2.1. Target Fabrication and Irradiation
After the careful adjustment of the beam intensity and the beam profile to the currently used conditions, the produced barium-131 shows reproducible activity values in the expected quantity. The used 80 mg of CsCl target material corresponds to an area density of 70 mg/cm
2. The energy lost was calculated by SRIM (
www.srim.org), and it is 840 keV within the CsCl. Hence, the used energy range of 27.5–26.7 MeV is slightly below the optimal energy window (29–28 MeV) due to the current design of the solid target unit with its energy degrader. The small layer thickness is determined by the low thermal conductivity of the cesium salt and best possible avoidance of the corresponding (
p,
n)- and (
p,
2n)-reactions to stable barium-132 and long-lived barium-133.
The saturation yield of the barium-131 production for a thick target is 9.1·10
3 MBq/µA (
https://www-nds.iaea.org/radionuclides/csp31ba0.html). A maximal activity of 1.4 GBq could be achieved with a thick target and the used irradiation parameters (4 h, 15 µA). To estimate the maximal producible activity with our target condition, we estimated the activity of our target thickness as follows. The cross section for the
133Cs(
p,
3n)
131Ba reaction is shown in
Figure 3. The thick target yield is based on the cross section of the complete energy range from 0 to 27.5 MeV. Only the area between 26.7 to 27.5 MeV is used for the nuclear reaction in the used target. Hence, the activity for a thick target has to be scaled by the ratio of the integral of the cross section between the used energy range and the whole energy range. A theoretical maximum activity of 320 MBq could be achieved with the used target and irradiation parameters using Equation (1).
An influence of moisture on the target was not observed in the short time between preparation and irradiation. Except darkening in the irradiated area, the pressed cesium salt shows no significant changes after irradiation. The dark color disappears immediately during the addition of nitric acid. For further irradiations, a second solid target system without energy degrader and another careful increase in intensity seems to be possible. The target current can be increased by a factor of 2 without increasing the power density on the target by using the 30° solid target holder. Hence, the amount of 131Ba is possible to be more than doubled within the same irradiation time.
2.2. Barium-131 Purification
In average, a total barium-131 activity of (190 ± 26) MBq was obtained, as determined within four equal and independent irradiations analyzing the crude target solution. This corresponds to a saturation yield of (1.2 ± 0.2) GBq/µA. An energy of 0.84 MeV is absorbed in the CsCl layer of the target. Considering the thick target yield, a maximum activity of 320 MBq can be expected with the used beam parameters. Moreover, directly after the end of bombardment (EOB), the radionuclides chlorine-34m, cesium-131, cesium-132, barium-131m, barium-131, and barium-133m were detected. After two days of decay time for the short-lived side products, mainly chlorine-34m (approx. 5.7 GBq) and several co-produced gold isotopes in the Pt target backing, the target was worked-up to isolate the barium isotopes from the bulk cesium salt as well as the radioactive isotopes cesium-131/-132. Therefore, Sr Resin was used applying the method described in
Section 3.4. A barium-131 recovery of (80 ± 8) % of the initially measured barium-131 activity (directly before the work-up) was achieved after purification. The obtained elution profile of cesium and barium, portrayed by the volume-dependent activity of barium-131 and cesium-131 is displayed in
Figure 4.
Since the separation was completed, cesium was not detectable anymore in the product solution, and only barium-131 and barium-133m were found, whereby barium-133m is responsible for the formation of the isotopic barium impurity barium-133. Thereby we conclude, that our applied work-up method is applicable for removing both the bulk natural cesium and the cesium radioisotope impurities.
One of our major obstacles is the amount of barium-133 impurity, so the calculation of the barium-131/-133 ratio is necessary. Consequently, retention samples were measured three months after EOB, and the activity ratio was calculated to be approx. 7∙103 to 1 for all samples. This means that about 1 kBq of barium-133 is produced for each 7 MBq of barium-131, which corresponds with the injection dose for one mouse. We assume that roughly 100 MBq of barium-131 would be needed for human imaging in the human situation, leading to a coincident injection of 15 kBq barium-133 impurity. Taking into account that more than 80% of the injected dose should have been excreted within 24 h, less than 3 kBq barium-133 remains (same range as average kalium-40-content in the human body), which is possibly important for dosimetric assessment. Barium-133 excretes via the same metabolic pathway as barium-131 due to the same physiological behavior, which reduces its amount noticeably.
2.3. Radiolabeling
For quality control studies, radio-TLCs were prepared with the
131Ba- and
131Cs-stock solutions to visualize the eligibility of the above-mentioned separation method in addition to the quantification via γ-spectroscopy. Therefore, two different TLC systems, reverse and normal phase (RP/NP), were chosen. The NP system is based on a silica solid phase. The free radiometal ions in the stock solutions move along with the mobile phase (50 mM EDTA) as EDTA complexes (cf.
Figure 5A,B). The R
f values for Ba
2+ and Cs
+ are determined to be 0.35 and 0.71, respectively. The pH value of the mobile phase was chosen to be 7, close to the physiological pH value, but also low enough not to form insoluble barium hydroxy species.
