Cell Labeling Agents Research Articles

19F-perfluorocarbon-labeled human peripheral blood mononuclear cells can be detected in vivo using clinical MRI parameters in a therapeutic cell setting

A 19Fluorine (19F) perfluorocarbon cell labeling agent, when employed with an appropriate cellular MRI protocol, allows for in vivo cell tracking. 19F cellular MRI can be used to non-invasively assess the location and persistence of cell-based cancer vaccines and other cell-based therapies. This study was designed to determine the feasibility of labeling and tracking peripheral blood mononuclear cells (PBMC), a heterogeneous cell population. Under GMP-compliant conditions human PBMC were labeled with a 19F-based MRI cell-labeling agent in a manner safe for autologous re-injection. Greater than 99% of PBMC labeled with the 19F cell-labeling agent without affecting functionality or affecting viability. The 19F-labeled PBMC were detected in vivo in a mouse model at the injection site and in a draining lymph node. A clinical cellular MR protocol was optimized for the detection of PBMC injected both at the surface of a porcine shank and at a depth of 1.2 cm, equivalent to depth of a human lymph node, using a dual 1H/19F dual switchable surface radio frequency coil. This study demonstrates it is feasible to label and track 19F-labeled PBMC using clinical MRI protocols. Thus, 19F cellular MRI represents a non-invasive imaging technique suitable to assess the effectiveness of cell-based cancer vaccines.

Two-photon graphene quantum dot modified Gd2O3 nanocomposites as a dual-mode MRI contrast agent and cell labelling agent.

For obtaining high-resolution macroscopical anatomical information and high sensitivity microscopical optical signals, it is highly desirable to develop dual-modality magnetic resonance imaging (MRI) and fluorescent probes in medical imaging simultaneously. In this study, Gd2O3 nanoparticles were modified with two-photon graphene quantum dots (GQD), integrating a magnetic resonance imaging (MRI) contrast agent with two-photon imaging functionality into one nanoprobe. A photoluminescence study indicated that the GQD modification process integrated MRI properties with both one-photon and two-photon imaging properties. Gd2O3/GQD nanocomposites showed a significantly improved longitudinal relaxivity (r1 = 15.995 mM-1 s-1) in comparison with commercial Magnevist (Gd-DTPA, r1 = 4.5 mM-1 s-1) and some reported papers. Excellent water solubility and good biocompatibility make Gd2O3/GQD nanocomposites an ideal dual-modal imaging agent, suggesting their potential and significant biological and clinical applications in the future.

Synthesis of spherical Prussian blue with high surface area using acid etching

Prussian blue (PB, Fe(III)4[Fe(II)(CN)6]3) is a prototype of metal hexacyanoferrates with well-known magnetic properties and electrochromic (redox reaction between Fe2+ and Fe3+) and electrochemical behavior [1,2]. The shape-control of PB has drawn increasing attention owing to its shape-dependent properties such as porosity, storage, and magnetism with stacking order [3]. Wet chemical routes are usually considered a powerful method for top-down shape control by carefully tuning the reaction conditions. However, previous reports focused on chalcogenides, metals, and oxides with selective etching of the special facets of the nanomaterial [3].In this study, we report a hydrothermal synthesis method to control the shape of monodisperse PB from cube to sphere via selective acid etching. Polyvinylpyrrolidone (PVP), used as a surfactant, not only maintains a good dispersion of the particle without aggregation by stabilizing the PB particles during crystallization but also plays a key role in the etching process after mixing with strong acid. The size and shape of the resulting spherical PB particles were sensitive to the molar concentration of acid solution. Consequently, the spherical PB had a high specific surface area and the calcined PB exhibited superparamagnetic properties. The results show that the spherical PB can be utilized in applications such as magnetic-field-guided drug delivery, cell labeling, and contrast agents for magnetic resonance imaging. The chemical and physical properties of the obtained samples were characterized using X-ray diffraction, Brunauer–Emmett–Teller analysis, Raman spectroscopy, and scanning electron microscopy.

Characterization of Magneto-Endosymbionts as MRI Cell Labeling and Tracking Agents.

