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Abstract Background The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biannual highlight commentary to update the readership on trends in the field of radiopharmaceutical development. Main body This selection of highlights provides commentary on 19 different topics selected by each coauthoring Editorial Board member addressing a variety of aspects ranging from novel radiochemistry to first-in-human application of novel radiopharmaceuticals. Conclusion Trends in radiochemistry and radiopharmacy are highlighted. Hot topics cover the entire scope of EJNMMI Radiopharmacy and Chemistry, demonstrating the progress in the research field in many aspects.

Abstract This work aims at developing a diagnostic method based on Electron Paramagnetic Resonance (EPR) measurements of stable nitroxide radicals released from “EPR silent” liposomes. The liposome destabilisation and consequent radical release is enzymatically triggered by the action of phospholipase A2 (PLA2) present in the biological sample of interest. PLA2 are involved in a broad range of processes, and changes in their activity may be considered as a unique valuable biomarker for early diagnoses. The minimum amount of PLA2 measured “in vitro” was 0.09 U/mL. Moreover, the liposomes were successfully used to perform Overhauser-enhanced Magnetic Resonance Imaging (OMRI) in vitro at 0.2 T. The amount of radicals released by PLA2 driven liposome destabilization was sufficient to generate a well detectable contrast enhancement in the corresponding OMRI image.

Abstract Background The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biannual highlight commentary to update the readership on trends in the field of radiopharmaceutical development. Main Body This selection of highlights provides commentary on 21 different topics selected by each coauthoring Editorial Board member addressing a variety of aspects ranging from novel radiochemistry to first-in-human application of novel radiopharmaceuticals. Conclusion Trends in radiochemistry and radiopharmacy are highlighted. Hot topics cover the entire scope of EJNMMI Radiopharmacy and Chemistry, demonstrating the progress in the research field, and include new PET-labelling methods for 11C and 18F, the importance of choosing the proper chelator for a given radioactive metal ion, implications of total body PET on use of radiopharmaceuticals, legislation issues and radionuclide therapy including the emerging role of 161Tb.

Proton transporters play a key role in maintaining the acidic tumor microenvironment; hence, their inhibition has been proposed as a new therapeutic treatment, although few methods can accurately assess their effect in vivo. In this study, we investigated whether MRI-CEST (Magnetic Resonance Imaging—Chemical Exchange Saturation Transfer) tumor pH imaging can be a useful tool to evaluate in vivo the therapeutic efficacy of several Proton Pump Inhibitors (PPIs) in breast cancer. Cell viability and extracellular pH assays were carried out in breast cancer cells cultured at physiological pH (7.4) or acid-adapted (pH of 6.5 and 6.8) following the exposure to inhibitors of V-ATPase (Lansoprazole, Esomeprazole) or NHE1 (Amiloride, Cariporide) at several concentrations. Next, triple-negative breast cancer 4T1 tumor-bearing mice were treated with Lansoprazole or Amiloride and MRI-CEST tumor pH imaging was utilized to assess the in vivo efficacy. Only Lansoprazole induced, in addition to breast cancer cell toxicity, a significant inhibition of proton extrusion. A significant reduction in tumor volume, prolonged survival, and increase in extracellular tumor pH after 1 and 2 weeks were observed after Lansoprazole treatment, whereas no significant changes were detected upon Amiloride treatment. Our results suggested that MRI-CEST tumor pH imaging can monitor the therapeutic efficacy of PPIs in breast cancer murine models.

The carbonic anhydrase isoform IX (hCAIX) is one of the main players in extracellular tumor pH regulation, and it is known to be overexpressed in breast cancer and other common tumors. hCA IX supports the growth and survival of tumor cells, and its expression is correlated with metastasis and resistance to therapies, making it an interesting biomarker for diagnosis and therapy. The aim of this work deals with the development of an MRI imaging probe able to target the extracellular non-catalytic proteoglycan-like (PG) domain of CAIX. For this purpose, a specific nanoprobe, LIP_PepC, was designed by conjugating a peptidic interactor of the PG domain on the surface of a liposome loaded with Gd-bearing contrast agents. A Mouse Mammary Adenocarcinoma Cell Line (TS/A) was chosen as an in vitro breast cancer model to test the developed probe. MRI results showed a high selectivity and sensitivity of the imaging probe toward hCAI-expressing TS/A cells. This approach appears highly promising for the in vivo translation of a diagnostic procedure based on the targeting of hCA IX enzyme expression.