Additionally, a RP system was used to immobilize free ions at the baseline (cf.
Figure 5C,D). Due to the high acetonitrile content of the mobile phase (70 vol%), it is optimal to separate free metal ions and the radiolabeled organic ligand, which will move along with the mobile phase.
The radiolabeling with barium-131 and cesium-131 was investigated using the complexing agent macropa, a well-known ligand from the literature. Anyhow, only a few approaches with macropa and alkaline earth metal ions have been published so far [
22,
23]. Showing the HPLC stability for the macropa complex is challenging (obviously depending on stability issues in highly diluted systems), which is why we have developed the above mentioned two different TLC systems, one for providing information about the complexation of free barium, the other one for showing that only one reaction took place by having only one signal at the TLC plate. The nonradioactive Ba-macropa complex was fully characterized by Wilson and co-workers [
23]. Both stock solutions (
131Ba and
131Cs) were used for radiolabeling, which was performed according to the conditions described in
Section 3.5.
An efficient labeling was determined for macropa up to a concentration of 10
−4 M. The desired
131Ba complex was verified by the RP and NP TLC system. Considering the high amount of the competitive EDTA chelator in the NP system and the weak acceptor and donor properties of heavy alkaline earth metals, this is a satisfying result. Due to the promising radiolabeling with macropa, the
131Ba-macropa complex was tested against human serum. No release of the radiometal was determined by RP TLC within three days, even at a high serum concentration of 80 vol% (cf.
Figure 6A–C). Nevertheless, due to the high concentration of the competing chelator EDTA of 50 mM in the NP TLC system (cf.
Figure 6E–G), a slight dissociation of the complex was observed. However, both TLC systems proved a sufficient plasma stability of the
131Ba-macropa complex. The labeling of macropa with cesium-131, performed under equal conditions, did not lead to any TLC-detectable complex formation.
Furthermore, adsorption studies of barium 131 onto hydroxyapatite were performed, showing a rapid and efficient binding of [131Ba]Ba2+ with >99% within 5 min, which led us to the assumption that a rapid in vivo bone uptake will occur. After seven days, this 131Ba-labeled hydroxyapatite sample was centrifuged and the pellet was washed twice. The released cesium-131 was detected in the washing solutions, meaning, that there is no affinity of bone-like materials against the released cesium, even if the mother radionuclide has been stably captured. In contrast, once the barium-131 was stably bound to macropa, no adsorption onto hydroxyapatite was detected. From this observation, we conclude that this could be an important finding towards use of barium-131 in a non-calcimimetic way. Using the 131Cs-stock solution, no interaction with hydroxyapatite was detected.
2.4. SPECT Imaging
To select suitable photon energy windows for SPECT imaging of barium-131, an energy spectrum was recorded without multi-pinhole collimation, showing two suitable photopeaks at 124 keV and 216 keV, as well as two prominent high-energy peaks at 371 keV and 496 keV (
Figure 7A). However, installing the aperture with the multi-pinhole collimators, providing special information, decreased the relative heights of the suggested photopeaks compared to the high-energy peaks, and added considerable noise to the suggested photopeak windows (
Figure 7B). Most likely, these effects occurred since the attenuation of the high-energy photons in the collimator material is much less compared to the lower-energy photons within the photopeak windows.
Such effects have already been reported for iodine-123, a SPECT radionuclide that, besides the primary photopeak at 159 keV (83%), also emits high-energy photons, most abundantly at 529 keV (1.4%). Many of these high-energy photons are assumed to pass through the crystals of the SPECT camera and undergo Compton scattering from the components adjacent to the crystals (e.g., photo-multiplier tubes and associated electronics). These inelastically scattered photons are then detected by re-entering the crystal with a lower energy contaminating the projection data recorded within the photopeak windows [
29].
For barium-131, the percentage of high-energy photons, in particular at 371 keV (14%) and 496 keV (48%), and their contribution to image blurring and quantification errors is much higher compared to iodine-123. Due to the high intensity of noise interfering in particular with the 216 keV photopeak window, investigations on SPECT imaging of barium-131 were performed, recording only the 124 keV photopeak window.
SPECT imaging of a cylindrical syringe source filled with an aqueous solution of [
131Ba]Ba(NO
3)
2 showed that signals at the position of the source were surrounded by noise-derived artifacts accumulating in the periphery of the field of view (FOV) (peripheral artifacts) as well as along the central anterior–posterior axis of the FOV (central artifacts) (
Figure 7C). Such artifacts appear because the physical model used in the Tera-Tomo 3D reconstruction algorithm is inconsistent with the contaminated barium-131 projection data. Since there is less measured information content in the periphery of the FOV, it is more affected by this inconsistency, hence, stronger artefacts appear. Quantitative analyses showed that 52% of total signal intensity was located at the position of the source, whereas the remaining 48% were located within the ring of peripheral artifacts. Furthermore, voxel intensity profiles, measured across the central coronal slice image, showed inhomogeneity across the center of the source with variation up to 25%, due to overlap with the central artifacts (
Figure 7D). Moreover, the correlation analysis showed a significant positive relationship between voxel intensities of the source and the voxel intensities of peripheral artifacts (
rp = 0.79,
p < 0.001). These results demonstrate that SPECT imaging enables visualizing barium-131, with considerable limitations due to artifacts, deriving from high-energy photon emission. Use of high-energy collimators may allow for overcoming these limitations in future investigations. Until then, quantitative investigations on barium-131 in vivo rely on alternative techniques, such as activity measurement in tissue samples.
