Functionalized NaA nanozeolites labeled with 224,225Ra for targeted alpha therapy

The 223Ra, 224Ra, and 225Ra radioisotopes exhibit very attractive nuclear properties for application in radionuclide therapy. Unfortunately the lack of appropriate bifunctional ligand for radium is the reason why these radionuclides have not found application in receptor-targeted therapy. In the present work, the potential usefulness of the NaA nanozeolite as a carrier for radium radionuclides has been studied. 224Ra and 225Ra, α-particle emitting radionuclides, have been absorbed in the nanometer-sized NaA zeolite (30–70 nm) through simple ion exchange. 224,225Ra-nanozeolites exhibited very high stability in solutions containing physiological salt, EDTA, amino acids, and human serum. To make NaA nanozeolite particles dispersed in water their surface was modified with a silane coupling agent containing poly(ethylene glycol) molecules. This functionalization approach let us covalently attach a biomolecule to the NaA nanozeolite surface.


Introduction
It has been shown that ionizing radiation emitted by radionuclides is effective in the treatment of various types of cancers (Boyle and Levin 2008;Zoller et al. 2009). To minimize side effects, radiolabeled agents should be used that are selectively taken up by cancer cells, confining the radiation to the tumor, hence minimizing damage to normal cells (Carlsson et al. 2003;Cutler et al. 2013). For this purpose, tumorselective peptides, monoclonal antibodies (mAbs), or organ-specific proteins are used. Crucial for the success of this approach are bifunctional ligands which can be attached to a protein and can bind the radioactive metal cation rapidly and selectively (Schubiger et al. 1997;Volkert and Hoffman 1998;Neves et al. 2005). The vast majority of radionuclides used in targeted radiotherapy are metals with strong ability to form stable complexes. These metals can be attached to a biomolecule through multidentate cyclic or acyclic ligands. However, there are radionuclides with very attractive nuclear properties, which can be used in nuclear medicine, but cannot be stably bound to biomolecules by bifunctional ligands. To this group belong, for example, radionuclides of radium, which forms very weak complexes.
Radium radioisotopes: 223 Ra (t 1/2 = 11.43 days), 224 Ra (t 1/2 = 3.66 days), and 225 Ra (t 1/2 = 14.8 days) show very attractive nuclear properties for application in radionuclide therapy. These radionuclides decay through a cascade of short-lived a and bparticle emitters to stable lead and bismuth, releasing a high total energy of about 28 MeV (Fig. 1). There are four a particles emitted in each decay series, thus therapeutically relevant doses can be delivered from low amount of injected activity. The relatively long halflives give time for preparation, quality control, and shipment of the radiopharmaceuticals. Radium radionuclides can be obtained from their long-living parental radionuclides: 223 Ra from 227 Ac (t 1/2 = 21.8 years), 224 Ra from 228 Th (t 1/2 = 1.9 years), and 225 Ra from 229 Th (t 1/2 = 7,880 years). 227 Ac can be produced by neutron irradiation of natural radium, 229 Th by extraction from large stocks of 233 U, and 228 Th as the decay product of 232 U.
The lack of appropriate bifunctional ligand is the reason why radium radionuclides have not found application in receptor-targeted therapy. As already mentioned the Ra 2? cation, like other cations of group 2, does not form stable complexes, therefore labeling of biomolecules with 223,224,225 Ra is a very difficult task. Additionally, a chelating agent is required which could bind radium radionuclide, as well as control the fate of the daughter radionuclides and withstand the immense a-particle recoil energy of 100-200 keV, which is greater than the binding energy in macrocyclic metal ion complexes.
Previous studies on binding 223 Ra to biomolecules by its complexation with tetraazacarboxylic acids, cryptands, and calixarenes were unsuccessful (Henriksen et al. 2002). Also application of liposomes as carriers for 225 Ac, 227 Th, and 223 Ra, proposed by Henriksen and co-workers was not brought into practice because of the necessity to carry out many operations with radioactive liposomes (Henriksen et al. 2004).
Nanotechnology can help to overcome these limitations and bring fundamental changes in creating new diagnostic and therapeutic radiopharmaceuticals (Kairem et al. 2008;Ting et al. 2010;Hong et al. 2009). Radionuclides with specific emission properties can be incorporated into nanoparticles (NPs) and  Fig. 1 The decay series of radium radionuclides used for radionuclide therapy and radio-imaging. NPs are particularly suitable for the delivery of a or bemitters in radioimmunotherapy. The advantage of NPs is the potential for containing several radioactive atoms within a single carrier. This feature is useful when a target tissue has only a few receptors per cell, which limits the dose that can be delivered (McDevitt et al. 1998).
