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Improved Paramagnetic Chelate for Molecular Imaging with MRI
BRIEF ABSTRACTS
because of their negative surface charges. These particles are thus particularly well suited for cellular imaging in vivo. The aim of this study was to image splenic uptake of labelled lymphocytes, using hybridomas as a model on a 1.5 T clinical magnet.
MATERIALS & METHODS Cell labelling: Hybridomas cells were cultivated under standard conditions and incubated with the anionic particles composed of a maghemite core (␥-Fe2O3) with anionic citrate molecules adsorbed on the surface. —Time response study : Iron load was quantified at several time points (0, 10, 20, 60, 120, 240, 360 min) after incubation with the particles at a concentration of 1 and 15 mM. —Concentration response: Iron load was quantified at 20 and 120 min after incubation with different concentrations of nanoparticles of 0, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 15 mM. Iron quantification: All samples were measured both by Magnetophoresis (MP) and Electron Spin Resonance (ESR). MP measures the velocity of labelled cells submitted to a magnetic field gradient, which is related to the number of magnetic particles, and thus to the iron content. ESR is a more sensitive method. The ESR signal is obtained by sweeping the static field H and recording the microwave absorption for an excitation field at 9.2 GHz. MRI: Male Swiss Nude mice were injected intraperitoneally with 20 millions labelled hybridomas (2.5 pg Fe/ cell). MRI (1.5 Tesla GE, Buc, France) was performed 24 h later using Spin Echo (TR⫽2000 ms, TE⫽ 20 and 60 ms) and Gradient Echo (TR⫽20 ms, TE⫽4 ms, 30°) sequences. Nine mice were injected with labelled cells, and 7 others were used as controls.
RESULTS: MP and ESR gave similar results under all conditions. A maximum uptake was observed after 90 min in the time response study, and at 6 mM in the concentration response study, with a saturation effect in all cases.
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Academic Radiology, Vol 12, Suppl 1, May 2005
At MRI, a negative enhancement of the spleen was observed on T2 60 ms (⫺37%) and GRE (⫺16%) images, with a heterogeneous response among animals. When excluding the 3 non-enhancing spleens, maximum decrease could be observed up to ⫺45% on T2 and ⫺28% on GRE.
CONCLUSION: Anionic particles can efficiently label hybridomas, which become detectable in the spleen in vivo at 1.5 T. Such an approach could be extended to other cell types and used for therapeutic purposes. Improved Paramagnetic Chelate for Molecular Imaging with MRI Patrick M. Winter1, Phillip S. Athey2, Gary E. Kiefer2, Gyongyi Gulyas2, Ralph W. Fuhrhop1, J. David Robertson3, Samuel A. Wickline1, and Gregory M. Lanza1 1Washington University, St. Louis, MO; 2Dow Chemical Co., Freeport, TX; 3University of Missouri Research Reactor, Columbia, MO
Sensitive molecular imaging of sparse biochemical epitopes with magnetic resonance imaging (MRI) requires a targeted contrast agent with enormous paramagnetic influence to overcome partial-volume dilution. Tens of thousands of gadolinium chelates can be delivered to each binding site with lipid encapsulated perfluorocarbon nanoparticles, providing adequate T1-weighted contrast to detect microthrombi on ruptured plaques and cell surface receptors. These contrast agents can be improved, however, by optimizing the intrinsic relaxivity and transmetalation of the gadolinium chelate. The objective of this study was to evaluate three paramagnetic chelates for use as targeted MRI contrast agents. Paramagnetic nanoparticles were produced by microfluidization incorporating one of three different chelates onto the lipid surface (Gd-DTPA-BOA, Gd-MeODOTA-PE or Gd-MeO-DOTA-Triglycine-PE). Nanoparticles were diluted with distilled deionized water and proton longitudinal relaxation rates (1/T1) of each sample were measured at 40° C and a field strength of 0.47 Tesla. The [Gd3⫹] of each sample was measured via neutron activation techniques, while the concentration of nanoparticles was calculated from the known amount of perfluorocarbon in the formulation and the nominal particle size determined by elastic laser light
