An imageable retinoid acid derivative to detect human cancer xenografts and study therapeutic dosing to reduce its toxicity

Developing agents with ‘seek, treat and see’ capability is critical for personalized molecular medicine. Those agents will specifically target the disease markers for diagnosis and apply the biologically effective dose for treatment. Retinoids regulate a multitude of biological processes. In addition, retinoic acid can reverse premalignancy, significantly decrease second primary tumors and provide a treatment benefit in head and neck, lung, esophagus, colon and bladder cancer. These data suggest that cancer cells can take up retinoids. Therefore, retinoids are potential tumor‐imaging agents. We developed near‐infrared (NIR)‐labeled retinoid agents to detect human cancers, visualize drug redistribution within the body, determine the optimal biological dose and reduce systemic toxicity. Our data demonstrate that the retinoid agent, but not the free dye, binds to the human tumor cells and is internalized, where it permits the imaging of human cancer xenografts. The high dose of retinoid agent is significantly associated with systemic toxicity. In summary, synthetic NIR‐labeled retinoid agents can be used to detect multiple human cancer xenografts as the agent is internalized by cancer cells. The binding of the agent to the tumor xenografts is dependent on the redistribution of the agent. Therapeutic agents labeled with reporters will interrogate tumor–drug interactions and permit analysis of biodistribution, pharmacokinetics and pharmacodynamics in real time. At the same time, we can apply the biologically effective dose for therapy, instead of the traditional maximum tolerated dose, to reduce systemic toxicity. Copyright © 2010 John Wiley & Sons, Ltd.     

In vivo near‐infrared fluorescence targeting of T cells: comparison of nanobodies and conventional monoclonal antibodies

The large size of conventional antibodies impedes tissue penetration and renal elimination, resulting in suboptimal in vivo targeting. Here we assess the utility of nanobodies and nanobody‐Fc‐fusion proteins as alternatives to monoclonal antibodies as theranostics, using T cell ADP–ribosyltransferase 2 (ART2) as a model antigen for specific targeting of lymph nodes. ART2‐specific monovalent nanobody s + 16a (17 kDa), a bivalent Fc‐fusion protein of s + 16a (s + 16‐mFc, 82 kDa), and conventional antibody Nika102 (150 kDa) were labeled with AlexaFluor680. In vitro binding and inhibitory properties of the three AF680 conjugates were assessed by flow cytometry. For in vivo imaging experiments, AF680 conjugates were intravenously injected in mice lacking (KO) or overexpressing (TG) ART2. We monitored circulating and excreted AF680 conjugates in plasma and urine and performed in vivo near‐infrared fluorescence imaging. Nanobody s + 16a⁶⁸⁰ and s + 16mFc⁶⁸⁰ labeled and inhibited ART2 on T cells in lymph nodes within 10 min. In contrast, mAb Nika102⁶⁸⁰ required 2 h for maximal labeling without inhibition of ART2. In vivo imaging revealed specific labeling of ART2‐positive lymph nodes but not of ART2‐negative lymph nodes with all AF680 conjugates. Even though bivalent s + 16mFc⁶⁸⁰ showed the highest labeling efficiency in vitro, the best lymph node imaging in vivo was achieved with monovalent nanobody s + 16a⁶⁸⁰, since renal elimination of unbound s + 16a⁶⁸⁰ significantly reduced background signals. Our results indicate that small single‐domain nanobodies are best suited for short‐term uses, such as noninvasive imaging, whereas larger nanobody‐Fc‐fusion proteins are better suited for long‐term uses, such as therapy of inflammation and tumors. Copyright © 2014 John Wiley & Sons, Ltd.     

The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with ferucarbotran using various protocols: cells were labeled with 100 µg Fe/ml for 4 and 18 h and additional samples were cultured for 6 or 12 days after the 18 h labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP‐spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared with unlabeled controls by staining with Alcian blue and Hematoxylin and Eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3 T was investigated on days 1 and 14 of chondrogenic differentiation by measuring signal‐to‐noise ratios on T₂‐SE and T₂*‐GE sequences. Iron uptake was significant for all labeling protocols (p < 0.05). The uptake was highest after transfection with protamine sulfate (25.65 ± 3.96 pg/cell) and lowest at an incubation time of 4 h without transfection (3.21 ± 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4 h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose‐dependent. MR imaging showed markedly lower SNR values of all labeled cells compared with the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose‐dependent manner when using simple incubation techniques. When decreasing the incubation time to 4 h, inhibition of chondrogenesis was not significant. Copyright © 2009 John Wiley & Sons, Ltd.     

