Hong Shan

Lewis Acid-Assisted Isotopic 18F-19F Exchange in BODIPY Dyes: Facile Generation of Positron Emission Tomography/Fluorescence Dual Modality Agents for Tumor Imaging

Positron emission tomography (PET) is a powerful technique for imaging biological pathways in vivo, particularly those that are key targets in disease processes. In contrast, fluorescence imaging has demonstrated to be a superior method for image-guided surgery, such as tumor removal. Although the integration of PET and optical imaging could provide an attractive strategy for patient management, there is a significant shortage of established platforms/methods for PET/optical probe construction. In this study, various reaction conditions were explored to develop a simple and fast method allowing for the introduction of [18F]-fluoride into BODIPY dyes. Through a systematic optimization of the reaction conditions, we found that BODIPY dyes, including commercial amine-reactive BODIPY succinimidyl esters, may be converted into their radioactive analogues in the matter of minutes via a 18F-19F isotopic exchange reaction promoted by a Lewis acid such as SnCl4. An integrin-targeting RGD peptide was also conjugated with [18F]BODIPY® R6G , derived from the commercially available BODIPY® R6G fluorescent tag, to provide a [18F]-RGD conjugate in 82% yield. In vivo evaluation of this imaging probe showed a discernible tumor uptake in the U87MG xenograft model. The dual modality imaging properties of the probe was confirmed by ex vivo fluorescence and microPET imaging experiments. In summary, in the matter of minutes, BODIPY dyes were converted into their “hot” radioactive analogues via a 18F-19F isotopic exchange reaction promoted by a Lewis acid. This approach, which can be applied to commercial BODIPY dyes, provides easy access to positron emission tomography/fluorescence dual modality imaging agents.

The Efficient Synthesis and Biological Evaluation of Novel Bi-Functionalized Sarcophagine for 64Cu Radiopharmaceuticals

Purpose We and others have reported that Sarcophagine-based bifunctional chelators could be effectively used in the syntheses of 64Cu radiopharmaceuticals. The resulted 64Cu-Sarcophagine complexes demonstrated great in vivo stability. The goal of this study was to further derivatize Sarcophagine cage with amino and maleimide functional groups for conjugation with bioligands. Methods Starting from DiAmSar, three novel chelators (AnAnSar, BaMalSar, and Mal2Sar) with two functional groups have been synthesized. Among those, BaMalSar and Mal2Sar have been conjugated with cyclic peptide c(RGDyC) (denoted as RGD) and the resulted conjugates, BaMalSar-RGD and Mal2Sar-RGD2 have been labeled with 64Cu. The tumor targeting efficacy of 64Cu-labeled RGD peptides were evaluated in a subcutaneous U87MG glioblastoma xenograft model. Results The conjugates, BaMalSar-RGD and Mal2Sar-RGD2 could be labeled with 64CuCl2 in 10 min with high purity (>98%) and high radiochemical yield (>90%). Both 64Cu-BaMalSar-RGD and 64Cu-Mal2Sar-RGD2 exhibited high tumor uptake and tumor-to-normal tissue ratios. Conclusion Three novel chelators with two functional groups have been developed based on Sarcophagine cage. The platform developed in this study could have broad applications in the design and synthesis of 64Cu-radiopharmaceuticals.

MR tracking of magnetically labeled mesenchymal stem cells in rats with liver fibrosis

PURPOSEnIn vivo magnetic resonance (MR) tracking of magnetically labeled bone marrow mesenchymal stem cells (BMSCs) administered via the mesenteric vein to rats with liver fibrosis.nnnMATERIALS AND METHODSnRat BMSCs were labeled with superparamagnetic iron oxide (SPIO) and the characteristics of the BMSCs after labeling were investigated. Eighteen rats with CCL4-induced liver fibrosis were randomized to three groups to receive SPIO-labeled BMSCs (BMSC-labeled group), cell-free SPIO (SPIO group), or unlabeled BMSCs (control group). MR imaging of the liver was performed at different time points, and signal-to-noise ratio (SNR) of the liver was measured. In vivo distribution of delivered BMSCs was assessed by histological analysis.nnnRESULTSnLabeling of BMSCs with SPIO did not significantly alter cell viability and proliferation activity. In BMSC-labeled group, the liver SNR immediately decreased from 8.56+/-0.26 to 3.53+/-0.41 at 1 h post injection and remained at a significantly lower level till 12 days (P<.05 versus the level before). By contrast, the liver SNR of the SPIO group almost recovered to the preinjection level (P=.125) at 3 days after a transient decrease. In control group, the liver SNR demonstrated no significant difference at the tested time points. Additionally, Prussian blue-positive cells were mainly distributed in the liver parenchyma, especially in injured areas.nnnCONCLUSIONnThe magnetically labeled BMSCs infused through the mesenteric vein can be detected in the fibrotic liver of rats using in vivo MR imaging up to 12 days after injection.

