Kexian Liang

Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression

Integrins mediate many biological processes, including tumor-induced angiogenesis and metastasis. The arginine–glycine–aspartic acid (RGD) peptide sequence is a common recognition motif by integrins in many proteins and small peptides. While evaluating a small library of RGD peptides for imaging αVβ3 integrin (ABI)-positive tumor cell line (A549) by optical methods, we discovered that conjugating a presumably inactive linear hexapeptide GRDSPK with a near-infrared carbocyanine molecular probe (Cypate) yielded a previously undescribed bioactive ligand (Cyp-GRD) that targets ABI-positive tumors. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay with A549 cells showed that Cyp-GRD was not cytotoxic up to 100 μM in cell culture. The compound was internalized by cells, and this internalization was blocked by coincubation with a cyclic RGD peptide (cyclo[RGDfV], f is d-phenylalanine) that binds ABI with high affinity. In vivo, Cyp-GRD selectively accumulated in tumors relative to surrounding normal tissues. Blocking studies with cyclo[RGDfV] inhibited the in vivo uptake of Cyp-GRD, suggesting that both compounds target the same active site of the protein. A strong correlation between the Cyp-GRD peptide and mitochondrial NADH concentration suggests that the new molecule could also report on the metabolic status of cells ex vivo. Interestingly, neither a Cypate-labeled linear RGD peptide nor an 111In-labeled DOTA-GRD conjugate was selectively retained in the tumor. These results clearly demonstrate the synergistic effects of Cypate and GRD peptide for molecular recognition of integrin expression and suggest the potential of using carbocyanines as optical scaffolds for designing biologically active molecules.

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 (

Preparation and Biological Evaluation of 64Cu-CB-TE2A-sst2-ANT, a Somatostatin Antagonist for PET Imaging of Somatostatin Receptor–Positive Tumors

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.

In Vitro and In Vivo Evaluation of 64Cu-Labeled SarAr-Bombesin Analogs in Gastrin-Releasing Peptide Receptor–Expressing Prostate Cancer

Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst2-ANT was determined to have a high affinity for SSTR2. Additionally, 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst2-ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64Cu-CB-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst2-ANT (64Cu-CB-TE2A-sst2-ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors. Methods: Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64Cu-CB-TE2A-sst2-ANT was compared with that of the 64Cu-labeled agonist 64Cu-CB-TE2A-tyrosine3-octreotate (64Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and small-animal PET/CT of 64Cu-CB-TE2A-sst2-ANT was performed. Results: The dissociation constant value for the radiopharmaceutical was determined to be 26 ± 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64Cu-CB-TE2A-sst2-ANT showed significantly less internalization than did 64Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64Cu-CB-TE2A-sst2-ANT from the blood was rapid, whereas the clearance of 64Cu-CB-TE2A-sst2-ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64Cu-CB-TE2A-sst2-ANT than those for 64Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64Cu-CB-TE2A-sst2-ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h. Conclusion: The PET radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptor–positive tumors.

Rational Approach to Select Small Peptide Molecular Probes Labeled with Fluorescent Cyanine Dyes for in vivo Optical Imaging

Bombesin is a 14–amino-acid amphibian peptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR), which is overexpressed on a variety of solid tumors. It has been demonstrated that bombesin analogs can be radiolabeled with a variety of radiometals for potential diagnosis and treatment of GRPR-positive tumors. In this regard, several studies have used different chelators conjugated to the 8 C-terminal amino acids of bombesin(7-14) for radiolabeling with 64Cu. These analogs have demonstrated GRPR-specific small-animal PET of tumors but have various advantages and disadvantages. The objective of this study was to conjugate the previously described (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) (SarAr) chelator to bombesin(7-14), radiolabel the conjugate with 64Cu, and evaluate in vitro and in vivo. Methods: SarAr was synthesized as previously published and conjugated to bombesin(7-14) by solid-phase peptide synthesis using standard Fmoc chemistry. Succinic acid (SA), 8-aminooctanoic acid (Aoc), and Gly-Ser-Gly (GSG) were used as linkers between SarAr and bombesin(7-14) to yield the resulting SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSG-bombesin(7-14) peptides. The unlabeled peptides were evaluated in a competitive binding assay using PC-3 prostate cancer cells and 125I-Tyr4-bombesin to determine the inhibitory concentration of 50%. The peptides were radiolabeled with 64Cu and evaluated for internalization into PC-3 cells in vitro and for in vivo tumor uptake in mice bearing PC-3 xenografts using biodistribution and small-animal PET/CT studies. Results: The competitive binding assay demonstrated that both SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSG-bombesin(7-14) bound with high affinity to GRPR with an inhibitory concentration of 50% of 3.5 and 4.5 nM, respectively. Both peptides were radiolabeled with 64Cu at room temperature without further purification and demonstrated similar internalization into PC-3 cells. In vivo, the radiolabeled peptides demonstrated tumor-specific uptake (13.0 and 8.5 percentage injected dose per gram for 64Cu-SarAr-SA-Aoc-bombesin(7-14) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h) and imaging that was comparable to, or better than, that of the previously reported 64Cu-labeled bombesin analogs. The 64Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) had more rapid blood clearance and lower tumor and normal-tissue uptake than 64Cu-SarAr-SA-Aoc-bombesin(7-14), resulting in similar tumor-to-blood ratios for each analog (15.1 vs. 11.3 for 64Cu-SarAr-SA-Aoc-bombesin(7-14) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h). Conclusion: These studies demonstrate that 64Cu-SarAr-SA-Aoc-bombesin(7-14) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) bound with high affinity to GRPR-expressing cells and that these peptides can be used for PET of GRPR-expressing prostate cancer.

