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1.  Synthesis and characterization of a porphyrazine–Gd(III) MRI contrast agent and in vivo imaging of a breast cancer xenograft model 
Porphyrazines (Pz), or tetraazaporphyrins, are being studied for their potential use in detection and treatment of cancer. Here, an amphiphilic Cu–Pz–Gd(III) conjugate has been prepared via azide-alkyne Huisgen cycloaddition or ‘click’ chemistry between an azide functionalized Pz and alkyne functionalized DOTA–Gd(III) analog for use as an MRI contrast agent. This agent, Cu–Pz–Gd(III), is synthesized in good yield and exhibits solution-phase ionic relaxivity (r1 = 11.5 mm−1 s−1) that is approximately four times higher than that of a clinically used monomeric Gd (III) contrast agent, DOTA–Gd(III). Breast tumor cells (MDA-MB-231) associate with Cu–Pz–Gd(III) in vitro, where significant contrast enhancement (9.336 ± 0.335 contrast-to-noise ratio) is observed in phantom cell pellet MR images. This novel contrast agent was administered in vivo to an orthotopic breast tumor model in athymic nude mice and MR images were collected. The average T1 of tumor regions in mice treated with 50 mg kg−1 Cu–Pz–Gd (III) decreased relative to saline-treated controls. Furthermore, the decrease in T1 was persistent relative to mice treated with the monomeric Gd(III) contrast agent. An ex vivo biodistribution study confirmed that Cu–Pz–Gd(III) accumulates in the tumors and is rapidly cleared, primarily through the kidneys. Differential accumulation and T1 enhancement by Cu–Pz–Gd(III) in the tumor's core relative to the periphery offer preliminary evidence that this agent would find application in the imaging of necrotic tissue.
doi:10.1002/cmmi.1577
PMCID: PMC4327828  PMID: 24706615
tumor imaging; porphyrazine; MRI contrast agent; click chemistry
2.  Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics† 
Various nanoparticles have raised significant interest over the past decades for their unique physical and optical properties and biological utilities. Here we summarize the vast applications of advanced nanoparticles with a focus on carbon nanotube (CNT)-based or CNT-catalyzed contrast agents for photoacoustic (PA) imaging, cytometry and theranostics applications based on the photothermal (PT) effect. We briefly review the safety and potential toxicity of the PA/PT contrast nanoagents, while showing how the physical properties as well as multiple biological coatings change their toxicity profiles and contrasts. We provide general guidelines needed for the validation of a new molecular imaging agent in living subjects, and exemplify these guidelines with single-walled CNTs targeted to αvβ3, an integrin associated with tumor angiogenesis, and golden carbon nanotubes targeted to LYVE-1, endothelial lymphatic receptors. An extensive review of the potential applications of advanced contrast agents is provided, including imaging of static targets such as tumor angiogenesis receptors, in vivo cytometry of dynamic targets such as circulating tumor cells and nanoparticles in blood, lymph, bones and plants, methods to enhance the PA and PT effects with transient and stationary bubble conjugates, PT/PA Raman imaging and multispectral histology. Finally, theranostic applications are reviewed, including the nanophotothermolysis of individual tumor cells and bacteria with clustered nanoparticles, nanothrombolysis of blood clots, detection and purging metastasis in sentinel lymph nodes, spectral hole burning and multiplex therapy with ultrasharp rainbow nanoparticles.
doi:10.1002/cmmi.455
PMCID: PMC4282188  PMID: 22025336
photoacoustic molecular imaging; photothermal theranostics; nanotechnology; carbon nanotubes; flow cytometry; blood test; cancer; metastasis
3.  Nanoparticle Contrast Agents for Computed Tomography: A Focus on Micelles 
Computed tomography (CT) is an X-ray based whole body imaging technique that is widely used in medicine. Clinically approved contrast agents for CT are iodinated small molecules or barium suspensions. Over the past seven years there has been a great increase in the development of nanoparticles as CT contrast agents. Nanoparticles have several advantages over small molecule CT contrast agents, such as long blood-pool residence times, and the potential for cell tracking and targeted imaging applications. Furthermore, there is a need for novel CT contrast agents, due to the growing population of renally impaired patients and patients hypersensitive to iodinated contrast. Micelles and lipoproteins, a micelle-related class of nanoparticle, have notably been adapted as CT contrast agents. In this review we discuss the principles of CT image formation and the generation of CT contrast. We discuss the progress in developing non-targeted, targeted and cell tracking nanoparticle CT contrast agents. We feature agents based on micelles and used in conjunction with spectral CT. The large contrast agent doses needed will necessitate careful toxicology studies prior to clinical translation. However, the field has seen tremendous advances in the past decade and we expect many more advances to come in the next decade.