Magneto-endosymbionts (MEs) show promise as living magnetic resonance imaging (MRI) contrast agents for in vivo cell tracking. Here we characterize the biomedical imaging properties of ME contrast agents, in vitro and in vivo. By adapting and engineering magnetotactic bacteria to the intracellular niche, we are creating magneto-endosymbionts (MEs) that offer advantages relative to passive iron-based contrast agents (superparamagnetic iron oxides, SPIOs) for cell tracking. This work presents a biomedical imaging characterization of MEs including: MRI transverse relaxivity (r 2) for MEs and ME-labeled cells (compared to a commercially available iron oxide nanoparticle); microscopic validation of labeling efficiency and subcellular locations; and in vivo imaging of a MDA-MB-231BR (231BR) human breast cancer cells in a mouse brain. At 7T, r 2 relaxivity of bare MEs was higher (250s-1mM-1) than that of conventional SPIO (178s-1mM-1). Optimized in vitro loading of MEs into 231BR cells yielded 1-4pg iron/cell (compared to 5-10pg iron/cell for conventional SPIO). r 2 relaxivity dropped by a factor of ~3 upon loading into cells, and was on the same order of magnitude for ME-loaded cells compared to SPIO-loaded cells. In vivo, ME-labeled cells exhibited strong MR contrast, allowing as few as 100 cells to be detected in mice using an optimized 3D SPGR gradient-echo sequence. Our results demonstrate the potential of magneto-endosymbionts as living MR contrast agents. They have r 2 relaxivity values comparable to traditional iron oxide nanoparticle contrast agents, and provide strong MR contrast when loaded into cells and implanted in tissue.

Multivalent Biopolymer Capped Gold Nanoparticles as Stable and Non-Specific Endocytosis-Free Cell Labeling Agents

Multivalent Biopolymer Capped Gold Nanoparticles as Stable and Non-Specific Endocytosis-Free Cell Labeling Agents

Quantifications of in vivo labeled stem cells based on measurements of magnetic moments

Cells labeled by super paramagnetic iron-oxide (SPIO) nanoparticles are more easily seen in gradient echo MR images, but it has not been shown that the amount of nanoparticles or the number of cells can be directly quantified from MR images. This work utilizes a previously developed and improved Complex Image Summation around a Spherical or Cylindrical Object (CISSCO) method to quantify the magnetic moments of several clusters of SPIO nanoparticle labeled cells from archived rat brain images. With the knowledge of mass magnetization of the cell labeling agent and cell iron uptake, the number of cells in each nanoparticle cluster can be determined. Using a high pass filter with a reasonable size has little effect on each measured magnetic moment from the CISSCO method. These procedures and quantitative results may help improve the efficacy of cell-based treatments in vivo.

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging.

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

An enzyme-activatable and cell-permeable MnIII-porphyrin as a highly efficient T1 MRI contrast agent for cell labeling.

Magnetic resonance imaging (MRI) is a preferred technique for noninvasively monitoring the fate of implanted cells, such as stem cells and immune cells in vivo. Cellular MRI requires contrast agents (CAs) to label the cells of interest. Despite promising progress made in this emerging field, highly sensitive, stable and biocompatible T1 CAs with high cell permeability and specificity remains an unmet challenge. To address this need, a novel MnIII-porphyrin, MnAMP was designed and synthesized based on the modification of MnIIItetra(carboxy-porphyrin) (MnTCP), a small and highly stable non-Gd extracellular CA with good biocompatibility and high T1 relaxivity (r1 = 7.9 mM-1 s-1) at clinical field of 3 Tesla (T). Cell permeability was achieved by masking the polar carboxylates of MnTCP with acetoxymethyl-ester (AM) groups, which are susceptible to hydrolysis by intracellular esterases. The enzymatic cleavage of AM groups led to disaggregation of the hydrophobic MnAMP, releasing activated MnTCP with significant increase in T1 relaxivity. Cell uptake of MnAMP is highly efficient as tested on two non-phagocytic human cell lines with no side effects observed on cell viability. MRI of labeled cells exhibited significant contrast enhancement with a short T1 of 161 ms at 3 T, even though a relatively low concentration of MnAMP and short incubation time was applied for cell labeling. Overall, MnAMP is among the most efficient T1 cell labeling agents developed for cellular MRI.

Perylene bisimide-incorporated water-soluble polyurethanes for living cell fluorescence labeling

Perylene bisimides (PBIs) possess high fluorescence quantum yields, high extinction coefficients, and high chemical and photophysical stability. However, their poor water-solubility limits their potential biomedical applications. In this work, we show a facile one-step condensation method to synthesize PBI-incorporated polyurethanes (HDI-PBI-PEG) from hexamethylene diisocyanate (HDI), PBI, and poly(ethylene glycol) (PEG). Results show that copolymerization greatly improves the solubility of PBIs in polar–organic solvents and water. More importantly, HDI-PBI-PEG shows strong fluorescence in aqueous solutions and low cytotoxicity toward mammalian cells. Various living cells, including L929 cells, macrophages, and skov-3 cells, can internalize HDI-PBI-PEG; the labeled cells show intensive and stable light emission. Furthermore, folic acid-conjugated HDI-PBI-PEG exhibits significantly higher efficiency to label skov-3 cells than normal mammalian cells, thereby showing the possibility of achieving selective cell labeling. These results demonstrate the great potential of using HDI-PBI-PEG as a universal cell-labeling agent.