NPs can deliver radionuclides using either passive or active targeting strategy. The passive targeting accumulation of NPs takes place in non-specific way through enlarged gap junctions in tumor endothelial cells (Carmaliet and Jain 2000). This type of targeting, which enables macromolecules to selectively accumulate in the tumor tissue, is called enhanced permeation and retention (EPR). Unfortunately, local drug deposition is unfeasible for larger tumors with poor vascularization. Target specificity is then achieved through hybrid NPs produced by conjugating NPs with tumor-specific biomolecules, including mAbs, aptamers, peptides, or various receptor-specific substrates (Hamoudeh et al. 2008;Chanda et al. 2010).
In the present work, we propose to use nanozeolite whose channels and cavities will be filled with cations of 223 Ra,224 Ra,and 225 Ra by means of simple ion exchange, without any additional operations.
Zeolites are biocompatible crystalline aluminosilicates composed of tetrahedral structures which build open framework consisting of channels and cages of molecular dimensions. Each tetrahedron containing aluminum formally bears one unit of negative charge, because aluminum atom is connected to four oxygen atoms. To make the crystal electrically neutral metal cations (e.g., Na ? , K ? , Ca 2? ) are present in the interstices of the zeolitic framework. We can distinguish many different kinds of zeolite structures with different selectivity and ion-exchange properties (Sherry 1969). The use of nanozeolite with suitable structure should enable stable encapsulation of radium radionuclides inside the framework. In the light of the Eisenman-Sherry theory of cation-exchange selectivity, zeolites exhibit greater or smaller preference for one cation over another (Reichenberg 1966). Taking into account these rules, we can predict that zeolites with high aluminum content, like the A-type nanozeolite, will prefer Ra 2? than Na ? , K ? , Mg 2? , and Ca 2? , i.e., cations with high concentrations in physiological liquids.
Till now, only one paper was devoted to the application of radiolabeled nanozeolite in nuclear medicine. Tsotsalas et al. used nanozeolite L to fill it with c-emitter 111 In 3? , through simple ion exchange (Tsotsalas et al. 2010). The zeolite L has relatively large windows (r = 0.35 nm), in comparison with In 3? ionic radius (r i = 0.080 nm, CN = 6), which results in leakage of 111 In under physiological conditions. To avoid the leakage, the entrances to the channels were closed by specifically designed silanol stopcocks. The authors concluded that the obtained samples were highly stable in physiological liquids and may enable a large variety of functionalities on the external surface of nanozeolite L. However, the proposed procedure requires difficult post-labeling operations.
The aim of our work was to synthesize the NaA nanozeolite of small dimension (\100 nm), to load it with radium radionuclides, and to study stability of the labeled nanozeolite in various biological liquids.
Radionuclides 224 Ra was produced from a generator based on the 232 U/ 228 Th pair (Narbutt and Bilewicz 1998). About 5 MBq of 232 U was adsorbed in a small column on Teflon grains impregnated with HDEHP. Next, 224 Ra was eluted with 0.1 M HNO 3 , the eluate from the generator evaporated to dryness and the remaining residue containing radioactivity was re-dissolved in 200 ll of 0.1 M HCl. 225 Ra was obtained from the 229 Th source available at the Institute for Transuranium Elements, Karlsruhe, Germany. The source contains 214.5 mg of 229 Th (t 1/2 = 7,340 years), which makes possible to isolate 1.60 GBq of 225 Ra every 9 weeks. In order to isolate 225 Ra in the first step 225 Ra and 225 Ac were separated from 229 Th using ion exchange (Apostolidis et al. 2005), followed by separation of 225 Ra from 225 Ac using extraction chromatography (Zielinska et al. 2007). Briefly, thorium batch, consisting of 500 ml of Dowex 1 9 8, anion-exchange resin, was washed 5-6 times using each time 100 ml of 8 M HNO 3 . The combined washing solutions, containing 225 Ra, 225 Ac, and 3-5 % of 229 Th/ 232 Th, were evaporated to almost dryness, put into 20 ml of 8 M HNO 3 and filtered through a preliminary column (1.0 ml volume) to remove organic impurities. Next, the filtrate was loaded into an anion-exchange column (Dowex 1 9 8, 100-200 mesh, column volume 80 ml). 225 Ra and 225 Ac were eluted by washing with 250-300 ml of 8 M HNO 3 , and the eluate was evaporated to nearly dryness and put into 10 ml of 4 M HNO 3 for subsequent radium/actinium separation by extraction chromatography. Subsequently, the 225 Ra/ 225 Ac product from the anion-exchange separation in 4 M HNO 3 was loaded into a UTEVA/RE-resin column cascade (volume of each equal to 1.6 ml). Under these conditions, residual thorium was extracted on the UTEVA column. Next, 225 Ra was washed out from the column with no more than 20 ml of 4 M HNO 3 and evaporated to reduce its volume. Before each experiment, 225 Ra was purified from its decay products ( 225 Ac, 213 Bi) on a chelating resin. For this aim 1 ml of the solution containing 225 Ra with its decay products was adsorbed on a small column (d = 3 mm, h = 20 mm) filled with cation-exchange resin Chelex-100. Next, 225 Ra was quantitatively eluted with 0.001 M HCl solution.