doi:10.1002/cmmi.1551
PMCID: PMC3905628  PMID: 24470293
nanoparticle; micelle; computed tomography; X-ray; spectral CT; iodine; gold nanoparticle; lipoprotein; molecular imaging; bismuth
4.  Multicolor Computed Tomographic Molecular Imaging with Non-crystalline High Metal Density Nanobeacons 
Computed tomography (CT) is one of the most frequently pursued radiology technologies applied in the clinics today and in the preclinical field of biomedical imaging. Myriad advancement has been made to make this technique more powerful with improved signal sensitivity, rapid image acquisition and faster reconstruction. Synergistic development of novel nanoparticles has been adopted as the next generation CT contrasts agents for imaging specific biological markers. Nanometer-sized agents are anticipated to play a critical part in the prospect of medical diagnostics owing to their capabilities of targeting specific biological markers, extended blood circulation time and defined biological clearance. This review paper introduces the readers to the fundamental design principles of nanoparticulate CT contrast agents with a special emphasis on the molecular Imaging with non-crystalline high metal density nanobeacons.
doi:10.1002/cmmi.1571
PMCID: PMC4076970  PMID: 24470291
Computed tomographic imaging; nanoparticle; iodine; bismuth; gold; ytterbium; tantalum; fibrin; angiogenesis; sentinel lymph node; thrombus
5.  Radiolabelled GLP-1 analogues for in vivo targeting of insulinomas 
For peptide receptor targeting usually internalizing agonists are selected. There is increasing evidence that non-internalizing receptor antagonists can be used for this purpose. We investigated whether the glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin(9-39) can be used for in vivo targeting of GLP-1R expressing tumours and compared the in vitro and in vivo characteristics to the GLP-1R agonists exendin-3 and exendin-4.
The binding and internalization kinetics of labelled [Lys40(DTPA)]exendin-3, [Lys40(DTPA)]exendin-4 and [Lys40(DTPA)]exendin(9-39) were determined in vitro using INS-1 cells. The in vivo targeting properties of [Lys40(111In-DTPA)]exendin-3, [Lys40(111In-DTPA)]exendin-4 and [Lys40(111In-DTPA)]exendin(9-39) were examined in BALB/c nude mice with subcutaneous INS-1 tumours. natIn-labelled [Lys40(DTPA)]exendin-3, [Lys40(DTPA)]exendin-4 and [Lys40(DTPA)]exendin(9-39) exhibited similar IC50 values (13.5, 14.4 and 13.4 nM, respectively) and bound to 26 × 103, 41 × 103 and 37 × 103 receptors/cell, respectively. [Lys40(111In-DTPA)]exendin-3 and [Lys40(111In-DTPA)]exendin-4 showed rapid in vitro binding and internalization kinetics, whereas [Lys40(111In-DTPA)]exendin(9-39) showed lower binding and minimal internalization in vitro. In mice, high specific uptake of [Lys40(111In-DTPA)]exendin-3 (25.0 ± 6.0 %ID/g) in the tumour was observed at 0.5 h p.i. with similar uptake up to 4 h p.i.. [Lys40(111In-DTPA)]exendin-4 showed higher tumour uptake at 1 and 4 h p.i. (40.8 ± 7.0 and 41.9 ± 7.2 %ID/g, respectively). Remarkably, [Lys40(111In-DTPA)]exendin(9-39) showed only low specific uptake in the tumour at 0.5 h p.i. (3.2 ± 0.7 %ID/g), rapidly decreasing over time.
