Antibody-Functionalized SERRS Nanoprobe Based Imaging: A Highly Sensitive Raman-imaging Technique to Detect Metastatic Ovarian Cancer In Vivo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Gold Nanostar Core Synthesis

NOTE: Gold nanostars are used as cores for both flavors of SERRS nanoprobes used in this experiment.

1. Prepare 800 mL of 60 mM ascorbic acid (C₆H₈O₆) solution in deionized (DI) water and 8 mL of 20 mM tetrachloroauric acid (HAuCl₄) solution in DI water. Cool to 4 °C.
2. Perform this reaction step at 4 °C. Place a conical flask containing 800 mL of the ascorbic acid solution on a magnetic stir plate and induce a steady vortex. Quickly add 8 mL of the tetrachloroauric acid solution into the vortex. Within seconds, nanostars will form and the solution will assume a dark blue color. If the color at any time becomes pink or purple, signifying the formation of nanospheres, the suspension should be discarded and the synthesis reattempted.
3. Pour the nanostar suspension into 50 mL conical tubes and centrifuge for 20 min (4 °C, 3,220 x g). Aspirate the supernatant leaving approximately 200 µL of the solution in each tube. Pay caution not to disturb the pellet of nanoparticles at the bottom of the tube.
   NOTE: The supernatant should have a blue tint because of the remaining suspended nanostars.
4. Using a micropipette, agitate the solution to suspend and collect the nanoparticles from each tube. Part of the pellet may be compacted on the bottom of the tube and will not resuspend even with vigorous pipetting – discard this part.
5. Transfer the nanoparticle suspension to a dialysis cassette (MWCO 3.5 kDa) and dialyze at least three days against 2 L of DI water, changing the water daily. Store nanostars in dialysis at 4 °C for up to a month with water changes every 3-4 days.
   NOTE: The nanostars should be kept in dialysis until required for the silication reaction, as described in section 2.

2. Formation of the Silica Shell

NOTE: Two flavors of Raman nanoprobes are synthesized. The synthesis procedure is the same for both, with the only difference being the Raman reporter molecule (dye) used. In this experiment, IR780 perchlorate and IR140 are used. The reaction should always be performed in plastic containers. The synthesis is highly scalable and can be adjusted for the desired amount of injectate required. Here, a medium batch synthesis is described, which can be scaled linearly to lower or higher volumes as needed, with the same concentrations and reaction times. The reactions for the two SERRS nanoprobe types can be performed in parallel. Pay attention to avoid cross-contamination. Sonication should be performed for the redispersion of nanoparticle pellets after centrifugation during washing steps or after the nanoparticles were stored for longer than one hour. Sonication should be performed until the nanoparticles are clearly suspended into the solution, typically for 1 s.

1. In Tube A (a 50 mL conical tube), mix 10 mL of isopropanol, 500 µL of TEOS, 200 µL of DI water, and 60 µL of dye (IR780 perchlorate or IR140, 20 mM in DMF (dimethylformamide)).
2. In Tube B (a 15 mL conical tube), mix 3 mL of ethanol and 200 µL of ammonium hydroxide. Sonicate the nanostars from Step 1.4 to disperse any clusters in solution and add 1.2 mL of nanostars to the tube.
   NOTE: the ammonium hydroxide solution is highly volatile and hard to pipette accurately. Store it at 4 °C, until needed, to facilitate pipetting.
3. Place Tube A on a vortex mixer and induce a steady vortex. Rapidly add the contents of Tube B into the vortex and keep mixing for about 5 s. Immediately transfer to a shaker and allow it to react for 15 min while shaking at 300 rpm, at room temperature.
4. After the 15 min incubation, quench the reaction by adding ethanol to fill the 50 mL tube. Centrifuge for 20 min at 3,220 x g and 4 °C.
5. Aspirate the supernatant, leaving about 0.5 mL of solution, being careful not to disturb the pellet. Add 1 mL of ethanol and agitate with a pipette to resuspend the nanoparticles. Transfer to a 1.5 mL centrifuge tube and wash 4 times with ethanol by centrifuging at 11,000 x g for 4 min, aspirating the supernatant, and resuspending the pellet by ultrasonication for approximately 1 s.
6. NOTE: At this stage, the silicated nanoparticles can be functionalized, as described in section 3, or resuspended in DI water with an extra washing step, for storage at 4 °C for up to a week.

3. Surface Functionalization

NOTE: IR780 SERRS nanoprobes will be conjugated with folate receptor-targeting antibodies via a PEG crosslinker to form αFR-NPs; IR140 SERRS control nanoprobes will be conjugated with a passivating PEG monolayer, for nt-NPs. Both flavors are formed via a thiol-maleimide reaction in separate but parallel reactions.