Instrumentations
The particle size and morphology of the NaA nanozeolite were determined by scanning electron microscopy (SEM, Zeiss) and transmission electron microscopy (TEM, LEO 912AB). Identification and determination of crystallinity of the nanozeolite were carried out by X-ray diffraction (XRD, D8 Advance) using Vantec detector with Cu Ka radiation (1.54 Å ) operated at voltage 40 kV and current 20 mA. The average size and zeta potential (f) were measured by dynamic light scattering (DLS, Malvern, UK). Thermal stability of modified and unmodified samples was determined by thermogravimetric analysis (TGA, Q500, TA Instruments, US).
Gamma-spectrometry was carried out using a calibrated intrinsic Ge detector (crystal active volume 100 cm 3 ) and PC-based Multichannel Analyzer (MCA, Canberra). The detector had a resolution of 0.8 at 5.9 keV, 1.0 at 123 keV, and 1.9 at 1,332 keV. Radioactivity of 224 Ra and 212 Pb was quantified by their 237.75 keV c-ray, and by the 725.29 keV c-ray from 212 Bi. Radioactivity of 225 Ra was quantified by its 40 keV c-ray, by the 99.8 keV c-ray from 225 Ac, by the 217.86 keV c-ray from 221 Fr, and by the 439.8 keV c-ray from 213 Bi. The type of the decay product was confirmed by calculation of T 1/2 from the decay curves.
Synthesis of NaA nanozeolite Nanocrystalline NaA zeolite was prepared according to modified procedures described in the papers (Wang et al. 2002;Hu et al. 2009 The synthesis procedure was divided into two steps. In the first step, sodium aluminate was added to aqueous sodium hydroxide solution and kept under stirring to complete dissolution. Then the required amount of tetramethylammonium hydroxide (template) was slowly added and the solution stirred continuously for 24 h at room temperature. The second step was the hydrothermal treating. The silica gel was added dropwise to the solution under stirring and the mixture was immediately placed in an oil bath equipped with magnetic stirrer and temperature controller. Hydrothermal crystallization was carried out for 48 h, at 100°C with rotation rate 750 rpm. The obtained nanocrystals were cooled to room temperature and washed with distilled water by centrifugation at 13,500 rpm (2-16P Spincontrol Universal, Germany) until pH\8. Next, the sample was calcinated at 500°C for 3 h to remove the template.

Synthesis of silane-PEG modified NaA nanozoelite
Modified NaA nanozeolite has been synthesized according to the procedure described in the book (Hermanson 2008). The 50 ml of solution containing 4 % water in ethanol (v/v) was prepared and its pH was adjusted to 4.5-5.5 with acetic acid. 2 ml of silane-PEG solution was added dropwise to the acidic water/ethanol solution under stirring at room temperature. The reagents were stirred continuously for 5 min. After that, 50 mg of nanozeolite NaA was added to the solution and stirring was continued for 1 h, at room temperature. The obtained product was washed several times with ethanol to remove excess silane-PEG compound and dried at 110°C for 30 min.