In conclusion, the GLP-1R agonists [Lys40(DTPA)]exendin-3 and [Lys40(DTPA)]exendin-4 labelled with 111In could be useful for in vivo GLP-1R targeting, whereas [Lys40(DTPA)]exendin(9-39) is not suited for in vivo targeting of the GLP-1R.
doi:10.1002/cmmi.475
PMCID: PMC4246000  PMID: 22434628
GLP-1; exendin; antagonist; agonist; insulinoma
6.  Optical imaging of progenitor cell homing to patient-derived tumors 
Capitalizing on cellular homing to cancer is a promising strategy for targeting malignant cells for diagnostic, monitoring and therapeutic purposes. Murine C17.2 neural progenitor cells (NPC) demonstrate a tropism for cell line-derived tumors, but their affinity for patient-derived tumors is unknown. We tested the hypothesis that NPC accumulate in patient-derived tumors at levels detectable by optical imaging. Mice bearing solid tumors after transplantation with patient-derived leukemia cells and untransplanted controls received 106 fluorescent DiR-labeled NPC daily for 1–4 days, were imaged, then sacrificed. Tissues were analyzed by immunofluorescence and flow cytometry to detect tumor cell engraftment (CD45) and NPC (FITC-β galactosidase or DiR). Tumors consisted primarily of CD45-positive cells and demonstrated mild fluorescence, corresponding to frequent clusters of FITC-β gal-positive cells. Both transplanted and control mice demonstrated the highest fluorescent signal in the spleens and other tissues of the reticuloendothelial activating system. However, only rare FITC-β gal-positive cells were detected in the mildly engrafted transplanted spleens and none in the control spleens, suggesting that their high DiR signal reflects the sequestration of DiR-positive debris. The mildly engrafted transplanted kidneys demonstrated low fluorescent signal and rare FITC-β gal-positive cells whereas control kidneys were negative. Results indicate that NPC accumulate in tissues containing patient-derived tumor cells in a manner that is detectable by ex vivo optical imaging and proportional to the level of tumor engraftment, suggesting a capacity to home to micrometastatic disease. As such, NPC could have significant clinical applications for the targeted diagnosis and treatment of cancer.
doi:10.1002/cmmi.1485
PMCID: PMC4091721  PMID: 22991319
cellular imaging; homing; fluorescent probes; C17.2; chronic myeloid leukemia; neural progenitor cells; humanized cancer model; immunofluorescence; FACS
7.  Assessment of the biodistribution of an [18F]FDG-loaded perfluorocarbon double emulsion using dynamic micro-PET in rats 
Perfluorocarbon (PFC) double emulsions loaded with a water-soluble, therapeutic agent can be triggered by ultrasound in a process known as acoustic droplet vaporization (ADV). Elucidating the stability and biodistribution of these sonosensitive vehicles and encapsulated agents are critical in developing targeted drug delivery strategies using ultrasound. [18F]fluorodeoxyglucose (FDG) was encapsulated in a PFC double emulsion and the in vitro diffusion of FDG was assessed using a Franz diffusion cell. Using dynamic micro positron emission tomography (micro-PET) and direct tissue sampling, the biodistribution of FDG administered as a solution (i.e. non-emulsified) or as an emulsion was studied in Fisher 344 rats (n = 6) bearing subcutaneous 9L gliosarcoma. Standardized uptake values (SUVs) and area under the curve of the SUV (AUCSUV) of FDG were calculated for various tissues. The FDG flux from the emulsion decreased by up to a factor of 6.9 compared to the FDG solution. FDG uptake, calculated from the AUCSUV, decreased by 36% and 44% for brain and tumor, respectively, when comparing FDG solution versus FDG emulsion (p < 0.01). Decreases in AUCSUV in highly metabolic tissues such as brain and tumor demonstrated retention of FDG within the double emulsion. No statistically significant differences in lung AUCSUV were observed, suggesting minimal accumulation of the emulsion in the pulmonary capillary bed. The liver AUCSUV increased by 356% for the FDG emulsion, thus indicating significant hepatic retention of the emulsion.