1. Wash nanoparticles twice by centrifuging at 11,000 x g for 4 min, aspirating the supernatant, and resuspending the pellet in 1 mL of ethanol by ultrasonication. Repeat the washing step once more, but redisperse in 1 mL of 85% ethanol, 10% 3-MPTMS (3-mercaptopropyltrimethoxysilane), and 5% DI water. Incubate at room temperature for 1-2 h to introduce thiols on the particle surface.
2. Wash the thiol-functionalized nanoparticles by centrifuging at 11,000 x g for 4 min, aspirating the supernatant and resuspending the pellet by ultrasonication, twice in ethanol, twice in DI, and finally in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (10 mM, pH 7.1), and set aside.
3. NOTE: MES (2-(N-morpholino) ethanesulfonic acid) buffer or HEPES should be used. Buffers with higher salinity, such as PBS (phosphate-buffered saline), may induce nanoparticle aggregation.
4. For the antibody functionalized αFR-NPs, react 200 µg of antibodies (anti-folate binding protein antibody clone [LK26]) with tenfold molar excess of PEG crosslinker (poly(ethylene glycol)(N-hydroxysuccinimide 5-pentanoate) ether N′-(3-maleimidopropionyl) aminoethane (CAS: 851040-94-3), in dimethyl sulfoxide (DMSO)) in 500 µL of MES buffer (10 mM, pH 7.1) for 30 min.
5. Remove excess crosslinker and concentrate antibody by centrifuging the antibody-PEG solution in a centrifugal filter (MWCO 100 kDa). For the centrifugal filters used in this study, perform centrifugation for 10 min at 14,000 x g and 23 °C. Recover the conjugated antibodies in a fresh tube by inverting the filter and centrifuging at 1,000 x g for 2 min.
6. Pipette the IR780 nanoparticles from Step 3.2 into the tube with the antibodies and agitate with the pipette to mix. Incubate the mixture for at least 30 min at room temperature or at 4 °C overnight to form the αFR-NPs.
7. To form nt-NPs, add 1% w/v methoxy-terminated (m)PEG5000-maleimide (CAS: 99126-64-4) dissolved in DMSO to the IR140 SERRS nanoparticles from Step 3.2 and let react in 500 µL MES buffer (10 mM, pH 7.1) for at least 30 min at room temperature, or at 4 °C overnight.
8. For administration to mice (section 4), spin down both nanoprobe flavors at 11,000 x g for 4 min, aspirate the supernatant to remove the solution with free unreacted antibodies/PEG, and redisperse each flavor in MES buffer (10 mM, pH 7.1) at 600 pM concentrations. When resuspending the nanoparticles, minimize unnecessary exposure to the ultrasound to prevent denaturation of the antibody.

4. Nanoprobe Injection and Imaging

1. Prepare nanoprobes (αFR-NPs and nt-NPs) as described in sections 1-3 and mix at a 1:1 ratio, for a final concentration of 300 pM of each type in MES buffer (10 mM, pH 7.1). Optionally, prepare reference standards of 30 pM of each of the nanoprobe flavors in small (100 µL) conical tubes.
2. Inject intraperitoneally 1 mL of the nanoparticle suspension in each mouse and gently massage the belly to distribute the nanoparticles within the peritoneal cavity. Return the mouse to its housing. After 25 or more minutes, euthanize the mouse via CO₂ asphyxiation.
3. Fasten the mouse on a surgical platform, at the supine position (for whole abdomen imaging, the platform needs to be mountable onto the upright microscope stage).
   1. Using serrated forceps and dissection scissors, remove the skin to expose the peritoneum and perform a large sagittal incision (between 2 and 3 cm in length) to expose the whole abdomen. Attach the peritoneal flaps onto the platform. Wash the inside of the peritoneal cavity with at least 60 mL of PBS using a plastic squirt bottle.
      NOTE: To enable unobstructed imaging of the whole abdomen, the intestines need to be mobilized or excised. For excision, resect with a ligation of the mesenteric vessels in order to reduce hemorrhage into the abdominal cavity.
   2. Alternatively, to image specific organs, excise them after the PBS wash, and place them onto a microscope slide.
4. Transfer the platform or slide to a Raman microspectrophotometer with upright optical configuration and a motorized stage for imaging; use a commercial system with a 300 mW 785 nm diode laser, with a grating of 1,200 grooves per mm, centered at 1,115 cm^-1.
   1. Focus on the area of interest using white light optics, parfocal with the Raman laser. Select the area to be imaged and the desired resolution; in this report a high-speed acquisition mode was used (spectra acquired under continuous laser illumination with the microscope stage constantly moving, with effective spatial resolution 14.2 µm by 200 µm; at 5x magnification, 100 mW power at objective, and <100 ms exposure per point).
      NOTE: The tubes with the reference nanoprobes from Step 4.1 can be placed within the imaged area if desired, to provide internal reference standards for the subsequent analysis. Make sure no extraneous light sources other than the laser reach the objective.
5. Optionally, prepare the sample for histological processing and validation by fixation in 4% paraformaldehyde in PBS overnight at 4 °C. Rinse with PBS at 4 °C for 15 min at least twice. Keep the sample in 70% ethanol in water until standard histological processing and paraffin embedding. For histological validation of the tumors, sections (5 µm thick) from different depths of the paraffin block can be stained with hematoxylin and eosin (H&E).