Name: surface-enhanced resonance raman scattering nanoprobe ratiometry for detecting microscopic ovarian cancer via folate receptor targeting
Uploaded: 2019-03-25T13:00:00
Description: Watch this Scientific Journal Video about Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting at JoVE.com

The following funding sources (to M.F.K.) are acknowledged: NIH R01 EB017748, R01 CA222836 and K08 CA16396; Damon Runyon-Rachleff Innovation Award DRR-29-14, Pershing Square Sohn Prize by the Pershing Square Sohn Cancer Research Alliance, and MSKCC Center for Molecular Imaging &#38; Nanotechnology (CMINT) and Technology Development Grants. Acknowledgments are also extended to the grant-funding support provided by the MSKCC NIH Core Grant (P30-CA008748).

All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center (#06-07-011).
1.&#160;Gold Nanostar Core Synthesis
NOTE: Gold nanostars are used as cores for both flavors of SERRS nanoprobes used in this experiment.
<ol>
	<li>Prepare 800 mL of 60 mM ascorbic acid (C6H8O6) solution in deionized (DI) water and 8 mL of 20 mM tetrachloroauric acid (HAuCl<sub...

<ol>
	<li>Oseledchyk, A., Andreou, C., Wall, M. A., Kircher, M. 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The protocol described here provides instruction for the synthesis of two &#34;flavors&#34; of SERRS nanoprobes, and their employment in mice for Raman imaging of ovarian tumor overexpressing the Folate Receptor, using a ratiometric algorithm. The main advantage of Raman imaging over other optical imaging techniques (such as fluorescence) is the high specificity of the nanoprobe signal that cannot be confounded with any signals of biological origin. In this embodiment of Raman imaging, the nanoparticles are not administe...

Raman imaging with &#39;surface enhanced Raman scattering&#39; (SERS) nanoparticles has shown great promise in delineating lesions in a variety of settings and for many different tumor types1,2,3,4. The main advantage of SERS nanoparticles is their fingerprint-like spectral signature, affording them unquestionable detection that is not confounded by biological background signals5. Additionally, the intensity of the emitted signal is further amplified with the use of reporter molecules (dyes) with absorbance maxima in line with the excitation laser, giving rise to &#39;surface enhanced resonance Raman scattering&#39; (SERRS) nanoparticles with even greater sensitivity6,7,8,9,10,11,12.
One barrier that needs to be addressed for the adoption of SE(R)RS nanoparticles13 and many other nanoparticle constructs14,15 for clinical use is their mode of administration, as intravenous injection causes systemic exposure of the agent, and necessitates extensive testing to exclude potential adverse effects. In this article, we present a different paradigm based on the application of nanoparticles locally in vivo, directly into the peritoneal cavity during surgery, followed by a washing step to remove any unbound nanoparticles1. This approach is in line with novel therapeutic approaches that are currently under investigation that also make use of local instillation of agents into the peritoneal cavity, called hyperthermic intraperitoneal chemotherapy (HIPEC). Thus, the principle itself should be relatively easy to integrate into a clinical workflow. We have studied the biodistribution of the nanoparticles after intraperitoneal application, and have not observed any detectable absorption into the systemic circulation1. Additionally, the local application approach circumvents the sequestration of nanoparticles by the reticuloendothelial system, so the numbers of nanoparticles required are markedly reduced. However, when applied topically, antibody-functionalized nanoparticles tend to adhere onto the visceral surfaces even in the absence of their target. In order to minimize false positive signals due to non-specific nanoparticle adhesion, we pursue a ratiometric approach, where a molecularly targeted nanoprobe provides the specific signal, and a non-targeted control nanoprobe, with different Raman spectrum, accounts for non-specific background16,17. We have demonstrated this methodology of topically applied surface enhanced resonance Raman ratiometric spectroscopy recently in a mouse model of diffuse ovarian cancer1.
The overall goal of this method is to develop two SERRS nanoprobes, one targeted and one non-specific, to be applied locally in mouse models, in order to image the prevalence/overexpression of a cancer related biomarker using ratiometric detection of the two probes via Raman imaging. In this work, the folate receptor (FR) was chosen as the target, as this is a marker upregulated in many ovarian cancers18,19. Raman microimaging with SERS-based nanoparticles has also been demonstrated for cancer cell identification20. Two distinct &#34;flavors&#34; of Raman nanoparticles are synthesized, each deriving its fingerprint from a different organic dye. The nanoparticles consist of a star-shaped gold core surrounded by a silica shell and demonstrate surface plasmon resonance at approximately 710 nm. The Raman reporter (organic dye) is deposited concurrently with the formation of silica shell. Finally, for the FR-targeted nanoprobes (&#945;FR-NPs) the silica shell is conjugated with antibodies, whereas the non-targeted nanoprobes (nt-NPs) are passivated with a monolayer of polyethylene glycol (PEG).
This technique was successfully used to map microscopic tumors in a mouse xenograft model of diffuse metastatic ovarian cancer (SKOV-3), demonstrating its applicability for in vivo use. It can also be extended for use in excised tissues, for tumor phenotyping, or margin determination after debulking as shown in a cognate study21.
SERRS nanoprobes provide a robust platform for the creation of multiple targeted tags for biomarkers, synthesized with straightforward chemical reactions as outlined schematically in Figure 1. Here, we present the protocol for the synthesis of the two types of SERRS nanoprobes (sections 1-3), the development of a suitable ovarian cancer mouse model (section 4), the administration of nanoprobes and imaging (section 5), and finally the data analysis and visualization (section 6).

