Neutron Activated 153 Sm Sealed in Carbon Nanocapsules for In Vivo Imaging and Tumor Radiotherapy

: Radiation therapy along with chemotherapy and surgery remain the main cancer treatments. Radiotherapy can be applied to patients externally (external beam radiotherapy) or internally (brachytherapy and radioisotope therapy). Previously, nanoencapsulation of radioactive crystals within carbon nanotubes, followed by end-closing, resulted in the formation of nanocapsules that allowed ultrasensitive imaging in healthy mice. Herein we report on the preparation of nanocapsules initially sealing ‘cold’ isotopically enriched samarium ( 152 Sm), which can then be activated on demand to their ‘hot’ radioactive form ( 153 Sm) by neutron irradiation. The use of ‘cold’ isotopes avoids the need for radioactive facilities during the preparation of the nanocapsules, reduces radiation exposure to personnel, prevents the generation of nuclear waste and evades the time constraints imposed by the decay of radionuclides. A very high specific radioactivity is achieved by neutron irradiation (up to 11.37 GBq/mg), making the ‘hot’ nanocapsules useful not only for in vivo imaging but also therapeutically effective against lung cancer metastases after intravenous injection. The high in vivo stability of the radioactive payload, selective toxicity to cancerous tissues and the elegant preparation method offer a paradigm for application of nanomaterials in radiotherapy. 153 Sm@MWNT. bioluminescence treated with

ABSTRACT: Radiation therapy along with chemotherapy and surgery remain the main cancer treatments. Radiotherapy can be applied to patients externally (external beam radiotherapy) or internally (brachytherapy and radioisotope therapy). Previously, nanoencapsulation of radioactive crystals within carbon nanotubes, followed by end-closing, resulted in the formation of nanocapsules that allowed ultrasensitive imaging in healthy mice. Herein we report on the preparation of nanocapsules initially sealing 'cold' isotopically enriched samarium ( 152 Sm), which can then be activated on demand to their 'hot' radioactive form ( 153 Sm) by neutron irradiation. The use of 'cold' isotopes avoids the need for radioactive facilities during the preparation of the nanocapsules, reduces radiation exposure to personnel, prevents the generation of nuclear waste and evades the time constraints imposed by the decay of radionuclides. A very high specific radioactivity is achieved by neutron irradiation (up to 11.37 GBq/mg), making the 'hot' nanocapsules useful not only for in vivo imaging but also therapeutically effective against lung cancer metastases after intravenous injection. The high in vivo stability of the radioactive payload, selective toxicity to cancerous tissues and the elegant preparation method offer a paradigm for application of nanomaterials in radiotherapy.
Advances of nanomedicine in cancer diagnosis and therapy require the production of 'small' and 'smart' agents that can offer specific targeting, adequate detection sensitivity, efficient therapeutic effects and ideally favorable biocompatibility. [1][2][3][4][5] Carbon nanotubes (CNTs) have been exploited as delivery systems for theranostic applications. [6][7][8][9][10] Their needle-like structure enables efficient cell penetration. [11][12][13][14] Moreover, a selected biomedically relevant payload can be loaded onto the CNT structure either by chemical modification of the largely available external surface (exohedral), or by filling their interior space (endohedral). Surface engineering is a more straightforward approach and has been widely investigated. 15,16 Although less explored, confinement of imaging and therapeutic agents into CNTs is attracting an increased attention. 17,18 After encapsulation, the external walls remain available to anchor biocompatible and/or targeting ligands. One advantage of the nanoencapsulation approach is the protection that the carbon coating (CNT walls) offers to the encapsulated materials from enzymatic degradation once exposed to biological environment. It has been already demonstrated that only highly defective, oxidized CNTs are degraded by peroxidases. 19 Apart from CNTs, a variety of materials are being employed for the formation of nanocapsules that shield a selected payload. [20][21][22][23][24] In the vast majority of studies, release of the encapsulated cargo is necessary to achieve a therapeutic effect via the delivery of bioactive agents such as drugs. This is however not the case for nuclear imaging and targeted radiotherapy applications where departure of the encaged imaging and/or therapeutic radionuclides from the nanocapsule is neither required nor desired. From the library of nanocapsules available, the properties of carbon nanotubes make them unparalleled for the permanent sealing of radionuclides. [25][26][27][28] The graphene shells that constitute the walls of the CNTs can perfectly separate the core from the external environment, forming impermeable nanocapsules when the ends of the CNTs are closed. 29,30 Having closed ends allows the removal of non-encapsulated material, after the filling step, under harsh washing conditions 31 and even the retention of gases in their interior. 32 Mutual protection is therefore afforded to both biological systems and the nanotube cargo delivered. 7 When radionuclides are sealed, the action of ionizing particles exerts through the walls of CNTs 25,27,28 and it spans from micron to millimeter distances depending on the characteristics of the chosen isotopes. It is worth noting that the internal radionuclides and external moieties (biocompatible/targeting molecules) can be independently changed thus giving versatility to this approach.
