In Vivo Tumor Detection in Small Animals by Hematoporphyrin-Mediated Fluorescence Imaging.

Photomed Laser Surg. 2010 Jul 28;

Authors: Autiero M, Cozzolino R, Laccetti P, Marotta M, Quarto M, Riccio P, Roberti G

Abstract Objective: Noninvasive in vivo imaging of human tumors implanted in mice provides a reliable and economic tool for the investigation of tumor progression and metastasis and of the effectiveness of the antiblastic drugs on them. The purpose of this study is to report on the performance achievable by the well-known and extensively investigated HP-FRI (HematoPorphyrin (HP)-mediated Fluorescence Reflectance Imaging) when a high-quality image-acquisition device is used. Background Data: Previous articles of ours showed that HP-FRI still represents a useful, simple and reliable optical imaging technique to detect surface tumors. Therefore, it is particularly suitable to be used in combination with other imaging modalities in a multimodal imaging system endowed with diagnostic capabilities much better than each separate modality. Materials and Methods: Six-week-old Crl:CD-1 nude mice were subcutaneously inoculated with tumor cells. Tumor-bearing mice were irradiated in vivo by a frequency-doubled pulsed Nd:YAG laser (lambda = 532 nm). A cooled CCD digital camera recorded fluorescence light emitted by HP injected in mice through a cut-on long-wavelength pass filter. Results: The system we developed allows in vivo imaging of surface tumors on small animals with a large field of view, high photometric sensitivity, adequate space resolution, and short measurement time. The estimated spatial resolution is 730 mum for a fluorescence source placed about 0.5 mm under the mouse skin. The first exploration of the capabilities of this HP-FRI setup on few mice shows that it allows the detection of (a) both types of investigated tumors, (b) early stage and late stage but visually unrecognizable tumors, (c) the gross structure of tumors, and (d) the discrimination of necrotic and nonnecrotic tumor regions.

PMID: 20666573 [PubMed - as supplied by publisher]

4-dimensional intravital microscopy: a new model for studies of leukocyte recruitment and migration in hepatocellular cancer in mice.

J Gastrointest Surg. 2010 May;14(5):867-72

Authors: Takeichi T, Engelmann G, Mocevicius P, Schmidt J, Ryschich E

INTRODUCTION: Although it is accepted that the immune system plays a role in the prognosis of hepatocellular carcinoma (HCC), the exact mechanisms of leukocyte recruitment into HCC are poorly understood. Progress in the study of this aspect has been hindered by technical limitations. MATERIALS AND METHODS: In the present study, we describe the use of 4D intravital microscopy which represents an advantageous technology for the investigation of the microvascular system and leukocyte migration in HCC. To establish 4D intravital microscopy, we used a HCC tumor model in transgenic mice expressing enhanced green fluorescent protein in specific leukocyte subpopulations and combined digital time-lapse recording, laser scanning confocal microscopy, and 3D reconstruction. Using this technology, we studied the intra- and extravascular leukocyte adhesion and migration in HCC in vivo at the single-cell level. RESULTS: We showed that although vessel density in HCC was lower than in normal liver, tumor tissue was moderately infiltrated with leukocytes of lymphoid and myeloid origin. Most tumor-infiltrating leukocytes migrated in a random manner frequently changing direction of migration in the tumor tissue. The migration velocity of myeloid and lymphoid leukocytes in HCC tissue was not different. DISCUSSION: These results demonstrated that 4D intravital microscopy has potential to be a powerful tool in the study of mechanisms of leukocyte recruitment and intratumoral migration in HCC.

PMID: 20229071 [PubMed - indexed for MEDLINE]

In vivo angiogenesis imaging of solid tumors by {alpha}v{beta}3-targeted, dual-modality micellar nanoprobes.