Magnetic Resonance Imaging of Iron Oxide-Labeled Human Embryonic Stem Cell-Derived Cardiac Progenitors.

Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 μg/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI. The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts, highlighting its utility in future cardiac stem cell therapy trials.

Compartmentalization of Gd liposomes: the quenching effect explained.

Cationic liposomes carrying high [Gd] can be used as efficient cell-labeling agents. In a compartmentalized state, Gd can cause signal loss (relaxivity quenching). The contributions of liposomal [Gd], size and compartmentalization state to relaxivity quenching were assessed. The dependency of signal intensity (SI) on intraliposomal [Gd] was assessed comparing three different [Gd] (0.3, 0.6 and 1.0 M Gd) in both small (80 nm) and large (120 nm) cationic liposomes. In addition, five compartmentalization states were compared: free Gd, intact Gd liposomes, ruptured Gd liposomes, Gd liposomes in intact cells and Gd liposomes in ruptured cells (simulating cell death). Gd also causes R2 effects, which is often overlooked. Therefore, both R1 and R2 relaxation rates of a dilution range were measured by T1 and T2 mapping on a 7 T clinical scanner. Less is more. As the unidirectional water efflux rate (outbound across the liposome membrane, κle) is proportional to the surface:volume ratio, smaller liposomes yielded a consistently higher R1 than larger liposomes. For equal voxel [Gd] less concentrated liposomes (0.3 M Gd) yielded higher R1/R2 ratio because of the higher extraliposomal water fraction (vl ). Gd exhibits a dualistic behavior: from hypointensity to hyperintensity to hypointensity, with decreasing [Gd]. Regarding compartmentalization, fewer membrane barriers means a higher R1 /R2 ratio. Gd liposomes exhibit a versatile contrast behavior, dependent on the compartmentalization state, liposomal size, intraliposomal [Gd] and liposome number. Both R1 and R2 effects contribute to this. The versatility allows one to tailor the optimal liposomal formulation to desired goals in cell labeling and tracking.

Novel (89)Zr cell labeling approach for PET-based cell trafficking studies.

BackgroundWith the recent growth of interest in cell-based therapies and radiolabeled cell products, there is a need to develop more robust cell labeling and imaging methods for in vivo tracking of living cells. This study describes evaluation of a novel cell labeling approach with the positron emission tomography (PET) isotope 89Zr (T1/2 = 78.4 h). 89Zr may allow PET imaging measurements for several weeks and take advantage of the high sensitivity of PET imaging.MethodsA novel cell labeling agent, 89Zr-desferrioxamine-NCS (89Zr-DBN), was synthesized. Mouse-derived melanoma cells (mMCs), dendritic cells (mDCs), and human mesenchymal stem cells (hMSCs) were covalently labeled with 89Zr-DBN via the reaction between the NCS group on 89Zr-DBN and primary amine groups present on cell surface membrane protein. The stability of the label on the cell was tested by cell efflux studies for 7 days. The effect of labeling on cellular viability was tested by proliferation, trypan blue, and cytotoxicity/apoptosis assays. The stability of label was also studied in in vivo mouse models by serial PET scans and ex vivo biodistribution following intravenous and intramyocardial injection of 89Zr-labeled hMSCs. For comparison, imaging experiments were performed after intravenous injections of 89Zr hydrogen phosphate (89Zr(HPO4)2).ResultsThe labeling agent, 89Zr-DBN, was prepared in 55% ± 5% decay-corrected radiochemical yield measured by silica gel iTLC. The cell labeling efficiency was 30% to 50% after 30 min labeling depending on cell type. Radioactivity concentrations of labeled cells of up to 0.5 MBq/106 cells were achieved without a negative effect on cellular viability. Cell efflux studies showed high stability of the radiolabel out to 7 days. Myocardially delivered 89Zr-labeled hMSCs showed retention in the myocardium, as well as redistribution to the lung, liver, and bone. Intravenously administered 89Zr-labeled hMSCs also distributed primarily to the lung, liver, and bone, whereas intravenous 89Zr(HPO4)2 distributed to the liver and bone with no activity in the lung. Thus, the in vivo stability of the radiolabel on the hMSCs was evidenced.ConclusionsWe have developed a robust, general, and biostable 89Zr-DBN-based cell labeling strategy with promise for wide applications of PET-based non-invasive in vivo cell trafficking.

