Proton Therapy, Magnetic Nanoparticles and Hyperthermia as Combined Treatment for Pancreatic BxPC3 Tumor Cells.

We present an investigation of the effects on BxPC3 pancreatic cancer cells of proton therapy combined with hyperthermia, assisted by magnetic fluid hyperthermia performed with the use of magnetic nanoparticles. The cells' response to the combined treatment has been evaluated by means of the clonogenic survival assay and the estimation of DNA Double Strand Breaks (DSBs). The Reactive Oxygen Species (ROS) production, the tumor cell invasion and the cell cycle variations have also been studied. The experimental results have shown that the combination of proton therapy, MNPs administration and hyperthermia gives a clonogenic survival that is much smaller than the single irradiation treatment at all doses, thus suggesting a new effective combined therapy for the pancreatic tumor. Importantly, the effect of the therapies used here is synergistic. Moreover, after proton irradiation, the hyperthermia treatment was able to increase the number of DSBs, even though just at 6 h after the treatment. Noticeably, the magnetic nanoparticles' presence induces radiosensitization effects, and hyperthermia increases the production of ROS, which contributes to cytotoxic cellular effects and to a wide variety of lesions including DNA damage. The present study indicates a new way for clinical translation of combined therapies, also in the vision of an increasing number of hospitals that will use the proton therapy technique in the near future for different kinds of radio-resistant cancers.

Fast and non-destructive neutron activation analysis for simultaneous determination of TiO2 and SiO2 in sunscreens with attention to regulatory and research issues.

A new instrumental neutron activation analysis (INAA) for the simultaneous determination of titanium (TiO2) and silica (SiO2) dioxide as UV-filters in sunscreens is described. Samples are encapsulated, neutron irradiated (30 s) and after a suitable decay (3 min), the induced 51Ti (T1/2 = 5.76 min) and 29Al (T1/2 = 6.56 min) radionuclides are measured for the emitted γ-ray fingerprint. Three applications were carried out: (i) screening study (analysis of commercial sunscreens in combination with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS); (ii) research study (development of innovative UV-filters such as titanium dioxide or bismuth titanate loaded inorganic mesoporous silica nanoparticles, MSN); (iii) validation study (intercalibration of a spectrochemical method - inductively coupled plasma optical emission spectrometry, ICP-OES). Collectively, the nuclear method appears a powerful tool adequate for quantifying TiO2 and SiO2 in the above studies. The limited accessibility at the nuclear reactor for neutron activation is probably one of the reasons why the excellent characteristics of the nuclear technique are not always fully known and exploited in the industrial and research chemical world.

Decommissioning procedure and induced activation levels, calculations and measurements in an 18 MeV medical cyclotron.

The present article describes the decommissioning of a self-shielded 18 MeV medical cyclotron IBA Cyclone 18/9 after 14 years of operation. A Monte Carlo simulation of the possible nuclear reactions was performed in order to plan the decommissioning activities. During the cyclotron dismantling, the activities of the cyclotron components, concrete wall and floor samples were measured. Residual activities were analysed by means of an HPGe detector and liquid scintillation counting, and compared with simulation data. Dosimetry of the staff involved in the decommissioning procedure was monitored by individual TL dosimeters and/or digital dosimeter. The cyclotron component analysis confirmed the presence of gamma and pure beta emitters,22Na,54Mn,60Co,65Zn,207Bi,55Fe,63Ni at different values of specific activity, depending on the positioning of the sample point and on the alloy of the sampled part. In these components the presence of gamma and pure beta emitters was measured 5 years after the shutdown at levels far above clearance limits as defined by the 'Recommended radiological protection criteria for the recycling of metals from the dismantling of nuclear installations' (RP89) guidelines. The simulation, carried out by FLUKA Code (version 2020.0.5) on the cyclotron components, provided good agreement with measurements, with a maximum discrepancy of the same order as the uncertainties. Four engineers of the cyclotron maintenance staff were involved in the dismounting of the hottest components and rigging of the cyclotron in the deposit 6 months after shutdown and two engineers were involved during the drilling phase 3.5 years after shutdown. The measured dose from external exposure of the involved staff was lower than 100µSv person-1during the first phase and lower than 20µSv person-1during the final drilling phase. Measured doses from intake were negligible. In conclusion, the decommissioning of the 18 MeV cyclotron does not represent a risk for the involved staff, but, due to the presence of long-lived radioisotopes, the cyclotron components are to be treated as low level radioactive waste, and stored in an authorised storage area for at least 25 years after shutdown.

