High-speed Raman-encoded molecular imaging of freshly excised tissue surfaces with topically applied SERRS nanoparticles.

Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) are increasingly being engineered for a variety of disease-detection and treatment applications. For example, we have previously developed a fiber-optic Raman-encoded molecular imaging (REMI) system for spectral imaging of biomarker-targeted SERS NPs topically applied on tissue surfaces to identify residual tumors at surgical margins. Although accurate tumor detection was achieved, the commercial SERS NPs used in our previous studies lacked the signal strength to enable high-speed imaging with high pixel counts (large fields of view and/or high spatial resolution), which limits their use for certain time-constrained clinical applications. As a solution, we explored the use of surface-enhanced resonant Raman scattering (SERRS) NPs to enhance imaging speeds. The SERRS NPs were synthesized de novo, and then conjugated to HER2 antibodies to achieve high binding affinity, as validated by flow cytometry. Under identical tissue-staining and imaging conditions, the targeted SERRS NPs enabled reliable identification of HER2-overexpressed tumor xenografts with 50-fold-enhanced imaging speed compared with our standard targeted SERS NPs. This enables our REMI system to image tissue surfaces at a rate of 150 cm2 per minute at a spatial resolution of 0.5 mm.

Microscopic investigation of" topically applied nanoparticles for molecular imaging of fresh tissue surfaces.

Previous studies have shown that functionalized nanoparticles (NPs) topically applied on fresh tissues are able to rapidly target cell-surface protein biomarkers of cancer. Furthermore, studies have shown that a paired-agent approach, in which an untargeted NP is co-administered with a panel of targeted NPs, controls for the nonspecific behavior of the NPs, enabling quantitative imaging of biomarker expression. However, given the complexities in nonspecific accumulation, diffusion, and chemical binding of targeted NPs in tissues, studies are needed to better understand these processes at the microscopic scale. Here, fresh tissues were stained with a paired-agent approach, frozen, and sectioned to image the depth-dependent accumulation of targeted and untargeted NPs. The ratio of targeted-to-untargeted NP concentrations-a parameter used to distinguish between tumor and benign tissues-was found to diminish with increasing NP diffusion depths due to nonspecific accumulation and poor washout. It was then hypothesized and experimentally demonstrated that larger NPs would exhibit less diffusion below tissue surfaces, enabling higher targeted-to-untargeted NP ratios. In summary, these methods and investigations have enabled the design of NP agents with improved sensitivity and contrast for rapid molecular imaging of fresh tissues.

Multiplexed Optical Imaging of Tumor-Directed Nanoparticles: A Review of Imaging Systems and Approaches.

In recent decades, various classes of nanoparticles have been developed for optical imaging of cancers. Many of these nanoparticles are designed to specifically target tumor sites, and specific cancer biomarkers, to facilitate the visualization of tumors. However, one challenge for accurate detection of tumors is that the molecular profiles of most cancers vary greatly between patients as well as spatially and temporally within a single tumor mass. To overcome this challenge, certain nanoparticles and imaging systems have been developed to enable multiplexed imaging of large panels of cancer biomarkers. Multiplexed molecular imaging can potentially enable sensitive tumor detection, precise delineation of tumors during interventional procedures, and the prediction/monitoring of therapy response. In this review, we summarize recent advances in systems that have been developed for the imaging of optical nanoparticles that can be heavily multiplexed, which include surface-enhanced Raman-scattering nanoparticles (SERS NPs) and quantum dots (QDs). In addition to surveying the optical properties of these various types of nanoparticles, and the most-popular multiplexed imaging approaches that have been employed, representative preclinical and clinical imaging studies are also highlighted.

Raman-Encoded Molecular Imaging with Topically Applied SERS Nanoparticles for Intraoperative Guidance of Lumpectomy.

Intraoperative identification of carcinoma at lumpectomy margins would enable reduced re-excision rates, which are currently as high as 20% to 50%. Although imaging of disease-associated biomarkers can identify malignancies with high specificity, multiplexed imaging of such biomarkers is necessary to detect molecularly heterogeneous carcinomas with high sensitivity. We have developed a Raman-encoded molecular imaging (REMI) technique in which targeted nanoparticles are topically applied on excised tissues to enable rapid visualization of a multiplexed panel of cell surface biomarkers at surgical margin surfaces. A first-ever clinical study was performed in which 57 fresh specimens were imaged with REMI to simultaneously quantify the expression of four biomarkers HER2, ER, EGFR, and CD44. Combined detection of these biomarkers enabled REMI to achieve 89.3% sensitivity and 92.1% specificity for the detection of breast carcinoma. These results highlight the sensitivity and specificity of REMI to detect biomarkers in freshly resected tissue, which has the potential to reduce the rate of re-excision procedures in cancer patients. Cancer Res; 77(16); 4506-16. ©2017 AACR.

In vivo multiplexed molecular imaging of esophageal cancer via spectral endoscopy of topically applied SERS nanoparticles.

The biological investigation and detection of esophageal cancers could be facilitated with an endoscopic technology to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and investigation through the sensitive and multiplexed detection of cell-surface biomarkers. Here, we demonstrate that the topical application and endoscopic imaging of a multiplexed cocktail of receptor-targeted SERS NPs enables the rapid detection of tumors in an orthotopic rat model of esophageal cancer. Antibody-conjugated SERS NPs were topically applied on the lumenal surface of the rat esophagus to target EGFR and HER2, and a miniature spectral endoscope featuring rotational scanning and axial pull-back was employed to comprehensively image the NPs bound on the lumen of the esophagus. Ratiometric analyses of specific vs. nonspecific binding enabled the visualization of tumor locations and the quantification of biomarker expression in agreement with immunohistochemistry and flow cytometry validation data.

Rapid ratiometric biomarker detection with topically applied SERS nanoparticles.

Multiplexed surface-enhanced Raman scattering (SERS) nanoparticles (NPs) offer the potential for rapid molecular phenotyping of tissues, thereby enabling accurate disease detection as well as patient stratification to guide personalized therapies or to monitor treatment outcomes. The clinical success of molecular diagnostics based on SERS NPs would be facilitated by the ability to accurately identify tissue biomarkers under time-constrained staining and detection conditions with a portable device. In vitro, ex vivo and in vivo experiments were performed to optimize the technology and protocols for the rapid detection (0.1-s integration time) of multiple cell-surface biomarkers with a miniature fiber-optic spectral-detection probe following a brief (5 min) topical application of SERS NPs on tissues. Furthermore, we demonstrate that the simultaneous detection and ratiometric quantification of targeted and nontargeted NPs allows for an unambiguous assessment of molecular expression that is insensitive to nonspecific variations in NP concentrations.