Hydroxyapatite and Titanium Dioxide Nanoparticles: Radiolabelling and In Vitro Stability of Prospective Theranostic Nanocarriers for 223Ra and 99mTc

Hydroxyapatite and titanium dioxide are widely used materials in a broad spectrum of branches. Due to their appropriate properties such as a large specific surface area, radiation stability or relatively low toxicity, they could be potentially used as nanocarriers for medicinal radionuclides for diagnostics and therapy. Two radiolabelling strategies of both nanomaterials were carried out by 99mTc for diagnostic purposes and by 223Ra for therapeutic purposes. The first one was the radionuclide sorption on ready-made nanoparticles and the second one was direct radionuclide incorporation into the structure of the nanoparticles. Achieved labelling yields were higher than 94% in all cases. Afterwards, in vitro stability tests were carried out in several solutions: physiological saline, bovine blood plasma, bovine blood serum, 1% and 5% human albumin solutions. In vitro stability studies were performed as short-term (59 h for 223Ra and 31 h for 99mTc) and long-term experiments (five half-lives of 223Ra, approx. 55 days). Both radiolabelled nanoparticles with 99mTc have shown similar released activities (about 20%) in all solutions. The best results were obtained for 223Ra radiolabelled titanium dioxide nanoparticles, where overall released activities were under 6% for 59 h study in all matrices and under 3% for 55 days in a long-term perspective.


Introduction
The progress in the development of nanomaterial technology has a significant influence on all scientific and everyday life applications [1,2]. The massive expansion of nanomaterials is also seen in medicine, such as in bandages with antimicrobial nanosilver or with antibiotic capsules and nanoparticles (NPs), which are applicable for delivering drugs, light, heat, etc. [3]. In nuclear medicine, inorganic NPs could be used as one of the possible carriers for diagnostics or therapeutic radionuclides. At present, hydroxyapatite (HAp) [4], BaSO 4 -NPs [5], Ag-NPs [6], LaPO 4 -NPs [7], TiO 2 [8], superparamagnetic iron oxide NPs [9] are under examination. An important advantage of nanoparticles is a large specific surface area and radiation stability which allows them to resorb ions and also to retain recoil radionuclides [10]. For these reasons, hydroxyapatite (nHAp) and titanium dioxide nanoparticles (nTiO 2 ) were selected.
Hydroxyapatite is a natural material occurring in bones and teeth. Its artificial analogue is used in medicine as a part of bone and tooth implants [11,12]. Other HAp applications, for example as This work aims to verify the radiolabelled nanoparticles and to provide evidence as to whether they can be used as radionuclide carriers in nuclear medicine. The work was mainly focused on the NPs radiolabelling and the behaviour and stability of the labelled NPs in different media. Firstly a suitable and fast radiolabelling strategy with appropriate yield was developed. For this purpose, two strategies of radiolabelling were selected. The first strategy was the radionuclide sorption on ready-made NPs and the second one was the intrinsic labelling, where the radionuclide was incorporated into the structure of NPs at the stage of their preparation. The second task was to determine the influence of biologically relevant media on the in vitro stability of the radionuclide-NP carrier system. For this in vitro study physiological saline, bovine blood plasma and serum, 1% and 5% albumin solution were used. These data will allow to determine if the nanocarriers could be used for further in vivo experiments, and if the radionuclides remain with the carriers and the radionuclides are not released to the surrounding tissues.

Materials and Methods
All chemicals were of analytical grade and were used without further purification: tetrabutyl orthotitanate (TBOT), propane-2-ol (IPA), ammonium hydroxide, calcium nitrate tetrahydrate, sodium chloride, diammonium hydrogen phosphate, and sodium azide purchased from Merck (Darmstadt, Germany); SnCl 2 purchased from The British drug houses Ltd. (Poole, UK), and bovine plasma and serum, albumin lyophilised (pH 7) purchased from Biowest (Nuaillé, France). Demineralized water of 18 MΩ/cm −1 was obtained from Millipore (Burlington, MA, USA) water purification system. Gamma spectra were measured with a HPGe detector and analysed using the Maestro Software (ORTEC, Oak Ridge, TN, USA). Overall activities were measured with a well-type NaI(Tl) crystal CII CRC-55tW (CAPINTEC, Ramsey, NJ, USA). For mixing of samples, Stuart SSM3 rocker (Cole-Parmer Ltd., Vernon Hills, IL, USA) was used and separation was made on VWR Micro Star 12 centrifuge (VWR International, Radnor, PA, USA). All experiments were performed under aseptic conditions in laminar box Airflow 150 UV (Esi FLUFRANCE, Arcueil, France).

