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1.  Imaging of Her2-Targeted Magnetic Nanoparticles for Breast Cancer Detection: Comparison of SQUID-detected Magnetic Relaxometry and MRI 
Contrast media & molecular imaging  2012;7(3):10.1002/cmmi.499.
Both magnetic relaxometry and magnetic resonance imaging (MRI) can be used to detect and locate targeted magnetic nanoparticles, non-invasively and without ionizing radiation. Magnetic relaxometry offers advantages in terms of its specificity (only nanoparticles are detected) and the linear dependence of the relaxometry signal on the number of nanoparticles present. In this study, detection of single-core iron oxide nanoparticles by Superconducting Quantum Interference Device (SQUID)-detected magnetic relaxometry and standard 4.7 T MRI are compared. The nanoparticles were conjugated to a Her2 monoclonal antibody and targeted to Her2-expressing MCF7/Her2-18 breast cancer cells); binding of the nanoparticles to the cells was assessed by magnetic relaxometry and iron assay. The same nanoparticle-labeled cells, serially diluted, were used to assess the detection limits and MR relaxivities. The detection limit of magnetic relaxometry was 125,000 nanoparticle-labeled cells at 3 cm from the SQUID sensors. T2-weighted MRI yielded a detection limit of 15,600 cells in a 150 μl volume, with r1 = 1.1 mM−1s−1 and r2 = 166 mM−1s−1. Her2-targeted nanoparticles were directly injected into xenograft MCF7/Her2-18 tumors in nude mice, and magnetic relaxometry imaging and 4.7 T MRI were performed, enabling direct comparison of the two techniques. Co-registration of relaxometry images and MRI of mice resulted in good agreement. A method for obtaining accurate quantification of microgram quantities of iron in the tumors and liver by relaxometry was also demonstrated. These results demonstrate the potential of SQUID-detected magnetic relaxometry imaging for the specific detection of breast cancer and the monitoring of magnetic nanoparticle-based therapies.
PMCID: PMC3883306  PMID: 22539401
Magnetite; Nanoparticle; Magnetorelaxometry; SQUID; Magnetic Resonance Imaging; Magnetometry; Magnetic Susceptibility; Antibody targeting
2.  Rapid Spectrophotometric Technique for Quantifying Iron in Cells Labeled with Superparamagnetic Iron Oxide Nanoparticles: Potential Translation to the Clinic 
Labeling cells with superparamagnetic iron oxide (SPIO) nanoparticles provides the ability to track cells by Magnetic Resonance Imaging. Quantifying intracellular iron concentration in SPIO labeled cells would allow for the comparison of agents and techniques used to magnetically label cells. Here we describe a rapid spectrophotometric technique (ST) to quantify iron content of SPIO labeled cells, circumventing the previous requirement of an overnight acid digestion. Following lysis with 10% SDS of magnetically labeled cells, quantification of SPIO doped or labeled cells was performed using commonly available spectrophotometric instrument(s) by comparing absorptions at 370 and 750 nm with correction for turbidity of cellular products to determine iron content of each sample. Standard curves demonstrated high linear correlation (R2 = 0.998) between absorbance spectra of iron oxide nanoparticles and concentration in known SPIO doped cells. Comparisons of the ST to ICP-MS or NMR relaxometric (R2) determinations of intracellular iron contents in SPIO containing samples resulted in significant linear correlation between the techniques (R2 vs. ST, R2>0.992, p<0.0001, ST vs. ICP-MS, R2>0.995, p<0.0001) with the limit of detection of ST for iron = 0.66μg/ml. We have developed a rapid straightforward protocol that does not require overnight acid digestion for quantifying iron oxide content in magnetically labeled cells using readily available analytic instrumentation that should greatly expedite advances in comparing SPIO agents and protocols for labeling cells.
PMCID: PMC3490434  PMID: 23109392
Relaxometry; Iron Content; Cell Labeling; Stem Cells; Superparamagnetic Iron Oxide Nanoparticles; Ferumoxides; Ferumoxytol; ICP-MS; Spectrophotometry; Prussian Blue; MRI
3.  Enhanced relaxivity of Gd3+-based contrast agents geometrically confined within porous nanoconstructs 
Contrast media & molecular imaging  2012;7(6):10.1002/cmmi.1480.
