Journal of Nanoparticle Research

, Volume 12, Issue 7, pp 2435–2443

Radiolabelling of TiO2 nanoparticles for radiotracer studies

Authors

  • Kamel Abbas
    • European Commission, Joint Research Centre, Institute for Health and Consumer Protection T.P. 500
  • Izabela Cydzik
    • European Commission, Joint Research Centre, Institute for Health and Consumer Protection T.P. 500
  • Riccardo Del Torchio
    • European Commission, Joint Research Centre, Institute for Health and Consumer Protection T.P. 500
  • Massimo Farina
    • European Commission, Joint Research Centre, Institute for Health and Consumer Protection T.P. 500
  • Efrat Forti
    • European Commission, Joint Research Centre, Institute for Health and Consumer Protection T.P. 500
  • Neil Gibson
    • European Commission, Joint Research Centre, Institute for Health and Consumer Protection T.P. 500
    • European Commission, Joint Research Centre, Institute for Health and Consumer Protection T.P. 500
  • Federica Simonelli
    • European Commission, Joint Research Centre, Institute for Health and Consumer Protection T.P. 500
  • Wolfgang Kreyling
    • Comprehensive Pneumology Center, Institute of Lung Biology and DiseaseHelmholtz Zentrum Muenchen, German Research Center for Environmental Health (GmbH)
Research Paper

DOI: 10.1007/s11051-009-9806-8

Cite this article as:
Abbas, K., Cydzik, I., Del Torchio, R. et al. J Nanopart Res (2010) 12: 2435. doi:10.1007/s11051-009-9806-8

Abstract

Industrially manufactured titanium dioxide nanoparticles have been successfully radiolabelled with 48V by irradiation with a cyclotron-generated proton beam. Centrifugation tests showed that the 48V radiolabels were stably bound within the nanoparticle structure in an aqueous medium, while X-ray diffraction indicated that no major structural modifications to the nanoparticles resulted from the proton irradiation. In vitro tests of the uptake of cold and radiolabelled nanoparticles using the human cell line Calu-3 showed no significant difference in the uptake between both batches of nanoparticles. The uptake was quantified by Inductively Coupled Plasma Mass Spectrometry and high resolution γ-ray spectrometry for cold and radiolabelled nanoparticles, respectively. These preliminary results indicate that alterations to the nanoparticles’ properties introduced by proton bombardment can be controlled to a sufficient extent that their further use as radiotracers for biological investigations can be envisaged and elaborated.

Keywords

NanoparticlesRadiolabellingTitanium dioxideIn vitroCell uptakeNanomanufacturingNanomedicine

Introduction

Nanotechnology and nanoparticles hold enormous potential in many areas, from environmental remediation, to energy efficiency, novel consumer products and more efficient treatment of disease. However, many of the same physico-chemical properties that give nanomaterials such promising possibilities, also open the possibility that they could have adverse effects on human health and the environment (Balbus et al. 2007). For example, the ability of certain nanoparticles (NPs) to penetrate through cell membranes and into the cell nucleus (Pantarotto et al. 2004), to bind and potentially damage DNA (Zhao et al. 2005) makes them a promising vehicle to deliver therapeutic agents into specific cells and body compartments (Kam et al. 2005; Florence and Hussain 2001). On the other hand, the possibility that industrially fabricated NPs, which are released into the environment from consumer products or waste, and that unintentionally penetrate into and accumulate in the body, and/or pass through biological barriers or cell membranes could turn the benefit of this new technology into a major risk (Balbus et al. 2007; Reijnders 2009). Since the exploitation of nanoparticulate materials for commercial applications is rapidly increasing (e.g. OECD 2008; Roco 2008; Aitken et al. 2006; Anselmann 2001), as is the concentration of NPs in the environment (Handy et al. 2008a; Wiesner et al. 2006; Owen and Depledge 2005; Kreyling et al. 2004), a sound risk assessment of this new technology is overdue (Keller 2007; Maynard et al. 2006). Indeed, governments, industries and research organizations are beginning to address how the benefits of nanotechnologies can be realized while minimizing their potential risks (Maynard et al. 2006; OECD 2006). However, this attempt is hampered by scientific knowledge gaps. The lack of toxicological data and the absence of adequate testing methodologies has triggered much research world-wide on in vitro and in vivo testing methods in order to assess possible health effects of NPs to humans and has created a new scientific term: nanotoxicology (Oberdörster et al. 2005).