Exp Biol Med (Maywood). 2010 Aug 1;235(8):957-65

Authors: Kessinger CW, Khemtong C, Togao O, Takahashi M, Sumer BD, Gao J

The objective of this study was to develop and evaluate an alpha(v)beta(3)-specific nanoprobe consisting of fluorescent superparamagnetic polymeric micelles (FSPPM) for in vivo imaging of tumor angiogenesis. Spherical micelles were produced using poly(ethylene glycol)-b-poly(d,l-lactide) co-polymers conjugated with tetramethylrhodamine, a fluorescent dye, and loaded with superparamagnetic iron oxide nanoparticles. The resulting micelle diameter was 50-70 nm by dynamic light scattering and transmission electron microscopy measurements. Micelles were encoded with an alpha(v)beta(3)-specific peptide, cyclic RGDfK, and optimized for maximum fluorescence and targeting in alpha(v)beta(3)-overexpressing cells in vitro. In mice, cRGD-FSPPM-treated animals showed alpha(v)beta(3)-specific FSPPM accumulation in human lung cancer subcutaneous tumor xenografts. Together with the histological validation, the three-dimensional gradient echo magnetic resonance imaging (MRI) data provide high spatial resolution mapping and quantification of angiogenic vasculature in an animal tumor model using targeted, ultrasensitive MRI nanoprobes.

PMID: 20660096 [PubMed - in process]

Detection and Treatment of Diseases Using Light.

IFMBE Proc. 2010 Jan 11;27:19-22

Authors: Pham W

Fluorescence light is a natural phenomenon that has been employed recently in medical imaging and therapy. The implication of this technology in clinical studies is largely dependent on the chemical design of organic dyes or photosensitizers. The novel design and application of the probes is the subject of this discussion. In the past decade, significant improvements have yielded innovative dyes for in vivo imaging of numerous diseases in a mouse model while improvements in distribution enable photosensitizers to be used in photodynamic therapy.

PMID: 20657754 [PubMed - as supplied by publisher]

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Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy.

Gut. 2010 Aug;59(8):1046-55

Authors: Foersch S, Kiesslich R, Waldner MJ, Delaney P, Galle PR, Neurath MF, Goetz M

Background Vascular endothelial growth factor (VEGF) is a therapeutic target in gastrointestinal cancer (GiC). However, its in vivo visualisation could not be achieved to date with endoscopic techniques. Confocal laser endomicroscopy (CLE) is a novel imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. The aim of the study was to evaluate CLE for in vivo molecular imaging of VEGF in GiC. Methods Molecular imaging of tumours in APCmin mice, in xenograft models and in surgical specimens of patients with colorectal cancer (CRC) was achieved after application of labelled antibodies. The tumour sites were scanned with the probe for the strongest specific fluorescent signal. From all tumour sites examined with CLE in vivo, targeted specimens were obtained for histology, immunohistochemistry (IHC) and fluorescence microscopy. Results A VEGF-specific signal was visualised in vivo in 13/15 APCmin mice and in 9/10 xenograft tumours. CLE enabled the cytoplasmatic distribution of VEGF to be displayed due to its subcellular resolution. In human tissue, a VEGF-specific signal was observed in 12/13 malignant specimens and in 10/11 samples from healthy mucosa from the patients (p<0.03). CLE findings correlated well with ex vivo microscopy. Conclusion In vivo molecular imaging with specific targeting of VEGF is possible in murine tumours, human xenografts and tissue specimens using CLE. CLE with similar probes can be performed in human colonoscopy. Therefore-from a technical point of view-in vivo molecular imaging is transferable to stratification of patients with CRC during endoscopy even today. CLE could contribute to the identification of lesions at risk and potentially predict response to targeted treatment.

PMID: 20639250 [PubMed - in process]

Complete regression of human malignant mesothelioma xenografts following local injection of midkine promoter-driven oncolytic adenovirus.

J Gene Med. 2010 Jul 14;

Authors: Kubo S, Kawasaki Y, Yamaoka N, Tagawa M, Kasahara N, Terada N, Okamura H

