Introduction The goal of this work is to advance the production and use of 52Mn (t½ = 5.6 d, β+: 242 keV, 29.6%) as a radioisotope for in vivo preclinical nuclear imaging. More specifically, the aims of this study were: (1) to measure the excitation function for the natCr(p,n)52Mn reaction at low energies to verify past results [1–4]; (2) to measure binding constants of Mn(II) to aid the design of a method for isolation of Mn from an irradiated Cr target via ion-exchange chromatography, building upon previously published methods [1,2,5–7]; and (3) to perform phantom imaging by positron emission tomography/magnetic resonance (PET/MR) imaging with 52Mn and non-radioactive Mn(II), since Mn has potential dual-modality benefits that are beginning to be investigated [8]. Material and Methods Thin foils of Cr metal are not available commercially, so we fabricated these in a manner similar to that reported by Tanaka and Furukawa [9]. natCr was electroplated onto Cu discs in an industrial-scale electroplating bath, and then the Cu backing was digested by nitric acid (HNO3). The remaining thin Cr discs (~1 cm diameter) were weighed to determine their thickness (~ 75–85 μm) and arranged into stacked foil targets, along with ~25 μm thick Cu monitor foils. These targets were bombarded with ~15 MeV protons for 1–2 min at ~1–2 μA from a CS-15 cyclotron (The Cyclotron Corporation, Berkeley, CA, USA). The beamline was perpendicular to the foils, which were held in a machined 6061-T6 aluminum alloy target holder. The target holder was mounted in a solid target station with front cooling by a jet of He gas and rear cooling by circulating chilled water (T ≈ 2–5 °C). Following bombardment, these targets were disassembled and the radioisotope products in each foil were counted using a high-purity Ge (HPGe) detector. Cross-sections were calculated for the natCr(p,n)52Mn reaction. Binding constants of Mn(II) were measured by incubating 54Mn(II) (t½ = 312 d) dichloride with anion- or cation-exchange resin (AG 1-X8 (Cl− form) or AG 50W-X8 (H+ form), respectively; both: 200–400 mesh; Bio-Rad, Hercules, CA) in hydrochloric acid (HCl) ranging from 10 mM-8 M (anion-exchange) and from 1 mM-1 M (cation-exchange) or in sulfuric acid (H2SO4) ranging from 10 mM-8 M on cation-exchange resin only. The amount of unbound 54Mn(II) was measured using a gamma counter, and binding constants (KD) were calculated for the various concentrations on both types of ion-exchange resin. We have used the unseparated product for preliminary PET and PET/MR imaging. natCr metal was bombarded and then digested in HCl, resulting in a solution of Cr(III)Cl3 and 52Mn(II)Cl2. This solution was diluted and imaged in a glass scintillation vial using a microPET (Siemens, Munich, Germany) small animal PET scanner. The signal was corrected for abundant cascade gamma-radiation from 52Mn that could cause random false coincidence events to be detected, and then the image was reconstructed by filtered back-projection. Additionally, we have used the digested target to spike non-radioactive Mn(II)Cl2 solutions for simultaneous PET/MR phantom imaging using a Biograph mMR (Siemens) clinical scanner. The phantom consisted of a 4×4 matrix of 15 mL conical tubes containing 10 mL each of 0, 0.5, 1.0, and 2.0 mM concentrations of non-radioactive Mn(II)Cl2 with 0, 7, 14, and 27 μCi (at start of PET acquisition) of 52Mn(II)Cl2 from the digested target added. The concentrations were based on previous MR studies that measured spin-lattice relaxation time (T1) versus concentration of Mn(II), and the activities were based on calculations for predicted count rate in the scanner. The PET/MR imaging consisted of a series of two-dimensional inversion-recovery turbo spin echo (2D-IR-TSE) MR sequences (TE = 12 ms; TR = 3,000 ms) with a wide range of inversion times (TI) from 23–2,930 ms with real-component acquisition, as well as a 30 min. list-mode PET acquisition that was reconstructed as one static frame by 3-D ordered subset expectation maximization (3D-OSEM). Attenuation correction was performed based on a two-point Dixon (2PD) MR sequence. The DICOM image files were loaded, co-registered, and windowed using the Inveon Research Workplace software (Siemens).