BRIEF ABSTRACTS
Academic Radiology, Vol 12, Suppl 1, May 2005
scattering. The T1 relaxivity (r1) was determined from the slope of the linear least-squares regression of longitudinal relaxation rate versus Gd3⫹ (i.e., ion relaxivity) or nanoparticle (i.e., particle relaxivity) concentrations and are reported in units of (s⫺1mM)⫺1. The transmetalation was determined at 40° C and 0.47 Tesla using ZnCl as the competing ionic species. Gd-DTPA-BOA nanoparticles had higher ionic relaxivity (21.3 s⫺1mM⫺1) than typical MRI contrast agents, such as Gd-DTPA (⬃4 s⫺1mM⫺1). Because each particle caries more than 30,000 Gd3⫹ chelates, the particle-based relaxivity is orders of magnitude higher than any other paramagnetic contrast agent (700,000 s⫺1mM⫺1). Further improvements in ionic and particle-based relaxivity were observed with the GdMeO-DOTA-PE (29.8 and 979,000 s⫺1mM⫺1, respectively) and Gd-MeO-DOTA-Triglycine-PE (33.0 and 1,080,000 s⫺1mM⫺1, respectively) nanoparticles. The transmetalation of the DOTA chelates was also much more favorable than the DTPA chelate. Extrapolating to the amount of Gd3⫹ still bound to the chelate at time equal to infinity, the Gd-DTPA-BOA nanoparticles only retain 75.2%, while the Gd-MeO-DOTA-PE and Gd-MeO-DOTA-Triglycine-PE nanoparticles retain 91.2% and 90.6%, respectively. These results demonstrate the importance of paramagnetic chelates for optimizing molecular imaging contrast agents for MRI. The DOTA chelates demonstrated 40 – 55% higher relaxivity and ⬃20% lower transmetalation. These findings agree with well-documented advantages of DOTA chelates over DTPA chelates. In addition, the use of the PE moiety may improve water interaction in the DOTA chelates compared to the BOA utilized for the DTPA chelate. The insertion of the triglycine linker may also improve water interaction leading to increased relaxivity. In the context of molecular imaging, these improvements can lead to more sensitive detection of pathology and improved safety of the contrast agents.
Figure 2. Transmetalation of Gd-DTPA-BOA (DTPA), Gd-MeODOTA-PE (DOTA) and Gd-MeO-DOTA-Triglycene-PE (DOTA-PE) paramagnetic nanoparticles.
An Inexpensive Vascularized Tumor Model for Vascular Imaging Max Wu, PhD1, Jacqueline Corbeil1, Judith Varner, PhD2, David Vera, PhD1, Robert Mattrey, MD1 University of California, San Diego Departments of Radiology (1) and Medicine (2).
PURPOSE: Although many molecular imaging agents, particularly those aimed at imaging intracellular or cellular targets, can be tested in cell cultures, they as well as those agents aimed at the tumor matrix or angiogenesis require a solid tumor model for proof of principle studies. Solid tumors require a host, typically nude mice, to grow and be imaged. The handling, particularly during imaging, and the cost of animals can become prohibitive when screening potential agents. We propose an inexpensive model to screen new agents. The chick embryo is immunologically naı¨ve. Its vascularized chorioallantoic membrane (CAM) responds to angiogenic stimulation to provide vessels and nutrients to implanted tumor cells. The purpose of this study is to assess the feasibility of the CAM model, for contrast administeration, and tumor imaging before and after contrast administration.
METHODS AND MATERIALS
Figure 1. Ionic relaxivity of Gd-DTPA-BOA (DTPA), Gd-MeODOTA-PE (DOTA) and Gd-MeO-DOTA-Triglycene-PE (DOTA-PE) paramagnetic nanoparticles at 0.47 Tesla.
The CAM of 10-day-old chick embryos were exposed and 1.5x107 human HT29 colon carcinoma cells placed on the CAM surface. After eight days, the resulting tumor was removed, chopped, and transferred to the CAM of a second 10-day-old chick embryo. Approximately 6 days later, eggs were imaged using a 1.5T Siemens Symphony
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because of their negative surface charges. These particles are thus particularly well suited for cellular imaging in vivo. The aim of this study was to image splenic uptake of labelled lymphocytes, using hybridomas as a model on a 1.5 T clinical magnet.