Gadolinium chelates, which are currently approved for clinical MRI use, provide relaxivities well below their theoretical limit, and they also lack tissue specificity. Recently, the geometrical confinement of Gd3+-based contrast agents (CAs) within porous structures has been proposed as a novel, alternative strategy to improve relaxivity without chemical modification of the CA. Here, we have characterized and optimized the performance of MRI nanoconstructs obtained by loading [Gd(DTPA)(H2O)]2− (Magnevist®) into the pores of injectable mesoporous silicon particles. Nanoconstructs with three different pore sizes were studied, and at 60 MHz, they exhibited longitudinal relaxivities of ~ 24 mM−1s−1 for 5 – 10 nm pores and ~ 10 mM−1s−1 for 30 – 40 nm pores. No enhancement in relaxivity was observed for larger pores sizes. Using an outer-sphere compound, [GdTTHA]−3, and mathematical modeling, it was demonstrated that the relaxivity enhancement is due to the increase in rotational correlation times (CA adsorbed on the pore walls) and diffusion correlation times (reduced mobility of the water molecules), as the pore sizes decreases. It was also observed that extensive CA adsorption on the outer surface of the silicon particles negates the advantages offered by nanoscale confinement. Upon incubation with HeLa cells, the nanoconstructs did not demonstrate significant cytotoxicity for up to 3 days post incubation, at different particle/cell ratios. In addition, the nanoconstructs showed complete degradation after 24h of continuous agitation in PBS. These data support and confirm the hypothesis that the geometrical confinement of Gd3+-chelate compounds into porous structures offers MRI nanoconstructs with enhanced relaxivity (up to 6 times for [Gd(DTPA)(H2O)]2−, and 4 times for [GdTTHA]−3) and, potentially, improved stability, reduced toxicity and tissue specificity.
PMCID: PMC3846290  PMID: 22991316
Gadolinum chelates; MRI contrast agents; relaxivity enhancement; mesoporous silicon particles
4.  cGMP-Compatible Preparative Scale Synthesis of Near-Infrared Fluorophores 
Image-guided surgery using optical imaging requires the availability of large quantities of clinical-grade fluorophores. We describe the cGMP-compatible synthesis of the zwitterionic heptamethine indocyanine near-infrared fluorophore ZW800-1 at the 10-g scale (≈ 1,000 patient doses) using facile and efficient solvent purification, and without the need for column chromatography. ZW800-1 has > 90% yield at the final step and > 99% purity as measured by fluorescence and evaporative light scatter detection. We describe an analytical framework for qualifying impurities, as well as a detailed analysis of counterion identities. Finally, we report the unique in vivo properties of ZW800-1 in large animals approaching the size of humans, thus laying the foundation for rapid clinical translation of these methods.
PMCID: PMC3448123  PMID: 22991318
cGMP-Compatible Synthesis; Near-Infrared Fluorophore; Near-Infrared Fluorescence Imaging; Optical Imaging
5.  Comparison of Dynamic Contrast Enhanced MRI and Quantitative SPECT in a Rat Glioma Model 
Pharmacokinetic modeling of dynamic contrast enhanced (DCE)-MRI data provides measures of the extracellular volume fraction (ve) and the volume transfer constant (Ktrans) in a given tissue. These parameter estimates may be biased, however, by confounding issues such as contrast agent and tissue water dynamics, or assumptions of vascularization and perfusion made by the commonly used model. In contrast to MRI, radiotracer imaging with SPECT is insensitive to water dynamics. A quantitative dual-isotope SPECT technique was developed to obtain an estimate of ve in a rat glioma model for comparison to the corresponding estimates obtained using DCE-MRI with a vascular input function (VIF) and reference region model (RR). Both DCE-MRI methods produced consistently larger estimates of ve in comparison to the SPECT estimates, and several experimental sources were postulated to contribute to these differences.
PMCID: PMC3458584  PMID: 22991315
MRI; dynamic contrast; DCE; tumor; pharmacokinetic modeling; SPECT; radionuclide; diffusion
6.  Assessment and comparison of magnetic nanoparticles as MRI contrast agents in a rodent model of human hepatocellular carcinoma 
The purpose of this study was to synthesize, characterize and tailor the surface properties of magnetic nanoparticles with biocompatible copolymer coatings and to evaluate the efficiency of the resulting nanoconjugates as magnetic resonance imaging (MRI) contrast agents for liver imaging.
Magnetic nanoparticles with core diameters of 10 and 30 nm were synthesized by pyrolysis and were subsequently coated with a copolymer containing either carboxyl (SHP) or methoxy groups (SMG) as termini. All four formulas, and ferumoxides (Feridex I.V.®), were individually injected intravenously into separate, normal Balb/C mice (at 2.5, 1.0, and 0.56 mg Fe/kg), and the animals underwent T2-weighted MRI at multiple time points post injection (p.i.) to evaluate the hepatic uptake and clearance. Furthermore, we compared the abilities of the new formulas and Feridex to detect tumors in an orthotropic Huh7 tumor model.
