In vivo imaging of siRNA delivery and silencing in tumors

With the increased potential of RNA interference (RNAi) as a therapeutic strategy, new noninvasive methods for detection of siRNA delivery and silencing are urgently needed. Here we describe the development of dual-purpose probes for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution magnetic resonance imaging (MRI) and near-infrared in vivo optical imaging (NIRF). These probes consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model or therapeutic targets. Additionally, these nanoparticles were modified with a membrane translocation peptide for intracellular delivery. We show the feasibility of in vivo tracking of tumor uptake of these probes by MRI and optical imaging in two separate tumor models. We also used proof-of-principle optical imaging to corroborate the efficiency of the silencing process. These studies represent the first step toward the advancement of siRNA delivery and imaging strategies, essential for cancer therapeutic product development and optimization.     

An oxygen imaging system for medical applications: preliminary results.

The examination of functional processes in tissue is gaining importance in medical research. As a result the imaging and monitoring of biochemical parameters in vivo is the goal of many imaging methods. One key parameter in photodynamic therapy (PDT) is the molecular oxygen concentration. Two-dimensional monitoring of oxygen is demanded for PDT but has not yet been achieved. The use of optical methods provides a possible means of measuring molecular oxygen. The basis of this method is the measurement of the luminescence lifetime of a dye that is quenched by molecular oxygen. The molecular oxygen concentration can be monitored two-dimensionally by pixel-wise determination of the luminescence lifetime with a CCD-camera. A new O(2)-imaging system based on this principle is presented in this article. The dye Ru(bpy)(3)(2+) is quenched by molecular oxygen and was used in the first experiments with the system.     

In vivo molecular and single cell imaging

Molecular imaging is used to improve the disease diagnosis, prognosis, monitoring of treatment in living subjects. Numerous molecular targets have been developed for various cellular and molecular processes in genetic, metabolic, proteomic, and cellular biologic level. Molecular imaging modalities such as Optical Imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and Computed Tomography (CT) can be used to visualize anatomic, genetic, biochemical, and physiologic changes in vivo. For in vivo cell imaging, certain cells such as cancer cells, immune cells, stem cells could be labeled by direct and indirect labeling methods to monitor cell migration, cell activity, and cell effects in cell-based therapy. In case of cancer, it could be used to investigate biological processes such as cancer metastasis and to analyze the drug treatment process. In addition, transplanted stem cells and immune cells in cell-based therapy could be visualized and tracked to confirm the fate, activity, and function of cells. In conventional molecular imaging, cells can be monitored in vivo in bulk non-invasively with optical imaging, MRI, PET, and SPECT imaging. However, single cell imaging in vivo has been a great challenge due to an extremely high sensitive detection of single cell. Recently, there has been great attention for in vivo single cell imaging due to the development of single cell study. In vivo single imaging could analyze the survival or death, movement direction, and characteristics of a single cell in live subjects. In this article, we reviewed basic principle of in vivo molecular imaging and introduced recent studies for in vivo single cell imaging based on the concept of in vivo molecular imaging.     

Intraoperative imaging of positron emission tomographic radiotracers using Cerenkov luminescence emissions

Imaging the location and extent of cancer provides invaluable information before, during, and after surgery. The majority of "image-guided" methods that use, for example, positron emission tomography (PET) involve preoperative imaging and do not provide real-time information during surgery. It is now well established that the inherent optical emissions (Cerenkov radiation) from various β-emitting radionuclides can be visualized by Cerenkov luminescence imaging (CLI). Here we report the full characterization of CLI using the positron-emitting radiotracer 89Zr-DFO-trastuzumab for target-specific, quantitative imaging of HER2/neu-positive tumors in vivo. We also provide the first demonstration of the feasibility of using CLI for true image-guided, intraoperative surgical resection of tumors. Analysis of optical CLIs provided accurate, quantitative information on radiotracer biodistribution and tissue uptake that correlated well with the concordant PET images. CLI, PET, and biodistribution studies revealed target-specific uptake of 89Zr-DFO-trastuzumab in BT-474 (HER2/neu positive) versus MDA-MB-468 (HER2/neu negative) xenografts in the same mice. Competitive inhibition (blocking) studies followed by CLI also confirmed the in vivo immunoreactivity and specificity of 89Zr-DFO-trastuzumab for HER2/neu. Overall, these results strongly support the continued development of CLI as a preclinical and possible clinical tool for use in molecular imaging and surgical procedures for accurately defining tumor margins.     