The mechanistic basis of exposure and effect are poorly understood in many cases (Handy et al. 2008b) and experimental methods to investigate absorption, distribution, metabolism and excretion of NPs are incomplete or insufficient. Recent investigations of the toxicity of nanomaterials in cell cultures and animals have shown that size, surface area, surface chemistry, solubility and shape may all affect the toxicological potential of NPs (Poland et al. 2008; Maynard et al. 2006; Oberdörster et al. 2005). In addition, it has been observed that NPs can translocate from the tissue where they have been absorbed to other target tissues adding further complexity to the assessment of their potential toxicity (Semmler-Behnke et al. 2008; Oberdörster et al. 2005). In this context, the development of fast and reliable in vitro and in vivo test methods is essential, and for this purpose the use of radiolabelled NPs as tracers can be very advantageous. Radiolabelling of NPs for biological and toxicological experiments is however a challenge because modifications of their physical, chemical and surface properties have to be avoided in order to exclude unwanted effects on their bio-distribution and cell uptake characteristics. Moreover, for certain studies regarding the safety of industrially fabricated NPs the radiolabelled particles should not be synthesized in laboratory experiments from radioactive precursors, but should be used as they emerge from the industrial production process. Under such circumstances the only way of radiolabelling, apart from chelation of a suitable radiotracer which might modify the NP behaviour or indeed present stability problems, is via neutron or ion bombardment of the NPs in reactors or accelerators. Both methods involve subjecting the NPs to significant radiation fields and high rates of energy deposition.

The present work deals with radiolabelling of NPs by irradiation with a proton beam produced by a cyclotron and the comparison of the uptake of radiolabelled and cold (as received) NPs in vitro using a human cell line. Industrially fabricated TiO2-NPs have been examined due to their favourable physical properties and because protocols for in vitro testing using radioactive and cold NPs have been developed earlier to assess their skin toxicity, since they are used as an important component of sun creams (Di Gioacchino et al. 2007; Di Giampaolo et al. 2004). It can therefore be expected that the in vitro cell uptake obtained with cold and proton irradiated TiO2-NPs can be compared with high accuracy. Moreover, recent studies on the cytotoxicity of TiO2-NPs in fish (Handy et al. 2008b; Vevers and Jha 2008) raise questions about their ecotoxicological risk, and the large quantities involved in industrial production processes suggest that a further assessment of some aspects of airborne exposure in occupational health is necessary (Liao et al. 2009; Garabant et al. 1987).

The in vitro model chosen for this study was Calu-3, a human cell line derived from a bronchial adenocarcinoma (Forbes 2000). Calu-3 is known as a good cell model of the airway epithelium, as cells are able to form a polarized confluent monolayer with tight junctions and the production of mucous under air-interfaced culture conditions (Grainger et al. 2006; Steimer et al. 2005; Wan et al. 2000; Berger et al. 1999). The presence of the main characteristics of the airway epithelium in Calu-3 suggests the relevance of this cell line as in vitro model for studying the effect of micro- and nano-sized particles on the lung.