TEM revealed a narrow size distribution of both the 10 nm and 30 nm nanoparticles, in contrast to a wide size distribution of Feridex. MTT, apoptosis and Cyclin/DNA flow cytometry assays showed that the polymer coated nanoparticles had no adverse effect on cell growth. Among all the tested formulas, including Feridex, SHP-30 showed the highest macrophage uptake at the in vitro level. In vivo MRI studies on normal mice confirmed the superiority of SHP-30 in inducing hypointensities in the liver tissue, especially at clinical dose (0.56 mg Fe/kg) and 3T field. SHP-30 showed better contrast-to-noise ratio (CNR) than Feridex on the orthotropic Huh7 tumor model.
SHP-30 was found to be an efficient contrast agent for liver MR imaging. The success of this study suggests that by improving the synthetic approach and by tuning the surface properties of IONPs, one can arrive at better formulas than Feridex for clinical practice.
PMCID: PMC3759163  PMID: 22649042
Iron oxide nanoparticle (IONP); hepatocarcinoma (HCC); magnetic resonance imaging (MRI); liver contrast agent
7.  Effect of a Mesitylene-Based Ligand Cap on the Relaxometric Properties of Gd(III) Hydroxypyridonate MRI Contrast Agents[1] 
A series of new Gd(III) hydroxypyridonate complexes featuring a mesitylene (ME)-derived ligand cap has been prepared. Relaxometric characterization reveals that the complexes tend to form large aggregates in solution with slow tumbling rates, as estimated from NMRD analysis, and unique pH-dependent relaxivities. The solution behavior and relaxometric properties are compared to those observed for analogous TREN-capped compounds, and the potential for use of these new ME-capped complexes as pH-responsive MRI contrast agents is explored.
PMCID: PMC3732192  PMID: 19839031
hydroxypyridinone (HOPO); mesityl; gadolinium; MRI contrast agent; relaxivity; aggregation
8.  Toward absolute quantification of iron oxide nanoparticles as well as cell internalized fraction using multiparametric MRI 
Iron oxide nanoparticles (IONPs) are widely used as MR contrast agents because of their strong magnetic properties and broad range of applications. The contrast induced by IONPs typically depends on concentration, water accessibility, particle size, and heterogeneity of IONP distribution within the microenvironment. Although the latter could be a tool to assess local physiological effects at the molecular level, it renders IONP quantification from relaxation measurements challenging. This study aims to quantify IONP concentration using susceptibility measurements. In addition, further analysis of relaxation data is proposed to extract quantitative information about the IONP spatial distribution.
Mesenchymal stem cells were labeled with IONPs and the IONP concentration measured by mass spectroscopy. MR relaxation parameters (T1, T2, T2*) as well as magnetic susceptibility of cylindrical samples containing serial dilutions of mixtures of free and cell-internalized IONPs were measured and correlated with IONP concentration.
Unlike relaxation data, magnetic susceptibility was independent of whether IONPs were free or internalized, making it an excellent candidate for IONP quantification. Using IONP concentration derived from mass spectroscopy and measured relaxation times, free and internalized IONP fractions were accurately calculated.
Magnetic susceptibility was shown to be a robust technique to measure IONP concentration in this preliminary study. Novel imaging based susceptibility mapping techniques could prove to be valuable tools to quantify IONP concentration directly by MRI, for samples of arbitrarily shape. Combined with relaxation time mapping techniques, especially T2 and T2*, this could be an efficient way to measure both IONP concentration and the internalized IONP fraction in vivo using MRI, to gain insight into tissue function and molecular imaging paradigms.
PMCID: PMC3383460  PMID: 22649047
iron oxide nanoparticle; quantification; magnetic susceptibility; relaxation; responsive agent; cellular imaging
9.  Improved measurement of labile proton concentration-weighted chemical exchange rate (kws) with experimental factor-compensated and T1-normalized quantitative chemical exchange saturation transfer (CEST) MRI 
Chemical exchange saturation transfer (CEST) MRI enables measurement of dilute CEST agents and microenvironment properties such as pH and temperature, holding great promise for in vivo applications. However, because of confounding concomitant RF irradiation and relaxation effects, the CEST-weighted MRI contrast may not fully characterize the underlying CEST phenomenon. We postulated that the accuracy of quantitative CEST MRI could be improved if the experimental factors (labeling efficiency and RF spillover effect) were estimated and taken into account. Specifically, the experimental factor was evaluated as a function of exchange rate and CEST agent concentration ratio, which remained relatively constant for intermediate RF irradiation power levels. Hence, the experimental factors can be calculated based on the reasonably estimated exchange rate and labile proton concentration ratio, which significantly improved quantification. The simulation was confirmed with Creatine phantoms of serially varied concentration titrated to the same pH, whose reverse exchange rate (kws) was found to be linearly correlated with the concentration. In summary, the proposed solution provides simplified yet reasonably accurate quantification of the underlying CEST system, which may help guide the ongoing development of quantitative CEST MRI.
