A hyperspectral method to assay the microphysiological fates of nanomaterials in histological samples.

Nanoparticles are used extensively as biomedical imaging probes and potential therapeutic agents. As new particles are developed and tested in vivo, it is critical to characterize their biodistribution profiles. We demonstrate a new method that uses adaptive algorithms for the analysis of hyperspectral dark-field images to study the interactions between tissues and administered nanoparticles. This non-destructive technique quantitatively identifies particles in ex vivo tissue sections and enables detailed observations of accumulation patterns arising from organ-specific clearance mechanisms, particle size, and the molecular specificity of nanoparticle surface coatings. Unlike nanoparticle uptake studies with electron microscopy, this method is tractable for imaging large fields of view. Adaptive hyperspectral image analysis achieves excellent detection sensitivity and specificity and is capable of identifying single nanoparticles. Using this method, we collected the first data on the sub-organ distribution of several types of gold nanoparticles in mice and observed localization patterns in tumors.

A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer.

PURPOSE: Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes. PROCEDURES: We developed and used a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice. RESULTS: This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model. CONCLUSIONS: The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection.

A real-time clinical endoscopic system for intraluminal, multiplexed imaging of surface-enhanced Raman scattering nanoparticles.

The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.

A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors.

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy.

High-sensitivity, real-time, ratiometric imaging of surface-enhanced Raman scattering nanoparticles with a clinically translatable Raman endoscope device.

Topical application and quantification of targeted, surface-enhanced Raman scattering (SERS) nanoparticles offer a new technique that has the potential for early detection of epithelial cancers of hollow organs. Although less toxic than intravenous delivery, the additional washing required to remove unbound nanoparticles cannot necessarily eliminate nonspecific pooling. Therefore, we developed a real-time, ratiometric imaging technique to determine the relative concentrations of at least two spectrally unique nanoparticle types, where one serves as a nontargeted control. This approach improves the specific detection of bound, targeted nanoparticles by adjusting for working distance and for any nonspecific accumulation following washing. We engineered hardware and software to acquire SERS signals and ratios in real time and display them via a graphical user interface. We report quantitative, ratiometric imaging with nanoparticles at pM and sub-pM concentrations and at varying working distances, up to 50 mm. Additionally, we discuss optimization of a Raman endoscope by evaluating the effects of lens material and fiber coating on background noise, and theoretically modeling and simulating collection efficiency at various working distances. This work will enable the development of a clinically translatable, noncontact Raman endoscope capable of rapidly scanning large, topographically complex tissue surfaces for small and otherwise hard to detect lesions.

A small animal Raman instrument for rapid, wide-area, spectroscopic imaging.

Raman spectroscopy, amplified by surface enhanced Raman scattering (SERS) nanoparticles, is a molecular imaging modality with ultra-high sensitivity and the unique ability to multiplex readouts from different molecular targets using a single wavelength of excitation. This approach holds exciting prospects for a range of applications in medicine, including identification and characterization of malignancy during endoscopy and intraoperative image guidance of surgical resection. The development of Raman molecular imaging with SERS nanoparticles is presently limited by long acquisition times, poor spatial resolution, small field of view, and difficulty in animal handling with existing Raman spectroscopy instruments. Our goal is to overcome these limitations by designing a bespoke instrument for Raman molecular imaging in small animals. Here, we present a unique and dedicated small-animal Raman imaging instrument that enables rapid, high-spatial resolution, spectroscopic imaging over a wide field of view (> 6 cm(2)), with simplified animal handling. Imaging of SERS nanoparticles in small animals demonstrated that this small animal Raman imaging system can detect multiplexed SERS signals in both superficial and deep tissue locations at least an order of magnitude faster than existing systems without compromising sensitivity.

A Raman-based endoscopic strategy for multiplexed molecular imaging.

Endoscopic imaging is an invaluable diagnostic tool allowing minimally invasive access to tissues deep within the body. It has played a key role in screening colon cancer and is credited with preventing deaths through the detection and removal of precancerous polyps. However, conventional white-light endoscopy offers physicians structural information without the biochemical information that would be advantageous for early detection and is essential for molecular typing. To address this unmet need, we have developed a unique accessory, noncontact, fiber optic-based Raman spectroscopy device that has the potential to provide real-time, multiplexed functional information during routine endoscopy. This device is ideally suited for detection of functionalized surface-enhanced Raman scattering (SERS) nanoparticles as molecular imaging contrast agents. This device was designed for insertion through a clinical endoscope and has the potential to detect and quantify the presence of a multiplexed panel of tumor-targeting SERS nanoparticles. Characterization of the Raman instrument was performed with SERS particles on excised human tissue samples, and it has shown unsurpassed sensitivity and multiplexing capabilities, detecting 326-fM concentrations of SERS nanoparticles and unmixing 10 variations of colocalized SERS nanoparticles. Another unique feature of our noncontact Raman endoscope is that it has been designed for efficient use over a wide range of working distances from 1 to 10 mm. This is necessary to accommodate for imperfect centering during endoscopy and the nonuniform surface topology of human tissue. Using this endoscope as a key part of a multiplexed detection approach could allow endoscopists to distinguish between normal and precancerous tissues rapidly and to identify flat lesions that are otherwise missed.

