Cell Labeling Agents Research Articles

Synthesis and characterization of fluorescent perylene bisimide-containing glycopolymers for Escherichia coli conjugation and cell imaging

Fluorescent glycopolymers were prepared via combined atom transfer radical polymerization (ATRP) and ‘Click Chemistry’ in one-pot synthesis, in the presence of 2-azidoethyl methacrylate (AzEMA), 2-propynyl α- d-mannopyranoside, N, N′-bis{2-[2-[(2-bromo-2-methylpropanoyl)oxy]ethoxy]ethyl}perylene-3,4,9,10-tetracarboxylic acid bisimide (PBI-Br), copper (I) bromide catalyst and pentamethyldiethylenetriamine (PMDETA) ligand. Simultaneous ATRP and ‘Click Chemistry’ is an attractive method for the synthesis of functional glycopolymers as the reaction conditions are compatible with ATRP of an azide monomer, as long as an alkynyl-functionalized carbohydrate is available for click coupling reaction. The fluorescent glycopolymers were characterized by 1H NMR, FT-IR, UV–visible absorption, fluorescence and X-ray photoelectron spectroscopies, as well as by gel permeation chromatography. Incubation of Escherichia coli (E. coli DH5 α) with the fluorescent glycopolymers yielded green fluorescent bacterial clusters. The low cytotoxicity level of the fluorescent glycopolymers was revealed by incubation with 3T3 fibroblasts, macrophages and KB cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays in vitro. Subsequently, the water-soluble, biocompatible and fluorescent glycopolymers were used as effective fluorescent cell labeling agents.

A Magnetofluorescent Nanoparticle for <I>Ex-Vivo</I> Cell Labeling by Covalently Linking the Drugs Protamine and Feraheme

We synthesized a nanoparticle (NP) for ex-vivo cell labeling and MRI tracking by covalently coupling the C-terminus of a rhodamine-labeled protamine (ProRho) to Feraheme (FH) in order to yield the nanoparticle denoted ProRho-FH. Since protamine can adsorb to certain charged surfaces, we confirmed a covalent interaction between ProRho and FH by heparin affinity chromatography. ProRho-FH lacks a net charge (zeta potential approximately 0) due to the combination of negative FH and positive ProRho charges. ProRho-FH was readily internalized by U87 cells and mouse mesenchymal stem cells as determined by FACS and MR relaxometry. Finally, some 4,000 stem cells were implanted in a mouse brain and imaged by MRI. Due to its lack of net surface charge, ProRho-FH relies on the internalizing properties of the surface guanidinium groups present in the arginine-rich protamine to induce NP uptake. ProRho-FH is a unique cell-labeling agent due to its synthesis using two approved drugs, magnetofluorescence, site-specific covalent attachment chemistry, and lack of surface charge.

A highly sensitive magnetite nanoparticle as a simple and rapid stem cell labelling agent for MRI tracking

A superparamagnetic iron oxide nanoparticle (SPION) was coupled to 2-aminoethyl-trimethyl ammonium (TMA) in a 2-step ligand exchange reaction to produce TMA–SPION. This particle, which has a strong positive charge, was investigated as the basis for a simple and efficient method for labelling human mesenchymal stem cells (hMSCs) for noninvasive monitoring by magnetic resonance imaging (MRI). TMA–SPION has a ζ potential of +40 mV and a hydrodynamic size of 101 nm. In addition to its long-term stability in an aqueous solution, TMA–SPION has a low cytotoxicity and favourable magnetic properties as a T2 contrast agent due to its high relaxivity. The T2 relaxivity of TMA–SPION is 4.4 times greater than the commercially available Feridex® I.V. magnetic resonance agent. Despite a short labelling time of 4 h, hMSCs are efficiently labelled with TMA–SPION without the need for a transfection agent. An in vivo MRI study of a brain infarction model confirmed the utility of TMA–SPION as an MRI tracking marker of administered hMSCs.