Preparation of Nanomaterials
The detailed description of both hydroxyapatite and titanium dioxide nanoparticles' synthesis and characterization was already published by Kukleva et al. [15]. Here, only synthesis is described briefly. The basic characteristic of prepared nanoparticles are summarised in Table 1. For the synthesis of nHAp, 1.2 M Ca(NO 3 ) 2 in demineralized water was used. For correct synthesis running, pH was set and maintained at 11 by ammonium hydroxide solution. Afterwards, the same volume of 0.7 M (NH 4 ) 2 HPO 4 was dropwise added and mixed overnight. Then, prepared nanoparticles were washed with demineralized water (3×) and dried under vacuum.
The selected preparation method of nTiO 2 was the hydrolysis of TBOT. The mixture of TBOT and IPA (1:4, respectively) was dropwise added into demineralized water in ultrasonic generator and the solution was mixed at the laboratory temperature. After that, prepared nanoparticles were washed with physiological saline (3×) and IPA (1×) and dried under vacuum.

Preparation of 223 Ra and 99m Tc Stock Solutions
The stock 223 Ra solution was obtained from 227 Ac/ 227 Th/ 223 Ra generator prepared at the Department of Nuclear Chemistry at our laboratory according to Guseva et al. [43]. The column was filled with anion-exchanger Dowex-1 × 8 resin. The elution of 223 Ra was performed by 0.7 M HNO 3 in 80% methanol. Gamma-spectrometric analysis was used for radionuclide purity detection of 223 Ra(NO 3 ) 2 solutions (breakthrough of parents' radionuclides in the eluate). The eluted 223 Ra(NO 3 ) 2 solutions were dried and reconstituted in demineralised water [25].
The 99m Tc solution stock was gained from commercial generator DRYTEC TM (GE Healthcare LTD, Chicago, IL, USA). The elution was performed by physiological saline.

Radiolabelling Procedure
Two strategies of radiolabelling with both 223 Ra and 99m Tc were selected. The first strategy was the surface radiolabelling, where the radionuclide was sorbed on the ready-made NPs' surface. The previously prepared NPs (5 mg) were dispersed in 300 µL of physiological saline for 223 Ra radiolabelling or in 500 µL of fresh stannous chloride solution (480 mg/L) for 99m Tc radiolabelling. Consequently, 223 Ra or 99m Tc solutions were added. The radioactivity of 223 Ra was added from 5 to 10 kBq and the radioactivity of 99m Tc solution was from 60 to 100 MBq. The samples were mixed for one hour at laboratory temperature and then washed with physiological saline (3×).
The second strategy was the intrinsic labelling, where the radionuclide was incorporated directly into the NPs' structure. Radiolabelled nHAp were prepared in the following way: 35 µL of 1.2 M Ca(NO 3 ) 2 was added to 500 µL of demineralized water, then pH was set to 11 by 1 M ammonium hydroxide solution. Then, the 223 Ra solution was added in a small volume so that the activity added was in the same range as for surface labelling. Finally, 35 µL of 0.7 M (NH 4 ) 2 HPO 4 was added and the mixture was stirred for 1 h at laboratory temperature. In the case of 99m Tc radiolabelling, the procedure was basically the same, but instead of 223 Ra solution, 99m Tc was added. The samples were washed with physiological saline (3×).
Radiolabelled nTiO 2 was prepared in the following way: tetrabutyl orthotitanate in IPA (1:4, 70 µL) was dropwise hydrolysed in physiological saline (300 µL) with already-added 223 Ra solution or in stannous chloride solution (500 µL) with added 99m Tc. The radioactivity was in a similar range to in the previous case. The samples were mixed for 1 h at laboratory temperature and washed with physiological saline (3×).
All experiments for both radiolabelling strategies for both materials were repeated six times and resulting yields were calculated.

