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Synergetic action of ceria nanoparticles and doxorubicin on the early development of two
fish species, Danio rerio and Puntius tetrazona
E. Yu. Krysanov1, T. B. Demidova1, O. S. Ivanova2, K. G. Ordzhonikidze3, A. B. Shcherbakov4, V. K. Ivanov2'5
1Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, 119071 Russia 2Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences, Moscow, 119991 Russia 3Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia 4Zabolotny Institute of Microbiology and Virology,NAS of Ukraine, Kiev, D 03680 Ukraine 5National Research Tomsk State University, Tomsk, 634050 Russia
krysanov@sevin.ru, van@igic.ras.ru PACS 87.18.h, 89.60.k DOI 10.17586/2220-8054-2019-10-3-289-302
The combined action of ceria nanoparticles and doxorubicin on the early stages of ontogenesis of Danio rerio and Puntius tetrazona was studied.
Results obtained indicate that there is a synergetic effect of CeO2 nanoparticles and doxorubicin which is demonstrated by a high incidence of
embryonic malformations in fish. This synergetic effect is more pronounced in tiger barbs than in zebrafish, and depends strongly on the synthetic
route of ceria nanoparticles' preparation, the most notable effects being registered for citrate-stabilized nanoparticles.
Keywords: cerium dioxide, nanotoxicology, zebrafish, doxorubicin, embryotoxicity.
Received: 23 May 2019
Revised: 1 June 2019
1. Introduction
Nanoscale cerium oxide (nanoceria) is widely used in modern technological applications as a key component of catalysts, an abrasive, a corrosion inhibitor and a constituent of healthcare and cosmetics products [1,2].
Nanoceria applications in biomedicine have been extensively discussed in several reviews [3-6]. It has been demonstrated that ceria nanoparticles are capable of playing the role of reactive oxygen species (ROS) level regulator and free radicals scavenger, both in vitro and in vivo. The usefulness of nanoceria for biomedical applications can be attributed to two main factors: its oxygen nonstoichiometry and its relatively low toxicity. The first factor determines the ability of nanoceria to affect the redox processes in the living cell, in particular under oxidative stress conditions caused by ROS. The second factor offers the prospect of comparative safety for in vivo applications of ceria nanoparticles.
Another specific property of nanoceria is the ability to regenerate its oxygen nonstoichiometry: after participating in a redox process, ceria nanoparticles are able to return to their initial state in a relatively short period of time; this feature is responsible for a prolonged therapeutic effect of nanosized ceria inside the living body [7].
Current data on nanoceria effects on living beings are still somewhat controversial. Some papers have reported on the pro-oxidant properties of nanoceria [8-10], while others have demonstrated its antioxidant behavior [11-14]. Recent in vitro experiments have demonstrated the fine interplay between the synthesis conditions of nanoceria and its toxicity [15].
The data on nanoceria's effects on aquatic organisms are also quite controversial [16]. Different aquatic species demonstrate different sensitivity rates towards nanoceria. For instance, nanoceria did not show any toxic effects in an acute test with Daphnia magna for concentrations from 10 to 1000 mg/l [17], yet it appeared to be toxic for two other genus representatives, with significant interspecies differences. Toxicity for D. similis was 350 times as high as for D. pulex [18].
No toxic effects were observed upon microinjection of nanoceria into yolk of Danio rerio embryos [17,19]. A recent study [20] revealed that CeO2 nanoparticles are non-hazardous to D.rerio embryos, both under visible light and UV-A irradiation; also, nanoceria does not exhibit any UV-A-induced phototoxic effects on zebrafish.