Embryonic and neonatal phenotyping of genetically engineered mice

Considerable progress has been made in adapting existing and developing new technologies to enable increasingly detailed phenotypic information to be obtained in embryonic and newborn mice. Sophisticated methods for imaging mouse embryos and newborns are available and include ultrasound and magnetic resonance imaging (MRI) for in vivo imaging, and MRI, vascular corrosion casts, micro-computed tomography, and optical projection tomography (OPT) for postmortem imaging. In addition, Doppler and M-mode ultrasound are useful noninvasive tools to monitor cardiac and vascular hemodynamics in vivo in embryos and newborns. The developmental stage of the animals being phenotyped is an important consideration when selecting the appropriate technique for anesthesia or euthanasia and for labeling animals in longitudinal studies. Study design also needs to control for possible differences between inter- and intralitter variability, and for possible long-term developmental effects caused by anesthesia and/or procedures. Noninvasive or minimally invasive intravenous or intracardiac injections or blood sampling, and arterial pressure and electrocardiography (ECG) measurements are feasible in newborns. Whereas microinjection techniques are available for embryos as young as 6.5 days of gestation, further advances are required to enable minimally invasive fluid or tissue samples, or blood pressure or ECG measurements, to be obtained from mouse embryos in utero. The growing repertoire of techniques available for phenotyping mouse embryos and newborns promises to accelerate knowledge gained from studies using genetically engineered mice to understand molecular regulation of morphogenesis and the etiology of congenital diseases.     

Lipoprotein nanoplatform for targeted delivery of diagnostic and therapeutic agents

Low-density lipoprotein (LDL) provides a highly versatile natural nanoplatform for delivery of visible or near-infrared fluorescent optical and magnetic resonance imaging (MRI) contrast agents and photodynamic therapy and chemotherapeutic agents to normal and neoplastic cells that overexpress low-density lipoprotein receptors (LDLRs). Extension to other lipoproteins ranging in diameter from about 10 nm (high-density lipoprotein [HDL]) to over a micron (chylomicrons) is feasible. Loading of contrast or therapeutic agents onto or into these particles has been achieved by protein loading (covalent attachment to protein side chains), surface loading (intercalation into the phospholipid monolayer), and core loading (extraction and reconstitution of the triglyceride/cholesterol ester core). Core and surface loading of LDL have been used for delivery of optical imaging agents to tumor cells in vivo and in culture. Surface loading was used for delivery of gadolinium-bis-stearylamide contrast agents for in vivo MRI detection in tumor-bearing mice. Chlorin and phthalocyanine near-infrared photodynamic therapy agents (相似文献   

Cellular imaging of inflammation in atherosclerosis using magnetofluorescent nanomaterials

OBJECTIVE: Magnetofluorescent nanoparticles (MFNPs) offer the ability to image cellular inflammation in vivo. To better understand their cellular targeting and imaging capabilities in atherosclerosis, we investigated prototypical dextran-coated near-infrared fluorescent MFNPs in the apolipoprotein E-deficient (apo E-/-) mouse model. METHODS AND RESULTS: In vitro MFNP uptake was highest in activated murine macrophages (p < .001). Apo E-/- mice (n = 11) were next injected with the MFNP (15 mg/kg iron) or saline. In vivo magnetic resonance imaging (MRI) demonstrated strong plaque enhancement by the MFNPs (p < .001 vs. saline), which was confirmed by multimodality ex vivo MRI and fluorescence reflectance imaging. On fluorescence microscopy, MFNPs were found in cellular-rich areas of atheroma and colocalized with immunofluorescent macrophages over endothelial cells and smooth muscle cells (p < .001). CONCLUSIONS: Here we show that (1) the in vitro and in vivo cellular distribution of atherosclerosis-targeted MFNPs can be quantified by using fluorescence imaging methods; (2) in atherosclerosis, dextranated MFNPs preferentially target macrophages; and (3) MFNP deposition in murine atheroma can be noninvasively detected by in vivo MRI. This study thus provides a foundation for using MFNPs to image genetic and/or pharmacological perturbations of cellular inflammation in experimental atherosclerosis and for the future development of novel targeted nanomaterials for atherosclerosis.     