BACKGROUND: Malignant mesothelioma is a highly aggressive tumor with poor prognosis. We hypothesized that the tumor-specific midkine (Mdk) promoter could confer transcriptional targeting to oncolytic adenoviruses for effective treatment of malignant mesothelioma. METHODS: We analysed Mdk expression by quantitative reverse transcription-polymerase chain reaction in six human mesothelioma cell lines, and tested Mdk promoter activity by luciferase reporter assay. On the basis of these data, we constructed a replication-selective oncolytic adenovirus designated AdMdk-E1-iresTK. This virus contains a Mdk promoter-driven adenoviral E1 gene and herpes simplex virus-thymidine kinase (TK) suicide gene and cytomagalovirus promoter-driven enhanced green fluorescent protein marker gene. Selectivity of viral replication and cytolysis were characterized in normal versus mesothelioma cells in vitro, and intratumoral spread and antitumor efficacy were investigated in vivo. RESULTS: Mdk promoter activity was restricted in normal cells, but highly activated in mesothelioma cell lines. AdMdk-E1-iresTK was seen to efficiently replicate, produce viral progeny and spread in multiple mesothelioma cell lines. Lytic spread of AdMdk-E1-iresTK mediated the efficient killing of these mesothelioma cells, and its in vitro cytocidal effect was significantly enhanced by treatment with the prodrug, ganciclovir. Intratumoral injection of AdMdk-E1-iresTK caused complete regression of MESO4 and MSTO human mesothelioma xenografts in athymic mice. In vivo fluorescence imaging demonstrated intratumoral spread of AdMdk-E1-iresTK-derived signals, which vanished after tumor eradication. CONCLUSIONS: These data indicate that transcriptional targeting of viral replication by the Mdk promoter represents a promising general strategy for oncolytic virotherapy of cancers with up-regulated Mdk expression, including malignant mesothelioma. Copyright (c) 2010 John Wiley & Sons, Ltd.

PMID: 20635326 [PubMed - as supplied by publisher]

Tumor targeting efficiency of bare nanoparticles does not mean the efficacy of loaded anticancer drugs; Importance of Radionuclide Imaging for Optimization of Highly Selective Tumor Targeting Polymeric Nanoparticles with or without Drug.

J Control Release. 2010 Jul 9;

Authors: Lee BS, Park K, Park S, Kim GC, Kim HJ, Lee S, Kil H, Oh SJ, Chi D, Kim K, Kwon IC, Kim SY

The better understanding of polymeric nanoparticles as a drug delivery carrier is a decisive factor to get more efficient therapeutic response in vivo. Here, we report the non-invasive imaging of bare polymeric nanoparticles and drug loaded polymeric nanoparticles to evaluate biodistribution in tumor bearing mice. To make nano-sized drug delivery carrier, glycol chitosan was modified with different degree of hydrophobic N-acetyl histidine (NAcHis-GC-1,-2, and -3). The biodistribution of polymeric nanoparticles and drug were confirmed by using gamma camera with (131)I-labeled-NAcHis-GC and (131)I-labeled Doxorubicin (DOX) and by using in vivo live animal imaging with near-infrared fluorescence Cy5.5-labeled-NAcHis-GC. Among bare nanoparticles, NAcHis-GC3 (7.8% NAcHis content) showed much higher tumor targeting efficiency than NAcHis-GC1 (3.3% NAcHis content) and NAcHis-GC2 (6.8% NAcHis content). In contrast, for drug-loaded nanoparticles, DOX-NAcHis-GC1 displayed two-fold higher tumor targeting property than DOX-NAcHis-GC3. These data imply that the biodistribution and tumor targeting efficiency between bare and drug-loaded nanoparticles may be greatly different. Therapeutic responses for NAcHis-GC nanoparticles after drug loading were also evaluated. In xenograft animal model, we could find out that DOX-NAcHis-GC1 with higher tumor targeting of DOX has more excellent therapeutic effect than DOX-NAcHis-GC3 and free DOX. These results mean that the hydrophobic core stability might be critical factor for tumor targeting efficiency of nanoparticles. The present study indicates that by using molecular imaging, we can select more appropriate nanoparticles with the highest tumor targeting properties, leading to exerting more excellent therapeutic results in cancer therapy.

PMID: 20624433 [PubMed - as supplied by publisher]

Quantitative mitochondrial redox imaging of breast cancer metastatic potential.

J Biomed Opt. 2010 May-Jun;15(3):036010

Authors: Xu HN, Nioka S, Glickson JD, Chance B, Li LZ

Predicting tumor metastatic potential remains a challenge in cancer research and clinical practice. Our goal was to identify novel biomarkers for differentiating human breast tumors with different metastatic potentials by imaging the in vivo mitochondrial redox states of tumor tissues. The more metastatic (aggressive) MDA-MB-231 and less metastatic (indolent) MCF-7 human breast cancer mouse xenografts were imaged with the low-temperature redox scanner to obtain multi-slice fluorescence images of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp). The nominal concentrations of NADH and Fp in tissue were measured using reference standards and used to calculate the Fp redox ratio, Fp(NADH+Fp). We observed significant core-rim differences, with the core being more oxidized than the rim in all aggressive tumors but not in the indolent tumors. These results are consistent with our previous observations on human melanoma mouse xenografts, indicating that mitochondrial redox imaging potentially provides sensitive markers for distinguishing aggressive from indolent breast tumor xenografts. Mitochondrial redox imaging can be clinically implemented utilizing cryogenic biopsy specimens and is useful for drug development and for clinical diagnosis of breast cancer.