In the majority of papers, the toxic effects were only observed at high concentrations of nanoparticles. In turn, estimated nanoceria concentrations in the environment have been fairly low [21,22], and in aquatic systems the majority of nanoparticles precipitate [23] and only 1.3 % remain suspended in water. For nanoceria concentrations below 1 mg/l, the toxicity has only been observed in one investigation [24]. The technique of suspension preparation also affects toxicity of nanoceria. Suspensions prepared using magnetic stirring did not show any toxic effects in C. dubia, while the same suspension prepared under sonication caused death (EC50 = 11.9 - 25.3 mg/l) [25]. Another significant
factor that predetermines toxicity of nanoceria is the composition of precursors for CeO2 synthesis. CeO2 nanoparti-cles prepared with hexamethylenetetramine (HMT) caused toxic effects in Daphnia during a 48-hour test [26], while HMT itself did not. It should be noted that, in this case, the formation of ceria nanoparticles was accompanied by hydrolysis of HMT, with the release of harmful formaldehyde. Some authors have suggested that nanoceria effects on living organisms depend on the synthesis conditions [4] and type of stabilizers used [27]. For example, in the experiment with fibroblasts, CeO2 nanoparticles stabilized by citrate species interacted more actively with cells and were able to penetrate them [27]. It is worth noting that, in the presence of dissolved organic substances, stability of ceria suspensions increases and aggregate size decreases [17,28,29]. It is fair to assume that synergetic effects of nanoceria and dissolved ecotoxic agents may also be observed in a natural water environment.
The present paper aims to investigate the synergetic action of CeO2 nanoparticles and doxorubicin on the early stages of fish ontogenesis. The effects of antibiotics on the early stages of embryogenesis have been quite well investigated. Doxorubicin is a strong cardiotoxin, and the choice of doxorubicin in our experiments was due to the fact that its cardiotoxic effect is easily observable and can be distinguished from the effects caused by nanoparticles [30]. As the toxic effects of nanoceria-doxorubicin composition can exhibit interspecies differences, we used two fish species in our study, namely zebrafish (Danio rerio) and tiger barb (Puntius tetrazona), whose embryogenesis features are different.
2. Materials and methods
We used three types of ceria nanoparticle, including two aqueous sols and water-dispersible nanopowder. A non-stabilized 6 nm ceria aqueous sol was synthesized using a previously reported technique of hydrothermal-microwave treatment of the colloid solution formed upon anionite treatment of a cerium (III) nitrate aqueous solution [31]. Briefly, Amberlite IRA 410 CL ion-exchange resin (Aldrich, #216569), preliminarily converted to the OH-form, was gradually added to a 0.01 M cerium (III) nitrate (Aldrich, #238538) solution until pH reached 10.0. Sols formed in this way were separated from the resin by filtering, immediately transferred to 100 ml polytetrafluoroethylene autoclaves (filled to 50 %) and subjected to hydrothermal-microwave treatment in a Berghof Speedwave MWS-3+ setup at 190 °C for 3 h. Upon completion of the synthesis, the autoclaves were withdrawn from the microwave oven and cooled down to room temperature in air. A citrate-stabilized ceria sol was obtained from the "naked" one by addition of an equimolar quantity of citric acid, and through careful neutralizing of the solution with ammonia (Chimmed, Russia). In addition, a CeO2 nanopowder was used (Sigma Aldrich, particle size < 25 nm).
Lyophilized doxorubicin hydrochloride powder (Pharmachemi, Netherlands) was purchased from a local distributor.
Particle size measurements by dynamic light scattering and Z-potential measurements by automatic titration were carried out on the Malvern Zetasizer Nano ZS analyser (Malvern Instruments, UK). The light source used was a helium-neon laser, (the radiation wavelength was 632.8 nm).
Tests were carried out in zebrafish (Danio rerio) and tiger barb (Puntius tetrazona) embryos. The fish were kept under standard conditions in 20 litre aquaria at 26 °C with a 12 h light / 12 h dark time-schedule. The eggs were collected immediately after spawning; the quality was estimated under a stereomicroscope. The embryos were incubated in embryo media (5 mmol NaCl; 0.17 mmol KCl; 0.33 mmol CaCl2; 0.33 mmol MgSO4; pH = 7.2 - 7.3) in 24-well plates.
In a preliminary experiment, we assessed the action of pure doxorubicin (concentrations from 0.1 to 50 mg/l) on the development of zebrafish using ceria nanoparticles alone (0.001, 0.01, 0.1, 1.0 and 10 mg/l), doxorubicin alone (1.0, 5.0, 10, 20, 30 and 50 mg/l) and mixtures of nanoparticles and doxorubicin, wherein a single concentration of doxorubicin (10 mg/l) was used with nanoceria sols of different concentrations (from 0.001 to 10 mg/l), or a single concentration of nanoceria sols (10 mg/l) was used with 1.0 and 5.0 mg/l of doxorubicin.
