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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folate-Targeted+Surface-Enhanced+Resonance+Raman+Scattering+Nanoprobe+Ratiometry+for+Detection+of+Microscopic+Ovarian+Cancer.">Folate-Targeted Surface-Enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detection of Microscopic Ovarian Cancer.</a> ACS Nano. 11 (2), 1488-1497 (2017).</li><li>Andreou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+Liver+Tumors+Using+Surface-Enhanced+Raman+Scattering+Nanoparticles.">Imaging of Liver Tumors Using Surface-Enhanced Raman Scattering Nanoparticles.</a> ACS Nano. 10 (5), 5015-5026 (2016).</li><li>Karabeber, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guiding+brain+tumor+resection+using+surface-enhanced+Raman+scattering+nanoparticles+and+a+hand-held+Raman+scanner.">Guiding brain tumor resection using surface-enhanced Raman scattering nanoparticles and a hand-held Raman scanner.</a> ACS Nano. 8 (10), 9755-9766 (2014).</li><li>Kircher, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brain+tumor+molecular+imaging+strategy+using+a+new+triple-modality+MRI-photoacoustic-Raman+nanoparticle.">A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle.</a> Nature Medicine. 18 (5), 829-834 (2012).</li><li>Andreou, C., Kishore, S. A., Kircher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-Enhanced+Raman+Spectroscopy:+A+New+Modality+for+Cancer+Imaging.">Surface-Enhanced Raman Spectroscopy: A New Modality for Cancer Imaging.</a> Journal of Nuclear Medicine. 56 (9), 1295-1299 (2015).</li><li>Harmsen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+chalcogenopyrylium-based+surface-enhanced+resonance+Raman+scattering+nanoprobe+with+attomolar+sensitivity.">Rational design of a chalcogenopyrylium-based surface-enhanced resonance Raman scattering nanoprobe with attomolar sensitivity.</a> Nature Communications. 6, 6570 (2015).</li><li>Harmsen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-enhanced+resonance+Raman+scattering+nanostars+for+high-precision+cancer+imaging.">Surface-enhanced resonance Raman scattering nanostars for high-precision cancer imaging.</a> Science Translational Medicine. 7 (271), 271ra277 (2015).</li><li>Harmsen, S., Wall, M. A., Huang, R., Kircher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+imaging+using+surface-enhanced+resonance+Raman+scattering+nanoparticles.">Cancer imaging using surface-enhanced resonance Raman scattering nanoparticles.</a> Nature Protocols. 12 (7), 1400-1414 (2017).</li><li>Huang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Precision+Imaging+of+Microscopic+Spread+of+Glioblastoma+with+a+Targeted+Ultrasensitive+SERRS+Molecular+Imaging+Probe.">High Precision Imaging of Microscopic Spread of Glioblastoma with a Targeted Ultrasensitive SERRS Molecular Imaging Probe.</a> Theranostics. 6 (8), 1075-1084 (2016).</li><li>Iacono, P., Karabeber, H., Kircher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+&amp;#34;schizophotonic&amp;#34;+all-in-one+nanoparticle+coating+for+multiplexed+SE(R)RS+biomedical+imaging.">A &amp;#34;schizophotonic&amp;#34; all-in-one nanoparticle coating for multiplexed SE(R)RS biomedical imaging.</a> Angewandte Chemie, International Edition in English. 53 (44), 11756-11761 (2014).</li><li>Spaliviero, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Lymph+Node+Metastases+with+SERRS+Nanoparticles.">Detection of Lymph Node Metastases with SERRS Nanoparticles.</a> Molecular Imaging and Biology. 18 (5), 677-685 (2016).</li><li>Nayak, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+factor-specific+ultra-bright+SERRS+nanostars+for+Raman+detection+of+pulmonary+micrometastases.">Tissue factor-specific ultra-bright SERRS nanostars for Raman detection of pulmonary micrometastases.</a> Nanoscale. 9 (3), 1110-1119 (2017).</li><li>Thakor, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+and+toxicity+of+Raman-active+silica-gold+nanoparticles+in+mice.">The fate and toxicity of Raman-active silica-gold nanoparticles in mice.