Efficient Construction of PET/Fluorescence Probe Based on Sarcophagine Cage: An Opportunity to Integrate Diagnosis with Treatment

PurposeDue to the shortage of established platforms/methods for multimodality probe construction, in this study, we developed a heterofunctional chelator, BaAn(Boc)Sar, from sarcophagine cage as a general platform for dual-modality probe construction.ProceduresA dual-modality probe for positron-emission tomography (PET) and fluorescence imaging was synthesized using the developed BaAn(Boc)Sar chelator. The c(RGDyK)2 peptide (denoted as RGD2) and fluorescence dye Cy5.5 were conjugated with BaAn(Boc)Sar to form BaAnSar-RGD2-Cy5.5. Then, BaAnSar-RGD2-Cy5.5 was labeled with 64Cu in ammonium acetate buffer. PET and fluorescent imaging were carried out to evaluate 64Cu-BaAnSar-RGD2-Cy5.5 in nude mice bearing U87MG glioblastoma xenograft.ResultsThe BaAnSar-RGD2-Cy5.5 was labeled with 64Cu very efficiently in 0.1xa0M NH4OAc buffer within 10xa0min at 37xa0°C in the yield of 86.7u2009±u20094.4xa0% (nu2009=u20093). The specific activity of 64Cu-BaBaSar-RGD2 was controlled at 50–200xa0mCi/μmol for the consideration of both PET and optical imaging. MicroPET quantification analysis shows that the U87MG tumor uptake is 6.41u2009±u20090.28, 6.51u2009±u20091.45, and 5.92u2009±u20091.57xa0%ID/g at 1, 4, and 20xa0h postinjection, respectively. Good correlation was obtained between the tumor to muscle ratios measured by the radioactivity and fluorescence intensity. As a proof of concept, an animal surgery study demonstrated that this dual-modality probe would greatly benefit the patients because the PET moiety could be used for tumor detection, and the fluorescent moiety would allow image-guided surgery.ConclusionsOur findings demonstrated the effectiveness and feasibility of preparing dual-modality imaging probes based on the sarcophagine scaffold. The resulting PET and fluorescent imaging probe also holds a great potential for clinical translation.

Evaluation of 18F-labeled BODIPY dye as potential PET agents for myocardial perfusion imaging☆

INTRODUCTIONnDespite the great potential of positron emission tomography/computed tomography (PET/CT) in cardiovascular disease imaging, one of the major limitations is the availability of PET probes with desirable half-lives and reasonable cost. In this report, we hypothesized that lipophilic cationic BODIPY dye could be selectively accumulated in cardiac muscle, possibly for the development of novel PET myocardial perfusion imaging (MPI) probes.nnnMETHODSnA (18)F-labeled BODIPY dye ([(18)F]1) was synthesized efficiently through a fluoride exchange reaction catalyzed by the Lewis acid tin chloride (SnCl₄). The compound was first evaluated by a cellular uptake assay in vitro, followed by biodistribution and microPET imaging studies in vivo.nnnRESULTSn[(18)F]1 was obtained in more than 90% labeling yield, with >98% radiochemical purity. The HEK-293 cellular uptake assay showed that the preferential uptake of [(18)F]1 could be related to the cell membrane potential. The biodistribution data demonstrated high levels of [(18)F]1 accumulation in the heart. In the biodistribution study in mice, the radioactivity uptake in the heart, blood, liver and lung was 3.01 ± 0.44, 0.39 ± 0.09, 0.69 ± 0.07, 1.71 ± 0.27%ID/g, respectively, at 3h post-injection (p.i.). The heart-to-lung and heart-to-liver ratios are 1.76 ± 0.14 and 4.37 ± 0.51 at 3h p.i., respectively. Volume-of-interest analysis of the microPET images correlated well with the biodistribution studies in mice. The heart was clearly visualized in normal rats, with 0.72 ± 0.18, 0.69 ± 0.18, 0.67 ± 0.20 and 0.59 ± 0.17%ID/g uptake at 0.5, 1, 2 and 4h p.i., respectively.nnnCONCLUSIONSn(18)F-labeled BODIPY dye showed good heart uptake and heart-to-blood and heart-to-lung contrast. A (18)F-labeled BODIPY dyes may represent a new category of cationic PET agents for myocardial perfusion imaging.