Introduction 52Mn (t½ =5.59 d, β+ = 29.6%, Eβmax = 0.58 MeV) has great potential as a long lived PET isotope for use in cell tracking studies, observation of immunologic response to disease states, or as an alternative to manganese-based MRI contrast agents. Its favorable max positron energy leads to superb imaging resolution, comparable to that of 18F.[1] Manganese is naturally taken up by cells via a multitude of pathways including the divalent metal transporter (DMT1), ZIP8, transferrin receptors (TfR), store-operated Ca2+ channels (SOC-Ca2+), and ionotropic glutamate receptor Ca2+ channels (GluR).[2] These natural transport mechanisms make 52Mn an attractive isotope for applications necessitating non-perturbative cell uptake. In particular, cell tracking is critical to the development and translation of stem cell therapies in regenerative medicine. Alternative-ly, 52Mn could be used in immunotherapy techniques such as adoptive cellular therapy (ACT) to evaluate the ability of external immune cells to reach their intended target. Material and Methods 52Mn was produced by natCr(p,x)52Mn using 16 MeV protons. The average thick target production yield was 0.23 mCi/µA-h with less than 0.25% co-production of 54Mn. Small amounts of 51Cr were observed in the target, but were absent from the radiochemically separated product. Target construction consisted of a water jet cooled chromium disc (3/4” diameter, 0.4” thick). Targets were purchased from Kamis Inc, and are 99.95% pure. Targets withstood beam currents of 30 µA with no visible aberration. Chromium targets were etched by concentrated HCl following bombardment. Mn2+ ions were extracted from 9M HCl to 0.8M trioctylamine in cyclohexane leaving the bulk chromium in the aqueous phase. After isolating the organic phase, 0.001M NH4OH was used to back-extract the Mn2+ ions to aqueous phase. This purification cycle was conducted a total of three times for each 52Mn production. Results and Conclusion For a starting bulk chromium mass of 456 ± 1 mg, a post-separation chromium mass of 5.35 ± 0.04 ng was measured by microwave plasma atomic emission spectrometry (MP-AES). This mass reduction corresponds to an average separation factor of 440 for a single purification cycle. Each purification cycle had a 52Mn recovery efficiency of 73 ± 7 % (n = 6), resulting in an overall separation efficiency of approximately 35 %. These efficiencies and separation factors agree reasonably well with the work conducted by Lahiri et. al.[3] Prior to use, the product was passed through a C-18 Sep-Pak to remove any residual organic phase. After four target irradiations and etchings, some pitting became noticeable on the target face. These have not yet compromised the o-ring seal with the target deplater, but it is possible that target replacement after every 6–9 52Mn productions will be necessary moving forward. Following the successful separation of 52Mn from chromium, in vitro experiments were conducted to demonstrate the uptake of 52Mn by human stem cells and mouse tumor cells. A linear uptake response was observed as a function of the amount of activity exposed to the cells for both cell models. These experiments have shown great promise for 52Mn as a long-lived PET isotope in cell tracking studies. Details will be presented.

Introduction The goal of this work is to advance the production and use of 52Mn (t½ = 5.6 d, β+: 242 keV, 29.6%) as a radioisotope for in vivo preclinical nuclear imaging. More specifically, the aims of this study were: (1) to measure the excitation function for the natCr(p,n)52Mn reaction at low energies to verify past results [1–4]; (2) to measure binding constants of Mn(II) to aid the design of a method for isolation of Mn from an irradiated Cr target via ion-exchange chromatography, building upon previously published methods [1,2,5–7]; and (3) to perform phantom imaging by positron emission tomography/magnetic resonance (PET/MR) imaging with 52Mn and non-radioactive Mn(II), since Mn has potential dual-modality benefits that are beginning to be investigated [8]. Material and Methods Thin foils of Cr metal are not available commercially, so we fabricated these in a manner similar to that reported by Tanaka and Furukawa [9]. natCr was electroplated onto Cu discs in an industrial-scale electroplating bath, and then the Cu backing was digested by nitric acid (HNO3). The remaining thin Cr discs (~1 cm diameter) were weighed to determine their thickness (~ 75–85 μm) and arranged into stacked foil targets, along with ~25 μm thick Cu monitor foils. These targets were bombarded with ~15 MeV protons for 1–2 min at ~1–2 μA from a CS-15 cyclotron (The Cyclotron Corporation, Berkeley, CA, USA). The beamline was perpendicular to the foils, which were held in a machined 6061-T6 aluminum alloy target holder. The target holder was mounted in a solid target station with front cooling by a jet of He gas and rear cooling by circulating chilled water (T ≈ 2–5 °C). Following bombardment, these targets were disassembled and the radioisotope products in each foil were counted using a high-purity Ge (HPGe) detector. Cross-sections were calculated for the natCr(p,n)52Mn reaction. Binding constants of Mn(II) were measured by incubating 54Mn(II) (t½ = 312 d) dichloride with anion- or cation-exchange resin (AG 1-X8 (Cl− form) or AG 50W-X8 (H+ form), respectively; both: 200–400 mesh; Bio-Rad, Hercules, CA) in