</a> Science Translational Medicine. 3 (79), 79ra33 (2011).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Cd-based+Quantum+Dot+Exposure+on+the+Reproduction+and+Offspring+of+Kunming+Mice+over+Multiple+Generations.">Effects of Cd-based Quantum Dot Exposure on the Reproduction and Offspring of Kunming Mice over Multiple Generations.</a> Nanotheranostics. 1 (1), 23-37 (2017).</li><li>Wu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+studies+of+polymer+dots+on+pregnant+mice+and+fetuses.">The biocompatibility studies of polymer dots on pregnant mice and fetuses.</a> Nanotheranostics. 1 (3), 261-271 (2017).</li><li>Garai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+real-time,+ratiometric+imaging+of+surface-enhanced+Raman+scattering+nanoparticles+with+a+clinically+translatable+Raman+endoscope+device.">High-sensitivity real-time, ratiometric imaging of surface-enhanced Raman scattering nanoparticles with a clinically translatable Raman endoscope device.</a> Journal of Biomedical Optics. 18 (9), 096008 (2013).</li><li>Wang, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+ratiometric+biomarker+detection+with+topically+applied+SERS+nanoparticles.">Rapid ratiometric biomarker detection with topically applied SERS nanoparticles.</a> Technology (Singap World Sci). 2 (2), 118-132 (2014).</li><li>Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+development+and+metastasis.">Ovarian cancer development and metastasis.</a> American Journal of Pathology. 177 (3), 1053-1064 (2010).</li><li>Vergote, I. B., Marth, C., Coleman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+folate+receptor+in+ovarian+cancer+treatment:+evidence,+mechanism,+and+clinical+implications.">Role of the folate receptor in ovarian cancer treatment: evidence, mechanism, and clinical implications.</a> Cancer and Metastasis Reviews. 34 (1), 41-52 (2015).</li><li>Fasolato, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folate-based+single+cell+screening+using+surface+enhanced+Raman+microimaging.">Folate-based single cell screening using surface enhanced Raman microimaging.</a> Nanoscale. 8 (39), 17304-17313 (2016).</li><li>Wang, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman-Encoded+Molecular+Imaging+with+Topically+Applied+SERS+Nanoparticles+for+Intraoperative+Guidance+of+Lumpectomy.">Raman-Encoded Molecular Imaging with Topically Applied SERS Nanoparticles for Intraoperative Guidance of Lumpectomy.</a> Cancer Research. 77 (16), 4506-4516 (2017).</li><li>Andreou, C., Pal, S., Rotter, L., Yang, J., Kircher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+in+Nanotechnology+and+Theranostics.">Molecular Imaging in Nanotechnology and Theranostics.</a> Molecular Imaging and Biology. 19 (3), 363-372 (2017).</li><li>Chitgupi, U., Qin, Y., Lovell, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Nanomaterials+for+Phototherapy.">Targeted Nanomaterials for Phototherapy.</a> Nanotheranostics. 1 (1), 38-58 (2017).</li><li>Choi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iodinated+Echogenic+Glycol+Chitosan+Nanoparticles+for+X-ray+CT/US+Dual+Imaging+of+Tumor.">Iodinated Echogenic Glycol Chitosan Nanoparticles for X-ray CT/US Dual Imaging of Tumor.</a> Nanotheranostics. 2 (2), 117-127 (2018).</li><li>Dubey, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Hyaluronic+Acid+Conjugates+for+Dual+Nuclear+Imaging+and+Therapy+in+CD44-Expressing+Tumors+in+Mice+In+Vivo.">Novel Hyaluronic Acid Conjugates for Dual Nuclear Imaging and Therapy in CD44-Expressing Tumors in Mice In Vivo.</a> Nanotheranostics. 1 (1), 59-79 (2017).</li><li>Gupta, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+strategies+to+design+vascular+theranostic+nanoparticles.">Recent strategies to design vascular theranostic nanoparticles.</a> Nanotheranostics. 1 (2), 166-177 (2017).