In Vitro Stability Studies of Radiolabelled Materials
In vitro stability studies were performed in several matrices: physiological saline, bovine blood plasma and serum and human albumin solutions (1% and 5%). Nanoparticles were centrifuged after labelling and washing, and the solution was replaced with a new matrix. All samples were performed in triplets. The samples were incubated at laboratory temperature and shaken during the incubation. The matrices were replaced with the same but fresh solution after 2-7-12-17-26-35-59 h from the begging of the experiment (in the case of 99m Tc last interval was removed and the previous one was shortened from 9 to 5 h due to its shorter half-life). Samples and supernatants were measured on NaI(Tl) scintillation detector. The percentages of released radioactivity were specified separately for 223 Ra-NPs (short-term) and 99m Tc-NPs. Long-term stability studies were provided for 223 Ra, where the matrices were replaced every 11 days (T 1/2 ( 223 Ra) = 11.4 d) five times (total 55 days).

Radiolabelling
Two strategies of NP radiolabelling were tested: surface (S) and intrinsic (I) labelling. The radiolabelling yields were determined according to Equation (1) where A NPs is the final activity of radiolabelled nanoparticles and A init is an initial activity. The longest radionuclide of the decay chain, the 223 Ra, was measured. Daughter radionuclides were not specifically determined. All radiolabelling yields for both strategies and materials were higher than 94% ( Table 2). Some data about HAp radiolabelling were already published by other scientists. Spherical hydroxyapatite with diameter 900-1000 µm (10 mg or 19 spherical granules) were incubated 24 h with 223 Ra. The radiolabelling yield was approximately 80% [26]. Another paper focused on hydroxyapatite was by Albernaz et al. [4]. Hydroxyapatite powder (<210 µm) was radiolabelled with 99m Tc (approx. 3.7 MBq). Particles were incubated for 10 min and the average radiolabelling yield was 98.5%. These results are in good agreement with obtained experimental data.
To date, there has been no literature found dealing with 223 Ra radiolabelling of titanium dioxide. The presented results of nTiO 2 radiolabelling yields were slightly higher than nHAp yields and strategy of intrinsic labelling showed the same or even better yields than surface radiolabelling. However, the difference was statistically undetectable. It is important to note that all the results correspond with earlier published data from sorption and kinetic studies [38,39].
Both labelling strategies could be considered for radiopharmaceuticals preparation. Each type, with its advantages, could be appropriate for another utilisation. On the one hand, surface radiolabelling is preferable for the often-prepared radiopharmaceutical doses. Standardised kits with ready-made NPs of defined size can be prepared with required sterile and apyrogenic properties easily. The kit type radiopharmaceuticals are, at present, often used in nuclear medicine departments and could be stored and used in laboratory conditions. On the other hand, the intrinsic labelling may be used as rapid method, where both the nanoparticle preparation and the radiolabelling procedures are performed in a single step. This method could be useful for a variety of modifications of radiolabelled nanoparticles, which cannot be provided before radiolabelling.

In Vitro Stability Studies of Radiolabelled Nanomaterials
In vitro experiments were designed as pseudo-open systems, where used liquid was replaced in defined periods under free air conditions. All samples were shaken during incubation period. These in vitro stability tests deal with the radionuclide-carrier system only. The system was considered as stable if the radionuclide release from the material to the media was observed minimally. The colloidal stability of the nanoparticles themselves was not studied, however this important parameter requires further research, particularly for in vivo applications.
Both radiolabelled nanomaterials were studied in vitro in the following solutions: physiological saline, bovine blood plasma and bovine blood serum, 1% and 5% human albumin solution. The percentage of released activity, %A, was performed according to Equation (2) where A sup is activity of supernatant and A NPs is activity of labelled separated nanoparticles. Physiological saline is the most frequently used solution in medicine, therefore it was selected as a comparative standard solution. The blood proteins serve as transporters of various compounds, therefore, two different solutions of albumin were also used for examination. Further, it is necessary to study labelled nanoparticles' behaviour in plasma and serum. The plasma composition consists mostly of water and proteins such as albumins, globulins and fibrinogen, then ions, saccharides etc. The difference between blood plasma and serum is the presence of fibrinogen and clotting factors in plasma [44]. Commercially available plasma also contains sodium citrate as a stabilizing agent.