Activatable molecular systems using homologous near-infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in vivo.

We demonstrate that the structure of carbocyanine dyes, which are commonly used to label small peptides for molecular imaging and not the bound peptide, controls the rate of extravasation from blood vessels to tissue. By examining several near-infrared (NIR) carbocyanine fluorophores, we demonstrate a quantitative correlation between the binding of a dye to albumin, a model plasma protein, and the rate of extravasation of the probe into tissue. Binding of the dyes was measured by fluorescence quenching of the tryptophans in albumin and was found to be inversely proportional to the rate of extravasation. The rate of extravasation, determined by kurtosis from longitudinal imaging studies using rodent ear models, provided a basis for quantitative measurements. Structure-activity studies aimed at evaluating a representative library of NIR fluorescent cyanine probes showed that hydrophilic dyes with binding constants several orders of magnitude lower than their hydrophobic counterparts have much faster extravasation rate, establishing a foundation for rational probe design. The correlation provides a guideline for dye selection in optical imaging and a method to verify if a certain dye is optimal for a specific molecular imaging application.

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 generic approach to determine enzyme activities in vitro and monitor their functional status in vivo. Specifically, a method to generate donor (CbOH)-acceptor (Me2NCp) near-infrared (NIR) fluorescent dye pairs for preparing enzyme activatable molecular systems were developed based on the structural template of heptamethine cyanine dyes. Using caspase-3 as a model enzyme, we prepared two new caspase-3 sensitive compounds with high fluorescence quenching efficiency: Me2NCp-DEVD-K(CbOH)-OH (4) and AcGK(Me2NCp)-DEVD-APK(CbOH)-NH2 (5). The mechanism of quenching was based on combined effects of direct (classical) and reverse fluorescence resonance energy transfer (FRET). Caspase-3 cleavage of the scissile DEVD amide bond regenerated the NIR fluorescence of both donor and acceptor dyes. While both compounds were cleaved by caspase-3, substrate 5 was cleaved more readily than 4, yielding k(cat) and K(M), values of 1.02 +/- 0.06 s(-1) and 15 +/- 3 microM, respectively. Treatment of A549 tumor cells with paclitaxel resulted in > 2-fold increase in the fluorescence intensity by NIR confocal microscopy, suggesting the activation of pro-caspase-3 to caspase-3. A similar trend was observed in a mouse model, where the fluorescence intensity was nearly twice the value in caspase-3-rich tissue relative to the control. These results demonstrate the use of the same NIR activatable molecular systems for monitoring the activities of enzymes across a wide spatial scale ranging from in vitro kinetics measurements to in cellulo and in vivo localization of caspase-3 activation. The NIR activatable molecular probes provide an effective strategy to screen new drugs in vitro and monitor treatment response in living organisms.

Multimodal Fluorescence-Mediated Tomography and SPECT/CT for Small-Animal Imaging

The inability to identify microscopic tumors and assess surgical margins in real-time during oncologic surgery leads to incomplete tumor removal, increases the chances of tumor recurrence, and necessitates costly repeat surgery. To overcome these challenges, we have developed a wearable goggle augmented imaging and navigation system (GAINS) that can provide accurate intraoperative visualization of tumors and sentinel lymph nodes in real-time without disrupting normal surgical workflow. GAINS projects both near-infrared fluorescence from tumors and the natural color images of tissue onto a head-mounted display without latency. Aided by tumor-targeted contrast agents, the system detected tumors in subcutaneous and metastatic mouse models with high accuracy (sensitivity = 100%, specificity = 98% ± 5% standard deviation). Human pilot studies in breast cancer and melanoma patients using a near-infrared dye show that the GAINS detected sentinel lymph nodes with 100% sensitivity. Clinical use of the GAINS to guide tumor resection and sentinel lymph node mapping promises to improve surgical outcomes, reduce rates of repeat surgery, and improve the accuracy of cancer staging.

