Ján Kozempel

Radiolabelling of engineered nanoparticles for in vitro and in vivo tracing applications using cyclotron accelerators

We present in this article an outline of some cyclotron-based irradiation techniques that can be used to directly radiolabel industrially manufactured nanoparticles, as well as two techniques for synthesis of labelled nanoparticles using cyclotron-generated radioactive precursor materials. These radiolabelled nanoparticles are suitable for a range of different in vitro and in vivo tracing studies of relevance to the field of nanotoxicology. A basic overview is given of the relevant physics of nuclear reactions regarding both ion-beam and neutron production of radioisotopes. The various issues that determine the practicality and usefulness of the different methods are discussed, including radioisotope yield, nuclear reaction kinetics, radiation and thermal damage, and radiolabel stability. Experimental details are presented regarding several techniques applied in our laboratories, including direct light-ion activation of dry nanoparticle samples, neutron activation of nanoparticles and suspensions using an ion-beam driven activator, spark-ignition generation of nanoparticle aerosols using activated electrode materials, and radiochemical synthesis of nanoparticles using cyclotron-produced isotopes. The application of these techniques is illustrated through short descriptions of some selected results thus far achieved. It is shown that these cyclotron-based methods offer a very useful range of options for nanoparticle radiolabelling despite some experimental difficulties associated with their application. For direct nanoparticle radiolabelling, if care is taken in choosing the experimental conditions applied, useful activity levels can be achieved in a wide range of nanoparticle types, without causing substantial thermal or radiation damage to the nanoparticle structure. Nanoparticle synthesis using radioactive precursors presents a different set of issues and offers a complementary and equally valid approach when laboratory generation of the nanoparticles is acceptable for the proposed studies, and where an appropriate radiolabel can be incorporated into the nanoparticles during synthesis.

Cyclotron Production of Radioactive

Nowadays, a wide variety of nanoparticles (NPs) are applied in different fields such as medical science and industry. Due to their large commercial volume, the OECD Working Party on Manufactured Nanomaterials (NMs) has proposed to study a set of 14 nanomaterials, one of which being cerium oxide (CeO2). In particular, CeO2 based NPs are widely used in automotive industry, healthcare, and cosmetics. In this paper, we propose a method for the production of radioactive CeO2 NPs. We demonstrate that they maintain the same physicochemical characteristics as the “cold” ones in terms of size distribution and Zeta potential; we develop a new protocol to assess their cellular interaction in immortalized mouse fibroblast cell line Balb/3T3, a model for the study of basal cytotoxicity and carcinogenic potential induced by chemicals and in the present case by NPs. Experimental result of this work, which shows a quasi-linear concentration-uptake response of cells, can be useful as a reference dose-uptake curve for explaining effects following biological uptake after exposure to CeO2 NPs.

{\hbox{CeO}} _{2}

Abstract The biokinetics of a size-selected fraction (70u2009nm median size) of commercially available and 48V-radiolabeled [48V]TiO2 nanoparticles has been investigated in female Wistar-Kyoto rats at retention timepoints 1u2009h, 4u2009h, 24u2009h and 7 days after oral application of a single dose of an aqueous [48V]TiO2-nanoparticle suspension by intra-esophageal instillation. A completely balanced quantitative body clearance and biokinetics in all organs and tissues was obtained by applying typical [48V]TiO2-nanoparticle doses in the range of 30–80u2009μg•kg−1 bodyweight, making use of the high sensitivity of the radiotracer technique. The [48V]TiO2-nanoparticle content was corrected for nanoparticles in the residual blood retained in organs and tissue after exsanguination and for 48V-ions not bound to TiO2-nanoparticles. Beyond predominant fecal excretion about 0.6% of the administered dose passed the gastro-intestinal-barrier after one hour and about 0.05% were still distributed in the body after 7 days, with quantifiable [48V]TiO2-nanoparticle organ concentrations present in liver (0.09u2009ng•g−1), lungs (0.10u2009ng•g−1), kidneys (0.29u2009ng•g−1), brain (0.36u2009ng•g−1), spleen (0.45u2009ng•g−1), uterus (0.55u2009ng•g−1) and skeleton (0.98u2009ng•g−1). Since chronic, oral uptake of TiO2 particles (including a nano-fraction) by consumers has continuously increased in the past decades, the possibility of chronic accumulation of such biopersistent nanoparticles in secondary organs and the skeleton raises questions about the responsiveness of their defense capacities, and whether these could be leading to adverse health effects in the population at large. After normalizing the fractions of retained [48V]TiO2-nanoparticles to the fraction that passed the gastro-intestinal-barrier and reached systemic circulation, the biokinetics was compared to the biokinetics determined after IV-injection (Part 1). Since the biokinetics patterns differ largely, IV-injection is not an adequate surrogate for assessing the biokinetics after oral exposure to TiO2 nanoparticles.