Abstract Background The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biyearly highlight commentary to update the readership on trends in the field of radiopharmaceutical development. Results This commentary of highlights has resulted in 23 different topics selected by each member of the Editorial Board addressing a variety of aspects ranging from novel radiochemistry to first in man application of novel radiopharmaceuticals. Conclusion Trends in radiochemistry and radiopharmacy are highlighted demonstrating the progress in the research field being the scope of EJNMMI Radiopharmacy and Chemistry.

This study aims to explore whether the water exchange rate constants in tumor cells can act as a hallmark of pathology status and a reporter of therapeutic outcomes. It has been shown, using 4T1 cell cultures and murine allografts, that an early assessment of the therapeutic effect of doxorubicin can be detected through changes in the cellular water efflux rate constant kio. The latter has been estimated by analyzing the magnetization recovery curve in standard NMR T1 measurements when there is a marked difference in the proton relaxation rate constants (R1) between the intra- and the extra-cellular compartments. In cellular studies, T1 measurements were carried out on a relaxometer working at 0.5 T, and the required difference in R1 between the two compartments was achieved via the addition of a paramagnetic agent into the extracellular compartment. For in-vivo experiments, the large difference in the R1 values of the two-compartments was achieved when the T1 measurements were carried out at low magnetic field strengths. This task was accomplished using a Fast Field Cycling (FFC) relaxometer that was properly modified to host a mouse in its probe head. The decrease in kio upon the administration of doxorubicin is the result of the decreased activity of Na+/K+-ATPase, as shown in an independent test on the cellular uptake of Rb ions. The results reported herein suggest that kio can be considered a non-invasive, early and predictive biomarker for the identification of responsive patients immediately from the first doxorubicin treatment.

Novel anticancer treatments target the pH regulating system that plays a major role in tumor progression by creating an acidic microenvironment, although few studies have addressed their effect on tumor acidosis. In this study, we investigated in vivo several proton pump inhibitors (PPIs) targeting NHE-1 (Amiloride and Cariporide) and V-ATPase (Esomeprazole and Lansoprazole) proton transporters in the DU145 androgen-insensitive human prostate cancer model. In cellulo results showed that DU145 are sensitive, with decreasing efficacy, to Amiloride, Esomeprazole and Lansoprazole, with marked cell toxicity both in normoxia and in hypoxia, with almost any change in pH. In vivo studies were performed upon administration of Esomeprazole to assess both the acute and chronic effects, and Iopamidol-based tumor pH imaging was performed to evaluate tumor acidosis. Although statistically significant tumor pH changes were observed a few hours after Esomeprazole administration in both the acute study and up to one week of treatment in the chronic study, longer treatment resulted in a lack of changes in tumor acidosis, which was associated to similar tumor growth curves between treated and control groups in both the subcutaneous and orthotopic models. Overall, this study highlights MRI-CEST tumor pH imaging as a valid approach to monitoring treatment response to PPIs.

Nuclear Magnetic Resonance allows the non-invasive detection and quantitation of metabolites to be carried out in cells and tissues. This means that that metabolic changes can be revealed without the need for sample processing and the destruction of the biological matrix. The main limitation to the application of this method to biological studies is its intrinsic low sensitivity. The introduction of hyperpolarization techniques and, in particular, of dissolution-Dynamic Nuclear Polarization (d-DNP) and ParaHydrogen Induced Polarization (PHIP) is a significant breakthrough for the field as the MR signals of molecules and, most importantly, metabolites, can be increased by some orders of magnitude. Hyperpolarized pyruvate is the metabolite that has been most widely used for the investigation of metabolic alterations in cancer and other diseases. Although d-DNP is currently the gold-standard hyperpolarization method, its high costs and intrinsically slow hyperpolarization procedure are a hurdle to the application of this tool. However, PHIP is cost effective and fast and hyperpolarized pyruvate can be obtained using the so-called Side Arm Hydrogenation approach (PHIP-SAH). The potential toxicity of a solution of the hyperpolarized metabolite that is obtained in this way is presented herein. HP pyruvate has then been used for metabolic studies on different prostate cancer cells lines (DU145, PC3, and LnCap). The results obtained using the HP metabolite have been compared with those from conventional biochemical assays.