PMCID: PMC3415239  PMID: 22649044
Amide proton transfer (APT); Chemical exchange saturation transfer (CEST); MRI
10.  ß-cell Subcellular Localization of Glucose Stimulated Mn Uptake By X-ray Fluorescence Microscopy: Implications for Pancreatic MRI 
Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β-cells and showed that, in the presence of MnCl2, glucose activated pancreatic islets yield significant signal enhancement in T1-weigheted MR images. In this study, we exploited for the first time the unique capabilities of X-ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β-cells at cellular and sub-cellular levels. MIN-6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 ×10-11 μg/μm2, homogenously distributed across the cell. Exposure to 2mM glucose and 50 μM MnCl2 for 20 minutes resulted in non-glucose dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 ×10-11 μg/μm2. When cells were activated by incubation in 16mM glucose in the presence of 50 μM MnCl2, a significant increase in cytoplasmic Mn was measured reaching 2.57 ± 1.34 ×10-10 μg/μm2. A further rise in intracellular concentration was measured following KCl induced depolarization, with concentrations totaling 1.25 ± 0.33 ×10-9 and 4.02 ± 0.71 ×10-10 μg/μm2 in the cytoplasm and nuclei respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings confirming that Mn can be used as a functional imaging reporter of pancreatic β-cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast
PMCID: PMC3626438  PMID: 22144025
manganese; sub-cellular localization; pancreatic β-cells function; X-ray fluorescence microscopy; MRI
11.  Improving T1 and T2 magnetic resonance imaging (MRI) contrast agents through the conjugation of an esteramide dendrimer to high water coordination Gd(III) hydroxypyridinone (HOPO) complexes 
Commercial gadolinium magnetic resonance imaging (MRI) contrast agents are limited by low relaxivity (r1) and coordination to only a single water molecule (q = 1). Consequently, gram quantities of these agents must be injected to obtain sufficient diagnostic contrast. In this study, MRI contrast agents for T1 and T2 relaxivity were synthesized using hydroxypyridinone (HOPO) and terephthalamide (TAM) chelators with mesityl and 1,4,7-triazacyclononane (TACN) capping moieties. When covalently conjugated to a highly biocompatible esteramide dendrimer, T2 relaxation rates up to 52 mM-1s-1 and T1 relaxation rates up to 31 mM-1s-1 per gadolinium are observed under clinically relevant conditions. These values are believed to be brought about by using a dendritic macromolecule to decrease the molecular tumbling time of the small molecule complexes. These agents also show high aqueous solubility and low toxicity in vitro. In this study we report six new compounds: three discrete complexes and three dendrimer conjugates.
PMCID: PMC3607543  PMID: 22344885
MRI contrast agent; hydroxypyridinone (HOPO); relaxivity; 1,4,7-triazacyclononane (TACN); terephthalamide (TAM); dendrimer; esteramide (EA) dendrimer; drug delivery; gadolinium
12.  A fluorescent, paramagnetic and PEGylated gold/silica nanoparticle for MRI, CT and fluorescence imaging 
An important challenge in medical diagnostics is to design all-in-one contrast agents that can be detected with multiple techniques such as magnetic resonance imaging (MRI), X-ray computed tomography (CT), positron emission tomography (PET), single photon emission tomography (SPECT) or fluorescence imaging (FI). Although many dual labeled agents have been proposed, mainly for combined MRI/FI, constructs for three imaging modalities are scarce. Here gold/silica nanoparticles with a poly(ethylene glycol), paramagnetic and fluorescent lipid coating were synthesized, characterized and applied as trimodal contrast agents to allow for nanoparticle-enhanced imaging of macrophage cells in vitro via MRI, CT and FI, and mice livers in vivo via MRI and CT. This agent can be a useful tool in a multitude of applications, including cell tracking and target-specific molecular imaging, and is a step in the direction of truly multi-modal imaging.