In current clinical cancer therapy, half of all cancer patients are treated with radiation therapy either alone or combined with other types of therapies. Nanotechnology strategies however have been mainly driven by the use of chemotherapeutic agents. Notwithstanding, there is a growing interest in the development of nanomaterials bearing radioisotopes, since they offer a platform not only for molecular imaging but also to enhance the radiation response of tumors while reducing side effects. [33][34][35][36] CNTs have been shown to deliver positron/γ-radioemitters and alpha particles of interest for imaging and therapeutic applications. This has been mainly achieved via chelation of radionuclides onto the external walls [37][38][39] although the impregnation of radionuclides onto highly defective CNTs, which can be regarded as mesoporous materials, has also been investigated by Wilson et al. [40][41][42] As just mentioned, an attractive feature of CNTs is the possibility to hermetically seal radionuclides in their interior. The nanocapsule will not only prevent release of the chosen radionuclides into the biological milieu but also of their decay products, which might be of a different chemical nature and detach from the nanocarrier with the previously employed strategies.
We have previously reported a pioneering work using hermetically sealed Na 125 I-filled and externally glycosylated single-walled CNTs (SWNTs). 25 Specific tissue accumulation (lung) coupled with high in vivo stability prevented leakage of radionuclide to high-affinity organs (thyroid/stomach) or excretion, and resulted in ultrasensitive imaging and delivery of a high radiation dose density 25 Pascu et al. have recently given versatility to this approach and investigated the behavior of radiometal-filled ( 64 Cu) SWNTs in vitro and in vivo. 27 In this study the ends of the CNTs were sealed using fullerenes as corks to prevent release of the encapsulated radioemitter. 43,44 In contrast to these previous reports, where 'hot' radionuclides, in the form of Na 125 I or 64 Cu(OAc)2, were directly filled into SWNT, followed by end-closing, herein, we present an approach by which 'cold' non-radioactive 152 Sm is initially sealed into the cavities of both SWNT and multi-walled CNTs (MWNT) leading to the formation of closed-ended 152 Sm-filled carbon nanocapsules. The encapsulated and stable 152 Sm can then be activated into therapeutically active 'hot' 153 Sm by neutron irradiation through the walls of the CNTs (Figure 1a; Schematic representations comparing both strategies to achieve 'hot' nanocapsules are included in Figure   S1). After neutron activation, exceptionally high specific activities were obtained, compared to previous works on direct encapsulation of radionuclides, which allowed not only in vivo nuclear imaging but also lung tumor radiotherapy. 153 Sm is an attractive radionuclide as it emits beta particles at maximum energy of 810 keV (suitable for cell killing), and also releases gamma energy of 103 keV (allowing clinical imaging). 45, 46 153 Sm is used clinically in the form of 153 Sm-ethylene diamine tetramethylene phosphonate chelate ( 153 Sm-EDTMP, Quadramet®) for palliation of bone metastases. 47 It offers added advantages such as desirable half-life (46.3 h) and lower gamma energy over other therapeutic radionuclides currently being used in clinic such as iodine-131 or yttrium-90. 48 The encapsulation of 'hot' radionuclides into the cavity of CNTs requires a fast and safe manipulation of the material due to their constant decay. By using the neutron activation strategy (Figure 1a), both the filling and the removal of the non-encapsulated compound do not require the use of radioactive facilities thus reducing radiation exposure to the personnel. It also alleviates the time constraints imposed by the constant decay of radionuclides since the 'cold' nanocapsules can be shelf-stored and activated by neutron irradiation on demand. Neutron activation of nanoparticles incorporating neutron-activatable isotopes is well documented, with initial studies focused on the use of fullerenes and polymeric nanoparticles. [49][50][51][52][53] Di Pasqua et al. also proposed the use of inorganic mesoporous silica nanoparticles loaded with neutron-activatable holmium-165. 54,55 Taking advantage of the porosity of the nanocarrier, this was later expanded to chemoradiotherapy. 56 Other inorganic materials including holmium iron garnet, mesoporous carbon nanoparticles and graphene oxide have more recently been explored as holmium nanocarriers. [57][58][59] On the other hand, neutron activation of large particles (i.e. holmium-166 containing microspheres) has been employed in clinical trials for the treatment of liver tumor via intra-arterial injection leading to radioembolization. 60,61 In this work we propose the use of neutron irradiation of 'cold' isotopically enriched 152 Sm sealed within carbon nanotubes. The activated 'hot' 153 Sm nanocapsules allow both in vivo imaging and tumor radiotherapy.