PET Imaging of Colorectal and Breast Cancer by Targeting EphB4 Receptor with 64Cu-Labeled hAb47 and hAb131 Antibodies

Accumulating evidence suggests that ephrin type B receptor 4 (EphB4) plays a key role in the progression of numerous cancer types. In this study, we developed a series of 64Cu-labeled antibodies for PET imaging of tumor EphB4 expression. Methods: Anti-EphB4 antibodies (hAb47 and hAb131) were conjugated with the 64Cu-chelator DOTA through lysine, cysteine, or oligosaccharide on the antibody. DOTA-human IgG (hIgG) was also prepared as a control, which did not bind to EphB4. The EphB4 binding activity of these probes was evaluated through the bead-based binding assay with EphB4-alkaline phosphatase. The resulting PET probes were further evaluated in both HT29 (colorectal cancer) and MDA-MB-231 (breast cancer) xenografts. Results: All 3 conjugation methods retained most of the EphB4 binding activity of the antibodies (83.85% ± 3.82%, 76.25% ± 5.90%, 98.93% ± 3.75%, and 82.09% ± 4.14% for DOTA-Lys-hAb47, DOTA-Cys-hAb47, DOTA-Sug-hAb47, and DOTA-Lys-hAb131, respectively). Although DOTA-Sug-hAb47 demonstrated the highest receptor binding activity based on a EphB4 binding assay, the corresponding PET probe was trapped in the liver quickly in vivo. In HT29 xenografts, both 64Cu-DOTA-Lys-hAb47 and 64Cu-DOTA-Cys-hAb47 demonstrated prominent tumor accumulation, which reached a maximum at 48 h after injection (18.13 ± 1.73 percentage injected dose [%ID]/g and 11.81 ± 2.05 %ID/g, respectively). In contrast, 64Cu-DOTA-Lys-hIgG had a low tumor accumulation, thus demonstrating the target specificity of EphB4-antibody–based probes. Moreover, 64Cu-DOTA-Lys-hAb131 (29.48 ± 2.60 %ID/g) demonstrated significantly higher HT29 tumor accumulation than 64Cu-DOTA-Lys-hAb47. 64Cu-DOTA-Lys-hAb131 was also found to specifically accumulate in the MDA-MB-231 tumor model (12.96 ± 2.31 %ID/g). Conclusion: We have demonstrated that EphB4 can serve as a valid target for colorectal and breast cancer imaging. This approach would be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. Moreover, these newly developed probes may have important applications in other cancer types overexpressing EphB4.

The synthesis of lanthanide-doped GdVO4 ultrathin nanosheets with great optical and paramagnetic properties for FRET biodetection and in vivo MR imaging

The development of two dimension (2D) inorganic nanosheets (NSs) with unique physical and chemical properties has been the focus of intense study in recent years. Herein, we report the synthesis of lanthanide ion (Ln3+ = Eu3+, Dy3+)-doped GdVO4 2D tetragonal NSs (with a thickness of ∼5 nm and a width of ∼150 nm) using a facile solvothermal reaction. After the ligand exchange reaction with polyacrylic acid (PAA), the hydrophilic 2D NSs demonstrated a high fluorescence (absolute quantum yield (QY) of 24%) and excellent paramagnetic properties (longitudinal relaxivity reached 37.8 mM-1 s-1). Moreover, this carboxyl functionalized NS could be easily modified for bio-application and exhibited a low toxicity towards cells. Based on the bright fluorescence of the GdVO4:Dy NSs, a sensitive Förster resonance energy transfer (FRET) bioprobe was developed to quantitatively detect the presence of streptavidin. In parallel, the paramagnetic properties of the NSs allows the magnetic resonance imaging (MRI) of integrin α2β1 expression in human prostate cancer. Collectively, multifunctional NSs with a 2D nanostructure demonstrated unique fluorescence and paramagnetic properties, which could be used to construct high performance nanoprobes for biomedical applications.