8-Hydroxypyrene-1,3,6-trisulfonate (HPTS) is a small, hydrophilic fluorescent molecule. Since the pKa of the hydroxyl group is close to neutrality and quickly responds to pH changes, it is widely used as a pH-reporter in cell biology for measurements of intracellular pH. HPTS fluorescence (both excitation and emission spectra) at variable pH was measured in pure water in the presence of NaCl solution or in the presence of different buffers (PBS or hepes in the presence or not of NaCl) and in a solution containing BSA. pKa values have been obtained from the sigmoidal curves. Herein, we investigated the effect of mono-, di-, and trivalent cations (Na+, Ca2+, La3+, Gd3+) on fluorescence changes and proposed its use for the quantification of trivalent cations (e.g., gadolinium ions) present in solution as acqua-ions. Starting from the linear regression, the LoD value of 6.32 µM for the Gd3+ detection was calculated. The effects on the emission were also analyzed in the presence of a combination of Gd3+ at two different concentrations and the previously indicated mono and di-valent ions. The study demonstrated the feasibility of a qualitative method to investigate the intracellular Gd3+ release upon the administration of Gd-based contrast agents in murine macrophages.

ProceduresTo preliminary assess the relationship between Manganese Enhanced Magnetic Resonance Imaging (MEMRI) and the expression of calcium receptors in human prostate and breast cancer animal models.MethodsNOD/SCID mice were inoculated with MDA-MB-231 breast cancer cells and prostate PC3 cancer cells to develop orthotopic or pseudometastatic cancer animal models. Mice were studied on a clinical 3T scanner by using a prototype birdcage coil before and after intravenous injection of MnCl2. Assessment of receptor's status was carried out after the MR images acquisition by immunohistochemistry on excised tumours.ResultsManganese contrast enhancement in breast or prostate cancer animal models well correlated with CaSR expression (pConclusionOur preliminary results suggest that MEMRI appears an efficient tool to characterize human breast and prostate cancer animal models in the presence of different expression level of calcium receptors.

Procedures To preliminary assess the relationship between Manganese Enhanced Magnetic Resonance Imaging (MEMRI) and the expression of calcium receptors in human prostate and breast cancer animal models. Methods NOD/SCID mice were inoculated with MDA-MB-231 breast cancer cells and prostate PC3 cancer cells to develop orthotopic or pseudometastatic cancer animal models. Mice were studied on a clinical 3T scanner by using a prototype birdcage coil before and after intravenous injection of MnCl2. Assessment of receptor’s status was carried out after the MR images acquisition by immunohistochemistry on excised tumours. Results Manganese contrast enhancement in breast or prostate cancer animal models well correlated with CaSR expression (p

A novel, highly biocompatible, well soluble melanin-based probe obtained from the conjugation of melanin macromolecule to bovine serum albumin (BSA) was tested as a contrast agent for photoacoustic tumor imaging. Five soluble conjugates (PheoBSA A-E) were synthesized by oxidation of dopamine (DA) in the presence of variable amounts of BSA. All systems showed the similar size and absorbance spectra, being PheoBSA D (DA:BSA ratio 1:2) the one showing the highest photoacoustic efficiency. This system was then selected for the investigations as it showed a marked enhancement of the photoacoustic (PA) contrast in the tumor region upon iv injection. Biodistribution studies confirmed the accumulation of PheoBSA D within the tumor region and showed fast renal elimination, lack of cell toxicity, and good hemocompatibility. A higher PA contrast enhancement was observed in the case of PC3 prostate tumor xenograft when compared to the TS/A breast one, likely reflecting different vascularization/extravasation properties between the two tumor murine models. The improved PA properties shown by PheoBSA D allowed to set up a 3D dynamic contrast-enhanced (DCE) approach that demonstrated a persistent increase of the PA signal in the tumor region for a long period. Overall, the herein reported results demonstrate that PheoBSA D is a promising blood pool contrast agent for in vivo PA imaging, particularly useful for the set-up of 3D DCE-PA approaches to monitor tumor vascular properties.