Labeling NaA nanozeolite with 224 Ra and 225 Ra. Stability studies of 224/225 Ra-nanozeolites and 224/225 Ra-nanozeolites-silane-PEG 224 Ra and 225 Ra nanozeolites were prepared by exchanging Na ? for 224,225 Ra 2? . To this end, 100 mg of nanozeolite sample was introduced into the eppendorf tube, suspended in 2 ml of the 224 RaCl 2 solution [A & 0.5 MBq], and sonificated in ultrasound bath for 15 min. The eppendorf tube was gently shaken on circular stirrer for 2 h. After that time, the suspension was centrifuged for 10 min at 10,000 rpm (MiniSpin, Eppendorf, Poland). The supernatant was separated from the precipitate and its activity was measured. Afterward, the sample was washed several times with deionized water, then suspended in 1 ml of water and sonificated (Polsonic, Poland). In order to study stability, 100 ll of the 224 Ra-nanozeolite suspension was incubated in several solutions: saline, PBS buffer, EDTA, L-cysteine, and human serum (at 37°C) by shaking for 2 h. Then, the suspensions were centrifuged under the same conditions as aforementioned and activities of the supernatants were measured. Afterward, the precipitates were re-suspended in the supernatants and stability studies were extended to 24 h. The same procedure was used for 225 RaCl 2 (A & 100 kBq). The stability studies for 225 Ra were carried out for 4 days. Stability studies of NaA nanozeolites modified with PEG and radiolabeled either with 224 Ra or 225 Ra were carried out in exact conditions as described above.
The blank tests with the serum and radionuclide solution, but without nanozeolites were carried out to estimate the percentage of possible binding of free radionuclides ( 224,225 Ra 2? , 212 Pb 2? , 208 Tl ? , 212,213 Bi 3? , 225 Ac 3? , 221 Fr ? ) to proteins present in the serum. After certain time of incubation of blank sample, 1 ml of human serum, combined with 5 ll of 224,225 RaCl 2 solution, was centrifuged with the same speed as that used with nanozeolites samples (10,000 rpm) and the activity of the serum taken from the top of centrifuged sample was measured on gamma-spectrometer.

Results and discussion
The NaA nanozeolite was successfully prepared by hydrothermal synthesis. Figure 2 shows XRD patterns of the obtained sample. The NaA nanozeolite structure was identified by comparing relative intensities in its XRD powder pattern (Fig. 2a) with those reported in the literature (Fig. 2b) (Gramlich and Meier 1971). The two powder diffraction patterns were found to be in good agreement. The broadened XRD pattern peaks observed in the obtained sample are indicative of small size of the crystalline product. Crystallinity of the NaA nanozeolite was calculated using Eq. (1) (Cullity 1956): where I hkl is the connected integral XRD peak intensity and I b is the integral background intensity for the same plane. The obtained nanozoelite was found to exhibit about 90.9 % of crystallinity. Scherrer's formula (Eq. 2) was used to calculate the crystallite dimension (Cullity 1956): where B is the diffraction curve width measured at an intensity equal to half the maximum intensity in radius, k is the X-ray wavelength, and h B is the Bragg angle. The average crystallite size of the NaA nanozeolite is about 43 nm. Both calculations were made with reference to the most significant peak (200). Size distribution and shape of NaA nanozeolite particles were investigated by DLS (Fig. 3), SEM and TEM (Fig. 4).
It can be seen from the SEM images that the particles with shape similar to parallelepiped, with the size between 50 and 170 nm, were formed. The particles seem to have tendency to aggregate into the size from 200 nm to even 1 lm. However, the DLS analysis shows that the suspended in water NPs are of the size between 40 and 120 nm, which is in good agreement with the SEM results. Disintegration of the agglomerates has been obtained by sonification in an ultrasound bath for 15 min, followed by filtration on syringe filters with 0.22 lm pores (Millipore). To verify the size range of the NPs purified by sonification and filtration the TEM image was made (Fig. 4b). The TEM image shows that the filtration process is crucial for separating the small NPs from agglomerates and for getting a well-dispersed sample.