Experimental methods

Preparation of irradiated TiO2 nanoparticles

Titanium dioxide (TiO2) in nanoparticulate form is manufactured worldwide in large quantities for use in a wide range of applications, from paint to sunscreen, to food colouring. Two types of TiO2-NPs have been proton irradiated. The first batch was P25 (Degussa) with a stated average primary particle size of 21 nm. This batch was used for initial determinations of activation yield, and a small amount was also used for XRD (X-ray diffraction) comparison of as-received and proton irradiated material to examine if the irradiation caused gross structural changes to the sample. Whereas phase changes can be detected by XRD due to additional diffraction peaks appearing in the XRD spectra, radiation damage will manifest in a slight broadening of the XRD peaks. In addition, the full height half width of the XRD peaks depends inversely on crystal or grain size, i.e. in the present case on the NP size. In order to be able to recognize radiation damage and to discern it from the effect of small particle size, narrow XRD peaks and thus larger NPs are preferred to examine this problem. More interesting for the envisaged radiotracer applications are, however, particles smaller than 20 nm. Therefore, a second type TiO2-powder of 99.9% purity purchased from Alfa Aesar (Johnson Matthey) with a supposed size of 5 nm was activated. An XRD scan on this material indicated, however, an average crystallite size somewhat larger, between 15 and 20 nm. This means that the particle size used to address the alteration of the NP properties and that foreseen for the cell uptake experiments was so similar that no additional assessment of radiation effects on the Alfa Aesar NPs was required.

The irradiations were performed with the Scanditronix MC 40 cyclotron of the Joint Research Centre (Ispra, Italy), which is able to accelerate positive ions such as protons, deuterons, alphas and 3He2+ to variable energies. The radioisotope 48V with a half-live T1/2 = 15, 97 d can efficiently be produced by the nuclear reaction 48Ti(p,n)48V and its half-live is suitable for biological tracer experiments. In order to preserve the properties of the NPs during the proton bombardment it is essential to limit the heat load and radiation damage by a reasonable limitation of the particle beam intensity and by assuring efficient cooling. The samples were irradiated in aluminium capsules as shown in Fig. 1 with an inner diameter of 10 mm. The capsules were inserted in a holder that allowed direct water cooling from both the rear and the front side. Consequently the most critical cooling problem was the heat transport inside the NP volume to the confining aluminium surfaces.
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Fig. 1

The powder capsule (1) is completely filled with TiO2-NPs and sealed with an O-ring (2) when fixing the cover (3) carefully with its screws (4). The capsule holder (5) keeps the capsule in its irradiation position and assures all-around water cooling. The target body (7) is sealed by an O-ring (6) when the capsule holder is fixed in its proper position. The upper O-ring sealed water connection (8) directs the incoming cooling water on the powder capsule; the water flows back through the lower connection (8). All parts are fabricated from pure aluminium that is itself getting less activated by ion beams than Al alloys. The thickness of the components (1) and (5) is reduced to 0.3 mm each within a radius of 5 mm around the beam axis, referred to as windows, in order to keep the attenuation of the proton beam in energy and intensity at reasonable values and to allow for an efficient activation of the TiO2-NPs without compromising mechanical stability

The cyclotron was set to a proton energy of 25 MeV. Due to the attenuation of the proton beam in the two aluminium windows and the front face water cooling, the available beam energy for the activation of the NPs was reduced to about 15 MeV. This energy still covers the range of the maximum reaction cross section of the 48Ti(p,n)48V reaction (IAEA 2008, EXFOR database). The beam attenuation inside the TiO2-NP volume leads to a temperature increase of the NPs, especially since the density of the NP powder and the thermal conductivity of TiO2 are low. This implies that the NPs in the center of the volume that have the largest distance from the water cooled surfaces suffer the most. In order to reduce this risk and in view of the small quantities of NPs that are required for biological radiotracer experiments, the volume of the capsule was reduced since shorter distances (higher temperature gradients) facilitate heat transfer, and a more efficient cooling is achieved. Leaving the inner diameter of the capsule constant, its useful thickness was reduced from initially 2 to 0.4 mm corresponding to a reduction of the activated TiO2-NP mass from 75 mg to about 12–15 mg. In addition, the required activity could be concentrated in a smaller NP volume since protons with attenuated energy and hence reduced reaction cross section deposit their energy now in the well cooled metallic material of the capsule cover (see (3) in Fig. 1) after having passed the NP volume, instead of depositing their energy in the NPs while having low activation probability. Therefore, this modification increased both the efficiency of the NP activation and of their cooling.

