Gail Sudlow

Near-Infrared pH-Activatable Fluorescent Probes for Imaging Primary and Metastatic Breast Tumors

Highly tumor selective near-infrared (NIR) pH-activatable probe was developed by conjugating pH-sensitive cyanine dye to a cyclic arginine-glycine-aspartic acid (cRGD) peptide targeting α(v)β(3) integrin (ABIR), a protein that is highly overexpressed in endothelial cells during tumor angiogenesis. The NIR pH-sensitive dye used to construct the probe exhibits high spectral sensitivity with pH changes. It has negligible fluorescence above pH 6 but becomes highly fluorescent below pH 5, with a pK(a) of 4.7. This probe is ideal for imaging acidic cell organelles such as tumor lysosomes or late endosomes. Cell microscopy data demonstrate that binding of the cRGD probe to ABIR facilitated the endocytosis-mediated lysosomal accumulation and subsequent fluorescence enhancement of the NIR pH-activatable dye in tumor cells (MDA-MB-435 and 4T1/luc). A similar fluorescence enhancement mechanism was observed in vivo, where the tumors were evident within 4 h post injection. Moreover, lung metastases were also visualized in an orthotopic tumor mouse model using this probe, which was further confirmed by histologic analysis. These results demonstrate the potential of using the new integrin-targeted pH-sensitive probe for the detection of primary and metastatic cancer.

Breaking the depth dependency of phototherapy with Cerenkov radiation and low-radiance-responsive nanophotosensitizers

The combination of light and photosensitizers for phototherapeutic interventions such as photodynamic therapy has transformed medicine and biology. However, the shallow penetration of light in tissues and the reliance on tissue oxygenation to generate cytotoxic radicals have limited the method to superficial or endoscope-accessible lesions. Here, we report a way to overcome these limitations by using Cerenkov radiation from radionuclides to activate an oxygen-independent nanophotosensitizer, titanium dioxide (TiO2). We show that administration of transferrin-coated TiO2 nanoparticles and clinical grade radionuclides in mice and co-localization in tumours resulted in either complete tumour remission or increased their median survival. Histological analysis of tumour sections showed selective destruction of cancerous cells and high numbers of tumour infiltrating lymphocytes, suggesting that both free radicals and the activation of the immune system mediated the destruction. Our results offer a way to harness low radiance-sensitive nanophotosensitizers to achieve depth-independent Cerenkov radiation-mediated therapy.

Hands-free, Wireless Goggles for Near-infrared Fluorescence and Real-time Image-guided Surgery

BACKGROUND Current cancer management faces several challenges, including the occurrence of a residual tumor after resection, the use of radioactive materials or high concentrations of blue dyes for sentinel lymph node biopsy, and the use of bulky systems in surgical suites for image guidance. To overcome these limitations, we developed a real-time, intraoperative imaging device that, when combined with near infrared fluorescent molecular probes, can aid in the identification of tumor margins, guide surgical resections, map sentinel lymph nodes, and transfer acquired data wirelessly for remote analysis. METHODS We developed a new compact, wireless, wearable, and battery-operated device that allows for hands-free operation by surgeons. A charge-coupled device-based, consumer-grade night vision viewer was used to develop the detector portion of the device, and the light source portion was developed from a compact headlamp. This piece was retrofitted to provide both near infrared excitation and white light illumination simultaneously. Wireless communication was enabled by integrating a battery-operated, miniature, radio-frequency video transmitter into the system. We applied the device in several types of oncologic surgical procedures in murine models, including sentinel lymph node mapping, fluorescence-guided tumor resection, and surgery under remote expert guidance. RESULTS Unlike conventional imaging instruments, the device displays fluorescence information directly on its eyepiece. When employed in sentinel lymph node mapping, the locations of sentinel lymph nodes were visualized clearly, even with tracer level dosing of a near infrared fluorescent dye (indocyanine green). When used in tumor resection, tumor margins and small nodules invisible to the naked eye were visualized readily. In a simulated, point-of-care setting, tumors were located successfully and removed under remote guidance using the wireless feature of the device. Importantly, the total cost of this prototype system (

Tunable Ultrasmall Visible-to-Extended Near-Infrared Emitting Silver Sulfide Quantum Dots for Integrin-Targeted Cancer Imaging

1200) is substantially less than existing imaging instruments. CONCLUSION Our results demonstrate the feasibility of using the new device to aid surgical resection of tumors, map sentinel lymph nodes, and facilitate telemedicine.