Fluorine-18-labeled phospholipid quantum dot micelles for in vivo multimodal imaging from whole body to cellular scales

We have designed new nanoprobes applicable for both positron emission tomography (PET) and optical fluorescence in vivo imaging. Fluorine-18, which is commonly used for clinical imaging, has been coupled to phospholipid quantum dot (QD) micelles. This probe was injected in mice and we demonstrated that its dynamic quantitative whole body biodistribution and pharmacokinetics could be monitored using PET as well as the kinetics of their cellular uptake using in vivo fibered confocal fluorescence imaging. Phospholipid micelle encapsulation of QDs provides a highly versatile surface chemistry to conjugate multiple chemicals and biomolecules with controlled QD:molecule valency. Here, we show that, in contrast with several previous studies using other QD polymer coatings, these phospholipid QD micelles exhibit long circulation half-time in the bloodstream (on the order of 2 h) and slow uptake by reticulo-endothelial system.     

Simultaneous PET-MRI: a new approach for functional and morphological imaging

Noninvasive imaging at the molecular level is an emerging field in biomedical research. This paper introduces a new technology synergizing two leading imaging methodologies: positron emission tomography (PET) and magnetic resonance imaging (MRI). Although the value of PET lies in its high-sensitivity tracking of biomarkers in vivo, it lacks resolving morphology. MRI has lower sensitivity, but produces high soft-tissue contrast and provides spectroscopic information and functional MRI (fMRI). We have developed a three-dimensional animal PET scanner that is built into a 7-T MRI. Our evaluations show that both modalities preserve their functionality, even when operated isochronously. With this combined imaging system, we simultaneously acquired functional and morphological PET-MRI data from living mice. PET-MRI provides a powerful tool for studying biology and pathology in preclinical research and has great potential for clinical applications. Combining fMRI and spectroscopy with PET paves the way for a new perspective in molecular imaging.     

Non-invasive Imaging of Endothelial Progenitor Cells in Tumor Neovascularization Using a Novel Dual-modality Paramagnetic/Near-Infrared Fluorescence Probe

Objective

Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.

Methods

The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.

Results

Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.

Conclusions

This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.     

A novel near-infrared indocyanine dye-polyethylenimine conjugate allows DNA delivery imaging in vivo

Near-infrared (NIR) fluorescence light has been applied to monitor several biological events in vivo since it penetrates tissues more efficiently than visible light. Dyes exhibiting NIR fluorescence and having large Stokes shift are key elements for this promising optical imaging technology. Here, we report the synthesis of a novel conjugate between a near-infrared indocyanine dye and an organic polyamine polymer (polyethylenimine, PEI) (IR820-PEI) with high chemical stability and good optical properties. IR820-PEI absorbs at 665 nm, emits at 780 nm, and displays a large Stokes shift (115 nm). Moreover, the reported conjugate is able to bind DNA, and the delivery process can be monitored in vivo with noninvasive optical imaging techniques. These characteristics make IR820-PEI one of the most effective and versatile indocyanine dye polymeric-conjugate reported so far.     