For the preparation of the CeO2 nanoparticle suspension, the necessary quantity of stock colloidal solution of CeO2 was dispersed in an embryo medium by sonication for 10 min. Doxorubicin stock solution was prepared in distilled water. The nanoparticle suspension was mixed with fresh doxorubicin solution immediately after preparation, with subsequent sonication for 2 min.
The embryo development stages were examined using a stereomicroscope (Carl Zeiss, Stemi 2000C) and were matched according to [32].
2.1. Developmental biology of zebrafish and barb eggs
Barb eggs have a sticky layer, for substrate affixing, which hardens after fertilization. The eggs are transparent. Development up to hatching lasts for 24 h at 28 °C. The yolk sack resolves in 58 - 72 h after hatching. The embryogenesis period in barbs, when the embryo remains inside the egg and is protected by chorion, is shorter than with
zebrafish (24 h for the barb and 72 h for zebrafish). However, the larval period, when the larva swims but does not eat, is longer in tiger barbs than in zebrafish.
Fertilized eggs were collected in the first two hours after spawning. Embryos were transferred to a 24-well plate containing 2 ml of the solution being tested per well. Each embryo was transferred to a separate well and 24 embryos were used per test concentration. Embryos were incubated in a culture medium containing 0.29 g/l NaCl, 0.013 g/l KCl, 0.05 g/l CaCl2 x 7H2O, 0.0365 g/l CaCl2, 0.815 g/l MgCl2 x 6H2O; 7.0 - 7.5 pH. Embryos were kept in an incubator at 26 0C with a 14 h light / 10 h dark time-schedule. The medium was not replaced during the course of the experiment. Embryos were tested daily using a Zeiss Stemi 2000 stereomicroscope. Malformations and other teratogenic effects were scored. Survival rate, percentage of malformed embryos and rate of hatching were calculated at 96 hpf.
Statistical analysis was performed using a Chi-Square test in an SPSS 21 data analysis package.
3. Results
4. Doxorubicin toxicity in fish
Doxorubicin toxicity was assessed for concentrations of 0.1 to 50 mg/l. A significant increase in embryo death was registered for concentrations of 40 mg/l and higher (Fig. 1(a)). The death rate was 91.7 % for a concentration of 40 mg/l. Embryonic abnormalities began to increase at concentrations starting from 30 mg/l (Fig. 1(b)). Pericardial edema was one of the most frequent types among the registered abnormalities. For this reason, for subsequent tests, we chose a doxorubicin concentration of 10 mg/l, for which the rate of embryonic abnormalities and mortality did not differ significantly from that for the control group.
(a)
(b)
Fig. 1. Mortality rate (a) and malformation rate (b) of zebrafish embryos on exposure to different concentrations of doxorubicin
5. CeO2 nanoparticles colloids and suspensions toxicity for Danio rerio embryos
Our data indicate that CeO2 nanoparticles in a concentration of 10 mg/l did not, themselves, affect zebrafish embryonic development (Fig. 2). The survival rate did not differ from that for the control group, and was independent of the method of nanoparticle preparation (Fig. 2(a)). The rate of malformations also did not exceed the control group
rate (Fig. 2(b)). Ceria nanoparticles also did not affect hatching time. Hatching started in 48 hours and finished in 72 hours. No significant differences between ceria-treated and control groups were registered (Fig. 3).
(a)
(b)
Fig. 2. Danio rerio embryo survival rates (a) and the rate of Danio rerio embryos with developmental abnormalities (b) on exposure to doxorubicin or ceria nanoparticles (10 mg/l). DOX -doxorubicin, 1 - citrate-stabilized ceria, 2 - non-stabilized ceria, 3 - ceria nanopowder
Fig. 3. Hatching rate of Danio rerio embryos upon exposure to doxorubicin or ceria nanoparticles (10 mg/l). DOX - doxorubicin, 1 - citrate-stabilized ceria, 2 - non-stabilized ceria, 3 - ceria nanopowder
5.1. The synergetic effects of various CeO2 concentrations and fixed doxorubicin concentration (10 mg/l)
For the assessment of possible synergetic effects, CeO2 nanoparticles in concentrations from 0.001 to 10 mg/l were mixed with doxorubicin (10 mg/l). Various combinations of nanoceria and doxorubicin had no significant effect on either zebrafish or tiger barb embryo survival rates (Fig. 4). In turn, the rate of malformations began to increase as
the ceria concentrations increased (Fig. 5). This effect was observed for all types of ceria nanoparticles and for both species of fish. The majority of abnormalities were represented by pericardial edema.