doi:10.1002/cmmi.1532
PMCID: PMC3677228  PMID: 23613440
micro-PET; biodistribution; perfluorocarbon; emulsion; acoustic droplet vaporization; ultrasound; responsive agents
8.  Synthesis and evaluation of a polydisulfide with Gd-DOTA monoamide side chains as a biodegradable macromolecular contrast agent for MR blood pool imaging 
Macromolecular Gd(III) based contrast agents are effective for contrast enhanced blood pool and cancer MRI in preclinical studies. However, their clinical applications are impeded by potential safety concerns associated with slow excretion and prolonged retention of these agents in the body. To minimize the safety concerns of macromolecular Gd contrast agents, we have recently designed and developed biodegradable macromolecular Gd contrast agents based on polydisulfide Gd(III) complexes. In this study, we designed and synthesized a new generation of the polydisulfide Gd(III) complexes containing macrocyclic Gd(III) chelate, Gd-DOTA monoamide, to further improve the in vivo kinetic stability of the Gd(III) chelates of the contrast agents. (N6-Lysyl)lysine Gd-DOTA monoamide and 3-(2-carboxyethyldisulfanyl)propanoic acid copolymers (GODC) was synthesized by copolymerization of (N6-lysyl)lysine DOTA monoamide and dithiobis (succinimidylpropionate), followed by complexation with Gd(OAc)3. The GODC had an apparent molecular weight of 26.4 kDa and T1 relaxivity of 8.25 mM−1s−1 per Gd at 1.5 T. The polymer chains of GODC were readily cleaved by L-cysteine and the chelates had high kinetic stability against transmetallation in the presence of an endogenous metal ion Zn2+. In vivo MR study showed that GODC produced strong and prolonged contrast enhancement in the vasculature and tumor periphery of mice with breast tumor xenografts. GODC is a promising biodegradable macromolecular MRI contrast agent with high kinetic stability for MR blood pool imaging.
doi:10.1002/cmmi.1520
PMCID: PMC4060968  PMID: 23606425
Gd; biodegradable macromolecular MRI contrast agent; blood pool imaging; cancer imaging; kinetic stability
9.  Kinetic analysis of hyaluronidase activity using a bioactive MRI contrast agent 
One of the attractions of molecular imaging using ‘smart’ bioactive contrast agents is the ability to provide non-invasive data on the spatial and temporal changes in the distribution and expression patterns of specific enzymes. The tools developed for that aim could potentially also be developed for functional imaging of enzyme activity itself, through quantitative analysis of the rapid dynamics of enzymatic conversion of these contrast agents. High molecular weight hyaluronan, the natural substrate of hyaluronidase, is a major antiangiogenic constituent of the extracellular matrix. Degradation by hyaluronidase yields low molecular weight fragments, which are proangiogenic. A novel contrast material, HA-GdDTPA-beads, was designed to provide a substrate analog of hyaluronidase in which relaxivity changes are induced by enzymatic degradation. We show here a first-order kinetic analysis of the time-dependent increase in R2 as a result of hyaluronidase activity. The changes in R2 and the measured relaxivity of intact HA-GdDTPA-beads (r2B) and HA-GdDTPA fragments (r2D) were utilized for derivation of the temporal drop in concentration of GdDTPA in HA-GdDTPA-beads as the consequence of the release of HA-GdDTPA fragments. The rate of dissociation of HA-GdDTPA from the beads showed typical bell-shaped temperature dependence between 7 and 36 °C with peak activity at 25 °C. The tools developed here for quantitative dynamic analysis of hyaluronidase activity by MRI would allow the use of activation of HA-GdDTPA-beads for the determination of the role of hyaluronidase in altering the angiogenic microenvironment of tumor micro metastases.
doi:10.1002/cmmi.96
PMCID: PMC4035508  PMID: 17193686
hyaluronidase; bioactive contrast material; magnetic resonance imaging; angiogenesis
10.  Simultaneous determination of labile proton fraction ratio and exchange rate with irradiation radio frequency (RF) power dependent quantitative CEST MRI analysis 
Chemical exchange saturation transfer (CEST) imaging is sensitive to dilute proteins/peptides and microenvironmental properties, and has been increasingly evaluated for molecular imaging and in vivo applications. However, the experimentally measured CEST effect depends on the CEST agent concentration, exchange rate and relaxation time. In addition, there may be non-negligible direct radio-frequency (RF) saturation effects, particularly severe for diamagnetic CEST (DIACEST) agents due to their relatively small chemical shift difference from that of the bulk water resonance. As such, the commonly used asymmetry analysis only provides CEST-weighted information. Recently, it has been shown with numerical simulation that both labile proton concentration and exchange rate can be determined by evaluating the RF power dependence of DIACEST effect. To validate the simulation results, we prepared and imaged two CEST phantoms: a pH phantom of serially titrated pH at a fixed creatine concentration and a concentration phantom of serially varied creatine concentration titrated to the same pH, and solved the labile proton fraction ratio and exchange rate per-pixel. For the concentration phantom, we showed that the labile proton fraction ratio is proportional to the CEST agent concentration with negligible change in the exchange rate. Additionally, we found the exchange rate of the pH phantom is dominantly base-catalyzed with little difference in the labile proton fraction ratio. In summary, our study demonstrated quantitative DIACEST MRI, which remains promising to augment the conventional CEST-weighted MRI analysis.