Multi‐modal imaging for assessment of tissue‐engineered bone in a critical‐sized calvarial defect mouse model

Tissue‐engineered bone (TEB) analysis in vivo relies heavily on tissue histological and end‐point evaluations requiring the sacrifice of animals at specific time points. Due to differences in animal response to implanted tissues, the conventional analytical methods to evaluate TEB can introduce data inconsistencies. Additionally, the conventional methods increase the number of animals required to provide an acceptable statistical power for hypothesis testing. Alternatively, our non‐invasive optical imaging allows for the longitudinal analysis of regenerating tissue, where each animal acts as its own control, thus reducing overall animal numbers. In our 6 month feasibility study, TEB, consisting of a silk protein scaffold with or without differentiated mesenchymal stem cells, was implanted in a critical‐sized calvarial defect mouse model. Osteogenesis of the TEB was monitored through signal variation, using magnetic resonance imaging (MRI) and near‐infrared (NIR) optical imaging with IRDye® 800CW BoneTag^TM (800CW BT, a bone‐specific marker used to label osteogenically differentiated mesenchymal stem cells and mineralization). Histological endpoint measurements and computed tomography (CT) were used to confirm imaging findings. Anatomical MRI revealed decreased signal intensity, indicating mineralization, in the TEB compared to the control (i.e. silk scaffold only) at various growth stages. NIR optical imaging results demonstrated a signal intensity increase of the TEB compared to control. Interpretation of the imaging results were confirmed by histological analysis. Specifically, haematoxylin and eosin staining revealing de novo bone in TEB showed that 80% of the defect was covered by TEB, while only 40% was covered for the control. Taken together, these results demonstrate the potential of multi‐modal non‐invasive imaging to visualize and quantify TEB for the assessment of regenerative medicine strategies. Copyright © 2015 John Wiley & Sons, Ltd.     

Developments in near‐infrared‐guided hepatobiliary,pancreatic and other upper gastrointestinal surgery

With the development of near‐infrared (NIR) technology, real‐time utility of NIR and its fluorophores has gained vast interest among surgeons of various sub‐disciplines. The purpose of this review is to assess and explore the most recent developments in NIR‐guided surgery in an upper gastrointestinal (UGI) surgical setting. Queries of PubMed and Medline literature databases was performed for experimental and clinical studies relevant to NIR use in the context of UGI surgery. NIR‐guided UGI surgeries have been reported to be valuable in: (1) esophageal anastomosis; (2) sentinel lymph node biopsy in gastric cancer; (3) detection of liver and pancreatic tumors; and (4) detection of extra bile duct and bile duct injuries. Although NIR technology has shown tremendous promise in UGI surgery, its full clinical translation and wider adaptation remains to be seen. Copyright © 2013 John Wiley & Sons, Ltd.     

Use of perfluorocarbon nanoparticles for non‐invasive multimodal cell tracking of human pancreatic islets

In vivo imaging of engraftment and immunorejection of transplanted islets is critical for further clinical development, with ¹H MR imaging of superparamagnetic iron oxide‐labeled cells being the current premier modality. Using perfluorocarbon nanoparticles, we present here a strategy for non‐invasive imaging of cells using other modalities. To this end, human cadaveric islets were labeled with rhodamine‐perfluorooctylbromide (PFOB) nanoparticles, rhodamine‐perfluoropolyether (PFPE) nanoparticles or Feridex® as control and tested in vitro for cell viability and c‐peptide secretion for 1 week. ¹⁹F MRI, computed tomography (CT) and ultrasound (US) imaging was performed on labeled cell phantoms and on cells following transplantation beneath the kidney capsule of mice and rabbits. PFOB and PFPE‐labeling did not reduce human islet viability or glucose responsiveness as compared with unlabeled cells or SPIO‐labeled cells. PFOB‐ and PFPE‐labeled islets were effectively fluorinated for visualization by ¹⁹F MRI. PFOB‐labeled islets were acoustically reflective for detection by US imaging and became sufficiently brominated to become radiopaque allowing visualization with CT. Thus, perfluorocarbon nanoparticles are multimodal cellular contrast agents that may find applications in real‐time targeted delivery and imaging of transplanted human islets or other cells in a clinically applicable manner using MRI, US or CT imaging. Copyright © 2011 John Wiley & Sons, Ltd.     