Metallobiochemistry of ultratrace levels of bismuth in the rat II. Interaction of 205+206Bi3+ with tissue, intracellular and molecular components.

BACKGROUND: Knowledge on Bi metabolism in laboratory animals refers to studies at "extreme" exposures, i.e. pharmacologically relevant high-doses (mg kg-1 b.w.) in relation to its medical use, or infinitesimal doses (pg kg-1b.w.) concerning radiobiology protection and radiotherapeutic purposes. There are no specific studies on metabolic patterns of environmental exposure doses (ultratrace level, µg kg-1 b.w.), becoming in this context Bi a "heavy metal fallen into oblivion". We previously reported the results of the metabolic fate of ultratrace levels of Bi in the blood of rats [1]. In reference to the same study here we report the results of the retention and tissue binding of Bi with intracellular and molecular components. METHODS: Animals were intraperitoneally injected with 0.8 µg Bi kg-1 b.w. as 205+206Bi(NO)3, alone or in combination with 59Fe for the radiolabeling of iron proteins. The use of 205+206Bi radiotracer allowed the determination of Bi down to pg fg-1 in biological fluids, tissues, subcellular fractions, and biochemical components isolated by differential centrifugation, size exclusion chromatography, solvent extraction, precipitation, immunoprecipitation and dialysis. MAIN FINDINGS: At 24 h post injection the kidney contained by far the highest Bi concentration (10 ng g-1 wt.w.) followed by the thymus, spleen, liver, thyroid, trachea, femur, lung, adrenal gland, stomach, duodenum and pancreas (0.1 to 1.3 ng g-1 wt.w.). Brain and testis showed smaller but consistently significant concentrations of the element (0.03 ng g-1 wt.w). Urine was the predominant route of excretion. Intracellularly, liver, kidney, spleen, testis, and brain cytosols displayed the highest percentages (35%-58%) of Bi of homogenates. Liver and testis nuclei were the organelles with the highest Bi content (24 % and 27 %). However, when the recovered Bi of the liver was recorded as percent of total recovered Bi divided by percent of total recovered protein the lysosomes showed the highest relative specific activity than in other fractions. In the brain subcellular fractions Bi was incorporated by neuro-structures with the protein and not lipidic fraction of the myelin retaining 18 % of Bi of the total homogenate. After the liver intra-subcellular fractionation: (i) 65 % of the nuclear Bi was associated with the protein fraction of the nuclear membranes and 35 % with the bulk chromatin bound to non-histone and DNA fractions; (ii) about 50 % of the mitochondrial Bi was associated with inner and outer membranes being the other half recovered in the intramitochondrial matrix; (iii) in microsomes Bi showed a high affinity (close to 90 %) for the membranous components (rough and smooth membranes); (iv) In the liver cytosol three pools of Bi-binding proteins (molecular size > 300 kDa, 70 kDa and 10 kDa) were observed with ferritin and metallothionein-like protein identified as Bi-binding biomolecules. Three similar protein pools were also observed in the kidney cytosol. However, the amount of Bi, calculated in percent of the total cytosolic Bi, were significantly different compared to the corresponding pools of the liver cytosol. CONCLUSIONS: At the best of our knowledge the present paper represents the first in vivo study, on the basis of an environmental toxicology approach, aiming at describing retention and binding of Bi in the rat at tissue, intracellular and molecular levels.

Metallobiochemistry of ultratrace levels of bismuth in the rat I. Metabolic patterns of 205+206Bi3+ in the blood.