In vitro and in vivo evaluation of a 64Cu-labeled NOTA-Bn-SCN-Aoc-bombesin analogue in gastrin-releasing peptide receptor expressing prostate cancer

Spatial and temporal coregistration of nuclear and optical images can enable the fusion of the information from these complementary molecular imaging modalities. A critical challenge is in integrating the optical and nuclear imaging hardware. Flexible fiber–based fluorescence-mediated tomography (FMT) systems provide a viable solution. The various bore sizes of small-animal nuclear imaging systems can potentially accommodate the FMT fiber imaging arrays. In addition, FMT imaging facilitates coregistration of the nuclear and optical contrasts in time. Herein, we combine a fiber-based FMT system with a preclinical SPECT/CT platform. Feasibility of in vivo imaging is demonstrated by tracking a monomolecular multimodal imaging agent (MOMIA) during transport from the forepaw to the axillary lymph node region of a rat. Methods: The fiber-based, video-rate FMT imaging system is composed of 12 sources (785- and 830-nm laser diodes) and 13 detectors. To maintain high temporal sampling, the system simultaneously acquires ratio–metric data at each detector. A 3-dimensional finite element model derived from CT projections provides anatomically based light propagation modeling. Injection of a MOMIA intradermally into the forepaw of rats provided spatially and temporally coregistered nuclear and optical contrasts. FMT data were acquired concurrently with SPECT and CT data. The incorporation of SPECT data as a priori information in the reconstruction of FMT data integrated both optical and nuclear contrasts. Results: Accurate depth localization of phantoms with different thicknesses was accomplished with an average center-of-mass error of 4.1 ± 2.1 mm between FMT and SPECT measurements. During in vivo tests, fluorescence and radioactivity from the MOMIA were colocalized in spatially coincident regions with an average center-of-mass error of 2.68 ± 1.0 mm between FMT and SPECT for axillary lymph node localization. Intravital imaging with surgical exposure of the lymph node validated the localization of the optical contrast. Conclusion: The feasibility of integrating a fiber-based, video-rate FMT system with a commercial preclinical SPECT/CT platform was established. These coregistered FMT and SPECT/CT results with MOMIAs may facilitate the development of the next generation of preclinical and clinical multimodal optical–nuclear platforms for a broad array of imaging applications and help elucidate the underlying biologic processes relevant to cancer diagnosis and therapy monitoring.

Modulation of nuclear internalization of Tat peptides by fluorescent dyes and receptor-avid peptides

INTRODUCTION Bombesin (BN) is an amphibian peptide that binds to the gastrin-releasing peptide receptor (GRPR). It has been demonstrated that BN analogues can be radiolabeled for potential diagnosis and treatment of GRPR-expressing malignancies. Previous studies have conjugated various chelators to the eight C-terminal amino acids of BN [BN(7-14)] for radiolabeling with 64Cu. Recently, (1,4,7-triazacyclononane-1,4,7-triacetic acid) (NOTA) has been evaluated as the five-coordinate 64Cu complex, with results indicating GRPR-specific tumor uptake. This study aimed to conjugate S-2-(4-isothiocyanatobenzyl)-NOTA (p-SCN-Bn-NOTA) to BN(7-14) such that it could form a six-coordinate complex with 64Cu and to evaluate the resulting peptide. METHODS p-SCN-NOTA was conjugated to 8-aminooctanoic acid (Aoc)-BN(7-14) in solution to yield NOTA-Bn-SCN-Aoc-BN(7-14). The unlabeled peptide was evaluated in a cell binding assay using PC-3 prostate cancer cells and 125I-Tyr4-BN to determine the IC50 value. The peptide was radiolabeled with 64Cu and evaluated for internalization into PC-3 cells and for tumor uptake in mice bearing PC-3 xenografts using biodistribution and micro-positron emission tomography imaging studies. RESULTS The binding assay demonstrated that NOTA-Bn-SCN-Aoc-BN(7-14) bound with high affinity to GRPR with an IC50 of 1.4 nM. The radiolabeled peptide demonstrated time-dependent internalization into PC-3 cells. In vivo, the peptide demonstrated tumor-specific uptake and imaging that were comparable to those of previously reported 64Cu-labeled BN analogues. CONCLUSIONS These studies demonstrate that 64Cu-NOTA-Bn-SCN-Aoc-BN(7-14) binds to GRPR-expressing cells and that it can be used for imaging of GRPR-expressing prostate cancer.