Syntheses and preliminary evaluation of [18F]AlF‐NOTA‐G‐TMTP1 for PET imaging of high aggressive hepatocellular carcinoma

The goal of this study is to evaluate a new ¹⁸F‐labeled imaging agent for diagnosing high metastatic (aggressive) hepatocellular carcinoma using positron emission tomography (PET). The new ¹⁸F‐labeled imaging agent [¹⁸F]AlF‐NOTA‐G‐TMTP1 was synthesized and radiolabeled with ¹⁸F using NOTA‐AlF chelation method. The tumor‐targeting characteristics of [¹⁸F]AlF‐NOTA‐G‐TMTP1 was assessed in HepG2, SMCC‐7721, HCC97L and HCCLM3 xenografts. The total synthesis time was about 20 min with radiochemical yield of 25 ± 6%. The specific activity was about 11.1–14.8 GBq/µmol at the end of synthesis based on the amount of peptide used and the amount of radioactivity trapped on the C₁₈ column. The log P value of [¹⁸F]AlF‐NOTA‐G‐TMTP1 was ‐3.166 ± 0.022. [¹⁸F]AlF‐NOTA‐G‐TMTP1 accumulated in SMCC‐7721 and HCCLM3 tumors (high metastatic potential) in vivo and result in tumor/muscle (T/M) ratios of 4.5 ± 0.3 and 4.7 ± 0.2 (n = 4) as measured by PET at 40 min post‐injection (p.i.). Meanwhile, the tumor/muscle (T/M) ratios of HepG2 and HCC97L tumors (low metastatic potential) were1.6 ± 0.3 and 1.8 ± 0.4. The tumor uptake of [¹⁸F]AlF‐NOTA‐G‐TMTP1 could be inhibited 61.9% and 57.6% by unlabeled G‐TMTP1 in SMCC‐7721 and HCCLM3 xenografts at 40 min p.i., respectively. Furthermore, [¹⁸F]AlF‐NOTA‐G‐TMTP1 showed pretty low activity in the liver and intestines in all tumor bearing mice, such in vivo distribution pattern would be advantageous for the detection of hepatic carcinoma. Overall, [¹⁸F]AlF‐NOTA‐G‐TMTP1 may specifically target high metastatic or/and aggressive hepatocellular carcinoma with low background activity and, therefore, holds the potential to be used as an imaging agent for detecting tumor lesions within the liver area. Copyright © 2016 John Wiley & Sons, Ltd.     

Mesenchymal stem cell labeling and in vitro MR characterization at 1.5 T of new SPIO contrast agent: Molday ION Rhodamine‐B™

In vivo detection of transplanted stem cells is requisite for improving stem cell‐based treatments by developing a thorough understanding of their therapeutic mechanisms. MRI tracking of magnetically labeled cells is non‐invasive and is suitable for longitudinal studies. Molday ION Rhodamine‐B? (MIRB) is a new superparamagnetic iron oxide (SPIO) contrast agent specifically formulated for cell labeling and is readily internalized by non‐phagocytic cells. This investigation characterizes mesenchymal stem cell (MSC) labeling and MR imaging properties of this new SPIO agent. Effects of MIRB on MSC viability and differentiation as well as cellular loading properties were assessed for MSC labeled with MIRB at concentrations from 5 to 100 µg Fe/ml. Labeled MSC were evaluated, in vitro, on a clinical 1.5 T MRI. Optimal scanning sequences and imaging parameters were determined based on contrast‐to‐noise ratio and contrast modulation. Relaxation rates (1/T₂*) for gradient‐echo sequences were approximated and an idealized limit of detection was established. MIRB labeling did not affect MSC viability or the ability to differentiate into either bone or fat. Labeling efficiency was found to be approximately 95% for labeling concentrations at or above 20 µg Fe/ml. Average MIRB per MSC ranged from 0.7 pg Fe for labeling MIRB concentration of 5 µg Fe/ml and asymptotically approached a value of 20–25 pg Fe/MSC as labeling concentration increased to 100 µg Fe/ml. MRI analysis of MIRB MSC revealed long echo time, gradient echo sequences to provide the most sensitivity. Limit of detection for gradient echo sequences was determined to be less than 1000 MSC, with approximately 15 pg Fe/MSC (labeled at 20µg Fe/ml). These investigations have laid the groundwork and established feasibility for the use of this contrast agent for in vivo MRI detection of MSC. Properties evaluated in this study will be used as a reference for tracking labeled MSC for in vivo studies. Copyright © 2010 John Wiley & Sons, Ltd.     