In vivo targeting of cell death using a synthetic fluorescent molecular probe

A synthetic, near-infrared, fluorescent probe, named PSS-794 was assessed for its ability to detect cell death in two animal models. The molecular probe contains a zinc(II)-dipicolylamine (Zn²⁺-DPA) affinity ligand that selectively targets exposed phosphatidylserine on the surface of dead and dying cells. The first animal model used rats that were treated with dexamethasone to induce thymic atrophy. Ex vivo fluorescence imaging and histological analysis of excised organs showed thymus uptake of PSS-794 was four times higher than a control fluorophore that lacked the Zn²⁺-DPA affinity ligand. In addition, the presence of PSS-794 produced a delayed and higher build up of dead and dying cells in the rat thymus. The second animal model employed focal beam radiation to induce cell death in tumor-bearing rats. Whole-body and ex vivo imaging showed that the amount of PSS-794 in a radiation-treated tumor was almost twice that in a non-treated tumor. The results indicate that PSS-794 may be useful for preclinical optical detection of tumor cell death due to therapy.     

Brain imaging in nonhuman primates: insights into drug addiction

In vivo brain imaging enables the systematic examination of trait and state variables that contribute to the etiology of human diseases. This review highlights the use of in vivo imaging in nonhuman primate models of drug abuse. In efforts to translate findings from laboratory animals to humans, monkey models offer considerable advantages over those that use rodents and other species because of their neurobiological similarity to humans and their longer life span, which makes it possible to study individual subjects over several years. This article provides a brief overview of positron emission tomography (PET), magnetic resonance imaging (MRI)-based techniques, and encephalographic approaches, with a focus on methodological issues that investigators new to the field should consider. We discuss PET imaging studies involving the dopamine (DA) system, with a special emphasis on DA D2 receptors, and describe experimental approaches through which PET imaging data can provide information about the neuropharmacological and neurochemical actions of drugs that modify behavior. We also consider the use of imaging to understand the impact and interactions of genetic predispositions and environmental and physiological modulators on disease states. For MRI-based and encephalographic studies, we describe approaches that can provide new information about brain function. Although much work remains to be done to adapt and apply these techniques for routine use in nonhuman primates, there has been much progress. These techniques will provide the foundation for future studies aimed at developing behavioral and pharmacological treatments for many human diseases.     

Real time non-invasive imaging of receptor-ligand interactions in vivo

Non-invasive longitudinal detection and evaluation of gene expression in living animals can provide investigators with an understanding of the ontogeny of a gene's biological function(s). Currently, mouse model systems are used to optimize magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical imaging modalities to detect gene expression and protein function. These molecular imaging strategies are being developed to assess tumor growth and the tumor microenvironment. In addition, pre-labeling of progenitor cells can provide invaluable information about the developmental lineage of stem cells both in organogenesis and tumorigenesis. The feasibility of this approach has been extensively tested by targeting of endogenous tumor cell receptors with labeled ligand (or ligand analog) reporters and targeting enzymes with labeled substrate (or substrate analog). We will primarily discuss MRI, PET, and SPECT imaging of cell surface receptors and the feasibility of non-invasive imaging of gene expression using the tumor microenvironment (e.g., hypoxia) as a conditional regulator of gene expression.     

Molecular imaging of brain lipid environment of lymphocytes in amyotrophic lateral sclerosis using magnetic resonance imaging and SECARS microscopy

This paper highlights some of the key technologies of using two innovative molecular imaging modalites, magnetic resonance imaging (MRI) and nonlinear optical microscopy, for imaging intravenously injected ultra small paramagnetic iron oxide nanoparticles cross linked with antibodies (CLUSPIO) in the amyotrophic lateral sclerosis (ALS) experimental model in vivo or ex vivo, respectively. Intensive efforts have been made in investigating the causes of abnormalities in lipid metabolism, monitored in some neurodegenerative disorders systems. It has been shown that an abnormal accumulation of some common lipids in motor nerve cells may play a critical role in the development of amyotrophic lateral sclerosis. The presented experiments were performed on brain specimens from the transgenic rat model expressing multiple copies of mutated (G93A) human SOD-1 gene, after CD4+ lymphocytes were magnetically labeled with i.v.i. CLUSPIO antibodies. In vivo MRI revealed marked signal intensity enhancements in specific pathological regions of the ALS rat brain as compared to the wild type. Surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy indicated cellular interactions based on lipids association to anti-CD4 CLUSPIO.     