<table border="0" cellpadding="0" cellspacing="0" style="width:764px;" width="764">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Name of Reagent</td>
			<td style="width:161px;"></td>
			<td style="width:132px;"></td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">3-MPTMS</td>
			<td>Sigma-Aldrich</td>
			<td>175617</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ammonium hydroxide (28%)</td>
			<td>Sigma-Aldrich</td>
			<td>338818</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Anti-Folate Receptor antibody [LK26]&#160;</td>
			<td>AbCam</td>
			<td>ab3361</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right">276855</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dimethyl sulfoxide (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>792780</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">IR140</td>
			<td>Sigma-Aldrich</td>
			<td>260932</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">IR780 perchlorate*</td>
			<td>Sigma-Aldrich</td>
			<td>576409</td>
			<td>Discontinued*</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>650447</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">N.N.Dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PEG crosslinker</td>
			<td>Sigma-Aldrich</td>
			<td>757853</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PEG-maleimide</td>
			<td>Sigma-Aldrich</td>
			<td>900339</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Tetrachloroauric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>244597</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Tetraethyl Orthosilicate</td>
			<td>Sigma-Aldrich</td>
			<td>86578</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">*IR792</td>
			<td>Sigma-Aldrich</td>
			<td>425982</td>
			<td>*Alternative</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Name of Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dialysis cassette (3,500 MWCO)</td>
			<td>ThermoFIsher</td>
			<td align="right">87724</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Centrifugal filters</td>
			<td>Millipore</td>
			<td>UFC510096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">inVia confocal Raman microscope</td>
			<td>Renishaw</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB (v2014b)</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PLS Toolbox (v8.0)</td>
			<td>Eigenvector research</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

surface-enhanced resonance raman scattering nanoprobe ratiometry for detecting microscopic ovarian cancer via folate receptor targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

For quality control purposes, the nanoparticles can be characterized using a variety of methods during the synthesis process, including TEM, DLS, nanoparticle tracking analysis, and UV/Vis absorbance spectroscopy, as shown in Figure 2.
In this way, the size of the gold nanostar core (described in section 1), the formation of the silica shell (section 2) and subsequent surface functionalization (sect...

Watch this Scientific Journal Video about Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting at JoVE.com

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

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.

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Cancer Research

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