</li><li>Huang, Y. J., Hsu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRAIL-functionalized+gold+nanoparticles+selectively+trigger+apoptosis+in+polarized+macrophages.">TRAIL-functionalized gold nanoparticles selectively trigger apoptosis in polarized macrophages.</a> Nanotheranostics. 1 (3), 326-337 (2017).</li><li>Pal, S., Harmsen, S., Oseledchyk, A., Hsu, H. T., Kircher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUC1+Aptamer+Targeted+SERS+Nanoprobes.">MUC1 Aptamer Targeted SERS Nanoprobes.</a> Advanced Functional Materials. 27 (32), (2017).</li><li>Zanganeh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+oxide+nanoparticles+inhibit+tumour+growth+by+inducing+pro-inflammatory+macrophage+polarization+in+tumour+tissues.">Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues.</a> Nat Nanotechnol. 11 (11), 986-994 (2016).</li><li>Lee, J., Lee, Y. M., Kim, J., Kim, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin/Ce6-Loaded+Nanoparticle+Coated+with+Polymer+via+Singlet+Oxygen-Sensitive+Linker+for+Photodynamically+Assisted+Chemotherapy.">Doxorubicin/Ce6-Loaded Nanoparticle Coated with Polymer via Singlet Oxygen-Sensitive Linker for Photodynamically Assisted Chemotherapy.</a> Nanotheranostics. 1 (2), 196-207 (2017).</li><li>Li, R., Zheng, K., Yuan, C., Chen, Z., Huang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Be+Active+or+Not:+the+Relative+Contribution+of+Active+and+Passive+Tumor+Targeting+of+Nanomaterials.">Be Active or Not: the Relative Contribution of Active and Passive Tumor Targeting of Nanomaterials.</a> Nanotheranostics. 1 (4), 346-357 (2017).</li><li>Lin, S. Y., Huang, R. Y., Liao, W. C., Chuang, C. C., Chang, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+PEGylated+Albumin/IR780/Iron+Oxide+Nanocomplexes+for+Cancer+Photothermal+Therapy+and+MR+Imaging.">Multifunctional PEGylated Albumin/IR780/Iron Oxide Nanocomplexes for Cancer Photothermal Therapy and MR Imaging.</a> Nanotheranostics. 2 (2), 106-116 (2018).</li><li>Roberts, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonophore-enhanced+nanoemulsions+for+optoacoustic+imaging+of+cancer.">Sonophore-enhanced nanoemulsions for optoacoustic imaging of cancer.</a> Chemical Science (Royal Society of Chemistry: 2010). 9 (25), 5646-5657 (2018).</li><li>Liu, L., Ruan, Z., Yuan, P., Li, T., Yan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+Self-Sufficient+Amphiphilic+Polypeptide+Nanoparticles+Encapsulating+BODIPY+for+Potential+Near+Infrared+Imaging-guided+Photodynamic+Therapy+at+Low+Energy.">Oxygen Self-Sufficient Amphiphilic Polypeptide Nanoparticles Encapsulating BODIPY for Potential Near Infrared Imaging-guided Photodynamic Therapy at Low Energy.</a> Nanotheranostics. 2 (1), 59-69 (2018).</li><li>Liu, R., Tang, J., Xu, Y., Zhou, Y., Dai, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-sized+Indocyanine+Green+J-aggregate+as+a+One-component+Theranostic+Agent.">Nano-sized Indocyanine Green J-aggregate as a One-component Theranostic Agent.</a> Nanotheranostics. 1 (4), 430-439 (2017).</li><li>Sneider, A., VanDyke, D., Paliwal, S., Rai, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remotely+Triggered+Nano-Theranostics+For+Cancer+Applications.">Remotely Triggered Nano-Theranostics For Cancer Applications.</a> Nanotheranostics. 1 (1), 1-22 (2017).</li><li>Wall, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant-Free+Shape+Control+of+Gold+Nanoparticles+Enabled+by+Unified+Theoretical+Framework+of+Nanocrystal+Synthesis.">Surfactant-Free Shape Control of Gold Nanoparticles Enabled by Unified Theoretical Framework of Nanocrystal Synthesis.</a> Advanced Materials. 29 (21), (2017).</li><li>Sonali, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotheranostics:+Emerging+Strategies+for+Early+Diagnosis+and+Therapy+of+Brain+Cancer.">Nanotheranostics: Emerging Strategies for Early Diagnosis and Therapy of Brain Cancer.</a> Nanotheranostics. 2 (1), 70-86 (2018).</li></ol>

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 ...

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