Hydroxyapatite nanoparticles in figures are given always as (a) and (b) and nTiO 2 as (c) and (d). The first radiolabelling strategy, the surface labelling (S), is shown as (a) and (c) and the second one strategy, the intrinsic labelling (I), as (b) and (d). Error bars were not included in in vitro stability figures due to better clarity and ranged under 0.5%.
The short-term in vitro stability study of 223 Ra radiolabelled nanoparticles is shown in Figure 1. The difference between the two studied nanomaterials can be seen. In the case of nHAp, the total released activity was around 60% in saline and bovine blood plasma after 59 h from radiolabelling. The released activities in saline were unexpectedly high. However, it can be possibly explained by the lower stability of nHAp and its dissolution at lower pH or by the influence of relatively high concentration of the sodium ions (0.9% sodium chloride solution).
Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 12 where Asup is activity of supernatant and ANPs is activity of labelled separated nanoparticles. Physiological saline is the most frequently used solution in medicine, therefore it was selected as a comparative standard solution. The blood proteins serve as transporters of various compounds, therefore, two different solutions of albumin were also used for examination. Further, it is necessary to study labelled nanoparticles' behaviour in plasma and serum. The plasma composition consists mostly of water and proteins such as albumins, globulins and fibrinogen, then ions, saccharides etc. The difference between blood plasma and serum is the presence of fibrinogen and clotting factors in plasma [44]. Commercially available plasma also contains sodium citrate as a stabilizing agent.
Hydroxyapatite nanoparticles in figures are given always as (a) and (b) and nTiO2 as (c) and (d). The first radiolabelling strategy, the surface labelling (S), is shown as (a) and (c) and the second one strategy, the intrinsic labelling (I), as (b) and (d). Error bars were not included in in vitro stability figures due to better clarity and ranged under 0.5%.
The short-term in vitro stability study of 223 Ra radiolabelled nanoparticles is shown in Figure 1. The difference between the two studied nanomaterials can be seen. In the case of nHAp, the total released activity was around 60% in saline and bovine blood plasma after 59 h from radiolabelling. The released activities in saline were unexpectedly high. However, it can be possibly explained by the lower stability of nHAp and its dissolution at lower pH or by the influence of relatively high concentration of the sodium ions (0.9% sodium chloride solution). Despite this fact, short-term stability experiments in saline showed that labelled nanoparticles could be temporarily stored in saline before the injection. Plasma experiments showed high released Despite this fact, short-term stability experiments in saline showed that labelled nanoparticles could be temporarily stored in saline before the injection. Plasma experiments showed high released activities in comparison with serum results, which can be caused by proteins or sodium citrate interference. To eliminate the sodium citrate influence stability, studies with 1% solution were performed, but they did not show such a massive effect. It is important to note that the concentration of sodium citrate in plasma was unknown and was not provided by the manufacturer. Thus the influence of this well-known complexation agent cannot be excluded. The short-term stability of 223 Ra-nHAp in serum had a very promising performance.
The released activity from 223 Ra-nTiO 2 was lower than 6% for surface radiolabelling and lower than 2.5% for radionuclide incorporation. In opposite to nHAp, the lowest released activity was in saline and then in bovine blood serum for both radiolabelling strategies. The released activity in the rest of the biological matrices was slightly higher but the differences were negligible compared to statistical deviations. It could also be noticed that in the case of surface labelling, the worst results were obtained again in plasma. Another difference compared to nHAp is the slight contrast between the radiolabelling strategies. In the case of intrinsic labelling, the released activities are slightly lower than in the surface labelling.
Total released activities in long-term in vitro studies ( Figure 2) were similar to short-term studies, which can be explained by distribution coefficients and rather fast kinetics, where the equilibrium was reached before matrix replacement. Comparing the short-term and the long-term studies it could be seen that the ion-exchange and the resorption of radionuclides back on the nanoparticle surfaces play an important role. This effect could cause in vivo radionuclides' release in the case of NPs depo accumulation, where the radionuclides are retained in the tissue. Nanomaterials 2020, 10, x FOR PEER REVIEW 7 of 12 activities in comparison with serum results, which can be caused by proteins or sodium citrate interference. To eliminate the sodium citrate influence stability, studies with 1% solution were performed, but they did not show such a massive effect. It is important to note that the concentration of sodium citrate in plasma was unknown and was not provided by the manufacturer. Thus the influence of this well-known complexation agent cannot be excluded. The short-term stability of 223 Ra-nHAp in serum had a very promising performance. The released activity from 223 Ra-nTiO2 was lower than 6% for surface radiolabelling and lower than 2.5% for radionuclide incorporation. In opposite to nHAp, the lowest released activity was in saline and then in bovine blood serum for both radiolabelling strategies. The released activity in the rest of the biological matrices was slightly higher but the differences were negligible compared to statistical deviations. It could also be noticed that in the case of surface labelling, the worst results were obtained again in plasma. Another difference compared to nHAp is the slight contrast between the radiolabelling strategies. In the case of intrinsic labelling, the released activities are slightly lower than in the surface labelling.