Nanoparticles and Their Application for In Vitro Uptake Studies

A novel production method for n.c.a. 64Cu based on deuteron irradiation of 64Zn is presented. The production takes place through the 64Zn(d, 2p) 64Cu reaction using a deuteron beam of 19.5 MeV energy on highly enriched 64Zn disks. An average yield over three irradiations of 31 MBq/μA h (850 μCi/μA h) and saturation yield of 575 MBq/μA (15.5 mCi/μA) at the end of the beam (EOB) was measured by γ-ray spectrometry. Two of the three runs, of low irradiation charge, were used for radiochemistry. The copper isotopes were separated from other radionuclidic impurities by the combination of cation and anion exchange chromatography. An average radiochemical yield of 90% was estimated for the two runs performed in this study, and the specific activity as determined using flame atomic absorption spectrometry was about 4 MBq/μg, 2 hours after EOB. An extrapolation of the present results to production conditions (50 μA, 10 h) indicates approximately 8 GBq/μg (220 mCi/μg) of specific activity. The overall uncertainty in these values is estimated to 15%.

Quantitative biokinetics of titanium dioxide nanoparticles after oral application in rats: Part 2

Abstract The biokinetics of a size-selected fraction (70u2009nm median size) of commercially available and 48V-radiolabeled [48V]TiO2 nanoparticles has been investigated in healthy adult female Wistar-Kyoto rats at retention time-points of 1u2009h, 4u2009h, 24u2009h, 7u2009d and 28u2009d after intratracheal instillation of a single dose of an aqueous [48V]TiO2-nanoparticle suspension. A completely balanced quantitative biodistribution in all organs and tissues was obtained by applying typical [48V]TiO2-nanoparticle doses in the range of 40–240u2009μg·kg−1 bodyweight and making use of the high sensitivity of the radiotracer technique. The [48V]TiO2-nanoparticle content was corrected for residual blood retained in organs and tissues after exsanguination and for 48V-ions not bound to TiO2-nanoparticles. About 4% of the initial peripheral lung dose passed through the air-blood-barrier after 1u2009h and were retained mainly in the carcass (4%); 0.3% after 28u2009d. Highest organ fractions of [48V]TiO2-nanoparticles present in liver and kidneys remained constant (0.03%). [48V]TiO2-nanoparticles which entered across the gut epithelium following fast and long-term clearance from the lungs via larynx increased from 5 to 20% of all translocated/absorbed [48V]TiO2-nanoparticles. This contribution may account for 1/5 of the nanoparticle retention in some organs. After normalizing the fractions of retained [48V]TiO2-nanoparticles to the fraction that reached systemic circulation, the biodistribution was compared with the biodistributions determined after IV-injection (Part 1) and gavage (GAV) (Part 2). The biokinetics patterns after IT-instillation and GAV were similar but both were distinctly different from the pattern after intravenous injection disproving the latter to be a suitable surrogate of the former applications. Considering that chronic occupational inhalation of relatively biopersistent TiO2-particles (including nanoparticles) and accumulation in secondary organs may pose long-term health risks, this issue should be scrutinized more comprehensively.

A Novel Method for n.c.a. 64Cu Production by the 64Zn(d,2p)64Cu Reaction and Dual Ion-exchange Column Chromatography

Abstract A method for preparation of 67Cu based on deuteron irradiation of enriched 70Zn is presented. Cross-sections for 67Cu formation were determined by the stacked foil technique for deuteron energies in the range from 10 to 20 MeV for the first time. Irradiations of 70Zn foils were followed by radiochemical separation of 67Cu from the target material and co-produced radionuclidic impurities. The maximum cross-section value of 25.5 ± 2.2 mb was reached at 19 MeV. The integral yield in the energy window of 20 → 10 MeV on 95% enriched 70Zn is estimated at 4.2 MBq/μA h (110 μCi/μA h) or 375 MBq/μA (10 mCi/μA) at saturation.

Quantitative biokinetics of titanium dioxide nanoparticles after intratracheal instillation in rats: Part 3

Radiolabelled nanoparticles are useful tools for biodistribution or cellular uptake studies related to the risk assessment of nanomaterials. Such studies are ideally carried out with industrially manufactured nanoparticles. Irradiation of small quantities of such nanoparticles, in the form of dry powders, with neutrons or light ions allows radiolabelling while preserving their biologically relevant properties. However, nanoparticle powders exhibit poor thermal conductivity and may overheat under irradiation. Their effective thermal conductivity is not known and conventional temperature measurement methods are difficult to apply. Reasonably accurate temperature data could be derived from post-irradiation X-ray diffraction studies on anatase ST-01 TiO2-nanoparticles, with a primary particle size of 7xa0nm, subjected to proton beams of different intensities. The anatase-to-rutile phase transition starting at about 750xa0°C was identified by observing rutile peaks in X-ray diffraction patterns. The onset of growth of single diffracting TiO2-domains at around 200xa0°C was revealed by shape analysis of the diffraction peaks. Identifying these reference temperatures allowed a calibration of the calculated temperature profile. The effective thermal conductivity in the TiO2 powder target was found to be close to that of air trapped in interstices of the nanoparticulate powder. This suggests that the contribution of the nanoparticles to the heat removal from the target is negligible, thus necessitating the use of thin nanoparticle layers in the target in order to facilitate cooling and prevent thermally induced alterations of the nanoparticles.

