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

Summary

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.

Abstract

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.

Introduction

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 types^1,2,3,4. 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 'surface enhanced resonance Raman scattering' (SERRS) nanoparticles with even greater sensitivity^{6,7,8,9,10,11,12}.

One barrier that needs to be addressed for the adoption of SE(R)RS nanoparticles¹³ and many other nanoparticle constructs^14,15 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 background^16,17. 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 cancers^18,19. Raman microimaging with SERS-based nanoparticles has also been demonstrated for cancer cell identification²⁰. Two distinct "flavors" 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 (αFR-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).

Protocol

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. Prepare 800 mL of 60 mM ascorbic acid (C6H8O6) solution in deionized (DI) water and 8 mL of 20 mM tetrachloroauric acid (HAuCl<sub…

Representative Results

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…

Discussion

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…

Materials

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