The obtained sample of the NaA nanozeolite was used for absorption of 224,225 Ra radionuclides by ion exchange with Na ? ions, which are present in the channels and interstices of NPs. The percentage of absorption for both radionuclides was above 99.9 %. The studied nanozeolites exhibited high affinity for both radium isotopes, with the distribution coefficient exceeding 10 4 cm 3 g -1 . The distribution coefficient was calculated as the ratio of the equilibrium activity in 1 g of the solid phase to that in 1 ml of the aqueous phase. Stability of radiolabeled nanozeolites was studied in various solutions containing: physiological salt, complexing agent (EDTA), buffer (PBS), amino acid (L-cysteine), and human serum. In all studied solutions, the leakage of 224,225 Ra from the nanozeolites was below 0.5 %. In solution only the decay products of radium radionuclides were found. Results of the stability study are shown in Table 1. Table 1 shows the percentage of liberated activity from each daughter radionuclide, determined by its decay characteristic c-ray peak. The percentage values of each radionuclide refer to the absolute amount of radionuclide in the sample. The NaA nanozeolite labeled with 224 Ra is very stable in the physiological salt and in PBS buffer solutions. The activities of the samples were near background. The examined sample is less stable in solutions containing either complexing agent (EDTA) or amino acid (L-cysteine). The cradioactivity, which was found in the solutions, has been attributed to 212 Pb, 208 Tl, 212 Bi (the decay products of 224 Ra). When the decay series is taken into consideration, we can assume that due to radioactive equilibrium certain amounts of 208 Tl and 212 Bi originate from 212 Pb decay. But there are also small amounts of these radionuclides, which were eliminated from nanozeolitic structure, due to leaching by aqueous solution. Confirmation of high stability of 224 Ra-nanozeolite was made after human serum study, which showed that the percentage of released radionuclides was low. The extension of the incubation time to 24 h has brought no changes in the percentage of liberated radioactivity. 225 Ra has been purified from its decay products before labeling. The purification made possible to obtain NPs labeled only with radium cations, which are strongly bound inside nanozeolite structure. Therefore, there has been no leakage of radioactivity to the solution in 2 days. After that time, the first cradioactivity, due to the presence of 225 Ac, 221 Fr, and 213 Bi (the daughter radionuclides of 225 Ra), has been found. For this reason, stability studies have been of 212,213 Bi binds to proteins. Thus, the presented percentage values of stability studies with human serum were re-calculated and adequately increased.
In the experiments described above unmodified nanozeolites have been used. In order to make the surface of the NaA nanozeolite crystals more hydrophilic and biocompatible, a batch of the nanomaterials has been modified by covalent attachment of PEG molecules. The modification of the NaA nanozeolite surface was fast and simple (Scheme 1). The functionalization of the surface was made by using silane coupling agent with three methoxy groups and PEG molecules. Silane-PEG agent was attached to the surface of NPs by the siloxane bonds formation. The TGA was used to confirm the modification (Fig. 5).
It can be seen on both thermograms that there is a loss of mass (*5 ± 1.5 %) in the range from 80 to 100°C, which can be attributed to desorption of physically adsorbed water molecules. The consecutive loss of mass is observed between 100 and 800°C (*8 ± 2 %), what is associated with desorption of water occluded in the interstices and channels of nanozeolite. Additionally, on the modified nanozeolite's thermogram a loss of mass at about 390°C (*3 ± 1.5 %) is observed, which can be attributed to degradation of the silane-PEG molecules.
The zeta potential (n) was measured in order to give an additional confirmation of the successful modification of NaA nanozeolite surface. We found that the zeta potential has changed from -32.5 ± 1.7 mV for unmodified NPs to -13.4 ± 1.9 mV for modified ones. The zeta potential was measured in phosphate buffer (pH 7.4).
The labeling with 224,225 Ra and stability studies were also carried out for PEGylated nanozeolite (Table 2). No significant differences were observed between results obtained for functionalized NPs and for those without modification. This indicates that the access to pores and channels of NaA nanozeolite has not been blocked by covering the surface with PEG molecules.

Conclusions
Our preliminary results have shown that the NaA nanozeolite can be used as a carrier for 224 Ra and 225 Ra and for all of their daughter radionuclides. We have also shown that PEG molecules can be covalently attached to the NaA nanozeolite surface using the silanization approach. The modification of the surface makes the NPs stable and dispersed in water, which was confirmed by zeta potential measurement. Additionally, it is worth to notice that 225 Ra radionuclide is a soft bemitter, whereas the alpha emission originates from its decay products. Therefore, by formation of radiopharmaceuticals labeled with 225 Ra, previously separated from all of daughter radionuclides, we can obtain radiobioconjugate with very low bactivity. The significant emission of a-particle occurs from 225 Ac and its decay products after few days after injection. Thus, 225 Ra can be used to label macromolecules (for example mAbs) with slow pharmacokinetics and biodistribution time. For example, IgG molecules often reach maximum of accumulation after few days (Henriksen et al. 2004).
In future experiments we intend to use the same procedure of modification, in order to cover the surface with silane coupling agent containing N-hydroxysuccinimide ester and afterward attach targeting vectors like octreotide, substance P or mAbs.