MATERIALS & METHODS Cell labelling: Hybridomas cells were cultivated under standard conditions and incubated with the anionic particles composed of a maghemite core (␥-Fe2O3) with anionic citrate molecules adsorbed on the surface. —Time response study : Iron load was quantified at several time points (0, 10, 20, 60, 120, 240, 360 min) after incubation with the particles at a concentration of 1 and 15 mM. —Concentration response: Iron load was quantified at 20 and 120 min after incubation with different concentrations of nanoparticles of 0, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 15 mM. Iron quantification: All samples were measured both by Magnetophoresis (MP) and Electron Spin Resonance (ESR). MP measures the velocity of labelled cells submitted to a magnetic field gradient, which is related to the number of magnetic particles, and thus to the iron content. ESR is a more sensitive method. The ESR signal is obtained by sweeping the static field H and recording the microwave absorption for an excitation field at 9.2 GHz. MRI: Male Swiss Nude mice were injected intraperitoneally with 20 millions labelled hybridomas (2.5 pg Fe/ cell). MRI (1.5 Tesla GE, Buc, France) was performed 24 h later using Spin Echo (TR⫽2000 ms, TE⫽ 20 and 60 ms) and Gradient Echo (TR⫽20 ms, TE⫽4 ms, 30°) sequences. Nine mice were injected with labelled cells, and 7 others were used as controls.
RESULTS: MP and ESR gave similar results under all conditions. A maximum uptake was observed after 90 min in the time response study, and at 6 mM in the concentration response study, with a saturation effect in all cases.
S40
Academic Radiology, Vol 12, Suppl 1, May 2005
At MRI, a negative enhancement of the spleen was observed on T2 60 ms (⫺37%) and GRE (⫺16%) images, with a heterogeneous response among animals. When excluding the 3 non-enhancing spleens, maximum decrease could be observed up to ⫺45% on T2 and ⫺28% on GRE.
CONCLUSION: Anionic particles can efficiently label hybridomas, which become detectable in the spleen in vivo at 1.5 T. Such an approach could be extended to other cell types and used for therapeutic purposes. Improved Paramagnetic Chelate for Molecular Imaging with MRI Patrick M. Winter1, Phillip S. Athey2, Gary E. Kiefer2, Gyongyi Gulyas2, Ralph W. Fuhrhop1, J. David Robertson3, Samuel A. Wickline1, and Gregory M. Lanza1 1Washington University, St. Louis, MO; 2Dow Chemical Co., Freeport, TX; 3University of Missouri Research Reactor, Columbia, MO
Sensitive molecular imaging of sparse biochemical epitopes with magnetic resonance imaging (MRI) requires a targeted contrast agent with enormous paramagnetic influence to overcome partial-volume dilution. Tens of thousands of gadolinium chelates can be delivered to each binding site with lipid encapsulated perfluorocarbon nanoparticles, providing adequate T1-weighted contrast to detect microthrombi on ruptured plaques and cell surface receptors. These contrast agents can be improved, however, by optimizing the intrinsic relaxivity and transmetalation of the gadolinium chelate. The objective of this study was to evaluate three paramagnetic chelates for use as targeted MRI contrast agents. Paramagnetic nanoparticles were produced by microfluidization incorporating one of three different chelates onto the lipid surface (Gd-DTPA-BOA, Gd-MeODOTA-PE or Gd-MeO-DOTA-Triglycine-PE). Nanoparticles were diluted with distilled deionized water and proton longitudinal relaxation rates (1/T1) of each sample were measured at 40° C and a field strength of 0.47 Tesla. The [Gd3⫹] of each sample was measured via neutron activation techniques, while the concentration of nanoparticles was calculated from the known amount of perfluorocarbon in the formulation and the nominal particle size determined by elastic laser light