PMCID: PMC3586549  PMID: 20812290
molecular imaging; cellular uptake; MRI; CT; contrast agents; paramagnetic agents; fluorescent probes; trimodal probes; nanoparticles
13.  1H Chemical Shift Magnetic Resonance Imaging Probes with High Sensitivity for Multiplex Imaging 
Proton-based chemical shift imaging (CSI) probes were encapsulated inside nano-carriers to increase the sensivitity of the reporters. Co-encapsulation with a relaxation agent results in improved sensitivity and suppresses background signals. Simultaneous imaging of different chemical shift reporters allows multiplexed detection.
PMCID: PMC3321363  PMID: 22434641
14.  Nanoparticle-based PARACEST agents: The quenching effect of silica nanoparticles on the CEST signal from surface conjugated chelates 
Silica nanoparticles of average diameter 53 ± 3 nm were prepared using standard water-in-oil microemulsion methods. After conversion of the surface Si-OH groups to amino groups for further conjugation, the PARACEST agent, EuDOTA-(gly)4− was coupled to the amines via one or more side-chain carboxyl groups in an attempt to trap water molecules in the inner-sphere of the complex. Fluorescence and ICP analyses showed that ~1200 Eu3+ complexes were attached to each silica nanoparticle, leaving behind excess protonated amino groups. CEST spectra of the modified silica nanoparticles showed that attachment of the EuDOTA-(gly)4− to the surface of the nanoparticles did not result in a decrease in water exchange kinetics as anticipated but rather resulted in a complete elimination of the normal Eu3+-bound water exchange peak and broadening of the bulk water signal. This observation was traced to catalysis of proton exchange from the Eu3+-bound water molecule by excess positively charged amino groups on the surface of the nanoparticles.
PMCID: PMC3400155  PMID: 22344876
Silica nanoparticles; MRI; PARACEST agents; water exchange
15.  The pH sensitivity of −NH exchange in LnDOTA-tetraamide complexes varies with amide substituent 
The amide proton exchange rates in various lanthanide(III) DOTA-tetraamide complexes were investigated by CEST as a function of variable chemical structures and charges on the amide substituents. Comparisons were made between YbDOTA-(gly)4− (Yb-1), YbDOTA-(NHCH2PO3)45− (Yb-2), and YbDOTA-(NHCH2PO3Et2)43+ (Yb-3). The general shapes of the CEST versus pH profiles were similar for the three complexes but they showed maximum CEST intensities at different pH values, pH 8.3, 8.8 and 6.9 for Yb-1, Yb-2 and Yb-3, respectively. This indicates that a more negatively charged substituent on the amide helps stabilize the partial positive charge on the amide nitrogen and consequently more base is required to catalyze proton exchange. The chemical shifts of the −NH protons in Yb-1 and Yb-2 were similar (−17 ppm) while the −NH proton in Yb-3 was at −13 ppm. This shows that crystal field produced by the amide oxygen donor atoms in Yb-3 is substantially weaker than that in the other two complexes. In an effort to expand the useful range of pH values that might be measured using these complexes as CEST agents, the shapes of the CEST versus pH curves were also determined for two thulium(III) complexes with much larger hyperfine shifted −NH proton resonances. The ratio of CEST from −NH exchange in Tm-1 compared to CEST from −NH exchange in Tm-3 was found to be linear over an extended pH range, from 6.3 to 7.4. This demonstrates a potential advantage of using mixtures of lanthanide(III) DOTA-tetraamides for mapping tissue pH by use of ratiometric CEST imaging.
PMCID: PMC3233223  PMID: 22144023
MRI; contrast agents; pH-responsive agents; paramagnetic agents; Chemical Exchange Saturation Transfer (CEST) agents; amide proton exchange; ratiometric analysis
16.  Pharmacokinetics and Magnetic Resonance Imaging of Biodegradable Macromolecular Blood-Pool Contrast Agent PG-Gd in Non-Human Primates: A Pilot Study 
The purpose of this study was to evaluate poly(L-glutamic acid)-benzyl-DTPA-Gd (PG-Gd), a new biodegradable macromolecular magnetic resonance imaging contrast agent, for its pharmacokinetics and MRI enhancement in nonhuman primates. Studies were performed in rhesus monkeys at intravenous doses of 0.01, 0.02, and 0.08 mmol Gd/kg. T1-weighted MR images were acquired at 1.5T using fast spoiled gradient recalled echo and fast spin echo imaging protocols. The small-molecule contrast agent Magnevist was used as a control. PG-Gd in the monkey showed a bi-exponential disposition. The initial blood concentrations within 2 hours of PG-Gd administration were much higher than for those of Magnevist. The high blood concentration of PG-Gd was consistent with the MR imaging data, which showed prolonged circulation of PG-Gd in the blood pool. Enhancement of blood vessels and organs with a high blood perfusion (heart, liver, and kidney) was clearly visualized at 2 hours after contrast injection at the three doses used. A greater than proportional increase of the area under the blood concentration-time curve was observed when the administered single dose was increased from 0.01 mmol/kg to 0.08 mmol/kg. By 2 days after PG-Gd injection, the contrast agent was mostly cleared from all major organs, including kidney. The mean residence time was 15 hours at the 0.08 mmol/kg dose. A similar pharmacokinetic profile was observed in mice, with a mean residence time of 5.4 hours and a volume of distribution at steady-state of 85.5 mL/kg, indicating that the drug was mainly distributed in the blood compartment. Based on this pilot study, further investigations on potential systemic toxicity of PG-Gd in both rodents and large animals are needed before testing this agent in humans.