Dextran-coated fluorapatite nanorods doped with lanthanides in labelling and directing osteogenic differentiation of bone marrow mesenchymal stem cells.

Terbium (Tb) or europium (Eu)-doped fluorapatite nanorods (Tb/Eu-FHA) were prepared using a hydrothermal method. The structure, morphology, and luminescence of the products were determined using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and photoluminescence spectra (PL). The surfaces of the Tb/Eu-FHA nanorods were further conjugated with hydrophilic cationic polymers, such as dextran, to enhance hydrophilicity, biocompatibility and cell penetration. Then, the dextran-coated nanorods were cocultured with bone marrow mesenchymal stem cells (BMSCs). A luminescence signal in the cells was detected after 12 hours with a laser scanning confocal microscope (LSCM). Labelled BMSCs were shown to reproducibly exhibit osteogenic differentiation potential in real-time PCR and ELISA assays. In vivo cell-tracking experiments also suggested that dextran-coated Tb-FHA nanorods could be used as a stable tracer of BMSCs. Therefore, dextran-coated Tb-FHA nanorods can be utilised for tracking and monitoring BMSCs in vitro and in vivo. Our current work attempts to provide an excellent fluorescent cell labelling agent for BMSCs in bone tissue engineering.

Sensitive in vivo cell detection using size-optimized superparamagnetic nanoparticles

Magnetic nanoparticle (MNP) enabled cell visualization with magnetic resonance imaging (MRI) is currently an intensively studied area of research. In the present study, we have synthesized polyethylene glycolated (PEG) MNPs and validated their suitability as MR cell labeling agents in in vitro and in vivo experiments. The labeling of therapeutic potent mesenchymal stem cells (MSCs) with small core and large core MNPs was evaluated. Both MNPs were, in combination with a transfection agent, stably internalized into the MSCs and didn't show an effect on cell metabolism. The labeled cells showed high contrast in MRI phantom studies. For quantification purposes, the MRI contrast generating properties of cells labeled with small core MNPs were compared with large core MNPs and with the commercial contrast agent Endorem. MSCs labeled with the large core MNPs showed the highest contrast generating properties in in vitro phantom studies and in in vivo intracranial stereotactic injection experiments, confirming the size–relaxivity relationship in biological systems. Finally, the distribution of MSCs pre-labeled with large core PEGylated MNPs was visualized non-invasively with MRI in a glioma model.

Single Cell Tracking of Gadolinium Labeled CD4+ T Cells by Laser Ablation Inductively Coupled Plasma Mass Spectrometry

Cellular therapy is emerging as a promising alternative to conventional immunosuppression in the fields of hematopoietic stem cell (HSC) transplantation, autoimmune disease, and solid organ transplantation. Determining the persistence of cell-based therapies in vivo is crucial to understanding their regulatory function and requires the combination of an extremely sensitive detection technique and a stable, long-lifetime cell labeling agent. This paper reports the first application of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to perform single cell detection of T cell populations relevant to cellular immunotherapy. Purified human CD4(+) T cells were labeled with commercially available Gd-based magnetic resonance imaging (MRI) contrast agents, Omniscan and Dotarem, which enabled passive loading of up to 10(8) Gd atoms per cell. In mixed preparations of labeled and unlabeled cells, LA-ICP-MS was capable of enumerating labeled cells at close to the predicted ratio. More importantly, LA-ICP-MS single cell analysis demonstrated that the cells retained a sufficient label to remain detectable for up to 10 days post-labeling both in vitro and in vivo in an immunodeficient mouse model.

Nonviral Cell Labeling and Differentiation Agent for Induced Pluripotent Stem Cells Based on Mesoporous Silica Nanoparticles