A new optical and nuclear dual-labeled imaging agent targeting interleukin 11 receptor alpha-chain

Optical imaging has great potential for studying molecular recognitions both in vivo and in vitro, yet nuclear imaging is the most effective clinical molecular imaging modality. The combination of optical and nuclear imaging modalities may provide complementary information for improving diagnosis and management of diseases. In this study we developed an optical and nuclear dual-labeled imaging agent, 111In-DTPA-Bz-NH-SA-K(IR-783-S-Ph-CO)-c(CGRRAGGSC)NH2, called DLIA-IL11Ralpha. 111In-DTPA-Bz-NH-SA is the radiotracer moiety; a near-infrared dye IR-783-S-Ph-COOH serves as the fluorescent emitter; and the cyclic peptide c(CGRRAGGSC), which is known to target interleukin 11 receptor alpha-chain (IL-11Ralpha), delivers the desired imaging agent to its target. Experiments revealed that the cyclic peptide c(CGRRAGGSC) continued to possess the targeting capability to IL-11Ralpha after the conjugation of the optical and nuclear tracers. Furthermore, the presence of the metal isotope chelator did not cause quenching of fluorescence emission. The cross validation and direct comparison of optical and nuclear imaging of a tumor was achieved using a single injection, and the preliminary results show the conjugate has tumor targeting capabilities in vivo.     

Agonist-antagonist dilemma in molecular imaging: evaluation of a monomolecular multimodal imaging agent for the somatostatin receptor

The combination of different imaging modalities, each providing information according to its strengths, can be a powerful method for diagnosing diseases. We have synthesized a monomolecular multimodal imaging agent (MOMIA), LS172, containing a subtype-2 somatostatin receptor (SSTr2)-avid peptide (Y3-octreotate or Y3-TATE), a radiometal chelating group (DOTA) and a near-infrared (NIR) fluorescent dye (cypate). In addition to optical methods, radiolabeling LS172 with 64Cu and 177Lu provides a strategy for in vitro evaluation or in vivo multimodal imaging by positron emission tomography (PET) and single photon emission computed tomography (SPECT), respectively. Determination of the binding affinity of LS172, nat Cu- and nat Lu-LS172 in SSTr2-transfected A427 cells (A427-7) showed that they all displayed high binding affinity toward SSTr2 with K i values of 0.234 nM, 11.5 nM, and 2.15 nM respectively. In contrast to cypate-labeled Y3-TATE (cytate), fluorescence microscopy showed that LS172 and nat Cu-LS172 accumulate modestly in A427-7 cells by SSTr2-mediated endocytosis, in spite of their relatively high binding affinity. In vivo, the biodistribution of the SSTr2 receptor specific 64Cu- and 177Lu-LS172 in AR42J tumor-bearing rats exhibited low (90% ID/liver). Both optical and radionuclear biodistribution studies showed a similar in vivo distribution profile. Surprisingly, the strong binding of LS172 to SSTr2 did not translate into high SSTr2-mediated endocytosis in cells or uptake in tumor in vivo. Considering that LS172 is a putative antagonist, the poor accumulation of the labeled MOMIAs in SSTr2 positive tumor tissue supports the paradigm that agonists with their concomitant internalization favors appreciable target tissue accumulation of receptor-specific ligands.     