99mTc-tricarbonyl labeled agents for cell labeling: Development, biodistribution in normal mice and preliminary in vitro evaluation

We have developed four 99mTc(CO) 3-labeled lipophilic tracers as potential radiolabeling agents for cells based on a hexadecyl tail. 99mTc(CO) 3-hexadecylamino- N, N′-diacetic acid (negatively charged), 99mTc(CO) 3-hexadecylamino- N-α-picolyl- N′-acetic acid (uncharged), 99mTc(CO) 3- N, N′-dipicolylhexadecylamine (positively charged), 99mTc(CO) 3- N-hexadecylaminoethyl- N′-aminoethylamine (positively charged) were prepared in a radiolabeling yield: >90%. Preliminary cell uptake studies were performed in mixed blood cells with or without plasma and were compared with 99mTc-d,l-HMPAO and [ 18F]FDG. In plasma-free blood cells, maximum uptake (78%) was obtained for 99mTc(CO) 3- N-hexadecylaminoethyl- N′-aminoethylamine after 60 min incubation (compared to 55% and 23% for 99mTc-d,l-HMPAO and [ 18F]FDG, respectively) while in plasma-rich medium, 99mTc(CO) 3- N, N′-dipicolylhexadecylamine was best bound (54%, similar to the binding of 99mTc-d,l-HMPAO). Biodistribution in normal mice showed mainly hepatobiliary clearance of the agents and initial high lung uptake. The radiolabeled compounds showed good blood clearance with maximally 7.9% injected dose per gram at 60 min post injection. While the least lipophilic agent ( 99mTc(CO) 3- N, N′-dipicolylhexadecylamine, log P = 1.3) showed the best cell uptake, there appears to be no direct correlation between lipophilicity and tracer uptake in mixed blood cells. In view of its comparable cell uptake to well known cell labeling agent 99mTc-d,l-HMPAO, 99mTc(CO) 3- N, N′-dipicolylhexadecylamine merits further evaluation as a potential cell labeling agent.

Biological and Magnetic Contrast Evaluation of Shape-Selective Mn–Fe Nanowires

One-dimensional composite Mn-Fe oxide nanostructures of different sizes (nanoneedles, nanorods, and nanowires) were prepared by a linker-induced organization of manganese-doped iron oxide nanoparticles. The nanostructures were obtained by the treatment of MnFe(2)O(4) nanoparticles in the presence of cystamine. The average lengths of nanoneedle, nanorod, and nanowire are approximately 400, 800, and 1000 nm, respectively. High-resolution transmission electron microscopy (HR-TEM), energy-dispersive X-ray (EDX) spectroscopy, and inductively coupled plasma-optical emission spectroscopy (ICP-OES) were employed to characterize the morphologies and the elemental contents of the nanostructures. As an example of their potential applications, these nanostructures were explored as the cell-labeling agents for magnetic resonance imaging (MRI). The magnetic contrast properties of the nanostructures were characterized by a 1.5 T (Tesla) whole body MR system. 10 microg/mL of the nanostructures caused substantial negative contrast. After in vitro incubation, the nanostructures could be effectively incorporated into the cells of a monocyte/macrophage cell line (RAW264.7). These cells' viability and proliferation potential were not affected when the labeling concentration was less than 50 microg/mL.

One-Pot Synthesis of Water-Stable ZnO Nanoparticles via a Polyol Hydrolysis Route and Their Cell Labeling Applications

ZnO nanoparticles have been identified as a new generation of biofriendly cell labeling agents since they are nontoxic, less expensive, and chemically stable in air. However, ZnO nanoparticles show poor water stability due to high equilibrium concentration of Zn species in water in a wide pH range. In this work, a one-pot polyol hydrolysis method was developed for synthesizing water-stable ZnO nanoparticles with blue emission. The as-synthesized ZnO nanoparticles were hydrophilic and stable in water, even at basic or acidic aqueous conditions. The PL properties of the ZnO nanoparticles stored at various pH values (i.e., 4.5-11) could be preserved for at least 3 days. The good water stability of the ZnO nanoparticles was offered by the surface attachment of an ester compound, which was formed as a result of the reaction between the stearic acid and triethylene glycol (TREG). This method provides a new approach to synthesize water-stable ZnO nanoparticles. The resultant ZnO nanoparticles demonstrated promising applications in cell labeling.