EPR assessment of protein sites for incorporation of Gd(III) MRI contrast labels

We have engineered apolipoprotein A‐I (apoA‐I), a major protein constituent of high‐density lipoprotein (HDL), to contain DOTA‐chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism and regulation in vivo. This protein contrast agent was obtained by attaching the thiol‐reactive Gd[MTS‐ADO3A] label at Cys residues replaced at four distinct positions (52, 55, 76 and 80) in apoA‐I. MRI of infused mice previously showed that the Gd‐labeled apoA‐I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoA‐I are superior when the ADO3A moiety is located at position 55, compared with the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave X‐band (9 GHz) electron paramagnetic resonance (EPR) spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipid‐free with the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipid‐free and rHDL states correlate to the product of the X‐band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDL‐associated apoA‐I containing the Gd[MTS‐ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by X‐band EPR. While room temperature EPR requires >1 m m Gd(III)‐labeled and only >10 µ m nitroxide‐labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5 µl). Thus, X‐band EPR provides a practical assessment for the suitability of imaging candidates containing the site‐directed ADO3A contrast probe. Copyright © 2013 John Wiley & Sons, Ltd.     

Multimodal liposomes for SPECT/MR imaging as a tool for in situ relaxivity measurements

One of the major challenges of MR imaging is the quantification of local concentrations of contrast agents. Cellular uptake strongly influences different parameters such as the water exchange rate and the pool of water protons, and results in alteration of the contrast agent's relaxivity, therefore making it difficult to determine contrast agent concentrations based on the MR signal only. Here, we propose a multimodal radiolabeled paramagnetic liposomal contrast agent that allows simultaneous imaging with SPECT and MRI. As SPECT‐based quantification allows determination of the gadolinium concentration, the MRI signal can be deconvoluted to get an understanding of the cellular location of the contrast agent. The cell experiments indicated a reduction of the relaxivity from 2.7 ± 0.1 m m ^?1 s^?1 to a net relaxivity of 1.7 ± 0.3 m m ^?1 s^?1 upon cellular uptake for RGD targeted liposomes by means of the contrast agent concentration as determined by SPECT. This is not observed for nontargeted liposomes that serve as controls. We show that receptor targeted liposomes in comparison to nontargeted liposomes are taken up into cells faster and into subcellular structures of different sizes. We suggest that the presented multimodal contrast agent provides a functional readout of its response to the biological environment and is furthermore applicable in in vivo measurements. As this approach can be extended to several MRI‐based contrast mechanisms, we foresee a broader use of multimodal SPECT/MRI nanoparticles to serve as in vivo sensors in biological or medical research. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparison of near‐infrared fluorescent deoxyglucose probes with different dyes for tumor diagnosis in vivo

Glucose plays a central role in the cellular energy metabolism. Malignant tumors exhibit an elevated rate of glycolysis over normal tissues. In this study, two near‐infrared fluorescent dyes, Cypate and ICG‐Der‐02, with different water solubility, were conjugated to 2‐amino‐2‐deoxy‐d ‐glucose (2DG) to form Cypate‐2DG and ICG‐Der‐02‐2DG, respectively, for NIR fluorescent imaging of tumors in nude mice. The clear routes and tumor targeting abilities of the two NIR fluorescent 2DG probes were compared. Results showed that ICG‐Der‐02‐2DG with higher hydrophilicity was cleared faster by kidneys than the more lipophilic Cypate‐2DG. Cypate‐2DG had slower but stronger tumor targeting ability compared with ICG‐Der‐02‐2DG. To investigate the correlation between the targeting ability of the probe and the glucose transporter (GLUT1) expression levels of cancer cells, the accumulation of Cypate‐2DG in tumors was assessed in MCF‐7/estradiol, U87MG, MCF‐7 and MDA‐MB‐435 tumor xenografts, which express different levels of GLUT1. The results show that both Cypate‐2DG and ICG‐Der‐02‐2DG possess tumor targeting ability on all the tumors examined, with a proportional correlation to GLUT1 expression. The findings demonstrate the broad applicability of these molecular probes for optical imaging of tumors and glucose‐related pathologies. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescence lifetime imaging of activatable target specific molecular probes