Targeting the EphB4 receptor for cancer diagnosis and therapy monitoring.

Accumulating evidence suggests that EphB4 plays key roles in cancer progression in numerous cancer types. In fact, therapies focusing on EphB4 have become potentially important components of various cancer treatment strategies. However, tumor sensitivity to EphB4 suppression may not be uniform for different cancers. In this study, we developed near-infrared fluorescence (NIRF) probes for EphB4 targeted imaging, based on EphB4-specific humanized monoclonal antibody hAb47. NIRF dye Cy5.5 was introduced to hAb47 either through the reaction with amino groups (named hAb47-Cy5.5) or sulfhydryl groups (named hAb47-Cy5.5-Mal). The resulting probes were evaluated in both HT-29 xenograft and the mAb131 (anti-EphB4) treated models. Although these methods lead to modifications of both the heavy chain and light chain of the antibody, the majority of the EphB4 binding affinity was maintained (81.62 ± 2.08% for hAb47-Cy5.5 and 77.14 ± 2.46% for hAb47-Cy5.5-Mal, respectively). hAb47-Cy5.5 was then chosen for in vivo NIRF imaging of EphB4 expression. In HT29 colorectal tumor xenografts, hAb47-Cy5.5 demonstrated significantly higher tumor uptake compared with that of the hIgG-Cy5.5 control, which was further confirmed by immunofluorescent staining. Moreover, hAb47-Cy5.5 successfully imaged the decreased EphB4 expression (confirmed by Western blot) in EphB4-targeted immunotherapy using another EphB4-specific antibody, mAb131. Collectively, hAb47-Cy5.5 could be used as a specific NIRF contrast agent for noninvasive imaging of EphB4 expression, which may predict whether an individual tumor would likely respond to EphB4 targeted interventions, as well as monitor the therapeutic response.

Urokinase Plasminogen Activator Receptor (uPAR) Targeted Nuclear Imaging and Radionuclide Therapy

Urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein. Besides regulating proteolysis, uPAR could also activate many intracellular signaling pathways that promote cell motility, invasion, proliferation, and survival through cooperating with transmembrane receptors. uPAR is overexpressed across a variety of tumors and is associated with cancer invasion and metastasis. In order to meet the demand for a rapid development and potential clinical application of anti-cancer therapy based on uPA/uPAR system, it is desirable to develop non-invasive imaging methods to visualize and quantify uPAR expression in vivo. In this review, we will discuss recent advances in the development of uPAR-targeted nuclear imaging and radionuclide therapy agents. The successful development of molecular imaging probes to visualize uPAR expression in vivo would not only assist preclinical researches on uPAR function, but also eventually impact patient management.

Design, synthesis and validation of Axl-targeted monoclonal antibody probe for microPET imaging in human lung cancer xenograft

Accumulating experimental evidence indicates that overexpression of the oncogenic receptor tyrosine kinase, Axl, plays a key role in the tumorigenesis and metastasis of various types of cancer. The objective of this study is to design a novel imaging probe based on the monoclonal antibody, h173, for microPET imaging of Axl expression in human lung cancer. A bifunctional chelator, DOTA, was conjugated to h173, followed by radiolabeling with 64Cu. The binding of DOTA-h173 to the Axl receptor was first evaluated by a cell uptake assay and flow cytometry analysis using human lung cancer cell lines. The probe 64Cu-DOTA-h173 was further evaluated by microPET imaging, and ex vivo histology studies in the Axl-positive A549 tumors. In vitro cellular study showed that Axl probe, 64Cu-DOTA-h173, was highly immuno-reactive with A549 cells. Western blot analysis confirmed that Axl is highly expressed in the A549 cell line. For microPET imaging, the A549 xenografts demonstrated a significantly higher 64Cu-DOTA-h173 uptake compared to the NCI-H249 xenograft (a negative control model). Furthermore, 64Cu-DOTA-h173 uptake in A549 is significantly higher than that of 64Cu-DOTA-hIgG. Immuno-fluorescence staining was consistent with the in vivo micro-PET imaging results. In conclusion, 64Cu-DOTA-h173 could be potentially used as a probe for noninvasive imaging of Axl expression, which could collect important information regarding tumor response to Axl-targeted therapeutic interventions.