Internalization of mesoporous silica nanoparticles induces transient but not sufficient osteogenic signals in human mesenchymal stem cells

The biocompatibility of nanoparticles is the prerequisite for their applications in biomedicine but can be misleading due to the absence of criteria for evaluating the safety and toxicity of those nanomaterials. Recent studies indicate that mesoporous silica nanoparticles (MSNs) can easily internalize into human mesenchymal stem cells (hMSCs) without apparent deleterious effects on cellular growth or differentiation, and hence are emerging as an ideal stem cell labeling agent. The objective of this study was to thoroughly investigate the effect of MSNs on osteogenesis induction and to examine their biocompatibility in hMSCs. Uptake of MSNs into hMSCs did not affect the cell viability, proliferation and regular osteogenic differentiation of the cells. However, the internalization of MSNs indeed induced actin polymerization and activated the small GTP-bound protein RhoA. The MSN-induced cellular protein responses as believed to cause osteogenesis of hMSCs did not result in promotion of regular osteogenic differentiation as analyzed by cytochemical stain and protein activity assay of alkaline phosphatase (ALP). When the effect of MSNs on ALP gene expression was further examined by reverse transcriptase polymerase chain reaction, MSN-treated hMSCs were shown to have significantly higher mRNA expression than control cells after 1-hour osteogenic induction. The induction of ALP gene expression by MSNs, however, was absent in cells after 1-day incubation with osteogenic differentiation. Together our results show that the internalization of MSNs had a significant effect on the transient protein response and osteogenic signal in hMSCs, thereby suggesting that the effects of nanoparticles on diverse aspects of cellular activities should be carefully evaluated even though the nanoparticles are generally considered as biocompatible at present.

In vitro and in vivo cell tracking of chondrocytes of different origin by fluorescent PKH 26 and CMFDA

Tissue engineering techniques for cartilage re-pair to heal defects in joint surfaces is a clinical practice. Harvested autologous chondrocytes are expanded in culture and delivered in a suitable carrier medium back into the patient＞s joint de-fect. The defect is then subsequently filled by new cartilage. Whether the cells in the repair tissue originate from the engineered tissue of the host or are derived from the surrounding original cartilage remains a relevant question for the ap-plied therapy. To answer this several methods exist to track cells, such as transfection of cells with LacZ carrying viruses, radio labeling with 111 IN or 51 Cr or fluorescent labeling with FDA. However, these techniques have drawbacks such as they may influence cellular properties, are radioactive and or quickly lose their tracking ability. New fluorescent probes are easier to handle and do not to interfere with cells. PKH 26劌 is a relatively new cell-labeling agent, but few data exist on the application of this dye in chondrocytes in vitro and in vivo. 5-chloromethylfluorescein diacetate - CMFDA (¨cell tracker green〔) is an established fluores-cent probe for imaging the dynamic processes of cell proliferation in vitro and in vivo. Likewise, several studies exist on different cell types. However, little data are available for chondro-cytes. The first aim of the study was to evaluate qualitative differences in fluorescence pattern after labeling of articular, auricular and costal chondrocytes. Secondly, we evaluated the influ-ence of labeling with CMFDA on cellular adhe-sion properties. The third aim was to compare the duration of cell labeling of chondrocytes of different origin with established CMFDA as stan-dard and PKH 26潴 for 3 cell generations in vitro and 12 weeks in vivo. We show that chondro-cytes from different origin can be labeled effec-tively with both PKH 26潴 and CMFDA. The PKH 26潴 labeled articular chondrocytes maintained fluorescence longer than CMFDA in vitro and in vivo. A higher percentage of articular chondro-cytes remained stained at 63 days than auricular or costal chondrocytes.