In a series of test irradiations for fixed time, the proton beam current was escalated and the NP powder was checked for visible alterations afterwards. These tests were stopped at a beam current of \(10\,\upmu\hbox{A}\) without encountering any visible changes to the powder or any different behaviour when washing the NP powder out of the capsule and preparing an aqueous TiO2-NP suspension. In order to reduce radiation exposure during handling of the activated capsule, the whole holder was left for 1 day of radioactive decay of the mainly short-lived radioisotopes that are co-produced in the aluminium capsule, before the capsule was removed from the beam line.

For subsequent centrifugation tests and cellular uptake studies 12 mg of the Alpha Aesar material were irradiated. It should be noted that cold and irradiated particles would be agglomerated to some extent. This would make no difference to the XRD studies, but a large increase in agglomeration would be likely to show up in the cellular uptake studies. For the cellular uptake studies both cold and irradiated samples were suspended in exactly the same way prior to the in vitro comparative tests in water and mixed with PBS solution.

Post irradiation tests

The nanoparticle activation level was analyzed by γ-ray spectrometry using high purity germanium detectors calibrated in energy and efficiency by using certified radioactive standard sources. The calibration was performed in the same geometries used for the measurements of the irradiated TiO2-NP samples. The γ-ray spectra were analyzed using the Genie 2000 software package (CANBERRA, USA).

The irradiated capsules were handled in a glove box when opened, and activated TiO2-powder was washed out of the aluminium capsule and suspended in distilled water. For the NP sample (Alfa Aesar) destined for use in uptake experiments, the obtained suspension of 12 mg of irradiated TiO2-NPs in 2 mL of water was transferred into a quick seal vial (Beckman, Italy) and subjected to ultracentrifugation at 41,000 rpm at a temperature of 4 °C for 1 h to check for any leaching or ionic release of the 48V radiolabel into the aqueous phase.

In order to determine if any structural changes to the NPs were induced by the proton irradiation, X-ray diffraction was performed on the irradiated P25 material both before and after irradiation. The measurements were made on a dedicated glancing angle diffractometer by mixing a small amount of material with poly(methyl methacrylate), short PMMA and smearing this onto the surface of a silicon wafer over an area of about 0.5 cm2. Due to the high surface sensitivity of the glancing angle XRD technique, the amount of material analyzed could be maintained low enough so that no special precautions regarding sample radioactivity had to be taken during the XRD measurements. This limitation had, however, the disadvantage that the XRD signal-to-noise ratio was not optimal.

For the present uptake tests Calu-3 cells were chosen because they are readily available, in contrast to primary human cell lines of airway tissue, and differentiate into a monolayer of polarized cells. These cells form tight junctions, produce mucous and express many of the characteristics of human native epithelium despite being derived from a human adenocarcinoma. Calu-3 human bronchial epithelial cell line was purchased from American Type Culture Collection (ATCC, USA). Cells were maintained in Minimum Essential Medium Eagle (Sigma Aldrich), supplemented with 10% heat inactivated fetal bovine serum (Lonza, Italy), 0.1 mM non-essential amino acids (Sigma Aldrich), 1 mM sodium pyrovate (Sigma Aldrich), and \(100\,\hbox{I.U.}/100\,\upmu\hbox{g/mL}\) Pen/ Strep (Sigma Aldrich). The cells were grown at 37 °C in an atmosphere with 5% CO2 in a humidified incubator. For the experiments the cells were seeded on Transwell cell culture supports (Sigma Aldrich) at a density of 105 cells/cm2 in 0.25 mL medium and with 1 mL medium added to the basolateral compartment. Cells were grown in air-interfaced culture for 14 days before the uptake experiments were performed. Cold TiO2-NP or proton irradiated [48V]-TiO2-NP suspensions were freshly prepared in phosphate buffered saline (PBS, Sigma Aldrich) and were diluted to appropriate concentrations (200 and \(500\,\upmu\hbox{M})\) with PBS. The radioactivity concentration expressed in counts per ng of NPs was used to determine the nominal concentrations by radioactive counting. At the day of exposure, the freshly prepared NP suspensions were applied apically to the cells and fresh medium was added to the basolateral compartment. After 24 h of exposure, the cells were trypsinised and centrifuged and the obtained pellet was rinsed twice with PBS.