Preparation of 67Cu via deuteron irradiation of 70Zn

Abstract Submicrometer TiO2 particles, including nanoparticulate fractions, are used in an increasing variety of consumer products, as food additives and also drug delivery applications are envisaged. Beyond exposure of occupational groups, this entails an exposure risk to the public. However, nanoparticle translocation from the organ of intake and potential accumulation in secondary organs are poorly understood and in many investigations excessive doses are applied. The present study investigates the biokinetics and clearance of a low single dose (typically 40–400u2009μg/kg BW) of 48V-radiolabeled, pure TiO2 anatase nanoparticles ([48V]TiO2NP) with a median aggregate/agglomerate size of 70u2009nm in aqueous suspension after intravenous (IV) injection into female Wistar rats. Biokinetics and clearance were followed from one-hour to 4-weeks. The use of radiolabeled nanoparticles allowed a quantitative [48V]TiO2NP balancing of all organs, tissues, carcass and excretions of each rat without having to account for chemical background levels possibly caused by dietary or environmental titanium exposure. Highest [48V]TiO2NP accumulations were found in liver (95.5%ID after one day), followed by spleen (2.5%), carcass (1%), skeleton (0.7%) and blood (0.4%). Detectable nanoparticle levels were found in all other organs. The [48V]TiO2NP content in blood decreased rapidly after 24u2009h while the distribution in other organs and tissues remained rather constant until day-28. The present biokinetics study is part 1 of a series of studies comparing biokinetics after three classical routes of intake (IV injection (part 1), ingestion (part 2), intratracheal instillation (part 3)) under identical laboratory conditions, in order to test the common hypothesis that IV-injection is a suitable predictor for the biokinetics fate of nanoparticles administered by different routes. This hypothesis is disproved by this series of studies.

Radiolabelling of nanoparticles by proton irradiation: temperature control in nanoparticulate powder targets

The intensive use of nano-sized particles in many different applications necessitates studies on their risk assessment as there are still open questions on their safe handling and utilization. For reliable risk assessment, the interaction of nanoparticles (NP) with biological systems after various routes of exposure needs to be investigated using well-characterized NP. We report here on the generation of gold-NP (Au-NP) aerosols for inhalation studies with the spark ignition technique, and their characterization in terms of chemical composition, physical structure, morphology, and specific surface area, and on interaction with lung tissues and lung cells after 1xa0h inhalation by mice. The originally generated agglomerated Au-NP were converted into compact spherical Au-NP by thermal annealing at 600xa0°C, providing particles of similar mass, but different size and specific surface area. Since there are currently no translocation data available on inhaled Au-NP in the 10–50xa0nm diameter range, the emphasis was to generate NP as small as 20xa0nm for inhalation in rodents. For anticipated in vivo systemic translocation and dosimetry analyses, radiolabeled Au-NP were created by proton irradiating the gold electrodes of the spark generator, thus forming gamma ray emitting 195Au with 186xa0days half-life, allowing long-term biokinetic studies. The dissolution rate of 195Au from the NP was below detection limits. The highly concentrated, polydisperse Au-NP aerosol (1–2xa0×xa0107xa0NP/cm3) proved to be constant over several hours in terms of its count median mobility diameter, its geometric standard deviation and number concentration. After collection on filters particles can be re-suspended and used for instillation or ingestion studies.

Quantitative biokinetics of titanium dioxide nanoparticles after intravenous injection in rats: Part 1

The use of superparamagnetic iron oxide nanoparticles (SPIONs) and radiolabelled nanoparticles (NPs) has grown considerably over the recent years, and the SPIONs labelled with medicinal radionuclides offer new opportunities in multimodal diagnostics and in the drug-delivery systems for targeted alpha-particle therapy (TAT) driven by magnetic field gradient or by biologically active moieties bound on NPs shell. However, the mechanisms of NPs radiolabelling are not studied substantially and still remain unclear, even though the way of label attachment directly implies the stability of the label-nanoparticle construct. Since the 223Ra was the first clinically approved alpha-emitter, it is a promising nuclide for further development of its targeted carriers. We report here on the study of 223Ra uptake by the Fe3O4SPIONs, together with an attempt to propose the 223Ra uptake mechanism by the Fe3O4NPs in the presence of a phosphate bufferxa0a typical formulation medium, under the pseudo-equilibrium conditions. Further, the in vitro stability tests of the prepared [223Ra]Fe3O4NPs were performed to estimate the 223Ra label stability. The potential use of 223Ra-labelled SPIONs in theranostic applications is also discussed.Graphical abstract