doi:10.1002/cmmi.1524
PMCID: PMC4034529  PMID: 23606428
amide proton transfer (APT); chemical exchange saturation transfer (CEST); pH
11.  Imaging integrin alpha-v-beta-3 expression in tumors with an 18F-labeled dimeric RGD peptide 
Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine-glycine-aspartatic acid (RGD) peptide and integrin αvβ3, but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of 18F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with 18F in a simple one-pot process with a radiolabeling yield of 20%; the whole process lasting only 45 min. NODAGA-E-[c(RGDfK)]2 labeled with 18F at a specific activity of 1.8 MBq/nmol and a radiochemical purity of 100% could be achieved. Log P value of 18F-labeled NODAGA-E-[c(RGDfK)]2 was −4.26 ± 0.02. In biodistribution studies, 18F-NODAGA-E-[c(RGDfK)]2 cleared rapidly from the blood with 0.03 ± 0.01 %ID/g in the blood at 2 h p.i., mainly via the kidneys and showed good in vivo stability. Tumor uptake of 18F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID/g, 2 h p.i.) was significantly lower than that of reference compounds 68Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID/g; P <0.001) and 111In-labeled NODAGA-E-[c(RGDfK)]2 (4.99 ± 0.64 %ID/g; P < 0.01). Co-injection of an excess of unlabeled NODAGA-E-[c(RGDfK)]2 along with 18F-NODAGA-E-[c(RGDfK)]2 resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID/g). The αvβ3 integrin-expressing SK-RC-52 tumor could be successfully visualized by microPET with 18F-labeled NODAGA-E-[c(RGDfK)]2. In conclusion, NODAGA-E-[c(RGDfK)]2 could be labeled rapidly with 18F using a direct aqueous, one-pot method and it accumulated specifically in αvβ3 integrin-expressing SK-RC-52 tumors, allowing for visualization by microPET.
doi:10.1002/cmmi.1523
PMCID: PMC4034708  PMID: 23606427
Integrin alpha-v-beta-3; PET; radiofluorination; aluminum fluoride; RGD; NODAGA
12.  EPR assessment of protein sites for incorporation of Gd(III) MRI contrast labels 
We have engineered Apolipoprotein A-I (apoA-I), a major protein constituent of high-density lipoprotein (HDL), to contain DOTA-chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism, and regulation in vivo. This protein contrast agent was obtained by reacting the thiol-reactive Gd[MTS-ADO3A] label with Cys engineered at four distinct positions (52, 55, 76 and 80) in apoA-I. MRI of infused mice previously showed that the Gd-labeled apoA-I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoA-I are superior when the ADO3A moiety is located at position 55, compared to the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave X-band (9 GHz) EPR spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipid-free to the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipid-free and rHDL states correlate to the product of the X-band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDL-associated apoA-I containing the Gd[MTS-ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by X-band EPR. While room temperature EPR requires >1 mM Gd(III)-labeled and only >10 μM nitroxide-labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5μL). Thus, X-band EPR provides a practical assessment for the suitability of imaging candidates containing the site-directed ADO3A contrast probe.