Percoll gradient (obtained by ultracentrifugation) was used to remove particles that were extracellular and not bound to the cell membrane. Finally, viable cells were counted using a hemacytometer, then the TiO2 and [48V]-TiO2, i.e. the NP uptake, was determined by Inductively Coupled Plasma Mass Spectrometry (ICPMS) and high resolution γ-ray spectrometry, respectively. The ICPMS instrument used was an ICPMS SCIEX ELAN DRC II (Perkin Elmer) equipped with a Dynamic Reaction Cell (DRC). 99.99% pure argon (Air Liquide) and anhydrous NH3 of 99.99% purity (Sigma Aldrich) were used for the ICPMS and the DRC, respectively. The analysis was done in 2–3% HNO3. Before the instrumental analysis, the samples were mineralized in a microwave furnace (CEM-MSD 2000). In order to correct for matrix effects due to the different sources of samples and calibration standards 5 ppb of Re was added as internal standard to all biological samples, blank specimens and calibration standards.

The uptake of [48V]-TiO2 was determined by high resolution γ-ray spectrometry by quantifying the radioactivity of 48V emitted from the radiolabelled [48V]-TiO2-NPs using a high purity germanium detector. The acquisition and analysis of the γ-ray spectra was carried out by specific software (Nuclear Elements Digital Analysis, NEDA, Ascom, Milano). The measurements of the radiation emitted from the [48V]-label are expressed in counts per minute (cpm) and converted into mass of [48V]-TiO2-NPs. The calibration factor for this conversion was determined from a comparison with standard suspensions of well-known concentration of [48V]-TiO2-NPs and activity of 48V.

Results and discussion

Figure 2 shows the γ-ray spectra of the proton irradiated aluminium capsule containing the Alfa Aesar TiO2-NPs and the spectrum of these TiO2-NPs after they were removed from the capsule, respectively. The γ-ray peaks of radioisotopes such as 65Zn and 56Co result from the activation of impurities in the aluminium material of the capsule. They are not present in the spectrum of the [48V]-labelled TiO2-NPs extracted from the capsule. Under the present irradiation conditions with a beam intensity of \(10\,\upmu\hbox{A}\) about 850 kBq of 48V were obtained within 45 min of proton bombardment of 12 mg of TiO2-NPs. This activity concentration is more than enough for the in vitro uptake studies reported here. However, higher activities—up to 1 MBq/mg or more may be required for certain studies. Figure 3 shows two γ-ray spectra of the TiO2-NP pellet and the aqueous phase after ultra-centrifugation, respectively.
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Fig. 2

γ-ray spectra (spectra a and b) of the proton irradiated TiO2-powder. Spectrum a refers to the aluminium capsule containing the NPs, spectrum b refers to the TiO2-NPs removed from the capsule. The γ-ray peaks of 48V are well resolved in both spectra. The γ-ray peaks of 56Co and 65Zn visible in (a) are due to the impurities in the aluminium the capsule is made of. The recovered NPs (spectrum b) are not contaminated with such impurities

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Fig. 3

γ-ray spectra (spectra a and b) obtained after ultracentrifugation of the colloidal TiO2-NP suspension. Spectrum a refers to the TiO2-pellet while the spectrum b refers to the supernatant which shows only a minor fraction of the 48V activity

The quantitative evaluation of the γ-ray spectra of the pellet and the aqueous phase after suspending the NPs in water and subsequent ultracentrifugation shows that only a tiny fraction of <1% of the 48V activity was not retained in the NPs. The issue of radiolabel stability as a function of crystalline structure, primary particle size, and possible irradiation damage is a complex one however, and is best addressed on a case-by-case basis by experimental determination of radiolabel loss in appropriate environments. The simple test performed here indicated that in an aqueous environment only a little of the 48V was detached from the NPs. However, for follow-up studies in other environments, more suitable leaching tests would be appropriate.