The generation of induced pluripotent stem cells (iPSCs) is an innovative personalized-regenerative technology, which can transform own-self somatic cells into embryonic stem (ES)-like cells, which have the potential to differentiate into all cell types of three dermal lineages. However, how to quickly, efficiently, and safely produce specific-lineage differentiation from pluripotent-state cells and iPSCs is still an open question. The objective of the present study was to develop a platform of a nonviral gene delivery system of mesoporous silica nanoparticles (MSNs) to rapidly generate iPSC-derived definitive-lineage cells, including endodermal-differentiated cells. We also evaluated the feasibility and efficiency of FITC-conjugated MSNs (FMSNs) for labeling of iPSCs and utilized the multifunctional properties of FMSNs for a suitable carrier for biomolecule delivery. We showed that FMSNs of various surface charges could be efficiently internalized by iPSCs without causing cytotoxicity. The levels of reactive oxygen species and pluripotent status, including in vitro stemness signatures and in vivo teratoma formation, remained unaltered. Notably, positive-charged FMSN enhanced cellular uptake efficiency and retention time. Moreover, when using positive-charged FMSN to deliver hepatocyte nuclear factor 3β (HNF3β) plasmid DNA (pDNA), the treated iPSCs exhibited significantly improved definitive endoderm formation and further quickly differentiated into hepatocyte-like cells with mature functions (low-density lipoprotein uptake and glycogen storage) within 2 weeks in vitro. Double delivery of pHNF3β further improved mRNA expression levels of liver-specific genes. These findings reveal the multiple advantages of FMSNs to serve as ideal vectors not only for stem cell labeling but also for safe gene delivery to promote the production of hepatocyte-like cells from iPSCs.

Design and evaluation of theranostic perfluorocarbon particles for simultaneous antigen-loading and 19F-MRI tracking of dendritic cells

Perfluorocarbon (PFC) particles are currently on the rise as cell labeling agents for 19F-MRI tracking of dendritic cell (DC)-based vaccines. In this work, we design theranostic PFC particles for single-step loading of DCs with both antigenic protein and with a liquid PFC for 19F-MRI detection of the antigen-loaded cells. Upon addition to DCs in vitro, the antigen-loaded PFC particles are efficiently internalized, resulting in intracellular presence of up to 40pmol 19F atoms per cell. At the same time, the DCs become loaded with antigenic proteins, that can be efficiently processed, without important effects on cell viability or altering the DC's phenotype and the cell's capacity to respond to danger signals. In addition, antigen-loaded PFC particle containing DCs are capable of inducing extensive proliferation of antigen-specific CD8+ T cells in vitro. Importantly, the antigen-coated PFC particles allow in vitro19F-MRI-based detection of the antigen-containing DCs with detection limits as low as 103cellsμl−1. The dual-modality characteristics of the designed particles could assure that only those DCs that have taken up the antigen, and hence are responsible for an immune response, are traceable via 19F-MRI. Taken together, these novel dual-modality particles represent an interesting strategy in the development of a traceable DC vaccine.

Synthesis of coumarin-containing conjugated polymer for naked-eye detection of DNA and cellular imaging

Three novel coumarin-containing conjugated polyelectrolytes (P1–P3) containing coumarin, carbazole, fluorene and phenylene moieties were successfully synthesized by Suzuki coupling reaction. The narrow bandgap coumarin–carbazole units and the wide bandgap fluorene–phenylene units in P1–P3 might form energy donor–acceptor molecule architectures, in which coumarin–carbazole units serve as an acceptor of the fluorescent resonance energy transfer (FRET). The addition of calf thymus DNA resulted in efficient FRET from fluorene–phenylene (energy donor) to coumarin–carbazole (energy acceptor) unit for P1, which caused the solution fluorescent color to change from blue to light green. In addition, P1 can be utilized as a reliable fluorescent probe for cellular imaging of human adult skin fibroblast cells, where blue and green fluorescence were observed in the cytoplasm by use of different excitation wavelength. Subsequently, the water-soluble and fluorescent P1 were used as high sensitive DNA sensor and effective fluorescent cell labeling agents with dual emission pathways.

Controlled synthesis and enhanced luminescence of europium-doped fluorine-substituted hydroxyapatite nanoparticles

Europium-doped fluorine-substituted hydroxyapatite (Eu:FHAp) nanoparticles with different morphologies were synthesized by a simple hydrothermal method. The structure, composition, morphology, and luminescence of the products were investigated by means of X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), and fluorescence spectroscopy. The prepared Eu:FHAp had a nanorod-like shape and their aspect ratios dropped when the pH of the solution was increased from 3.0 to 11.0. The luminescence intensity of the products increased significantly with increasing substitution of OH− with F−. The nanoparticles did not adversely affect the growth of hela cells, which indicates that they have potential applications as fluorescent cell labeling agents.

Synthesis of NaYF4:Yb,Er/single-walled carbon nanohorns nanocomposite and its application as cells label

A novel nanocomposite synthesis method of amino-modified NaYF4:Yb,Er upconversion luminescent nanoparticles and single-walled carbon nanohorns was developed via covalent linkage for the first time. The nanocomposite was covalently coupled with rabbit anti-CEA8 antibody and then used successfully as a cell labeling agent for the immunolabeling and imaging of HeLa cells.