RESULTS AND DISCUSSION
Preparation and neutron activation of 152 Sm-filled nanocapsules to yield radioactive 153 Sm@CNTs. As-received CNTs were initially purified and shortened by combination of solution processing with strong acids followed by steam. 62 High temperature molten phase capillary filling was employed for the encapsulation of isotopically enriched 152 SmCl3 into SWNT and MWNT, leading to the closing of the nanotube ends on cooling 29,30 (Figure 1a). For ease of nomenclature, we will refer to these nanocapsules as 152 Sm@SWNT and 152 Sm@MWNT. We employed enriched samarium ( 152 Sm) to ensure an efficient activation to the clinically employed 153 Sm isotope upon neutron irradiation. 63 After the filling experiment, the excess of 152 SmCl3 external to the walls of the CNTs was removed by repeated washings and filtration of the sample in water. The length and external diameter distribution of the samples was determined after the filling step. The median length turned out be ca. 200 nm for SWNTs and ca. 330 nm for MWNTs (see Figure S2 and Table   S1 for statistical data), and the median external diameters 2.1 nm for SWNTs and 10.7 nm for MWNTs ( Figure S3, Table S2). The amount of samarium present was next quantitatively Therefore, no samarium waste is generated in the filling step.
Neutron activation is favored in radionuclide production because of its relatively simple process and the availability of various types of nuclear reactors. It also permits lower radiation exposure to the personnel since the preparation procedures such as encapsulation, purification and characterization can be conducted prior to irradiation. Exceptionally high specific radioactivities (SRA) with respect to the mass of the composite material were obtained after 96 h neutron irradiation of 152 Sm@SWNT (6.33 GBq/mg) and 152 Sm@MWNT (11.37 GBq/mg) ( Table 1). The SRA values are two orders of magnitude (differ by a factor of about 100) higher than those previously reported for neutron irradiated nanocarriers loaded with clinically relevant radiotherapeutic isotopes, which are in the range of ca. 1-80 MBq/mg (Table S3). 64  interior. Based on recent studies on non-enriched SmCl3 nanocapsules, over 99% of SmCl3 that is not sealed inside the CNTs can be easily dissolved by a short sonication (10 s) of the sample in water. 65 Therefore, to ensure the complete dissolution of 153 Sm from the inner cavities of those CNTs that could have undergone structural damage, a longer sonication step (several min) in water was performed. As shown in Table 1 and Figure S4  Additional TEM images for both 'cold' and 'hot' nanocapsules are included in Figure S5.
Structural changes of the encapsulated crystals are sometimes observed during electron microscopy imaging. [66][67][68][69][70][71] In the present case we observed that in some cases, SmCl3 nanowires were fragmented into smaller crystals ( Figure S6). Preservation of the payload encapsulation was further confirmed by high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) (Figure 1b, central panels). The SmCl3 filling (bright white lines) aligned nicely within the CNT lumen (dim grey contrast) even following irradiation also indicating the presence of filling material.
Energy dispersive X-ray (EDX) spectroscopy was conducted to assess the preservation of the payload content upon irradiation. The presence of samarium was detected before and after irradiation ( Figure 1b, bottom panels). Europium, the decay product of 153 Sm, was not detected by EDX because despite the high SRA less than 1% of 152 Sm nuclei underwent nuclear reaction to radioactive 153 Sm under the employed conditions.  Toxicity assessments post 153 Sm@CNT injection and after radiotherapy. During the radiotherapy period, variable whole body weight changes were observed within groups ( Figure   4a). Body weight loss seemed to occur for the 153 Sm@CNT treated mice starting from day 10 posttumor inoculations but there is no significant difference between the groups including the untreated mice. The weights of major organs were measured at sacrifice. Marginal changes were found between most of the untreated and 153 Sm@CNT treated organs, except for lungs (with tumors, ***p < 0.001) and spleen (***p < 0.01 or ***p < 0.001) (Figure 4b). As previously evidenced in Figure 3b, the aggressively growing tumors increased the weight of lung significantly for the untreated mice. We have previously demonstrated that cold metal-filled CNTs were biocompatible in vivo. 75 The significant weight loss in lungs from mice treated with 153 Sm@CNTs compared to untreated mice is likely a result of reduction of tumor nodules. Histological assessment performed on mice treated with low dose radioactivity (200 µg 153 Sm@CNTs/mouse, 1 MBq) showed no significant histological abnormalities in the selected major organs (heart, lung, kidneys, liver, and spleen) sampled at 24 h post injection ( Figure S9). This further confirms that the reduced lung weights were due to the radiotherapy effect and not from the CNTs per se.