PMID: 20615012 [PubMed - in process]

Optical recording of neuronal spiking activity from unbiased populations of neurons with high spike detection efficiency and high temporal precision.

J Neurophysiol. 2010 Jul 7;

Authors: Ranganathan GN, Koester HJ

Activity in populations of neurons is essential for cortical function, including signaling of information and signal transport. Previous methods have made advances in recording activity from many neurons, but have both technical and analytical limitations. Here we present an optical method, dithered random-access functional calcium imaging, to record somatic calcium signals from up to 100 neurons, in vitro and in vivo. We further developed a maximum-likelihood deconvolution algorithm to detect spikes and precise spike timings from the recorded calcium fluorescence signals. Spike detection efficiency and spike timing detection was determined in acute slices of juvenile mice. The results indicate that the combination of the two methods detected precise spiking activity from unbiased and spatially distributed populations of neurons in acute slices with high efficiency of spike detection (>97%), low rate of false positives (0.0023 spikes/s), and high temporal precision. The results further indicate that there is only a small window of excitation intensities where high spike detection can be achieved consistently.

PMID: 20610791 [PubMed - as supplied by publisher]

In vivo validation of MRI vessel caliber index measurement methods with intravital optical microscopy in a U87 mouse brain tumor model.

Neuro Oncol. 2010 Apr;12(4):341-50

Authors: Farrar CT, Kamoun WS, Ley CD, Kim YR, Kwon SJ, Dai G, Rosen BR, di Tomaso E, Jain RK, Sorensen AG

The vessel caliber index (VCI), a magnetic resonance imaging biomarker of the average blood vessel diameter, is increasingly being used as a tool for assessing tumor angiogenesis and response to antiangiogenic therapy. However, although the VCI has been correlated with histological vessel diameters, good quantitative agreement with histology has been lacking. In addition, no VCI validation studies have been performed in vivo where the structural deformations frequently associated with histological tissue preparation are not present. This study employs intravital optical microscopy (IVM) measurements of cerebral blood vessel diameters in a mouse orthotopic glioma model to provide the first such in vivo validation. Two VCI correlation models, both a linear and a 3/2-power dependence on the DeltaR2*/DeltaR2 ratio, were compared with the IVM data. The linear VCI model, determined from steady-state susceptibility contrast (SSC) images, was found to be in excellent quantitative agreement with the intravitally determined VCI for separate tumor size matched groups of mice. In addition, preliminary data indicate that the VCI is independent of whether a dynamic susceptibility contrast or SSC measurement method is used.

PMID: 20308312 [PubMed - indexed for MEDLINE]

Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain.

Br J Pharmacol. 2010 Feb;159(4):808-11

Authors: Ortolano F, Maffia P, Dever G, Rodolico G, Millington OR, De Simoni MG, Brewer JM, Bushell TJ, Garside P, Carswell HV

BACKGROUND AND PURPOSE: T-cells may play a role in the evolution of ischaemic damage and repair, but the ability to image these cells in the living brain after a stroke has been limited. We aim to extend the technique of real-time in situ brain imaging of T-cells, previously shown in models of immunological diseases, to models of experimental stroke. EXPERIMENTAL APPROACH: Male C57BL6 mice (6-8 weeks) (n= 3) received a total of 2-5 x 10(6) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled lymphocytes from donor C57BL6 mice via i.v. injection by adoptive transfer. Twenty-four hours later, recipient mice underwent permanent left distal middle cerebral artery occlusion (MCAO) by electrocoagulation or by sham surgery under isoflurane anaesthesia. Female hCD2-green fluorescent protein (GFP) transgenic mice that exhibit GFP-labelled T-cells underwent MCAO. At 24 or 48 h post-MCAO, a sagittal brain slice (1500 microm thick) containing cortical branches of the occluded middle cerebral artery (MCA) was dissected and used for multiphoton laser scanning microscopy (MPLSM). KEY RESULTS: Our results provide direct observations for the first time of dynamic T-cell behaviour in living brain tissue in real time and herein proved the feasibility of MPLSM for ex vivo live imaging of immune response after experimental stroke. CONCLUSIONS AND IMPLICATIONS: It is hoped that these advances in the imaging of immune cells will provide information that can be harnessed to a therapeutic advantage.