New positron emission tomography (PET) radioligand for imaging σ-1 receptors in living subjects.

σ-1 receptor (S1R) radioligands have the potential to detect and monitor various neurological diseases. Herein we report the synthesis, radiofluorination, and evaluation of a new S1R ligand 6-(3-fluoropropyl)-3-(2-(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one ([(18)F]FTC-146, [(18)F]13). [(18)F]13 was synthesized by nucleophilic fluorination, affording a product with >99% radiochemical purity (RCP) and specific activity (SA) of 2.6 ± 1.2 Ci/µmol (n = 13) at end of synthesis (EOS). Positron emission tomography (PET) and ex vivo autoradiography studies of [(18)F]13 in mice showed high uptake of the radioligand in S1R rich regions of the brain. Pretreatment with 1 mg/kg haloperidol (2), nonradioactive 13, or BD1047 (18) reduced the binding of [(18)F]13 in the brain at 60 min by 80%, 82%, and 81%, respectively, suggesting that [(18)F]13 accumulation in mouse brain represents specific binding to S1Rs. These results indicate that [(18)F]13 is a promising candidate radiotracer for further evaluation as a tool for studying S1Rs in living subjects.

A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle.

The difficulty in delineating brain tumor margins is a major obstacle in the path toward better outcomes for patients with brain tumors. Current imaging methods are often limited by inadequate sensitivity, specificity and spatial resolution. Here we show that a unique triple-modality magnetic resonance imaging-photoacoustic imaging-Raman imaging nanoparticle (termed here MPR nanoparticle) can accurately help delineate the margins of brain tumors in living mice both preoperatively and intraoperatively. The MPRs were detected by all three modalities with at least a picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to MPR accumulation and retention by the tumors, with no MPR accumulation in the surrounding healthy tissue, allowing for a noninvasive tumor delineation using all three modalities through the intact skull. Raman imaging allowed for guidance of intraoperative tumor resection, and a histological correlation validated that Raman imaging was accurately delineating the brain tumor margins. This new triple-modality-nanoparticle approach has promise for enabling more accurate brain tumor imaging and resection.

Raman's "effect" on molecular imaging.

Raman spectroscopy is an optical technique that offers unsurpassed sensitivity and multiplexing capabilities to the field of molecular imaging. In the past, Raman spectroscopy had predominantly been used as an analytic tool for routine chemical analysis, but more recently, researchers have been able to harness its unique properties for imaging and spectral analysis of molecular interactions in cell populations and preclinical animal models. Additionally, researchers have already begun to translate this optical technique into a novel clinical diagnostic tool using various endoscopic strategies.

Preclinical evaluation of Raman nanoparticle biodistribution for their potential use in clinical endoscopy imaging.

Raman imaging offers unsurpassed sensitivity and multiplexing capabilities. However, its limited depth of light penetration makes direct clinical translation challenging. Therefore, a more suitable way to harness its attributes in a clinical setting would be to couple Raman spectroscopy with endoscopy. The use of an accessory Raman endoscope in conjunction with topically administered tumor-targeting Raman nanoparticles during a routine colonoscopy could offer a new way to sensitively detect dysplastic lesions while circumventing Raman's limited depth of penetration and avoiding systemic toxicity. In this study, the natural biodistribution of gold surface-enhanced Raman scattering (SERS) nanoparticles is evaluated by radiolabeling them with (64) Cu and imaging their localization over time using micropositron emission tomography (PET). Mice are injected either intravenously (IV) or intrarectally (IR) with approximately 100 microcuries (µCi) (3.7 megabecquerel (MBq)) of (64) Cu-SERS nanoparticles and imaged with microPET at various time points post injection. Quantitative biodistribution data are obtained as % injected dose per gram (%ID g(-1)) from each organ, and the results correlate well with the corresponding microPET images, revealing that IV-injected mice have significantly higher uptake (p < 0.05) in the liver (5 h = 8.96% ID g(-1); 24 h = 8.27% ID g(-1)) than IR-injected mice (5 h = 0.09% ID g(-1); 24 h = 0.08% ID g(-1)). IR-injected mice show localized uptake in the large intestine (5 h = 10.37% ID g(-1); 24 h = 0.42% ID g(-1)) with minimal uptake in other organs. Raman imaging of excised tissues correlate well with biodistribution data. These results suggest that the topical application of SERS nanoparticles in the mouse colon appears to minimize their systemic distribution, thus avoiding potential toxicity and supporting the clinical translation of Raman spectroscopy as an endoscopic imaging tool.

Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy.

Raman spectroscopy is a newly developed, noninvasive preclinical imaging technique that offers picomolar sensitivity and multiplexing capabilities to the field of molecular imaging. In this study, we demonstrate the ability of Raman spectroscopy to separate the spectral fingerprints of up to 10 different types of surface enhanced Raman scattering (SERS) nanoparticles in a living mouse after s.c. injection. Based on these spectral results, we simultaneously injected the five most intense and spectrally unique SERS nanoparticles i.v. to image their natural accumulation in the liver. All five types of SERS nanoparticles were successfully identified and spectrally separated using our optimized noninvasive Raman imaging system. In addition, we were able to linearly correlate Raman signal with SERS concentration after injecting four spectrally unique SERS nanoparticles either s.c. (R(2) = 0.998) or i.v. (R(2) = 0.992). These results show great potential for multiplexed imaging in living subjects in cases in which several targeted SERS probes could offer better detection of multiple biomarkers associated with a specific disease.

Imaging of 186Re-liposome therapy in ovarian cancer xenograft model of peritoneal carcinomatosis.

This study determined the biodistribution of rhenium-186 ((186)Re) encapsulated in biotin-liposomes containing patent blue dye, injected intraperitoneally (IP) with avidin in an OVCAR-3 ovarian cancer xenograft model and evaluated tumor response of this therapy with fluorine-18-fluorodeoxyglucose ((18)F-FDG) microPET imaging. Treated rats (n = 8) received an IP injection of (186)Re-blue-biotin-liposomes (1000 MBq/kg) 30 min before an IP injection of avidin (5 mg), whereas control rats (n = 4) received a sham IP injection of saline. Scintigraphic images showed that (186)Re-blue-biotin liposomes/avidin were retained in the peritoneal cavity with 18% of the original activity remaining after 5 days. From 1 to 4 weeks post-treatment, peritoneal (18)F-FDG standard uptake values decreased 30% in treatment group, yet increased 44% in control group. Total number of cells in ascites was significantly higher in control versus treatment group. Omental fat in control rats had numerous tumor cells compared with treated rats. Results show the potential for (186)Re-blue-biotin-liposome/avidin system in treating advanced ovarian cancer involving peritoneal carcinomatosis.

Use of avidin/biotin-liposome system for enhanced peritoneal drug delivery in an ovarian cancer model.

The goal of this study was to determine the distribution of the avidin/biotin-liposome system in an ovarian cancer xenograft model. Optimal avidin/biotin-liposome injection sequence with enhanced liposome accumulation to the peritoneum was determined. Two weeks after NIH:OVCAR-3 cell inoculation, rats were divided into three groups. Group 1 (B-A) (n=4), received an intraperitoneal injection of (99m)Tc-blue-biotin-liposomes 30 min before an intraperitoneal injection of avidin. Group 2 (A-B) (n=4), received an intraperitoneal injection of avidin 30 min before an intraperitoneal injection of (99m)Tc-blue-biotin-liposomes. Group 3 (A-B 2h) (n=5), received an intraperitoneal injection of avidin 2h before an intraperitoneal injection of (99m)Tc-blue-biotin-liposomes. Three additional non-tumor nude rats served as controls in each group, and were subjected to the same injection sequences. Scintigraphic imaging commenced at various times post (99m)Tc-blue-biotin-liposome injection. After imaging, rats were euthanized at 23 h post-liposome injection for tissue biodistribution. Images showed no apparent difference in liposome distribution between control and tumor animals. Regional uptake analysis at 4h for tumor rats showed significantly higher lymphatic channel uptake in the A-B 2h group (p<0.05) and a trend of increased peritoneal uptake in A-B group. By 22 h, peritoneal and lymphatic channel uptake was similar for all groups. At necropsy, most activity was found in blue-stained omentum, diaphragm, mediastinal and abdominal nodes. Bowel activity was minimal. These results correlate with previous normal rat studies, and demonstrate potential use of this avidin/biotin-liposome system for prolonging drug delivery to the peritoneal cavity and associating lymph nodes in this ovarian cancer xenograft model.