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Cancer Research

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

Published: March 25th, 2019



1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2Department of Chemistry, The Graduate Center of the City University of New York, 3Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, 4Center for Molecular Imaging and Nanotechnology (CMINT), Memorial Sloan Kettering Cancer Center, 5Gerstner Sloan Kettering Graduate School of Biomedical Sciences, 6Department of Radiology, Weill Cornell Medical College of Cornell University, 7Dana-Farber Cancer Institute and Harvard Medical Center

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 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 'flavors' of such nanoprobes: an antibody-functionalized one that targets the folate receptor — overexpressed in many ovarian cancers — and a non-targeted control nanoprobe, with 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 — which can be done conveniently during the surgical procedure — 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.

Raman imaging with 'surface enhanced Raman scattering' (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 ....

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All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center (#06-07-011).

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 (HAuCl

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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.......

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The protocol described here provides instruction for the synthesis of two "flavors" 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.......

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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 & Nanotechnology (CMINT) and Technology Development Grants. Acknowledgments are also extended to the grant-funding support provided by the MSKCC NIH Core Grant (P30-CA008748).


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Name Company Catalog Number Comments
Name of Reagent
Ascorbic acid Sigma-Aldrich A5960
3-MPTMS Sigma-Aldrich 175617
Ammonium hydroxide (28%) Sigma-Aldrich 338818
Anti-Folate Receptor antibody [LK26]  AbCam ab3361
Dimethyl sulfoxide Sigma-Aldrich 276855
Dimethyl sulfoxide (anhydrous) Sigma-Aldrich 276855
Ethanol Sigma-Aldrich 792780
IR140 Sigma-Aldrich 260932
IR780 perchlorate* Sigma-Aldrich 576409 Discontinued*
Isopropanol Sigma-Aldrich 650447
N.N.Dimethylformamide Sigma-Aldrich 227056
PEG crosslinker Sigma-Aldrich 757853
PEG-maleimide Sigma-Aldrich 900339
Tetrachloroauric Acid Sigma-Aldrich 244597
Tetraethyl Orthosilicate Sigma-Aldrich 86578
*IR792 Sigma-Aldrich 425982 *Alternative
Name of Equipment
Dialysis cassette (3,500 MWCO) ThermoFIsher 87724
Centrifugal filters Millipore UFC510096
inVia confocal Raman microscope Renishaw
MATLAB (v2014b) Mathworks
PLS Toolbox (v8.0) Eigenvector research

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