PMID: 20015295 [PubMed - indexed for MEDLINE]

Novel fluorescent cycloheximide derivatives for the imaging of protein synthesis.

Biochem Biophys Res Commun. 2010 May 28;396(2):258-64

Authors: Paoletti F, Ainger K, Donati I, Scardigli R, Vetere A, Cattaneo A, Campa C

Cycloheximide (CHX) is one of the most interesting protein synthesis inhibitors. For this reason, fluorescent derivatives of CHX could find useful applications in cell biology. We report the successful synthesis of a set of novel fluorescent derivatives of CHX. The effect of different functional groups on the biological activity of CHX was studied upon their modification through suitable strategies, i.e., acetylation of the hydroxyl group and reductive amination of the ketone group. The first route induced a complete loss of biological activity, while the second approach allowed a retained inhibition of protein synthesis, as demonstrated by in vitro translation assays. Various fluorescent dyes for reductive amination were tested (i.e., ANTS, APTS, and Rhodamine-123), and the success of the syntheses was demonstrated by diverse analytical techniques. Cycloheximide labeling with fluorescent dyes is a promising approach for developing fluorescence reporters for various applications, both in vitro (fluorescence spectroscopy) and in vivo (live imaging).

PMID: 20399740 [PubMed - indexed for MEDLINE]

Arterial and aortic valve calcification inversely correlates with osteoporotic bone remodelling: a role for inflammation.

Eur Heart J. 2010 Jul 2;

Authors: Hjortnaes J, Butcher J, Figueiredo JL, Riccio M, Kohler RH, Kozloff KM, Weissleder R, Aikawa E

Aims Westernized countries face a growing burden of cardiovascular calcification and osteoporosis. Despite its vast clinical significance, the precise nature of this reciprocal relationship remains obscure. We hypothesize that cardiovascular calcification progresses with inflammation and inversely correlates with bone tissue mineral density (TMD). Methods and results Arterial, valvular, and bone metabolism were visualized using near-infrared fluorescence (NIRF) molecular imaging agents, targeting macrophages and osteogenesis. We detected significant arterial and aortic valve calcification in apoE(-/-) mice with or without chronic renal disease (CRD, 30 weeks old; n = 28), correlating with the severity of atherosclerosis. We demonstrated decreases in osteogenic activity in the femurs of apoE(-/-) mice when compared with WT mice, which was further reduced with CRD. Three-dimensional micro-computed tomography imaging of the cortical and cancellous regions of femurs quantified structural remodelling and reductions in TMD in apoE(-/-) and CRD apoE(-/-) mice. We established significant correlations between arterial and valvular calcification and loss of TMD (R(2) = 0.67 and 0.71, respectively). Finally, we performed macrophage-targeted molecular imaging to explore a link between inflammation and osteoporosis in vivo. Although macrophage burden, visualized as uptake of NIRF-conjugated iron nanoparticles, was directly related to the degree of arterial and valvular inflammation and calcification, the same method inversely correlated inflammation with TMD (R(2) = 0.73; 0.83; 0.75, respectively). Conclusion This study provides direct in vivo evidence that in arteries and aortic valves, macrophage burden and calcification associate with each other, whereas inflammation inversely correlates with bone mineralization. Thus, understanding inflammatory signalling mechanisms may offer insight into selective abrogation of divergent calcific phenomena.

PMID: 20601388 [PubMed - as supplied by publisher]

Establishment of in vivo fluorescence imaging in mouse models of malignant mesothelioma.