In vivo optical imaging using fluorescently labeled self‐quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target‐to‐background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self‐quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2⁺ and BALB/3T3 (HER2^?) cell lines. Changes in fluorescence lifetime correlated with temperature‐ and time‐dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2⁺ and BALB/3T3 (HER2^?)] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target‐specific activatable antibody–fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging. Copyright © 2010 John Wiley & Sons, Ltd.     

Fluorescent molecular imaging of metastatic lymph node using near‐infrared emitting low molecular weight heparin modified nanoliposome based on enzyme‐substrate interaction

Tumor metastatic lymph node mapping has been widely used to predict the metastatic spread of primary tumor and guide the lymph node dissection in clinical practice. In this research, a new near‐infrared (NIR)‐emitting low molecular weight heparin (LMWHEP)‐modified Cy7‐loaded nanoliposome (LMWHEP‐NLips/Cy7) was developed and had the particle size of about 80 nm and the fluorescence intensity of about 2300, which is optimal for metastatic lymph node uptake and imaging. The NIR‐emitting nanoliposomes were designed by LMWHEP coating on the surface of Cy7‐loaded nanoliposome (NLips/Cy7) according to electrostatic attraction. The LMWHEP‐NLips/Cy7 with negligible cytotoxicity for Hela and RAW264.7 cells and was found to be fluorescent stability compared with the Cy7‐free dye at room temperature. The BALB/c nude mice bearing tumor lymphatic metastasis was established at eighth week post‐injection by subcutaneously injecting Hela cells suspension. Heparanase (HPA) expression concentrations quantitatively measured by ELISA kit respectively were 237.42U/mL, 214.82U/mL and 128.45U/mL in the extracellular Hela cells, metastatic popliteal and iliac lymph node. LMWHEP‐NLips/Cy7 successfully increased fluorescence signal in the metastatic lymph node compared with normal lymph node and achieve in vivo and ex vivo high fluorescence signal within 10 min and retention time up to 4 h post‐injection. Maximum mean fluorescence intensity of the LMWHEP‐NLips/Cy7 group was significantly more than NLips/Cy7 group (increase 2‐fold in the metastatic popliteal lymph node and 4.8‐fold in the metastatic iliac lymph node, p < 0.05). The experimental results demonstrating LMWHEP‐NLips/Cy7 have the potential utility as specific, biosafe and stable near‐infrared imaging contrast agents for HPA‐expression tumor metastatic lymph node mapping. Copyright © 2016 John Wiley & Sons, Ltd.     

Modification of Aminosilanized Superparamagnetic Nanoparticles: Feasibility of Multimodal Detection Using 3T MRI, Small Animal PET, and Fluorescence Imaging

Purpose

The aim of our study was to modify an aminosilane-coated superparamagnetic nanoparticle for cell labeling and subsequent multimodal imaging using magnetic resonance imaging (MRI), positron emission tomography (PET), and fluorescent imaging in vivo.

Procedures

We covalently bound the transfection agent HIV-1 tat, the fluorescent dye fluorescein isothiocyanate, and the positron-emitting radionuclide gallium-68 to the particle and injected them intravenously into Wistar rats, followed by animal PET and MRI at 3.0 T. As a proof of principle hepatogenic HuH7 cells were labeled with the particles and observed for cell toxicity as well as detectability by MRI and biodistribution in vivo.

Results

PET imaging and MRI revealed increasing hepatic and splenic accumulation of the particles over 24 h. Adjacent in vitro studies in hepatogenic HuH7 cells showed a rapid intracellular accumulation of the particles with high labeling efficiency and without any signs of toxicity. In vivo dissemination of the labeled cells could be followed by dynamic biodistribution studies.

