyzed nanostars to disperse any clusters and add 1.2 milliliters of nanostars to the tube.
Place the 50-milliliter tube on a vortex mixer and induce a steady vortex. Rapidly add the nanostar solution to the 50-milliliter tube with about five more seconds of mixing before quickly transferring the tube to a shaker for 15 minutes at 300 RPM at room temperature. The two tubes must be combined quickly and under constant nixing to ensure that the silication reaction happens on the nanostars and to minimize free silica production.
At the end of the incubation fill the tube with ethanol to quench the reaction and collect the nanostars by centrifugation. Aspirate all but about the last 500 microliters of supernatant taking care not to disturb the pellet and resuspend the nanoparticles in one milliliter of fresh ethanol. Then transfer the solution to a 1.5-milliliter centrifuge tube for four one-milliliter of ethanol washes by centrifugation, resuspending the pellet by ultrasonication for approximately one second between each wash.
To introduce thiols to the particle surfaces, after the last wash resuspend the pellet in one milliliter of an 85%ethanol, 10%3-mercaptopropyl-trimethoxysilane, and 5%deionized water solution for a one-to two-hour incubation at room temperature. At the end of the incubation, wash the thiol functionalized nanoparticles by centrifugation as indicated, resuspending the pellet by ultrasonication between washes. For the antibody functionalized anti-folate receptor nanoprobes react 200 micrograms of anti-folate binding protein antibody with tenfold molar excess of PEG cross-linker in 500 microliters of HEPES buffer.
After 30 minutes concentrate the antibody by centrifugation in a centrifugal filter, recovering the antibody by inverting the filter into a new tube for an additional centrifugation. Then mix the thiol functionalized nanoparticles with the concentrated antibody and incubate the mixture for at least 30 minutes at room temperature. To form non-targeted nanoprobes add five milligrams methoxy-terminated PEG 5000 maleimide dissolved in DMSO to the thiol-functionalized nanoparticles in 500 microliters of HEPES buffer for an at least 30-minute incubation at room temperature.
For nanoprobe injection spin down both nanoprobe flavors and resuspend the pellets in fresh MES buffer at a 600 picomolar concentration. Mix the nanoparticles and load one milliliter of the resulting solution into one one-milliliter syringe equipped with a 26-gauge needle per mouse and intraperitoneally inject the entire volume into the abdomen of each mouse. Then gently massage the abdomen to distribute the nanoparticles throughout the peritoneal cavity.
After at least 25 minutes secure the limbs of a euthanized injected animal onto a surgical platform and use serrated forceps and dissection scissors to remove the skin to expose the peritoneum. Incise the peritoneum. Attach the peritoneal flaps to the platform and wash the inside of the peritoneal cavity with at least 60 milliliters of PBS.
Then transfer the platform to a Raman spectrophotometer with an upright optical configuration and a motorized stage and image the abdomen under the appropriate imaging parameters. This is crucial that focal plane for the Raman scan is on the surface of most of the viscera. If the organs are out of plane, the acquired signal will not be useful.
For quality control purposes the nanoparticles can be characterized using a variety of methods during the synthesis process, including transmission electron microscopy, dynamic light scattering, nanoparticle tracking analysis, and ultraviolet visible absorbent spectroscopy. Raman measurements reveal the presence of the unique spectra of each flavor of nanoparticle throughout the synthesis. Typical reaction yields and concentrations depend strongly on the pipetting technique during the wash steps.
The Raman spectra can be processed to remove the fluorescence and normalized to unit area to compensate for the signal strength before applying the classical least squares. Although the classical least-squared scores on each nanoprobe reference spectrum do not individually provide the specific locations of the tumors, the point-wise ratios reveal the presence of the disseminated microscopic spread. While attempting this procedure it's important to remember to validate the nanoparticles at every step of the synthesis as the quality of the nanoparticles strongly affects the end results.
This technique demonstrates the potential for researchers to use SERRS nanoprobes for the detection of tumor-related markers in animal models and excised patient tissues with a high degree of microscopic precision.