(a)
(b)
Fig. 4. The survival rate of zebrafish (D. rario) (a) and of tiger barb (P tetrazona) (b) embryos on exposure to doxorubicin alone (10 mg/l) and the mixture of ceria nanoparticles (0.001 - 10 mg/l) with doxorubicin (10 mg/l), N = 72 (a), N = 40 (b). 1 - citrate-stabilized ceria, 2 - non-stabilized ceria, 3 - ceria nanopowder
There was a significant difference between the effects of various types of ceria nanoparticles on zebrafish embryos. For the citrate-stabilized CeO2 sol, a significant increase in abnormalities was noted, even for a concentration of 1 mg/l (15.3 %), while, at a CeO2 concentration of 10 mg/l, the rate of malformations increased to 98.6 %. For the non-stabilized cerium dioxide sol at a concentration of 10 mg/l, the abnormality rate also increased, compared with the control group (51.4 %), but it was significantly lower than for citrate-stabilized nanoceria. The addition of the ceria nanopowder caused only minimal effects on the malformation rate; the malformation rate exceeded that caused by doxorubicin alone, but was lower than for CeO2 sols (31.9 %), with statistically significant differences.
In the experiments with the tiger barbs, only CeO2 nanopowder mixed with doxorubicin was used. Increasing the concentration of CeO2 to 10 mg/l caused a rise in embryonic abnormality rate to 90 %. Thus, significant differences in the ceria nanopowder-doxorubicin mixture effects were revealed for two different species of fish, with the malformation rate higher for the tiger barbs.
The high malformation rate did not affect the hatching of surviving embryos; the rate of hatched larvae exceeded 90 % for all groups (Fig. 6(a, b, c, d)). No significant differences were observed, either for the rate of hatched larvae or for the hatching time period.
5.2. Synergetic effects of a fixed concentration ofceria nanoparticles (10 mg/l) and various concentrations of doxorubicin (1 and 5 mg/l) on the embryonic development of zebrafish
For the next experimental series, ceria nanoparticles, at a concentration of 10 mg/l, were mixed with doxorubicin (1.0 or 5.0 mg/l). The data obtained demonstrate that survival and malformation rates and the hatching success of zebrafish upon exposure to low concentrations of doxorubicin mixed with ceria did not differ significantly from the control and doxorubicin alone groups (Table 1).
Thus, low doxorubicin concentrations in combination with high CeO2 nanoparticle concentrations had no significant effects on zebrafish embryo development.