Conclusions

We conclude that our modified superparamagnetic nanoparticles are stable under in vitro and in vivo conditions and are therefore applicable for efficient cell labeling and subsequent multimodal molecular imaging. Moreover, their multiple free amino groups suggest the possibility for further modifications and might provide interesting opportunities for various research fields.     

cGMP‐Compatible preparative scale synthesis of near‐infrared fluorophores

Image‐guided surgery using optical imaging requires the availability of large quantities of clinical‐grade fluorophores. We describe the cGMP‐compatible synthesis of the zwitterionic heptamethine indocyanine near‐infrared fluorophore ZW800‐1 at the 10 g scale (~1000 patient doses) using facile and efficient solvent purification, and without the need for column chromatography. ZW800‐1 has >90% yield at the final step and >99% purity as measured by fluorescence and evaporative light scatter detection. We describe an analytical framework for qualifying impurities, as well as a detailed analysis of counterion identities. Finally, we report the unique in vivo properties of ZW800‐1 in large animals approaching the size of humans, thus laying the foundation for rapid clinical translation of these methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Magnetic labeling of pancreatic β‐cells modulates the glucose‐ and insulin‐induced phosphorylation of ERK1/2 and AKT

This study was undertaken to investigate the effect of a magnetic resonance imaging (MRI) contrast agent, superparamagnetic iron oxide nanoparticle (SPIO), on signal transduction by glucose and insulin in pancreatic β‐cells. INS‐1 cells were labeled in culture medium containing clinically approved SPIO for 24 h. Labeled and unlabeled cells were stimulated with glucose (25 mM) or insulin (0.1–1 µM) for 12 h. The phosphorylation of extracellular signal‐regulated kinase1/2 (ERK1/2) and protein kinase B (AKT) and intracellular insulin protein levels were assessed by Western blotting. After labeling with increasing amounts of SPIO, cytotoxicity was not observed, yet the intracellular iron concentration increased in a dose‐dependent manner. SPIO labeling (200 µg Fe ml^?1) induced a significant increase in ERK1/2 and AKT phosphorylation (labeled vs unlabeled, p < 0.05), but significantly reduced the glucose‐stimulated phosphorylation of ERK1/2 and AKT and insulin‐stimulated phosphorylation of AKT (labeled vs unlabeled, p < 0.05). The level of intracellular insulin protein was found to be lower in labeled cells than unlabeled cells (labeled vs unlabeled, p < 0.05). This study demonstrates that SPIO labeling alters some fundamental functional variables, at least in INS‐1 cells, through modulation of the glucose‐ or insulin‐induced activation of ERK1/2 and AKT, which leads to insulin biosynthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Manganese cell labeling of murine hepatocytes using manganese(III)‐transferrin

Manganese(III)‐transferrin [Mn(III)–Tf] was investigated as a way to accomplish manganese‐labeling of murine hepatocytes for MRI contrast. It is postulated that Mn(III)–Tf can exploit the same transferrin‐receptor‐dependent and ‐independent metabolic pathways used by hepatocytes to transport the iron analog Fe(III)–Tf. More specifically, it was investigated whether manganese delivered by transferrin could give MRI contrast in hepatocytes. Comparison of the T₁ and T₂ relaxation times of Mn(III)–Tf and Fe(III)–Tf over the same concentration range showed that the r₁ relaxivities of the two metalloproteins are the same in vitro, with little contribution from paramagnetic enhancement. The degree of manganese cell labeling following incubation for 2–7 h in 31.5 µm Mn(III)–Tf was comparable to that of hepatocytes incubated in 500 µm Mn²⁺ for 1 h. The intrinsic manganese tissue relaxivity between Mn(III)–Tf‐labeled and Mn²⁺‐labeled cells was found to be the same, consistent with Mn(III) being released from transferrin and reduced to Mn²⁺. For both treatment regimens, manganese uptake by hepatocytes appeared to saturate in the first 1–2 h of the incubation period and may explain why the efficiency of hepatocyte cell labeling by the two methods appeared to be comparable in spite of the ～16‐fold difference in effective manganese concentration. Hepatocytes continuously released manganese, as detected by MRI, and this was the same for both Mn²⁺‐ and Mn(III)–Tf‐labeled cells. Manganese release may be the result of normal hepatocyte function, much in the same way that hepatocytes excrete manganese into the bile in vivo. This approach exploits a biological process—namely receptor binding, endocytosis and endosomal acidification—to initiate the release of an MRI contrast agent, potentially conferring more specificity to the labeling process. The ubiquitous expression of transferrin receptors by eukaryotic cells should make Mn(III)–Tf particularly useful for manganese labeling of a wide variety of cells both in culture and in vivo. Published in 2008 by John Wiley & Sons, Ltd.     