PMCID: PMC3435886  PMID: 21861289
Magnetic resonance imaging; blood pool; contrast media; polymers; non-human primate
17.  Photoacoustic tomography: fundamentals, advances and prospects 
Optical microscopy has been contributing to the development of life science for more than three centuries. However, due to strong optical scattering in tissue, its in vivo imaging ability has been restricted to studies at superficial depths. Advances in photoacoustic tomography (PAT) now allow multiscale imaging at depths from sub-millimeter to several centimeters, with spatial resolutions from sub-micrometer to sub-millimeter. Because of this high scalability and its unique optical absorption contrast, PAT is capable of performing anatomical, functional, molecular and fluid-dynamic imaging at various system levels, and is playing an increasingly important role in fundamental biological research and clinical practice. This Review discusses recent technical progress in PAT and presents corresponding applications. It ends with a discussion of several prospects and their technical challenges.
PMCID: PMC3205414  PMID: 22025335
Photoacoustic tomography; photoacoustic microscopy; photoacoustic computed tomography; photoacoustic endoscopy; multiscale imaging; optical absorption contrast
18.  Self-illuminating in vivo lymphatic imaging using a bioluminescence resonance energy transfer quantum dot nano-particle 
Autofluorescence arising from normal tissues can compromise the sensitivity and specificity of in vivo fluorescence imaging by lowering the target-to-background signal ratio. Since bioluminescence resonance energy transfer quantum dot (BRET-QDot) nano-particles can self-illuminate in near-infrared in the presence of the substrate, coelenterazine, without irradiating excitation lights, imaging using BRET-QDots does not produce any autofluorescence. In this study, we applied this BRET-QDot nano-particle to the in vivo lymphatic imaging in mice in order to compare with BRET, fluorescence or bioluminescence lymphatic imaging. BRET-QDot655, in which QDot655 is contained as a core, was injected at different sites (e.g. chin, ear, forepaws and hind paws) in mice followed by the intravenous coelenterazine injection, and then bioluminescence and fluorescence imaging were serially performed. In all mice, each lymphatic basin was clearly visualized in the BRET imaging with minimal background signals. The BRETsignal in the lymph nodes lasted at least 30 min after coelenterazine injections. Furthermore, the BRETsignal demonstrated better quantification than the fluorescence signal emitting from QDot655, the core of this BRET particle. These advantages of BRET-QDot allowed us to perform real-time, quantitative lymphatic imaging without image processing. BRET-Qdots have the potential to be a robust nano-material platform for developing optical molecular imaging probes.
PMCID: PMC3419257  PMID: 21351373
quantum dot; nanotechnology; fluorescence; bioluminescence; lymphatic imaging; bioluminescence resonance energy transfer (BRET)
19.  Fluorescence lifetime imaging of activatable target specific molecular probes 
In vivo optical imaging using fluorescently labeled self-quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target-to-background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self-quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2+ and BALB/3T3 (HER2−) cell lines. Changes in fluorescence lifetime correlated with temperature- and time-dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2+ and BALB/3T3 (HER2−)] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target-specific activatable antibody–fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging.