Total released activities in long-term in vitro studies ( Figure 2) were similar to short-term studies, which can be explained by distribution coefficients and rather fast kinetics, where the equilibrium was reached before matrix replacement. Comparing the short-term and the long-term studies it could be seen that the ion-exchange and the resorption of radionuclides back on the nanoparticle surfaces play an important role. This effect could cause in vivo radionuclides' release in the case of NPs depo accumulation, where the radionuclides are retained in the tissue. Despite similar values of the released activity (from 10 to 50%), 223 Ra-nHAp showed better stability in saline in the long-term than in the short-term study. The highest effect on the activity release had the albumin solution, which showed similar results for all four series of stability studies Despite similar values of the released activity (from 10 to 50%), 223 Ra-nHAp showed better stability in saline in the long-term than in the short-term study. The highest effect on the activity release had the albumin solution, which showed similar results for all four series of stability studies with 223 Ra-nHAp. Considering the serum experiments with total released activity of about 10%, it can be concluded, that the serum does not have such a negative impact on the radiolabelled materials, therefore nHAp still can be taken as a suitable material, however, this requires further studies with other radionuclides, a modified labelling strategy or the carrier modifications.
Released activities from nTiO 2 were lower than 3%, which may be caused by radionuclide resorption during the longer studied intervals. The lowest value was detected in the reference saline solution as in the previous case. Again, the differences among matrices for 223 Ra-nTiO 2 were negligible. In the case of nTiO 2, the effect of lower activity release for intrinsic labelling was observable.
To support the theranostic idea of inorganic nanoparticles as carriers, short-term in vitro stability study of 99m Tc radiolabelled nanoparticles was performed and the results are summarized in Figure 3. The behaviour of 99m Tc-labelled nanoparticles was different from the 223 Ra-labelled ones. Contrary to 223 Ra, the released activities of 99m Tc were lower in the case of nHAp and higher for nTiO 2 .
Nanomaterials 2020, 10, x FOR PEER REVIEW  8 of 12 with 223 Ra-nHAp. Considering the serum experiments with total released activity of about 10%, it can be concluded, that the serum does not have such a negative impact on the radiolabelled materials, therefore nHAp still can be taken as a suitable material, however, this requires further studies with other radionuclides, a modified labelling strategy or the carrier modifications. Released activities from nTiO2 were lower than 3%, which may be caused by radionuclide resorption during the longer studied intervals. The lowest value was detected in the reference saline solution as in the previous case. Again, the differences among matrices for 223 Ra-nTiO2 were negligible. In the case of nTiO2, the effect of lower activity release for intrinsic labelling was observable.
To support the theranostic idea of inorganic nanoparticles as carriers, short-term in vitro stability study of 99m Tc radiolabelled nanoparticles was performed and the results are summarized in Figure  3. The behaviour of 99m Tc-labelled nanoparticles was different from the 223 Ra-labelled ones. Contrary to 223 Ra, the released activities of 99m Tc were lower in the case of nHAp and higher for nTiO2. In the case of 99m Tc-nHAp, the released activities were under 20% for both radiolabelling strategies in 31 h. However, there was high released activity in blood plasma, which was around 60%. This could be caused by a sodium citrate present as a preservative agent. For better visualisation of obtained results, plasma data were not included in Figure 3 in the case of nHAp due to fast and high activity release, where it was higher than 50% after only 10 h from labelling. The lowest released activities were found in 5% albumin solution for both type of radiolabelling. It is also the enormous difference against the 223 Ra-nHAp. This difference between in vitro stability studies of both studied radionuclides may be caused especially by their dissimilarity, e.g., valence, reactivity and other In the case of 99m Tc-nHAp, the released activities were under 20% for both radiolabelling strategies in 31 h. However, there was high released activity in blood plasma, which was around 60%. This could be caused by a sodium citrate present as a preservative agent. For better visualisation of obtained results, plasma data were not included in Figure 3 in the case of nHAp due to fast and high activity release, where it was higher than 50% after only 10 h from labelling. The lowest released activities were found in 5% albumin solution for both type of radiolabelling. It is also the enormous difference against the 223 Ra-nHAp. This difference between in vitro stability studies of both studied radionuclides may be caused especially by their dissimilarity, e.g., valence, reactivity and other properties originating from type of metal, where the radium is alkaline earth metal and technetium is transition meal.