The average weight of spleen of 153 Sm@CNT treated mice was reduced compared to untreated tumor-bearing mice. This is less likely to be general toxicity of the CNTs but a consequence of the high spleen uptake of 153 Sm@CNTs leading to high radioactivity accumulation. Histological

Preparation of 'cold' nanocapsules ( 152 Sm@CNT). Both SWNTs and MWNTs (Elicarb ® )
were initially treated to shorten the tubes, open their ends and remove carbonaceous and metallic (catalyst) impurities. SWNTs were exposed to a combined piranha-steam treatment, whereas MWNTs underwent a combined H2SO4:HNO3-steam treatment following previously reported protocols. 62 Steam is very efficient in removing functional groups and highly defective carbon nanotubes. 62 Enriched 152 Sm2O3 (CIS-Bio International-Ion Beam Applications, France) was transformed to anhydrous 152 SmCl3 following the protocol reported for the synthesis of anhydrous SmCl3 with natural isotopic distribution (non-enriched). 80 The synthesized anhydrous 152 SmCl3 is highly hygroscopic and was handled under an inert atmosphere, and employed for filling CNTs.
SWNT or MWNT and 152 SmCl3 were ground together in a weight ratio 1:10 (CNTs: 152 SmCl3) inside an argon filled glove box, split in smaller fractions, placed inside silica tubes and sealed under vacuum. The mixtures were annealed for 12 h at 900 °C (SWNT) or 1200 °C (MWNT) thus leading to the formation of carbon nanocapsules (closed-ended filled CNTs). 29,30 Removal of the non-encapsulated 152 SmCl3 with 0.6 M HCl was followed by UV-Vis, until no more 152 SmCl3 was detected in the washings (0.2 μm Whatman ® polycarbonate membranes). 65 The length distribution of the resulting 152 Sm@SWNT and 152 Sm@MWNT was determined from SEM images, following a previously described methodology, 81  Tumor growth was monitored by bioluminescence imaging. The tumor growth data was expressed as mean ± SEM (standard error of the mean), with n denoting the number of animals.

Dosimetry simulation.
For targeted radionuclide therapy, the surviving fraction (SF) of the tumor cell population over time, , can be described by equation (2). 82 (2) where is the radiosensitivity parameter (Gy -1 ), is the extrapolated initial dose rate (Gy h -1 ), is the radionuclide effective uptake rate (h -1 ), is the radionuclide effective washout rate (h -1 ) and is the tumor cell proliferation rate (h -1 ). The effective rates of uptake and washout of the radionuclide account for both biological and physical processes and they are defined through and , respectively, where is the biological uptake half-life, is the biological washout half-life, and is the physical half-life of the radionuclide. The values of where is the number of tumor cells at . The rate constant in equation (4) is normally expressed as with being the tumor doubling time. Fit of the experimental data by equation (4) yields (see Figure 7a).
The extrapolated initial dose rate in the tumor, , entering equation (2) is estimated from equation where is the administered activity to the mice (20 MBq), is obtained from equation (3), is the fraction of the organ activity that is taken up by the tumor, and is the radiation absorbed dose to the tumor per radionuclide decay (Gy Bq -1 s -1 ). For 153 Sm and a tumor mass of 0.07 g, . 83 To obtain , it is assumed that the activity was distributed uniformly Substituting the above parameters in equation (2), the values and are obtained for MWNTs and SWNTs, respectively.
The radiosensitivity parameter α was determined empirically from cell survival data using the expression ,    Results are presented as mean ± S.D. (n = 9-10). No statistical differences were found in % whole body weight changes between different treatments. Mice treated with 153 Sm@CNTs showed significant weight reduction in lung and spleen. Significant differences were examined using oneway ANOVA followed by Tukey's multiple comparison test (**p < 0.01, ***p < 0.001). TABLES.

Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. We thank Thomas Swan Co. Ltd. for supplying CNT Elicarb ® samples. We are grateful to S. Sandoval (ICMAB) and C. Ramos (IQS-ICMAB) for assessing the length and diameter distribution of CNTs.