Figure 4 shows the XRD patterns obtained from the irradiated and non-irradiated P25 NPs, together with the background pattern due to the PMMA used to fix the NPs onto the Si surface. As expected the P25 material is composed mainly of anatase with a minor fraction of rutile (roughly 10% in this sample). There are no obvious major structural changes to the P25 TiO2-NPs caused by the irradiation, though minor differences in relative peak heights indicate that a small increase in the fraction of rutile (to roughly 20%) could have occurred. Due to the small amount of material analyzed and the resulting suboptimal signal-to-noise ratio of the XRD scans, this observation needs to be further investigated. Computer simulations of radiation displacement damage (see below) indicate that this is unlikely to account for any structural change. However, at high temperatures anatase converts to rutile, so the observed changes indicate some local heating of the NPs during irradiation. The thicker capsules initially used for the P25 irradiations with a large amount of material in the activated volume were in fact not optimal regarding cooling, so localized heating could have occurred in the center of the capsule volume, accounting for some anatase to rutile transformation. The cooling of the Alfa Aesar NPs that were activated in the thinner capsules, with much smaller sample volume and a much shorter beam path through the NPs, was much more efficient. Currently, even more efficient geometries for sample cooling are being developed, and further investigations of structural changes to NPs of different primary particle sizes, induced by ion-beam irradiation over a range of different irradiation parameters, are warranted, given the sensitivity of NPs to increased temperatures. This holds in particular if higher activity concentrations are required for subsequent experiments, as would be the case for some in vivo biokinetics studies.
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Fig. 4

X-ray diffraction patterns of as-received and proton irradiated P25 TiO2-NPs. Also shown is the background due to the PMMA used to fix the TiO2-NPs onto a Si surface. ‘A’ and ‘R’ indicate the positions of major anatase and rutile peaks, respectively. The patterns have been normalized to the same intensity value of the main anatase peak at 25.3° for easy comparison

Table 1 summarizes the average uptake of cold and radiolabelled NPs in Calu-3 cells under the conditions reported in Sect. Post irradiation tests. The data obtained at each concentration are the average of three independent uptake experiments with three replicas for each concentration with the standard deviation being presented as the measurement error. The results show that the radiolabelling by proton irradiation of the TiO2-NPs did not significantly alter the uptake behaviour in Calu-3 cells for both concentrations used. Within the experimental error, the results on the non-irradiated and irradiated materials are the same. We conclude that the irradiation did not massively affect the state of agglomeration of the NP powder. This should become visible in a significant difference between the uptake of cold and radioactive NPs otherwise treated in exactly the same way. However, it cannot be excluded that small differences caused by the irradiation are still hidden within the given error margins. Nevertheless, our results serve as a first demonstration that proton irradiation is a viable technique for nanoparticle radiolabelling, and that under the conditions used, no significant differences in uptake were observed. Further studies on a variety of different NP types are under way using a range of characterization methods in order to carefully study to what level the NPs may be activated before significant changes to the NP structure or in vitro behaviour are observed.
Table 1

Comparison of the uptake of cold TiO2 and radiolabelled [48V]-TiO2-NPs in Calu-3 cells in pg of TiO2 per cell after exposure to NP suspensions of different concentrations

Uptake

Concentration

pg TiO2/cell

TiO2

[48V]-TiO2

\(200\,\upmu\hbox{M}\)

1.15 ± 0.12

0.96 ± 0.11

\(500\,\upmu\hbox{M}\)

2.25 ± 0.33

1.98 ± 0.2

The error margin is given by the standard deviation of the average uptake determined in three independent experiments and three replicas for each concentration, respectively