The stability of 99m Tc-HAp was also studied by Albernaz et al. [4]. However, the stability experiments were performed only in reaction solutions containing stannous chloride in 24 h. The average overall released activity was 6%. This is in good agreement with obtained experimental results from short-term stability studies of 99m Tc-nHAp.
The highest released activities from nTiO 2 were around 15% in 5% albumin solution in the case of surface radiolabelling and even around 25% in bovine blood plasma in the case of intrinsic labelling. In all samples with titania, the lowest released activities were in physiological saline.
Our results showed that the sorption and kinetic experiments with 99m Tc on nHAp and nTiO 2 are necessary. They could bring the answers about mechanism of technetium uptake and identify the rate-controlling sorption process as it could have an influence on the in vitro stability of 99m Tc-NPs. In the case of 223 Ra, these experiments were already performed (see previous publications) and it was found that ion exchange mechanism plays the main role in sorption on nHAp and nTiO 2 .
In general, it is possible to conclude that the overall released activities were lower for 223 Ra-nTiO 2 , than for any other studied material. The best performance was shown by nTiO 2 with 223 Ra, however, overall released activities from 99m Tc radiolabelled nanoparticles were similar for both types of materials and radiolabelling strategies. Although the released activities from 223 Ra-nHAp were relatively high, it is still a promising carrier according to serum stability results. Previously published results [15,38,39] showed that both materials are suitable as carriers and both radiolabelling strategies showed high radiolabelling yields for inorganic nanomaterials.
The nanocarriers preparation and radiolabelling is the first step in new radiopharmaceutical development. Consequently, it could be necessary to upgrade the carrier to prepare core-shell structured particles or modify their surface with targeting and protecting substances, e.g., polyethylene glycol [6]. While the first step should prevent the radionuclide and its daughters from escape from the NPs, the latter should provide active targeting and better in vivo biodistribution. This concept could be also useful for nHAp modification to improve the behaviour of the radiolabelled nanoparticles in biologically relevant media.

Conclusions
Radiolabelling of nano-sized HAp and TiO 2 was studied with 99m Tc or 223 Ra using two labelling strategies, which were the labelling of ready-made particles (surface labelling) and radionuclides' incorporation into the structure of the nanomaterial (intrinsic labelling). Both methods showed high labelling yields (>94%) for both nanoparticle types and both radionuclides. Consequently, the in vitro stability studies of radiolabelled nanoparticles were performed in biologically relevant media: physiological saline, bovine blood plasma and serum and 1% and 5% human albumin solutions. The most stable in all media were the 223 Ra radiolabelled nTiO 2 , where the overall released activities in short-term stability experiments were under 6% in 59 h. Good results also were shown by both 99m Tc radiolabelled nanoparticles, where the overall released activities were under 20% in short-term aspect. In general, the worst stability shown by all materials was in plasma. From the long-term perspective, both types of NP showed good results, in some cases even better than from the short-term perspective. This may be caused by the resorption of radionuclides due to longer study periods. This, in fact, leads us to the conclusion that the stability experiments under static conditions-as usually performed and reported in the literature-may lead to false-positive results, that can be later compromised under the dynamic conditions, e.g., in animal in vivo models (see Figures 1 and 2).
Both labelling strategies can find their application in practice. Despite the several high released activities, overall activity release in all experiments was stable and both nanomaterials are still promising radionuclide carriers and surface modification, for example, could improve the stability of radiolabelled nanomaterials. Based on the obtained results, it could be concluded that nHAp is more appropriate for local application and controlled activity release and nTiO 2 is suitable for system application due to its good long-term stability.