BACKGROUND: The number of the applications of bismuth (Bi) is rapidly and remarkably increasing, enhancing the chance to increase the levels to which humans are normally daily exposed. The interest to Bi comes also from the potential of Bi-based nanoparticles (BiNPs) for industrial and biomedical purposes. Like other metal-based NPs used in nanomedicine, BiNPs may release ultratrace amounts of Bi ions when injected. The metabolic fate and toxicity of these ions still needs to be evaluated. At present, knowledge of Bi metabolism in laboratory animals refers almost solely to studies under unnatural "extreme" exposures, i.e. pharmacologically relevant high-doses (up to thousand mg kg-1) in relation to its medical use, or infinitesimal-doses (pg kg-1 as non-carrier-added Bi radioisotopes) for radiobiology protection, diagnostic and radiotherapeutic purposes. No specific study exists on the "metabolic patterns" in animal models exposed to levels of Bi, i.e. at "environmental dose exposure" that reflect the human daily exposure (µg kg-1). METHODOLOGY: Rats were intraperitoneally injected with 0.8 µg Bi kg-1 bw as 205+206Bi(NO)3 alone or in combination with 59Fe for radiolabelling of iron proteins. The use of 205+206Bi radiotracers allowed the detection and measurement down to pg fg-1 of the element in the blood biochemical compartments and protein fractions as isolated by differential centrifugation, size exclusion- and ion exchange chromatography, electrophoresis, solvent extraction, precipitation and dialysis. RESULTS: 24 h after the administration, the blood concentration of Bi was 0.18 ng mL-1, with a repartition plasma/red blod cells (RBC) in a ratio of 2:1. Elution profiles of plasma from gel filtration on Sephadex G-150 showed four pools of Bi-binder proteins with different molecular sizes (> 300 kDa, 160 kDa, 70 kDa and < 6.5 kDa). In the 70 kDa fraction transferrin and albumin were identified as biomolecule carriers for Bi. In red blood cells, Bi was distributed between cytosol and membranes (ghosts) in a ratio of about 5:1. In the cytosol, low molecular components (LMWC) and the hemoglobin associated the Bi in a ratio of about 1.8:1. In the hemoglobin molecule, Bi was bound to the beta polypeptide chain of the globin. In the ghosts, Bi was detected at more than one site of the protein fraction, with no binding with lipids. Dialysis experiments and the consistently high recovery (80-90 %) of 206Bi from chromatography of 206Bi-containing biocomponents suggest that Bi was firmly complexed at physiological pH with a low degree of breaking during the applications of experimental protocols for the isolation of the 206Bi-biocomplexes. These latter were sensitive to acid buffer pH 5, and to the presence of complexing agents in the dialysis fluid. CONCLUSIONS: On the basis of an environmental biochemical toxicology approach, we have undertaken a study on the metabolic patterns of Bi3+ ions in rats at tissue, subcellular and molecular level with the identification of cellular Bi-binding components. As a first part of the study the present work reports the results concerned with the metabolic fate of ultratrace levels of 205+206Bi(NO)3 in the blood.

Genotoxicity and Immunotoxicity of Titanium Dioxide-Embedded Mesoporous Silica Nanoparticles (TiO2@MSN) in Primary Peripheral Human Blood Mononuclear Cells (PBMC).

Background: TiO2 nanoparticles (TiO2 NPs) are the nanomaterial most produced as an ultraviolet (UV) filter. However, TiO2 is a semiconductor and, in nanoparticle size, is a strong photocatalyst, raising concerns about photomutagenesis. Mesoporous silica nanoparticles (MSN) were synthetized incorporating TiO2 NPs (TiO2@MSN) to develop a cosmetic UV filter. The aim of this study was to assess the toxicity of TiO2@MSN, compared with bare MSN and commercial TiO2 NPs, based on several biomarkers. Materials and Methods: Human peripheral blood mononuclear cells (PBMC) were exposed to TiO2@MSN, bare MSN (network) or commercial TiO2 NPs for comparison. Exposed PBMC were characterized for cell viability/apoptosis, reactive oxygen species (ROS), nuclear morphology, and cytokines secretion. Results: All the nanoparticles induced apoptosis, but only TiO2 NPs (alone or assembled into MSN) led to ROS and micronuclei. However, TiO2@MSN showed lower ROS and cytotoxicity with respect to the P25. Exposure to TiO2@MSN induced Th2-skewed and pro-fibrotic responses. Conclusions: Geno-cytotoxicity data indicate that TiO2@MSN are safer than P25 and MSN. Cytokine responses induced by TiO2@MSN are imputable to both the TiO2 NPs and MSN, and, therefore, considered of low immunotoxicological relevance. This analytical assessment might provide hints for NPs modification and deep purification to reduce the risk of health effects in the settings of their large-scale manufacturing and everyday usage by consumers.