Minimization of self‐quenching fluorescence on dyes conjugated to biomolecules with multiple labeling sites via asymmetrically charged NIR fluorophores

Self‐aggregation of dyes even at low concentrations poses a considerable challenge in preparing sufficiently bright molecular probes for in vivo imaging, particularly in the conjugation of near infrared cyanine dyes to polypeptides with multiple labeling sites. Such self‐aggregation leads to a significant energy transfer between the dyes, resulting in severe quenching and low brightness of the targeted probe. To address this problem, we designed a novel type of dye with an asymmetrical distribution of charge. Asymmetrical distribution prevents the chromophores from π‐stacking thus minimizing the energy transfer and fluorescence quenching. The conjugation of the dye to polypeptides showed only a small presence of an H‐aggregate band in the absorption spectra and, hence, a relatively high quantum efficiency. Copyright © 2014 John Wiley & Sons, Ltd.     

Spectral near‐infrared fluorescence imaging of curved surfaces using projection reconstruction algorithms

In vivo spectral fluorescence imaging has made it possible to non‐invasively visualize superficial curved structures as well as structures deep to the skin. However, the defocus created by blurring has been an obstacle to creating anatomically interpretable surface images. Herein we present a methodology to correct for blurring induced by curved structures during spectral fluorescence imaging using signal intensity projection algorithms. In a phantom and an animal model in which the lymphatic system was visualized after the interstitial injection of quantum dots with emission spectra in the near‐infrared (NIR) range, the planes of focus were sequentially adjusted to obtain a z‐stack of images which contains images acquired from multiple focal points. Maximum, minimum, median and average intensity projections were applied to the resulting images. Using the phantom, the minimum and the median intensity projection images demonstrated improved deblurring whereas during in vivo imaging the median intensity projection images more clearly visualized important structures than did the other projection techniques. Image stacking with subsequent application of appropriate projection techniques provides a simple method for deblurring in vivo optical images obtained from curved surfaces, thus improving their anatomic resolution. Copyright © 2007 John Wiley & Sons, Ltd.     

Fast clearing RGD‐based near‐infrared fluorescent probes for in vivo tumor diagnosis

A fast clearing hydrophilic near‐infrared (NIR) dye ICG‐Der‐02 was used to constitute tumor targeting contrast agents. Cell adhesion molecule integrin α_vβ₃ served as the target receptor because of its unique expression on almost all sprouting tumor vasculatures. The purpose of this study was to synthesize and compare the properties of integrin α_vβ₃‐targeted, fast clearing NIR probes both in vitro and in vivo for tumor diagnosis. ICG‐Der‐02 was covalently conjugated to three kinds of RGD peptide including linear, monoeric cyclic and dimeric RGD to form three RGD‐based NIR probes. The integrin receptor specificities of these probes were evaluated in vitro by confocal microscopy. The dynamic bio‐distribution and elimination ratse were in vivo real‐time monitored by a near‐infrared imaging system in normal mice. Further, the in vivo tumor targeting abilities of the RGD‐based NIR probes were compared in α_vβ₃‐positive MDA‐MB‐231, U87MG and α_vβ₃‐negtive MCF‐7 xenograft mice models. Three RGD‐based NIR probes were successfully synthesized with good optical properties. In vitro cellular experiments indicated that the probes have a clear binding affinity to α_υβ₃‐positive tumor cells, with a cyclic dimeric RGD probe owing the highest integrin affinity. Dynamic bio‐distributions of these probes showed a rapid clearing rate through the renal pathway. In vivo tumor targeting ability of the RGD‐based porbes was demonstrated on MDA‐MB‐231 and U87MG tumor models. As expected, the c(RGDyK)₂‐ICG‐Der‐02 probe displayed the highest tumor‐to‐normal tissue contrast. The in vitro and in vivo block experiments confirmed the receptor binding specificity of the probes. The hydrophilic dye‐labeled NIR probes exhibited a fast clearing rate and deep tissue penetration capability. Further, the α_υβ₃ receptor affinity of the three RGD‐based NIR probes followed the order of dimer cyclic > monomer cyclic > linear. The results demonstrate potent fast clearing probes for in vivo early tumor diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.