PMCID: PMC3404610  PMID: 20101762
molecular imaging; fluorescence lifetime; activatable fluorescence probe; cancer; near-infrared
20.  Use of perfluorocarbon nanoparticles for non-invasive multimodal cell tracking of human pancreatic islets 
In vivo imaging of engraftment and immunorejection of transplanted islets is critical for further clinical development, with 1H MR imaging of superparamagnetic iron oxide-labeled cells being the current premier modality. Using perfluorocarbon nanoparticles, we present here a strategy for non-invasive imaging of cells using other modalities. To this end, human cadaveric islets were labeled with rhodamine-perfluorooctylbromide (PFOB) nanoparticles, rhodamine-perfluoropolyether (PFPE) nanoparticles or Feridex® as control and tested in vitro for cell viability and c-peptide secretion for 1 week. 19F MRI, computed tomography (CT) and ultrasound (US) imaging was performed on labeled cell phantoms and on cells following transplantation beneath the kidney capsule of mice and rabbits. PFOB and PFPE-labeling did not reduce human islet viability or glucose responsiveness as compared with unlabeled cells or SPIO-labeled cells. PFOB- and PFPE-labeled islets were effectively fluorinated for visualization by 19F MRI. PFOB-labeled islets were acoustically reflective for detection by US imaging and became sufficiently brominated to become radiopaque allowing visualization with CT. Thus, perfluorocarbon nanoparticles are multimodal cellular contrast agents that may find applications in real-time targeted delivery and imaging of transplanted human islets or other cells in a clinically applicable manner using MRI, US or CT imaging.
PMCID: PMC3160722  PMID: 21861285
cell tracking; molecular imaging; fluorine MRI; ultrasound imaging; X-ray imaging; islet cell; diabetes
21.  Surface Functionalization of Magnetic Iron Oxide Nanoparticles for MRI Applications – Effect of Anchoring Group and Ligand Exchange Protocol 
Hydrophobic magnetite nanoparticles synthesized from thermal decomposition of iron salts must be rendered hydrophilic for their application as MRI contrast agents. This process requires refunctionalizing the surface of the nanoparticles with a hydrophilic organic coating such as polyethylene glycol. Two parameters were found to influence the magnetic behavior and relaxivity of the resulting hydrophilic iron oxide nanoparticles: the functionality of the anchoring group and the protocol followed for the functionalization. Nanoparticles coated with PEGs via a catecholate-type anchoring moiety maintain the saturation magnetization and relaxivity of the hydrophobic magnetite precursor. Other anchoring functionalities, such as phosphonate, carboxylate, and dopamine decrease the magnetization and relaxivity of the contrast agent. The protocol for functionalizing the nanoparticles also influences the magnetic behavior of the material. Nanoparticles refunctionalized according to a direct biphasic protocol exhibit higher relaxivity than those refunctionalized according to a two-step procedure which first involves stripping the nanoparticles. This research presents the first systematic study of both the binding moiety and the functionalization protocol on the relaxivity and magnetization of water-soluble coated iron oxide nanoparticles used as MRI contrast agents.
PMCID: PMC3123420  PMID: 21861279
MRI; contrast agent; MION; iron oxide nanoparticles; superparamagnetic agents; surface functionalization; relaxivity; magnetism
22.  Effect of Anesthesia Carrier Gas on In-Vivo Circulation Times of Ultrasound Microbubble Contrast Agents in Rats 
Microbubble contrast agents are currently implemented in a variety of both clinical and preclinical ultrasound imaging studies. The therapeutic and diagnostic capabilities of these contrast agents are limited by their short in-vivo lifetimes, and research to lengthen their circulation times is ongoing. In this manuscript, observations are presented from a controlled experiment performed to evaluate differences in circulation times for lipid shelled perfluorocarbon-filled contrast agents circulating within rodents as a function of inhaled anesthesia carrier gas.
The effects of two common anesthesia carrier gas selections - pure oxygen and medical air – were observed within five rats. Contrast agent persistence within the kidney was measured and compared for oxygen and air anesthesia carrier gas for six bolus contrast injections in each animal. Simulations were performed to examine microbubble behavior with changes in external environment gases.
A statistically significant extension of contrast circulation time was observed for animals breathing medical air compared to breathing pure oxygen. Simulations support experimental observations and indicate that enhanced contrast persistence may be explained by reduced ventilation/perfusion mismatch and classical diffusion, in which nitrogen plays a key role by contributing to the volume and diluting other gas species in the microbubble gas core. Conclusion: Using medical air in place of oxygen as the carrier gas for isoflurane anesthesia can increase the circulation lifetime of ultrasound microbubble contrast agents.