Int J Oncol. 2010 Aug;37(2):273-279

Authors: Yamaoka N, Kawasaki Y, Xu Y, Yamamoto H, Terada N, Okamura H, Kubo S

Malignant mesothelioma is a highly aggressive tumor with poor prognosis, and new treatment paradigms are urgently needed. For testing preclinical efficacy of new therapeutic agents, establishment of appropriate animal models is crucial. We developed in vivo fluorescence imaging models for human malignant mesothelioma in mice using tumor cells engineered to express fluorescent proteins (EGFP, mRFP, mCherry, and mPlum) by lentiviral vectors. Among these fluorescent proteins, the expression of mCherry protein in the transduced tumor cells was shown to be robust and stable both in vitro and in vivo. In both, peritoneally disseminated and orthotopic pleural mesothelioma models, mCherry-positive tumors were sensitively detected and tumor growth was successfully monitored. This represents the first study to achieve sensitive tumor detection and tracking of tumor growth and development in the malignant mesothelioma mouse models by non-invasive in vivo fluorescence imaging. These imaging models can be versatile and powerful tools to explore new treatment paradigms for malignant mesothelioma.

PMID: 20596654 [PubMed - as supplied by publisher]

Multimodality Imaging Reveals a Gradual Increase in Matrix Metalloproteinase Activity at Aneurysmal Lesions in Live Fibulin-4 Mice.

Circ Cardiovasc Imaging. 2010 Jun 30;

Authors: Kaijzel E, van Heijningen P, Wielopolski P, Vermeij M, Koning G, van Cappellen WA, Que I, Chan A, Dijkstra J, Ramnath N, Hawinkels L, Bernsen M, Löwik C, Essers J

BACKGROUND: -We imaged the protease activity of matrix metalloproteinases (MMPs) upregulated during aneurysm formation, using protease-activatable near-infrared fluorescence (NIRF) probes. We tested whether these protease-activatable sensors can directly report the in vivo activity of the key biomarkers in aneurysm using our genetically modified Fibulin-4 mouse models for aneurysm formation. Mice homozygous for the Fibulin-4 reduced expression allele (Fibulin-4(R/R)) show dilatation of the ascending aorta and a tortuous, stiffened aorta resulting from disorganized elastic fiber networks. Strikingly, even a moderate reduction in expression of Fibulin-4 in the heterozygous Fibulin-4(+/R) mice occasionally results in modest aneurysm formation. METHODS AND RESULTS: -Aorta transcriptome and protein expression analysis of Fibulin-4(+/R) and Fibulin-4(R/R) animals identified excessive TGFbeta signaling as the critical event in the pathogenesis of aneurysm formation. To determine whether perturbed elastin lamellar structure arose from induction of TGFbeta regulated MMPs, we performed gelatin-zymography and used a protease-activatable NIRF probe to monitor and quantify MMP upregulation in animals using various in vivo optical imaging modules and co-registration of the fluorescence signal with CT images of the same animals. Gelatin-zymography demonstrated a significant increase in the presence of the active form of MMP-9 in the aortic arch of Fibulin-4(R/R) mice. In vivo analysis of MMP upregulation using the NIRF probe and subsequent isosurface concentration mapping from reconstructed tomographic images from Fibulin-4(+/R) and Fibulin-4(R/R) mice revealed a graded increase in activation of MMPs within the aneurysmal lesions. CONCLUSIONS: -We aimed to develop molecular imaging procedures for faster, earlier and easier recognition of aortic aneurysms. Here we show that in vivo co-registration of MMP activity by non-invasive tomographic imaging methods allows the detection of increased MMP activity, even before the aneurysm has actually formed.

PMID: 20592247 [PubMed - as supplied by publisher]

Indocyanine green and lymphatic imaging: current problems.

Lymphat Res Biol. 2010 Jun;8(2):127-30

Authors: Gashev AA, Nagai T, Bridenbaugh EA

BACKGROUND: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has recently been presented as a comparatively easy and informative technique to image lymphatic channels in vivo. However, no data or references have been provided concerning the impact of ICG application on normal lymphatic contractility and lymph transport. Thus, the imaging agent and/or the method of administration may introduce a significant artifact. METHODS AND RESULTS: Standard pharmacological tests were performed to investigate the influence of ICG on the spontaneous contractility of isolated, cannulated, and pressurized rat mesenteric lymphatic vessels. The data demonstrate that non-irradiated ICG dramatically and dynamically influences the contractility of rat lymphatic vessels in both a dose- and diluent-dependent manner with low ICG concentrations principally altering contractile frequency and higher ICG concentrations completely blocking lymphatic contractility. CONCLUSIONS: Currently, both researchers and doctors should exercise caution in extrapolating the data obtained with ICG imaging to normal lymphatic function regardless of whether it was obtained in mice, pigs, or humans. Careful and extended pharmacological tests must be performed to evaluate the mechanism of action of ICG on the contractility and physiology of lymphatic vessels with consideration of dose, diluent, and duration of irradiation.