This review highlights the potential of using liposomes in bioassays. Liposomes consist of nano- or micro-sized, synthetically constructed phospholipid vesicles. Liposomes can be loaded with a number of reporting molecules that allow a dramatic amplification of the detection threshold in bioassays. Liposome-based sensors bind or react with the biological components of targets through the introduction of properly tailored vectors anchored on their external surface. The use of liposome-based formulations allows the set-up of bioassays that are rapid, sensitive, and often suitable for in-field applications. Selected applications in the field of immunoassays, as well as recognition/assessment of corona proteins, nucleic acids, exosomes, bacteria, and viruses are surveyed. The role of magnetoliposomes is also highlighted as an additional tool in the armory of liposome-based systems for bioassays.

Nowadays, magnetic resonance imaging (MRI) is one of the key, noninvasive modalities to detect and stage cancer which benefits from contrast agents (CA) to differentiate healthy from tumor tissue. An innovative class of MRI CAs is represented by Gd-loaded gold nanoparticles. The size, shape and chemical functionalization of Gd-loaded gold nanoparticles appear to affect the observed relaxation enhancement of water protons in their suspensions. The herein reported results shed more light on the determinants of the relaxation enhancement brought by Gd-loaded concave cube gold nanoparticles (CCGNPs). It has been found that, in the case of nanoparticles endowed with concave surfaces, the relaxivity is remarkably higher compared to the corresponding spherical (i.e., convex) gold nanoparticles (SPhGNPs). The main determinant for the observed relaxation enhancement is represented by the occurrence of a large contribution from second sphere water molecules which can be exploited in the design of high-efficiency MRI CA.

Leukemia is a type of hematopoietic stem/progenitor cell malignancy characterized by the accumulation of immature cells in the blood and bone marrow. Treatment strategies mainly rely on the administration of chemotherapeutic agents, which, unfortunately, are known for their high toxicity and side effects. The concept of targeted therapy as magic bullet was introduced by Paul Erlich about 100 years ago, to inspire new therapies able to tackle the disadvantages of chemotherapeutic agents. Currently, nanoparticles are considered viable options in the treatment of different types of cancer, including leukemia. The main advantages associated with the use of these nanocarriers summarized as follows: i) they may be designed to target leukemic cells selectively; ii) they invariably enhance bioavailability and blood circulation half-life; iii) their mode of action is expected to reduce side effects. FDA approval of many nanocarriers for treatment of relapsed or refractory leukemia and the desired results extend their application in clinics. In the present review, different types of nanocarriers, their capability in targeting leukemic cells, and the latest preclinical and clinical data are discussed.

INTRODUCTION:Ex vivo cell expansion under Good Manufacturing Practice (GMP) guidelines can be performed using medium additives containing human growth factors from platelets. These products can differently affect proliferation of adipose mesenchymal stromal stem cells (ASC). Qualification of medium additive performance is required for validation under GMP regulations: assessment of growth factor concentrations is not sufficient to predict the biological activity of the product batch. Proton nuclear magnetic resonance spectrometry (1H-NMR) and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) provide wide molecular characterization of samples. AIMS:We aimed to assess if 1H-NMR and MALDI-TOF MS techniques can be used as quality control test potentially predicting the impact of a medium additive on cell proliferation. METHODS:We tested the impact on ASC growth rate (cell proliferation assessment and cell morphology analysis) of four medium additives, obtained by different methods from human platelet apheresis product. In order to classify each medium additive, we evaluated growth factor concentrations and spectra obtained by 1H-NMR and by MALDI-TOF MS. RESULTS:Medium additive obtained by CaCl2 activation of platelet rich products induced higher proliferation rate vs additive derived from platelet depleted ones. Additives obtained by freeze-and-thaw methods weakly induced ASC proliferation. As expected, principal component analysis of growth factor concentrations did not unravel specific biochemical features characterizing medium additives in relation with their biological activity. Otherwise, while 1H-NMR showed a partial resolution capacity, analysis of MALDI-TOF MS spectra allowed unambiguous distinction between the medium additives we used to differently stimulate cell growth in vitro. DISCUSSION:MALDI-TOF and, despite limitations, 1H-NMR are promising cost effective and reliable quality controls to classify the potential of a medium additive to promote ASC growth. This can represent, after further investigations and appropriate validation, a significant advantage for GMP compliant manufacturing of advanced cell therapy products.