Hadron Therapy, Magnetic Nanoparticles and Hyperthermia: A Promising Combined Tool for Pancreatic Cancer Treatment.

A combination of carbon ions/photons irradiation and hyperthermia as a novel therapeutic approach for the in-vitro treatment of pancreatic cancer BxPC3 cells is presented. The radiation doses used are 0-2 Gy for carbon ions and 0-7 Gy for 6 MV photons. Hyperthermia is realized via a standard heating bath, assisted by magnetic fluid hyperthermia (MFH) that utilizes magnetic nanoparticles (MNPs) exposed to an alternating magnetic field of amplitude 19.5 mTesla and frequency 109.8 kHz. Starting from 37 °C, the temperature is gradually increased and the sample is kept at 42 °C for 30 min. For MFH, MNPs with a mean diameter of 19 nm and specific absorption rate of 110 ± 30 W/gFe3o4 coated with a biocompatible ligand to ensure stability in physiological media are used. Irradiation diminishes the clonogenic survival at an extent that depends on the radiation type, and its decrease is amplified both by the MNPs cellular uptake and the hyperthermia protocol. Significant increases in DNA double-strand breaks at 6 h are observed in samples exposed to MNP uptake, treated with 0.75 Gy carbon-ion irradiation and hyperthermia. The proposed experimental protocol, based on the combination of hadron irradiation and hyperthermia, represents a first step towards an innovative clinical option for pancreatic cancer.

On the production of 52gMn by deuteron irradiation on natural chromium and its radionuclidic purity.

The positron emitter 52gMn is used for the Positron Emission Tomography - PET imaging.In this work we investigate the nuclear reactions for production of 52gMn and 54Mn induced by deuteron beams on natural chromium targets at energies up to Ed = 28 MeV using the stacked-foils activation technique. We calculate the thick target yields for 52gMn and for the radionuclidic impurity 54Mn, and we compare the radionuclidic purity of 52gMn with that achievable in proton activation of Cr. The cross-sections of the reactions natCr(d,pxn)51Cr and natCr(d,x)48V are also presented.

Excitation function and yield for the 103Rh(d,2n)103Pd nuclear reaction: Optimization of the production of palladium-103.

Deuteron-induced nuclear reactions for the generation of 103Pd were investigated using the stacked-foil activation technique on rhodium targets at deuteron energies up to Ed=33MeV. The excitation functions of the reactions 103Rh(d,xn)101,103Pd, 103Rh(d,x)100g,cum,101m,g,102m,gRh and 103Rh(d,2p)103Ru have been measured, and the Thick-Target Yield for 103Pd has been calculated.

Uptake from water, internal distribution and bioaccumulation of selenium in Scenedesmus obliquus, Unio mancus and Rattus norvegicus: part B.

(75)Se-selenite transfer was investigated in a phytoplankton-mussel-rat food chain model consisting of Scenedesmus obliquus (Turpin) Kützing, Unio mancus Lamark and Rattus norvegicus Berkenhout. (75)Se-metabolized forms were investigated in order to identify potential critical steps in the food chain, as well as its relative bioavailability looking also at intracellular, cellular and organ partitioning. Tissue and intracellular distribution of (75)Se in mussels fed with (75)Se-S. obliquus was different compared to those exposed only to inorganic (75)Se-selenite. The intracellular distribution of (75)Se in the hepatopancreas and mantle of mussels fed (75)Se-microalgae was similar to hepatic and renal distributions in rats, suggesting that their stomach dissociated larger (75)Se-containing molecules. The (75)Se partitioned from water (culture medium) to microalgae showing a bioconcentration factor of 435. The bottleneck in the trophic transfer of (75)Se occurred between S. obliquus-U. mancus. From microalgae to mussels and subsequently to rats no bioaccumulation was verified.

Uptake from water, internal distribution and bioaccumulation of selenium in Scenedesmus obliquus, Unio mancus and Rattus norvegicus: part A.