Ultrasmall Mixed Ferrite Colloids as Multidimensional Magnetic Resonance Imaging, Cell Labeling, and Cell Sorting Agents

One area that has been overlooked in the evolution of magnetic nanoparticle technology is the possibility of introducing informational atoms into the iron oxide core of the coated colloid. Introduction of suitable atoms into the iron oxide core offers an opportunity to produce a quantifiable probe, thereby adding one or more dimensions to the magnetic colloid's informational status. Lanthanide-doped iron oxide nanoparticles have been synthesized to introduce informational atoms through the formation of colloidal mixed ferrites. These colloids are designated ultrasmall mixed ferrite iron oxides (USMIOs). USMIOs containing 5 mol % europium exhibit superparamagnetic behavior with an induced magnetization of 56 emu/g Fe at 1.5 T, a powder X-ray diffraction pattern congruent with magnetite, and R1 and R2 relaxivity values of 15.4 (mM s) (-1) and 33.9 (mM s) (-1), respectively, in aqueous solution at 37 degrees C and 0.47 T. USMIO can be detected by five physical methods, combining the magnetic resonance imaging (MRI) qualities of iron with the sensitive and quantitative detection of lanthanide metals by neutron activation analysis (NA), time-resolved fluorescence (TRF), X-ray fluorescence, along with detection by electron microscopy (EM). In addition to quantitative detection using neutron activation analysis, the presence of lanthanides in the iron oxide matrix confers attractive optical properties for long-term multilabeling studies with europium and terbium. These USMIOs offer high photostability, a narrow emission band, and a broad absorption band combining the high sensitivity of time-resolved fluorescence with the high spatial resolution of MRI. USMIO nanoparticles are prepared through modifications of traditional magnetite-based iron oxide colloid synthetic methods. A 5 mol % substitution of ferric iron with trivalent europium yielded a colloid with nearly identical magnetic, physical, and chemical characteristics to its magnetite colloid parent.

MRI Contrast Agents: Current Status and Future Perspectives

Magnetic Resonance Imaging (MRI) is increasingly used in clinical diagnostics, for a rapidly growing number of indications. The MRI technique is non-invasive and can provide information on the anatomy, function and metabolism of tissues in vivo. MRI scans of tissue anatomy and function make use of the two hydrogen atoms in water to generate the image. Apart from differences in the local water content, the basic contrast in the MR image mainly results from regional differences in the intrinsic relaxation times T(1) and T(2), each of which can be independently chosen to dominate image contrast. However, the intrinsic contrast provided by the water T(1) and T(2) and changes in their values brought about by tissue pathology are often too limited to enable a sensitive and specific diagnosis. For that reason increasing use is made of MRI contrast agents that alter the image contrast following intravenous injection. The degree and location of the contrast changes provide substantial diagnostic information. Certain contrast agents are predominantly used to shorten the T(1) relaxation time and these are mainly based on low-molecular weight chelates of the gadolinium ion (Gd(3+)). The most widely used T(2) shortening agents are based on iron oxide (FeO) particles. Depending on their chemical composition, molecular structure and overall size, the in vivo distribution volume and pharmacokinetic properties vary widely between different contrast agents and these largely determine their use in specific diagnostic tests. This review describes the current status, as well as recent and future developments of MRI contrast agents with focus on applications in oncology. First the basis of MR image contrast and how it is altered by contrast agents will be discussed. After some considerations on bioavailability and pharmacokinetics, specific applications of contrast agents will be presented according to their specific purposes, starting with non-specific contrast agents used in classical contrast enhanced magnetic resonance angiography (MRA) and dynamic contrast enhanced MRI. Next targeted contrast agents, which are actively directed towards a specific molecular target using an appropriate ligand, functional contrast agents, mainly used for functional brain and heart imaging, smart contrast agents, which generate contrast as a response to a change in their physical environment as a consequence of some biological process, and finally cell labeling agents will be presented. To conclude some future perspectives are discussed.