Gd-DO3A-diph and Gd-AAZTAC17 are lipophilic magnetic resonance imaging (MRI) agents that display high affinity for low-density lipoprotein (LDL) particles. However, on binding to LDL, Gd-DO3A-diph shows a decreased hydration that results in a lower enhancement of water proton relaxation rate. Conversely, GdAAZTAC17 displays a strong relaxation enhancement at the imaging fields. Each LDL particle can load up to 100 and 400 UNITS of Gd-DO3A-diph and Gd-AAZTAC17, respectively. Their LDL adducts are taken up by human hepatoblastoma G2 (HepG2) and melanoma B16 tumor cells when added to the incubation medium. T, measurements of the labeled cells indicate that Gd-AAZTAC17 is significantly more efficient than Gd-DO3A-diph. Furthermore, it has been found that HepG2 hepatoma cells can internalize higher amounts of Gd-AAZTAC17 than B16 cells and the involvement of LDL receptors (LDLRs) has been demonstrated in competition assays with free LDL. Gd-AAZTAC17/LDL adduct proved to be an efficient probe in the magnetic resonance (MR) visualization of subcutaneous tumors in animal models obtained by injecting B16 melanoma cells into the right flank of mice. Finally, confocal microscopy validation of the distribution of LDL-based probes in the tumor has been obtained by doping the Gd-AAZTAC17/LDL adduct with a fluorescent phospholipid moiety.

PURPOSE:The body concentration of iron is regulated by a fine equilibrium between absorption and losses of iron. Iron can be absorbed from diet as inorganic iron or as heme. Hemopexin is an acute phase protein that limits iron access to microorganisms. Moreover, it is the plasma protein with the highest binding affinity for heme and thus it mediates heme-iron recycling. Considering its involvement in iron homeostasis, it was postulated that hemopexin may play a role in the physiological absorption of inorganic iron. METHODS AND RESULTS:Hemopexin-null mice showed elevated iron deposits in enterocytes, associated with higher duodenal H-Ferritin levels and a significant increase in duodenal expression and activity of heme oxygenase. The expression of heme-iron and inorganic iron transporters was normal. The rate of iron absorption was assessed by measuring the amount of (57)Fe retained in tissues from hemopexin-null and wild-type animals after administration of an oral dose of (57)FeSO4 or of (57)Fe-labelled heme. Higher iron retention in the duodenum of hemopexin-null mice was observed as compared with normal mice. Conversely, iron transfer from enterocytes to liver and bone marrow was unaffected in hemopexin-null mice. CONCLUSIONS:The increased iron level in hemopexin-null duodenum can be accounted for by an increased iron uptake by enterocytes and storage in ferritins. These data indicate that the lack of hemopexin under physiological conditions leads to an enhanced duodenal iron uptake thus providing new insights to our understanding of body iron homeostasis.

The ability to visualize plasmid DNA entrapment in muscle cells undergoing an “in vivo” electroporation treatment was investigated on BALB/c mice using a 7-T magnetic resonance imaging (MRI) scanner using the paramagnetic Gd–DOTA–spd complex as imaging reporter. Gd–DOTA–spd bears a tripositively charged spermidine residue that yields a strong binding affinity toward the negatively charged DNA chain (6.4 kb, K a = 2.2 × 10 3 M −1 for approximately 2500 ± 500 binding sites). Cellular colocalization of Gd-DOTA-spd and plasmid DNA has been validated by histological analysis of excised treated muscle. In vivo MRI visualization of Gd-DOTA-spd distribution provides an excellent route to access the cellular entrapment of plasmid DNA upon applying an electroporation pulse.