The (75)Se internal bioavailability was investigated in microalgae, mussels and rats as biological experimental models. The (75)Se accumulation from freshwater to microalgae [Scenedesmus obliquus (Turpin) Kützing], from freshwater to mussels (Unio mancus Lamark) and, finally, per os to rats (Rattus norvegicus Berkenhout) was followed using (75)Se-labelled selenite looking at (75)Se uptake, retention, intracellular distribution and binding with cellular biocomplexes. After exposure to 10, 50 and 500 µg Se L(-1), the microalgae showed an inhibitory effect on population growth only at the highest concentration. Mussels exposed to 105 µg Se L(-1) showed an accumulation of the element with time in all tissues. Intracellularly, Se was present in all subcellular fractions, especially in the cytosol. Rats were treated via oral administration with 5 µg Se rat(-1). After 24 h, liver and kidney showed the highest Se concentration.

The excitation functions of (100)Mo(p,x)(99)Mo and (100)Mo(p,2n)(99m)Tc.

Proton-induced nuclear reactions for generation of (99)Mo and (99m)Tc radionuclides were investigated using the stacked-foil activation technique on 99.05% enriched (100)Mo targets at energies up to Ep=21MeV. Excitation functions of the reactions (100)Mo(p,x)(99)Mo and (100)Mo(p,2n)(99m)Tc have been measured.

Physical optimization of production by deuteron irradiation of high specific activity (177g)Lu suitable for radioimmunotherapy.

Deuteron-induced nuclear reactions for generation of no-carrier-added (NCA) Lu isotopes were investigated using the stacked-foil activation technique on natural Yb targets at energies up to Ed=18.18MeV. The decay curve of ¹77Yb, the growth curve of the cumulative (direct and indirect) and the direct production of (177g)Lu were determined. The analysis of these curves conducts to the evidence that the predominant route for the production of (177g)Lu is the indirect reaction ¹76Yb(d,p)¹77Yb, which decays to (177g)Lu. In the spectra acquired one year from the EOB the γ lines of (177m)Lu are not evident. A comparison between the calculated activity of (177g)Lu produced with a cyclotron and with a nuclear reactor is given.

Fukushima fallout at Milano, Italy.

The radionuclides (131)I, (137)Cs and (134)Cs were observed in the Milano region (45°) of Italy early after the nuclear accident in Fukushima, Japan. Increased atmospheric radioactivity was observed on an air filter taken on 30 March 2011, while the maximum activity of 467 µBq m(-3) for (131)I was recorded at April 3-4, 2011. The first evidence of Fukushima fallout was confirmed with (131)I and (137)Cs measured in precipitation at two sampling sites at Milano on 28 March, 2011, with the concentrations of (131)I and (137)Cs in the rainwater equal to 0.89 Bq L(-1) and 0.12 Bq L(-1), respectively. A sample of dry deposition that was collected 9 days after the first rainfall event of 27-28 March, 2011 showed that the dry deposition was more effective in the case of (137)Cs than it was for (131)I, probably because iodine was mainly in gaseous form whereas caesium was rapidly bound to aerosols and thus highly subject to dry deposition. The relatively high observed values of (137)Cs in grass, soil and fresh goat and cow milk samples were probably from Chernobyl fallout and global fallout from past nuclear tests rather than from the Fukushima accident. Finally, a dose assessment for the region of investigation showed clearly that the detected activities in all environmental samples were very far below levels of concern.

Excitation function for deuteron induced nuclear reactions on natural ytterbium for production of high specific activity 177g Lu in no-carrier-added form for metabolic radiotherapy.

Deuteron-induced nuclear reactions for generation of no-carrier-added Lu radionuclides were investigated using the stacked-foil activation technique on natural Yb targets at energies up to E(d)=18.18 MeV. Excitation functions of the reactions (nat)Yb(d,xn)(169,170,171,172,173,174g,174m,176m,177g)Lu and (nat)Yb(d,pxn)(169,175,177)Yb have been measured, among them three ((169)Lu, (174m)Lu and (176m)Lu) are reported for the first time. The upper limit of the contamination from the long-lived metastable level (177m)Lu was evaluated too. Thick-target yields for all investigated radionuclides are calculated.