PMCID: PMC3341737  PMID: 21246710
anesthesia; carrier gas; microbubble; lifetime; persistence; circulation time
23.  Biocompatible blood pool MRI contrast agents based on hyaluronan 
Biocompatible gadolinium blood pool contrast agents based on a biopolymer, hyaluronan, were investigated for magnetic resonance angiography application. Hyaluronan, a non-sulfated linear glucosaminoglycan composed of 2000–25,000 repeating disaccharide subunits of D-glucuronic acid and N-acetylglucosamine with molecular weight up to 20 MDa, is a major component of the extracellular matrix. Two gadolinium contrast agents based on 16 and 74 kDa hyaluronan were synthesized, both with R1 relaxivity around 5 mM−1 s−1 per gadolinium at 9.4 T at 25°C. These two hyaluronan based agents show significant enhancement of the vasculature for an extended period of time. Initial excretion was primarily through the renal system. Later uptake was observed in the stomach and lower gastrointestinal tract. Macromolecular hyaluronan-based gadolinium agents have a high clinical translation potential as hyaluronan is already approved by FDA for a variety of medical applications.
PMCID: PMC3032015  PMID: 21504061
hyaluronan; biocompatible contrast agents; magnetic resonance angiography
24.  Manganese Cell Labeling of Murine Hepatocytes Using Manganese(III)-Transferrin 
Manganese(III)-transferrin (Mn(III)-Tf) was investigated as a way to accomplish manganese-labeling of murine hepatocytes for MRI contrast. It is postulated that Mn(III)-Tf can exploit the same transferrin-receptor-dependent and -independent metabolic pathways used by hepatocytes to transport the iron analogue Fe(III)-Tf. More specifically, it was investigated whether manganese delivered by transferrin could give MRI contrast in hepatocytes. Comparison of the T1 and T2 relaxation times of Mn(III)-Tf and Fe(III)-Tf over the same concentration range showed that the r1 relaxivities of the two metalloproteins are the same in vitro; with little contribution from paramagnetic enhancement. The degree of manganese cell labeling following incubation for 2–7 h in 31.5 μM Mn(III)-Tf was comparable to that of hepatocytes incubated in 500 μM Mn2+ for 1 h. The intrinsic manganese tissue relaxivity between Mn(III)-Tf-labeled and Mn2+-labeled cells was found to be the same; consistent with Mn(III) being released from transferrin and reduced to Mn2+. For both treatment regimens, manganese uptake by hepatocytes appeared to saturate in the first 1–2 h of the incubation period and may explain why the efficiency of hepatocyte cell labeling by the two methods appeared to be comparable in spite of the ~16-fold difference in effective manganese concentration. Hepatocytes continuously released manganese, as detected by MRI, and this was the same for both Mn2+- and Mn(III)-Tf-labeled cells. Manganese release may be the result of normal hepatocyte function; much in the same way that hepatocytes excrete manganese into the bile in vivo. This approach exploits a biological process – namely receptor binding, endocytosis, and endosomal acidification – to initiate the release of an MRI contrast agent; potentially confering more specificity to the labeling process. The ubiquitous expression of transferrin receptors by eukaryotic cells should make Mn(III)-Tf particularly useful for manganese labeling of a wide variety of cells both in culture and in vivo.
PMCID: PMC3197774  PMID: 18546093
manganese-enhanced MRI; manganese(III)-transferrin; relaxivity; isolated hepatocytes; mouse hepatocytes; iron(III)-transferrin; transferrin receptor; endocytosis
25.  Cytotoxicity of iron oxide nanoparticles made from the thermal decomposition of organometallics and aqueous phase transfer with Pluronic F127 
Magnetic nanoparticles are promising molecular imaging agents due to their relative high relaxivity and the potential to modify surface functionality to tailor biodistribution. In this work we describe the synthesis of magnetic nanoparticles using organic solvents with organometallic precursors. This method results in nanoparticles that are highly crystalline, and have uniform size and shape. The ability to create a monodispersion of particles of the same size and shape results in unique magnetic properties that can be useful for biomedical applications with MR imaging. Before these nanoparticles can be used in biological applications, however, means are needed to make the nanoparticles soluble in aqueous solutions and the toxicity of these nanoparticles needs to be studied.
We have developed two methods to surface modify and transfer these nanoparticles to the aqueous phase using the biocompatible co-polymer, Pluronic F127. Cytotoxicity was found to be dependent on the coating procedure used. Nanoparticle effects on a cell-culture model was quantified using concurrent assaying; a LDH assay to determine cytotoxicity and an MTS assay to determine viability for a 24 hour incubation period. Concurrent assaying was done to insure that nanoparticles did not interfere with the colorimetric assay results.
This report demonstrates that a monodispersion of nanoparticles of uniform size and shape can be manufactured. Initial cytotoxicity testing of new molecular imaging agents need to be carefully constructed to avoid interference and erroneous results.
PMCID: PMC3020093  PMID: 20623517
MRI; molecular imaging; nanoparticles; superparamagnetic agents; cytotoxicity; Colorimetric Assay; Pluronics

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