Multimodality molecular imaging of disease progression in living subjects

The enormous advances in our understanding of the progression of diseases at the molecular level have been supplemented by the new field of ‘molecular imaging’, which provides for in vivo visualization of molecular events at the cellular level in living organisms. Molecular imaging is a noninvasive assessment of gene and protein function, protein–protein interaction and/or signal transduction pathways in animal models of human disease and in patients to provide insights into molecular pathogenesis. Five major imaging techniques are currently available to assess the structural and functional alterations in vivo in small animals. These are (i) optical bioluminescence and fluorescence imaging techniques, (ii) radionuclide-based positron emission tomography (PET) and single photon emitted computed tomography (SPECT), (iii) X-ray-based computed tomography (CT), (iv) magnetic resonance imaging (MRI) and (v) ultrasound imaging (US). Functional molecular imaging requires an imaging probe that is specific for a given molecular event. In preclinical imaging, involving small animal models, the imaging probe could be an element of a direct (‘direct imaging’) or an indirect (‘indirect imaging’) event. Reporter genes are essential for indirect imaging and provide a general integrated platform for many different applications. Applications of multimodality imaging using combinations of bioluminescent, fluorescent and PET reporter genes in unified fusion vectors developed by us for recording events from single live cells to whole animals with high sensitivity and accurate quantification are discussed. Such approaches have immense potential to track progression of metastasis, immune cell trafficking, stem cell therapy, transgenic animals and even molecular interactions in living subjects.     

Multi-Task Linear Programming Discriminant Analysis for the Identification of Progressive MCI Individuals

Accurately identifying mild cognitive impairment (MCI) individuals who will progress to Alzheimer''s disease (AD) is very important for making early interventions. Many classification methods focus on integrating multiple imaging modalities such as magnetic resonance imaging (MRI) and fluorodeoxyglucose positron emission tomography (FDG-PET). However, the main challenge for MCI classification using multiple imaging modalities is the existence of a lot of missing data in many subjects. For example, in the Alzheimer''s Disease Neuroimaging Initiative (ADNI) study, almost half of the subjects do not have PET images. In this paper, we propose a new and flexible binary classification method, namely Multi-task Linear Programming Discriminant (MLPD) analysis, for the incomplete multi-source feature learning. Specifically, we decompose the classification problem into different classification tasks, i.e., one for each combination of available data sources. To solve all different classification tasks jointly, our proposed MLPD method links them together by constraining them to achieve the similar estimated mean difference between the two classes (under classification) for those shared features. Compared with the state-of-the-art incomplete Multi-Source Feature (iMSF) learning method, instead of constraining different classification tasks to choose a common feature subset for those shared features, MLPD can flexibly and adaptively choose different feature subsets for different classification tasks. Furthermore, our proposed MLPD method can be efficiently implemented by linear programming. To validate our MLPD method, we perform experiments on the ADNI baseline dataset with the incomplete MRI and PET images from 167 progressive MCI (pMCI) subjects and 226 stable MCI (sMCI) subjects. We further compared our method with the iMSF method (using incomplete MRI and PET images) and also the single-task classification method (using only MRI or only subjects with both MRI and PET images). Experimental results show very promising performance of our proposed MLPD method.     

Oleyl-chitosan nanoparticles based on a dual probe for optical/MR imaging in vivo

Oleic acid-conjugated chitosan (oleyl-chitosan) is a powerful platform for encapsulating oleic acid-decorated iron oxide nanoparticles (ION), resulting in a good magnetic resonance imaging (MRI) probe. Oleyl-chitosan could self-assemble into core-shell structures in aqueous solution and provide the effective core compartment for loading ION. ION-loaded oleyl-chitosan nanoparticles showed good enhanced MRI sensitivity in a MR scanner. Cy5.5 dye was accessed to the oleyl-chitosan conjugate for near-infrared (NIR) in vivo optical imaging. After intravenous injection of ION-loaded Cy5.5-conjugated oleyl-chitosan (ION-Cy5.5-oleyl-chitosan) nanoparticles in tumor-bearing mice, both NIRF and MR imaging showed the detectable signal intensity and enhancement in tumor tissues via enhanced permeability and retention (EPR) effect. Tumor accumulation of the nanoparticles was confirmed through ex vivo fluorescence images and Prussian blue staining images in tumor tissues. It is concluded that ION-Cy5.5-oleyl-chitosan nanoparticle is highly an effective imaging probe for detecting tumor in vivo.