BRIEF ABSTRACTS
Academic Radiology, Vol 12, Suppl 1, May 2005
scattering. The T1 relaxivity (r1) was determined from the slope of the linear least-squares regression of longitudinal relaxation rate versus Gd3⫹ (i.e., ion relaxivity) or nanoparticle (i.e., particle relaxivity) concentrations and are reported in units of (s⫺1mM)⫺1. The transmetalation was determined at 40° C and 0.47 Tesla using ZnCl as the competing ionic species. Gd-DTPA-BOA nanoparticles had higher ionic relaxivity (21.3 s⫺1mM⫺1) than typical MRI contrast agents, such as Gd-DTPA (⬃4 s⫺1mM⫺1). Because each particle caries more than 30,000 Gd3⫹ chelates, the particle-based relaxivity is orders of magnitude higher than any other paramagnetic contrast agent (700,000 s⫺1mM⫺1). Further improvements in ionic and particle-based relaxivity were observed with the GdMeO-DOTA-PE (29.8 and 979,000 s⫺1mM⫺1, respectively) and Gd-MeO-DOTA-Triglycine-PE (33.0 and 1,080,000 s⫺1mM⫺1, respectively) nanoparticles. The transmetalation of the DOTA chelates was also much more favorable than the DTPA chelate. Extrapolating to the amount of Gd3⫹ still bound to the chelate at time equal to infinity, the Gd-DTPA-BOA nanoparticles only retain 75.2%, while the Gd-MeO-DOTA-PE and Gd-MeO-DOTA-Triglycine-PE nanoparticles retain 91.2% and 90.6%, respectively. These results demonstrate the importance of paramagnetic chelates for optimizing molecular imaging contrast agents for MRI. The DOTA chelates demonstrated 40 – 55% higher relaxivity and ⬃20% lower transmetalation. These findings agree with well-documented advantages of DOTA chelates over DTPA chelates. In addition, the use of the PE moiety may improve water interaction in the DOTA chelates compared to the BOA utilized for the DTPA chelate. The insertion of the triglycine linker may also improve water interaction leading to increased relaxivity. In the context of molecular imaging, these improvements can lead to more sensitive detection of pathology and improved safety of the contrast agents.
Figure 2. Transmetalation of Gd-DTPA-BOA (DTPA), Gd-MeODOTA-PE (DOTA) and Gd-MeO-DOTA-Triglycene-PE (DOTA-PE) paramagnetic nanoparticles.
An Inexpensive Vascularized Tumor Model for Vascular Imaging Max Wu, PhD1, Jacqueline Corbeil1, Judith Varner, PhD2, David Vera, PhD1, Robert Mattrey, MD1 University of California, San Diego Departments of Radiology (1) and Medicine (2).
PURPOSE: Although many molecular imaging agents, particularly those aimed at imaging intracellular or cellular targets, can be tested in cell cultures, they as well as those agents aimed at the tumor matrix or angiogenesis require a solid tumor model for proof of principle studies. Solid tumors require a host, typically nude mice, to grow and be imaged. The handling, particularly during imaging, and the cost of animals can become prohibitive when screening potential agents. We propose an inexpensive model to screen new agents. The chick embryo is immunologically naı¨ve. Its vascularized chorioallantoic membrane (CAM) responds to angiogenic stimulation to provide vessels and nutrients to implanted tumor cells. The purpose of this study is to assess the feasibility of the CAM model, for contrast administeration, and tumor imaging before and after contrast administration.
METHODS AND MATERIALS
Figure 1. Ionic relaxivity of Gd-DTPA-BOA (DTPA), Gd-MeODOTA-PE (DOTA) and Gd-MeO-DOTA-Triglycene-PE (DOTA-PE) paramagnetic nanoparticles at 0.47 Tesla.
The CAM of 10-day-old chick embryos were exposed and 1.5x107 human HT29 colon carcinoma cells placed on the CAM surface. After eight days, the resulting tumor was removed, chopped, and transferred to the CAM of a second 10-day-old chick embryo. Approximately 6 days later, eggs were imaged using a 1.5T Siemens Symphony
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