Rapid determination of (90)Sr impurities in freshly "generator eluted"(90)Y for radiopharmaceutical preparation.

(90)Y is one of the most useful radionuclides for radioimmunotherapeutic applications and has a half-life (t(1/2)=64.14h) suitable for most therapeutic applications, beta particles of high energy and decays to a stable daughter. It is significant that (90)Y is available conveniently and inexpensively from a radionuclide "generator" by decay of its parent, (90)Sr. Nevertheless, current and planned clinical applications with [(90)Y] labelled compounds employ activity levels that cannot be readily obtained from an in-house generator, but from commercial sources. We have evaluated Eichrom's Sr-resin, either as an "in-house" generator or as a fast QC method for analysis of (90)Y solutions. In particular, for the development as a generator, we investigated the percentage of the radio-Sr in the first 8M HNO(3) eluate: in this fraction the concentration of (90)Sr must be smaller than 10(-5)% (recommendations of the International Commission on Radiological Protection). For evaluation as a rapid QC method, we analyzed the concentration of (90)Y in all the fractions containing "only" radio-Sr: (90)Y should not be present in these eluates. After the collection of beta(-) and gamma spectra and analysis of them, we concluded that commercial Sr-resin minicolumn cannot give us the results expected; we developed an in-house system loaded with 4mL of Sr-resin which gave better results as a generator and a rapid QC method.

On the photon self-absorption correction for thin-target-yields vs. thick-target-yields in radionuclide production.

In the experimental determination of activity of low gamma energy emitters by HPGe spectrometry, after cyclotron irradiation of thick targets of high Z elements, it is mandatory to introduce a correction for gamma-ray self-absorption in the target. The amount of this correction is substantial in order to provide a statistically acceptable agreement with the thick-target-yield data obtained by integration of the experimental thin-target-yield data that are very slightly affected by self-absorption instead.

Improved determination of the position and activity of a radioactive point source inside a bulky medium using several detectors. The special case of a lung counter--a general view.

A method is described to locate a radioactive point source in a lung using a commercial array of lung detectors. The method takes advantage of the fact that modern commercial systems consist of several Ge detectors, each connected to a multi-channel analyzer, and the information from each detector can be analyzed separately. The system is calibrated with point sources placed in various locations in the lungs of a phantom. A vector method is presented for determining which of the calibration locations in the phantom is closest to the location of the unknown point source. It has been shown that the accuracy of the method increases with increasing number of the vector dimensions. A higher dimensionality can be achieved by analyzing counts of several gamma lines or by using more detectors.

On the "artificial" nature of Tc and the "carrier-free" nature of 99mTc from 99Mo/99mTc generators.

Two widespread misconceptions regarding Tc are dealt with. First, it is shown that although primordial Tc has decayed completely, Tc is still present in nature due to natural processes, in addition to substantial man-made quantities. Thus, Tc cannot be considered as a purely artificial element. Second, it is shown that (99m)Tc from (99)Mo/(99m)Tc generators is certainly not carrier-free (CF) and that, "no-carrier-added" (NCA) is a more appropriate designation. As a quantitative measure of the amount of long-lived (99g)Tc carrier present in (99m)Tc preparations from generators, the Isotope Dilution Factor (IDF) is used and calculated for some practical situations.

Preparation of "high specific activity" radiochemical forms of vanadium, manganese and thallium for metallo-toxicological studies.

In this paper are presented the production methods for very "high specific activity" radionuclides (HSA-RN) of vanadium, manganese and thallium which have been developed in our laboratories for labelling different chemical forms of these elements present in the echo-systems in ultra-trace amounts, for metallo-toxicological and bio-kinetic studies. Use was made of both cyclotron and thermal nuclear reactor. If the nuclear reaction product has atomic number different from irradiated target, it is possible separating the radioactive nuclide from irradiated target, without addition of isotopic carrier. This kind of radionuclide is named No Carrier Added, NCA, and his specific activity, As is very high and can reach values close to the theoretical Carrier Free one, CF. The experimental determination of specific activity, chemical and radiochemical purities is mandatory for all these kinds of applications.