PMID: 20583875 [PubMed - in process]

Near-Infrared Fluorescent Probe for Imaging of Pancreatic beta Cells.

Bioconjug Chem. 2010 Jun 29;

Authors: Reiner T, Kohler RH, Liew CW, Hill JA, Gaglia J, Kulkarni RN, Weissleder R

The ability to image and ultimately quantitate beta-cell mass in vivo will likely have far reaching implications in the study of diabetes biology, in the monitoring of disease progression or response to treatment, and for drug development. Here, using animal models, we report on the synthesis, characterization, and intravital microscopic imaging properties of a near-infrared fluorescent exendin-4 analogue with specificity for the GLP-1 receptor on beta cells (E4(K12)-Fl). The agent demonstrated subnanomolar EC(50) binding concentrations, with high specificity and binding that could be inhibited by GLP-1R agonists. Following intravenous administration to mice, pancreatic islets were readily distinguishable from exocrine pancreas, achieving target-to-background ratios within the pancreas of 6:1, as measured by intravital microscopy. Serial imaging revealed rapid accumulation kinetics (with initial signal within the islets detectable within 3 min and peak fluorescence within 20 min of injection), making this an ideal agent for in vivo imaging.

PMID: 20583828 [PubMed - as supplied by publisher]

Efficient and rapid labeling of transplanted cell populations with superparamagnetic iron oxide nanoparticles using cell surface chemical biotinylation for in vivo monitoring by MRI.

Cell Transplant. 2010;19(4):419-29

Authors: So PW, Kalber T, Hunt D, Farquharson M, Al-Ebraheem A, Parkes HG, Simon R, Bell JD

Determination of the dynamics of specific cell populations in vivo is essential for the development of cell-based therapies. For cell tracking by magnetic resonance imaging (MRI), cells need to internalize, or be surface labeled with a MRI contrast agent, such as superparamagnetic iron oxide nanoparticles (SPIOs): SPIOs give rise to signal loss by gradient-echo and T(2)-weighted MRI techniques. In this study, cancer cells were chemically tagged with biotin and then magnetically labeled with anti-biotin SPIOs. No significant detrimental effects on cell viability or death were observed following cell biotinylation. SPIO-labeled cells exhibited signal loss compared to non-SPIO-labeled cells by MRI in vitro. Consistent with the in vitro MRI data, signal attenuation was observed in vivo from SPIO-labeled cells injected into the muscle of the hind legs, or implanted subcutaneously into the flanks of mice, correlating with iron detection by histochemical and X-ray fluorescence (XRF) methods. To further validate this approach, human mesenchymal stem cells (hMSCs) were also employed. Chemical biotinylation and SPIO labeling of hMSCs were confirmed by fluorescence microscopy and flow cytometry. The procedure did not affect proliferation and multipotentiality, or lead to increased cell death. The SPIO-labeled hMSCs were shown to exhibit MRI signal reduction in vitro and was detectable in an in vivo model. In this study, we demonstrate a rapid, robust, and generic methodology that may be a useful and practical adjuvant to existing methods of cell labeling for in vivo monitoring by MRI. Further, we have shown the first application of XRF to provide iron maps to validate MRI data in SPIO-labeled cell tracking studies.

PMID: 20579412 [PubMed - in process]

Self-assembled gold nanoparticle molecular probes for detecting proteolytic activity in vivo.

ACS Nano. 2010 Mar 23;4(3):1511-20

Authors: Mu CJ, Lavan DA, Langer RS, Zetter BR

Target-activatable fluorogenic probes based on gold nanoparticles (AuNPs) functionalized with self-assembled heterogeneous monolayers of dye-labeled peptides and poly(ethylene glycol) have been developed to visualize proteolytic activity in vivo. A one-step synthesis strategy that allows simple generation of surface-defined AuNP probe libraries is presented as a means of tailoring and evaluating probe characteristics for maximal fluorescence enhancement after protease activation. Optimal AuNP probes targeted to trypsin and urokinase-type plasminogen activator required the incorporation of a dark quencher to achieve 5- to 8-fold signal amplification. These probes exhibited extended circulation time in vivo and high image contrast in a mouse tumor model.

PMID: 20146506 [PubMed - indexed for MEDLINE]