hydrochloric acid (HCl) ranging from 10 mM-8 M (anion-exchange) and from 1 mM-1 M (cation-exchange) or in sulfuric acid (H2SO4) ranging from 10 mM-8 M on cation-exchange resin only. The amount of unbound 54Mn(II) was measured using a gamma counter, and binding constants (KD) were calculated for the various concentrations on both types of ion-exchange resin. We have used the unseparated product for preliminary PET and PET/MR imaging. natCr metal was bombarded and then digested in HCl, resulting in a solution of Cr(III)Cl3 and 52Mn(II)Cl2. This solution was diluted and imaged in a glass scintillation vial using a microPET (Siemens, Munich, Germany) small animal PET scanner. The signal was corrected for abundant cascade gamma-radiation from 52Mn that could cause random false coincidence events to be detected, and then the image was reconstructed by filtered back-projection. Additionally, we have used the digested target to spike non-radioactive Mn(II)Cl2 solutions for simultaneous PET/MR phantom imaging using a Biograph mMR (Siemens) clinical scanner. The phantom consisted of a 4×4 matrix of 15 mL conical tubes containing 10 mL each of 0, 0.5, 1.0, and 2.0 mM concentrations of non-radioactive Mn(II)Cl2 with 0, 7, 14, and 27 μCi (at start of PET acquisition) of 52Mn(II)Cl2 from the digested target added. The concentrations were based on previous MR studies that measured spin-lattice relaxation time (T1) versus concentration of Mn(II), and the activities were based on calculations for predicted count rate in the scanner. The PET/MR imaging consisted of a series of two-dimensional inversion-recovery turbo spin echo (2D-IR-TSE) MR sequences (TE = 12 ms; TR = 3,000 ms) with a wide range of inversion times (TI) from 23–2,930 ms with real-component acquisition, as well as a 30 min. list-mode PET acquisition that was reconstructed as one static frame by 3-D ordered subset expectation maximization (3D-OSEM). Attenuation correction was performed based on a two-point Dixon (2PD) MR sequence. The DICOM image files were loaded, co-registered, and windowed using the Inveon Research Workplace software (Siemens).

Introduction 52Mn (t½ =5.59 d, β+ = 29.6%, Eβmax = 0.58 MeV) has great potential as a long lived PET isotope for use in cell tracking studies, observation of immunologic response to disease states, or as an alternative to manganese-based MRI contrast agents. Its favorable max positron energy leads to superb imaging resolution, comparable to that of 18F.[1] Manganese is naturally taken up by cells via a multitude of pathways including the divalent metal transporter (DMT1), ZIP8, transferrin receptors (TfR), store-operated Ca2+ channels (SOC-Ca2+), and ionotropic glutamate receptor Ca2+ channels (GluR).[2] These natural transport mechanisms make 52Mn an attractive isotope for applications necessitating non-perturbative cell uptake. In particular, cell tracking is critical to the development and translation of stem cell therapies in regenerative medicine. Alternative-ly, 52Mn could be used in immunotherapy techniques such as adoptive cellular therapy (ACT) to evaluate the ability of external immune cells to reach their intended target. Material and Methods 52Mn was produced by natCr(p,x)52Mn using 16 MeV protons. The average thick target production yield was 0.23 mCi/µA-h with less than 0.25% co-production of 54Mn. Small amounts of 51Cr were observed in the target, but were absent from the radiochemically separated product. Target construction consisted of a water jet cooled chromium disc (3/4” diameter, 0.4” thick). Targets were purchased from Kamis Inc, and are 99.95% pure. Targets withstood beam currents of 30 µA with no visible aberration. Chromium targets were etched by concentrated HCl following bombardment. Mn2+ ions were extracted from 9M HCl to 0.8M trioctylamine in cyclohexane leaving the bulk chromium in the aqueous phase. After isolating the organic phase, 0.001M NH4OH was used to back-extract the Mn2+ ions to aqueous phase. This purification cycle was conducted a total of three times for each 52Mn production. Results and Conclusion For a starting bulk chromium mass of 456 ± 1 mg, a post-separation chromium mass of 5.35 ± 0.04 ng was measured by microwave plasma atomic emission spectrometry (MP-AES). This mass reduction corresponds to an average separation factor of 440 for a single purification cycle. Each purification cycle had a 52Mn recovery efficiency of 73 ± 7 % (n = 6), resulting in an overall separation efficiency of approximately 35 %. These efficiencies and separation factors agree reasonably well with the work conducted by Lahiri et. al.[3] Prior to use, the product was passed through a C-18 Sep-Pak to remove any residual organic phase. After four target irradiations and etchings, some pitting became noticeable on the target face. These have not yet compromised the o-ring seal with the target deplater, but it is possible that target replacement after every 6–9 52Mn productions will be necessary moving forward. Following the successful separation of 52Mn from chromium, in vitro experiments were conducted to demonstrate the uptake of 52Mn by human stem cells and mouse tumor cells. A linear uptake response was observed as a function of the amount of activity exposed to the cells for both cell models. These experiments have shown great promise for 52Mn as a long-lived PET isotope in cell tracking studies. Details will be presented.