doi:10.1002/cmmi.1518
PMCID: PMC3633150  PMID: 23606429
apolipoprotein A-I; magnetic resonance imaging (MRI); electron paramagnetic resonance (EPR) spectroscopy; protein contrast agent; high-density lipoprotein (HDL); site-directed MRI label
13.  Influence of molecular parameters and increasing magnetic field strength on relaxivity of gadolinium- and manganese-based T1 contrast agents 
Simulations were performed to understand the relative contributions of molecular parameters to longitudinal (r1) and transverse (r2) relaxivity as a function of field, and to obtain theoretical relaxivity maxima over a range of fields to appreciate what relaxivities can be achieved experimentally. The field dependent relaxivity of a panel of gadolinium and manganese complexes with different molecular parameters: water exchange rates, rotational correlation times, hydration state, etc were measured to confirm that measured relaxivities were consistent with theory. The design tenets previously stressed for optimizing r1 at low fields (very slow rotational motion; chelate immobilized by protein binding; optimized water exchange rate) do not apply at higher fields. At 1.5T and higher fields, an intermediate rotational correlation time is desired (0.5 – 4 ns), while water exchange rate is not as critical to achieving a high r1. For targeted applications it is recommended to tether a multimer of metal chelates to a protein-targeting group via a long flexible linker to decouple the slow motion of the protein from the water(s) bound to the metal ions. Per ion relaxivities of 80, 45, and 18 mM−1s−1 at 1.5, 3, and 9.4T respectively are feasible for Gd3+ and Mn2+ complexes.
doi:10.1002/cmmi.267
PMCID: PMC3995751  PMID: 19177472
14.  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.
doi:10.1002/cmmi.499
PMCID: PMC3883306  PMID: 22539401
Magnetite; Nanoparticle; Magnetorelaxometry; SQUID; Magnetic Resonance Imaging; Magnetometry; Magnetic Susceptibility; Antibody targeting
15.  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.
doi:10.1002/cmmi.1493
PMCID: PMC3490434  PMID: 23109392
Relaxometry; Iron Content; Cell Labeling; Stem Cells; Superparamagnetic Iron Oxide Nanoparticles; Ferumoxides; Ferumoxytol; ICP-MS; Spectrophotometry; Prussian Blue; MRI
16.  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.
doi:10.1002/cmmi.1480
PMCID: PMC3846290  PMID: 22991316
Gadolinum chelates; MRI contrast agents; relaxivity enhancement; mesoporous silicon particles
17.  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.
doi:10.1002/cmmi.1484
PMCID: PMC3448123  PMID: 22991318
cGMP-Compatible Synthesis; Near-Infrared Fluorophore; Near-Infrared Fluorescence Imaging; Optical Imaging
18.  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.
doi:10.1002/cmmi.1479
PMCID: PMC3458584  PMID: 22991315
MRI; dynamic contrast; DCE; tumor; pharmacokinetic modeling; SPECT; radionuclide; diffusion
19.  Assessment and comparison of magnetic nanoparticles as MRI contrast agents in a rodent model of human hepatocellular carcinoma 
PURPOSE
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.
METHODS
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.
RESULTS
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.
CONCLUSION
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.
doi:10.1002/cmmi.494
PMCID: PMC3759163  PMID: 22649042
Iron oxide nanoparticle (IONP); hepatocarcinoma (HCC); magnetic resonance imaging (MRI); liver contrast agent
20.  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.
doi:10.1002/cmmi.281
PMCID: PMC3732192  PMID: 19839031
hydroxypyridinone (HOPO); mesityl; gadolinium; MRI contrast agent; relaxivity; aggregation
21.  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.
doi:10.1002/cmmi.1467
PMCID: PMC3383460  PMID: 22649047
iron oxide nanoparticle; quantification; magnetic susceptibility; relaxation; responsive agent; cellular imaging
22.  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.
doi:10.1002/cmmi.505
PMCID: PMC3415239  PMID: 22649044
Amide proton transfer (APT); Chemical exchange saturation transfer (CEST); MRI
23.  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
doi:10.1002/cmmi.447
PMCID: PMC3626438  PMID: 22144025
manganese; sub-cellular localization; pancreatic β-cells function; X-ray fluorescence microscopy; MRI
24.  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.
doi:10.1002/cmmi.483
PMCID: PMC3607543  PMID: 22344885
MRI contrast agent; hydroxypyridinone (HOPO); relaxivity; 1,4,7-triazacyclononane (TACN); terephthalamide (TAM); dendrimer; esteramide (EA) dendrimer; drug delivery; gadolinium
25.  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.
doi:10.1002/cmmi.376
PMCID: PMC3586549  PMID: 20812290
molecular imaging; cellular uptake; MRI; CT; contrast agents; paramagnetic agents; fluorescent probes; trimodal probes; nanoparticles

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