OP-1. Evaluation of Co-oxine, Co-tropolonate and Fe-oxine as potential cell labelling agents for PET imaging

OP-1. Evaluation of Co-oxine, Co-tropolonate and Fe-oxine as potential cell labelling agents for PET imaging

A comparative study of 99Tcm-d,l-HMPAO and 99Tcm-d,l-CBPAO as leukocyte-labelling agents

An attempt was made to use 99Tcm-d,l-cyclobutylpropylene amine oxime (99Tcm-d,l-CBPAO) to label leukocytes. The radiochemical purity of 99Tcm-d,l-CBPAO, labelling efficiency of leukocytes, cell viability of labelled leukocytes and stability of 99Tcm-d,l-CBPAO-labelled leukocytes were calculated. In comparison with the commercial cell-labelling agent 99Tcm-d,l-hexamethylpropylene amine oxime (99Tcm-d,l-HMPAO), (1) the radiochemical purity of 99Tcm-d,l-CBPAO was higher than that of 99Tcm-d,l-HMPAO immediately after and 1, 2, 4, 6, 8 and 24 h after 99Tcm labelling; (2) the labelling efficiency of 99Tcm-d,l-CBPAO-labelled leukocytes was lower than that of 99Tcm-d,l-HMPAO; (3) the viability of labelled white blood cells was high for both agents; and (4) the stability of 99Tcm-d,l-CBPAO-labelled leukocytes was better than that of 99Tcm-d,l-HMPAO after 1, 2, 4, 6, 8 and 24 h. In conclusion, although the labelling efficiency of 99Tcm-d,l-CBPAO is lower, it is more stable than 99Tcm-d,l-HMPAO and provides greater stability of 99Tcm-d,l-CBPAO-labelled leukocytes. Therefore, 99Tcm-d,l-CBPAO has the potential to replace 99Tcm-d,l-HMPAO as a commercial leukocyte-labelling agent.

The crystal and molecular structures of the blood cell labelling agents tris(2-hydroxy-2,4,6-cycloheptatrien-1-onato-O,O′)indium(III) and trans-{tris(4- iso-propyl-2-hydroxy-2,4,6-cycloheptatrien-1-onato-O,O′)indium(III)}

X-ray crystal structure analyses of tris chelate complexes of In(III) with tropolone [InT 3] and 4- iso-propyltropoloe [In(4iT) 3] show them to be monomeric with, in each case, three bidentate ligands bonded to a central metal atom. The coordination polyhedra are intermediate between trigonal prismatic and trigonal antiprismatic; mean twist angles: InT 3, 36.4°; In(4iT) 3, 36.6°. The structural features of InT 3 (which is routinely used as a blood cell labelling agent) and In(4iT) 3 (which has been investigated for labelling properties) are discussed in terms of their solution chemistry.

Blood Cell Labelling Experiments with Lipophilic Technetium Complexes

Blood cell labelling experiments were carried out with a series of 99mTc and 99m/99Tc nitridotechnetium(V) complexes containing dialkyldithiocarbamates, monoalkyldithiocarbamates or alkylxanthates as ligands. Distribution patterns of the radioactivity between plasma, erythrocytes and leukocytes/thrombocytes were determined suggesting that some of the compounds studied possess a potential as blood cell labelling agents. The chemical nature of the cell-bound technetium was found to be the intact TcN complex initially used.

Evaluation of 99mTc‐ECD as blood cell labeling agent

Evaluation of ^99mTc‐ECD as blood cell labeling agent

The99mTc-nitrido complex of tropolone: A highly lipophilic blood labelling agent

The preparation and biological behaviour of99mTcN-tropolone is described. This complex was found to be more lipophilic than the99mTc-tropolone complexes obtained using stannous chloride as a reducing agent and when injected i.v. into mice was found to label blood cells. The agent may have potential as a blood cell labelling agent.

Chelates of indium-111 as agents for labelling human granulocytes for clinical use

Chelates of indium-111 as agents for labelling human granulocytes for clinical use