Detection of MMP-2 and MMP-9 activity in vivo with a triple-helical peptide optical probe

The large size of many near-infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. In this study, we developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from 500 to 1200 nm and a QD core diameter between 1.5 and 9 nm. Conjugation of a tumor-avid cyclic pentapeptide (Arg-Gly-Asp-DPhe-Lys) resulted in monodisperse, water-soluble QDs (hydrodynamic diameter < 10 nm) without loss of the peptide’s high binding affinity to tumor-associated integrins (KI = 1.8 nM/peptide). Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and noncytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

Near-infrared fluorescence goggle system with complementary metal–oxide–semiconductor imaging sensor and see-through display

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.

Intraoperative detection of liver tumors aided by a fluorescence goggle system and multimodal imaging

Abstract. We have developed a near-infrared (NIR) fluorescence goggle system based on the complementary metal–oxide–semiconductor active pixel sensor imaging and see-through display technologies. The fluorescence goggle system is a compact wearable intraoperative fluorescence imaging and display system that can guide surgery in real time. The goggle is capable of detecting fluorescence of indocyanine green solution in the picomolar range. Aided by NIR quantum dots, we successfully used the fluorescence goggle to guide sentinel lymph node mapping in a rat model. We further demonstrated the feasibility of using the fluorescence goggle in guiding surgical resection of breast cancer metastases in the liver in conjunction with NIR fluorescent probes. These results illustrate the diverse potential use of the goggle system in surgical procedures.

Complementary fluorescence-polarization microscopy using division-of-focal-plane polarization imaging sensor

Real-time image guidance in the operating room is needed to improve instantaneous surgical decisions. Toward this goal, we utilized a new fluorescence goggle system and a near-infrared fluorescent dye approved for human use, indocyanine green, to demonstrate the feasibility of detecting liver tumors intraoperatively. The fluorescence goggle provided successful imaging of multifocal breast cancer metastases in mouse liver. Diffused tumor deposits as small as 0.8 mm in diameter were detected, which were not obvious without the fluorescence goggle. A combination of surface-weighted fluorescence imaging and deep tissue-sensitive ultrasound imaging allowed comprehensive image guidance with the fluorescence goggle system for tumor resection in a rabbit VX2 liver metastasis model. This multimodal detection and guided surgical intervention strategy using ultrasonic imaging and real-time intraoperative fluorescence guidance is a promising and innovative technology platform for improving surgical outcome of human patients with primary or metastatic liver cancer.

Video-rate fluorescence diffuse optical tomography for in vivo sentinel lymph node imaging

Abstract. Fluorescence microscopy offers high sensitivity for disease diagnosis. However, little structural information is revealed by this method, requiring another technique to localize the source of fluorescence. We developed a complementary fluorescence-polarization microscope. We used a division-of-focal-plane charge coupled device polarization sensor to enable real-time video rate polarization imaging without any moving parts. The polarization information provided by the microscope enabled detection of structural element and complements the fluorescence information. Application of this multimodal system for cancer imaging using a tumor selective molecular probe revealed the association of diminished structural integrity of tumor tissue with high fluorescence of the imaging agent compared to surrounding normal tissue. This study demonstrates a new paradigm to improve cancer detection and diagnosis.

Binocular Goggle Augmented Imaging and Navigation System provides real-time fluorescence image guidance for tumor resection and sentinel lymph node mapping

We have developed a fiber-based, video-rate fluorescence diffuse optical tomography (DOT) system for noninvasive in vivo sentinel lymph node (SLN) mapping. Concurrent acquisition of fluorescence and reference signals allowed the efficient generation of ratio-metric data for 3D image reconstruction. Accurate depth localization and high sensitivity to fluorescent targets were established in to depths of >10 mm. In vivo accumulation of indocyanine green (ICG) dye was imaged in the region of the SLN following intradermal injection into the forepaw of rats. These results suggest that video-rate fluorescence DOT has significant potential as a clinical tool for noninvasive mapping of SLN.