Despite the limitations regarding the SPECT imaging performance of barium-131, small animal SPECT/CT (computed tomography) imaging followed by removal of peripheral artifacts during post-processing provided sufficient image quality for visualizing the distribution of [
131Ba]Ba(NO
3)
2 and
131Ba labeled macropa in mice (
Figure 8A,D). After intravenous injection of [
131Ba]Ba(NO
3)
2 in mice, small animal SPECT/CT imaging showed an accumulation of barium-131 in the entire skeleton that established within 1 h after injection, and was still present after 24 h (
Figure 8A). Barium-131 showed the highest activity concentration in particular in bone regions with high metabolic activity such as epiphyses, vertebral bodies, and the upper jaw. Activity measurement in tissue samples confirmed the distribution pattern of [
131Ba]Ba(NO
3)
2 observed in SPECT images showing that barium-131 was excreted predominantly via the renal pathway (57% ID/organ within 24 h) and, to a lesser extent, also via the hepatobiliary pathway (14% ID/organ within 1 h) (
Figure 8B).
Organ distribution showed a rapid blood clearance and confirmed a rapid bone uptake of [
131Ba]Ba(NO
3)
2 as occurred in the femur within 5 min (SUV = 2.02), increased until 1 h (SUV = 2.94), and decreased again until 24 h (SUV = 2.41) (
Figure 8C). Activity levels of [
131Ba]Ba(NO
3)
2 accumulating in bones of mice are in accordance with previous reports [
2]. Similar to barium-131, bone accumulation in mice has also been reported for radium-223 after injection of [
223Ra]RaCl
2, with highest activity concentrations observed within the inorganic bone matrix of the epiphyses [
30]. For subsequent removal of barium-131 from the skeleton, several mechanisms have been discussed previously, such as physiological bone resorption, as well as successive replacement of barium by calcium followed by diffusion of the released barium ion through the calcified matrix, with some prospect of re-entry into blood vessels [
31,
32].
A rapid uptake of barium-131 is also observed in the thyroid within 5 min (SUV = 2.08), increased until 1 h (SUV = 2.44), and decreased again until 24 h (SUV = 1.38). This uptake possibly occurs through the sodium iodine symporter (NIS) system. Similar to [
131Ba]Ba(NO
3)
2, thyroid accumulation has been reported for radium-226 in humans [
33]. Otherwise, barium-131 may also have accumulated in parathyroid, a pair of small but important endocrine glands on both sides of the thyroid that synthesize parathyroid hormone regulating mainly phosphoric calcium metabolism. Parathyroid glands have been reported to accumulate isotopes of cesium, strontium, and iodine [
34]. However, the small organ size in mice does not allow for measuring the activity in tissue samples of the parathyroid separately.
After intravenous injection of
131Ba-labeled macropa in mice, small animal SPECT/CT imaging showed rapid passage of the radiolabeled complex through gallbladder and intestine within 1 h after injection, followed by only some accumulation in the skeleton and bigger joints, detectable after 24 h (
Figure 8D). Activity measurement in tissue samples confirmed the distribution pattern observed in SPECT images showing that, compared to [
131Ba]Ba(NO
3)
2, significantly (
p < 0.001) higher amounts of
131Ba-labeled macropa were excreted via the renal pathway (89% ID/organ within 24 h) and also via the hepatobiliary pathway (25% ID/organ within 1 h) (
Figure 8E).
Organ distribution showed rapid clearance of
131Ba-labeled macropa from blood and significantly (
p < 0.001) lower accumulation in bones compared to [
131Ba]Ba(NO
3)
2, as occurred in the femur within 5 min (SUV = 0.53) and slightly increased within 24 h (SUV = 0.65) (
Figure 8F). Furthermore, injection of
131Ba-labeled macropa resulted in significantly (
p < 0.001) lower accumulation of barium-131 in the thyroid or parathyroid compared to [
131Ba]Ba(NO
3)
2. These results demonstrate a striking difference of biodistribution behavior between
131Ba-labeled macropa and free [
131Ba]Ba
2+. The radiolabeled complex accumulates barely in bones and the thyroid/parathyroid. Especially the femoral uptake with 2.19%IA/g after 24 h for [
133Ba]Ba-macropa is comparable with [
223Ra]Ra-macropa (1.6%IA/g after 24 h), shown previously [
24]. These findings lead to the conclusion that barium-131 is a perfect radionuclide for a matched pair with radium-223/-224.