With regard to theoretical considerations, calculations of radiation damage based on a full cascade calculation, using the SRIM (Version 2008.03 by Ziegler et al. 2008), indicate that the direct displacement damage expected due to ion collision should be in the order of 6·10−4 dpa (displacements per atom) for an irradiation of 45 min at \(10\,\upmu\hbox{A}\) at a proton energy of 15 MeV (the incident beam energy is reduced from 25 to 15 MeV by the aluminium windows and water cooling), in a target with \(400\,\upmu\hbox{m}\) thickness and an assumed density of 0.5 g/cm3. This reflects therefore the approximate amount of damage calculated for the activity level used for the uptake experiments reported here, and would not be expected to induce any major structural change. However, if much higher [48V]-TiO2 activity levels are required, then damage levels might occur where several % of the NP atoms are displaced from their lattice positions. Likewise, if other NP types with lower activation cross sections, or irradiation with other particles such as deuterons or alphas is necessary, than it is clearly important to calculate and perhaps experimentally determine any possible structural damage to the NPs before using them for subsequent experiments. If thick targets are used where the ion beam is fully stopped in the nanoparticulate material, then the direct radiation damage near the end of the ion range may be several times higher than at the start of the trajectory. In addition, the obtainable activity concentration of the irradiated material will be much lower because a considerable fraction of the NPs will be irradiated far from the energy of maximum reaction cross section. Furthermore, significant heating can be expected to occur within the sample. Such a situation is clearly undesirable.

Another important consideration relates to the kinetics involved in the activation reaction. Conservation of momentum means that it is most probable that any radiolabels produced during the irradiation will not in fact remain on the lattice site occupied by the target atom involved. While this is not necessarily true for all reactions, it can be expected that in the majority of cases the radiolabels are either displaced within the target nanoparticle or are in fact ejected from their source nanoparticle and implanted into a nearby one. Stability of the radiolabel within the nanoparticle therefore is an important issue, and it is important to determine if any leaching of activity occurs in the environment associated with any subsequent experiments. In this study, we limited ourselves to simple centrifugation studies in water to determine if the 48V-radiolabels were well bound to the TiO2-NPs. In experiments going beyond the present simple uptake studies, e.g. for much longer exposure times or intracellular distribution or even more in in vivo studies, significant leaching would become a major concern and it is highly important to establish the stability of the radiolabelled NPs in appropriate media on longer time scales.

We activated un-coated non-functionalized dry TiO2-NP powder, which is expected to be rather resistant to radiation or thermal damage. Direct ion-beam activation of liquid suspensions or NP samples with organic coatings or functional layers will pose additional problems related to a lower incidence of ‘re-implantation’ of radioactive recoil nuclei in adjacent NPs, lower achievable activity concentrations, radiation damage to the organic component which is much more radiation sensitive than the non-organic core, and possible interaction of the NPs with the liquid medium under irradiation.

Conclusions

In the present study, we have demonstrated that industrially fabricated TiO2-NPs can successfully be radiolabelled with 48V, to useful activity levels for radiotracing purposes, by proton irradiation. The in vitro uptake study in Calu-3 cells shows that the NP properties that determine the cell uptake can essentially be preserved at the activity level used. For higher activity concentrations, which may easily be achieved by longer irradiations and/or higher beam currents it will be important to carefully control any possible thermal heating of the samples under irradiation, as well as investigating whether direct structural radiation damage occurs. The characterization methods we applied here were limited to those available in the cyclotron controlled area. It should be noted that several particle characteristics other than crystalline structure might affect in vitro or in vivo studies with radiolabelled NPs, including state of agglomeration, surface charge and dissolution rates. The present work demonstrates the feasibility of radiolabelling NPs by proton bombardment. Future work will address a systematic study of the activation of different NP types, additionally employing other characterization methods such as DLS and zeta-potential determination, as well as radiotracer stability studies under different conditions.

In summary, our results demonstrate that dry TiO2 NPs can be radiolabelled by direct proton irradiation in a suitable fashion for in vitro cellular uptake studies and other applications that require limited activity concentrations. For other NP types, activity concentrations and applications, it will be important to examine on a case-by-case basis if the radiolabelled material is effectively equivalent to the non-activated material for the required experiments. In addition, stability of the radiolabels within the NPs should be determined by appropriate tests for each application.

Acknowledgements

The authors would like thank W. Horstmann and F. Arroja for their continuous support in the fabrication and improvement of the design of the irradiation capsules and G. Cotogno for his IT support. This work has been partially funded by the European Commission’s 7th Framework Programme, ‘NeuroNano’ project (contract NMP4-SL-2008-214547).

Copyright information

© Springer Science+Business Media B.V. 2009