Ceria: a-0.001 mg/l s-0.01 mg/l n-O.lmg/1 s-1.0 mg/l m-10mg/l
(a)
100 90
| 40 rô 30
m
Egfriresai- I._i
control ■ - doxorubicin
(b)
Fig. 5. The malformation rate in the embryonic development of zebrafish (a) and tiger barbs (b) on exposure to doxorubicin alone (10 mg/l) and the mixture of ceria nanoparticles (0.001 - 10 mg/l) with doxorubicin (10 mg/l), N = 72 (a), N = 40 (b). 1 - citrate-stabilized ceria, 2 - non-stabilized ceria, 3 - ceria nanopowder
6. Discussion
Data obtained indicate that, regardless of the preparation method and stabilizer type, ceria nanoparticles alone in concentrations ranging from 0.001 to 10 mg/l did not demonstrate any toxic effects during acute tests in zebrafish. The survival rate of embryos, hatching success and malformation rate did not exceed control significantly. The data
Table 1. The effects of different types of CeO2 nanoparticles (10 mg/l) and doxorubicin on ze-brafish embryos
Control Doxorubicin Citrate-stabilized ceria Non-stabilized ceria Ceria nanopowder
Without doxorubicin With doxorubicin (1 mg/l) Without doxorubicin With doxorubicin (1 mg/l) Without doxorubicin With doxorubicin (1 mg/l) With doxorubicin (5 mg/l)
N 72 72 48 48 48 48 24 24 24
mortality 4.2 1.4 4.2 8.3 4.2 2.1 0 5.6 5.6
malformations 1.4 0 2.1 0 0 0 0 2.8 1.4
hatching 95.1 97.9 89.6 91.7 95.8 95.8 100 93.1 94.4
Fig. 6. The hatching success of zebrafish (a, b, c) and tiger barb (d) embryos on exposure to a mixture of citrate-stabilized ceria nanoparticles (0.001 - 10 mg/l) (a), non-stabilized ceria nanoparticles (0.001 - 10 mg/l) (b) and ceria nanopowder (0.001 - 10 mg/l) (c, d) with doxorubicin (10 mg/l) (N = 72 for (a, b, c) and N = 40 for (d))
obtained agree with data derived for zebrafish embryos and ceria nanoparticles at concentrations of 13 to 200 mg/l [17]. However, it was previously shown that TiO2 nanoparticles which were non-toxic in an acute test [33] influenced the behavior of larvae in a chronic test at nanoparticle concentrations of 1 mg/l [34]. Therefore, our data cannot exclude the possibility of chronic toxicity of nanoceria in fish.
The presence of ecotoxic agents in an aqueous environment can considerably influence the nanoparticle behavior and their effects on aquatic species. It has been shown in previous studies that metal oxide nanoparticles can increase the accumulation of diluted ecotoxicants in aquatic species or cause a synergetic effect. For example, in experiments on adult fish, the presence of TiO2 nanoparticles in water at a concentration of 10 mg/l increased As and Cd accumulation in carp (Cyprinus carpio) up to 30 - 140 % [35,36]. However, in the experiments on zebrafish, with the same TiO2 nanoparticles, at concentrations of 5 - 20 mg/l, no pronounced influence on Cd bioaccumulation was observed [37]. Similar results were obtained in Daphnia sp. The presence of 2 mg/l TiO2 nanoparticles increased Cd accumulation six-fold in D. magna. For TiO2 nanoparticles, the increase in Cu2+ accumulation and toxicity was also observed for D. magna [38]; the same effect was registered for As5+ accumulation in C. dubia [39]. Similarly, the introduction of CeO2 nanoparticles increased atrazine accumulation in Daphnia due to herbicide adsorption and transfer by the nanoparticles [40].
In the present study, doxorubicin was used as a model ecotoxic agent. Doxorubicin is known to have cardiotoxic effects on zebrafish development [41], and leads to embryonic malformations, including pericardial edema, in concentrations exceeding 10 mg/l. Doxorubicin concentrations were used which do not affect zebrafish development. Addition of ceria nanoparticles did not lead to embryonic death, but had a significant effect on doxorubicin toxicity. When mixed with all types of ceria nanoparticles (10 mg/l), doxorubicin taken in 10 mg/l concentration caused a
significant rise in embryonic malformations, mainly pericardial oedema. Lower concentrations of ceria nanoparticles in the mixtures did not lead to such effects.
It is well known that metal oxide nanoparticles in aqueous solutions easily adsorb molecules and ions of various organic and inorganic substances. Due to its specific chemical properties, cerium dioxide appears to be a very good sorbent for several substances. For instance, CeO2 nanoparticles adsorb 58 % of an initial concentration of Pb2+, which is considerably higher than for TiO2 and Fe3O4 nanoparticles [42]. Furthermore, CeO2 nanoparticles are good adsorbents for various antibiotics, due to their affinity to hydrophilic molecules [43]. Apparently, in experiments conducted for the current research, ceria nanoparticles could adsorb doxorubicin and consequently promote the increase in its local concentration in zebrafish chorion. This effect was registered only for certain concentrations of doxorubicin and CeO2 nanoparticles in the media.
Malformation rate in tiger barbs during embryogenesis was significantly higher than in zebrafish. Enhanced sensitivity of barb embryos is most likely to appear due to a shorter incubation period of the embryo in the egg: in contrast with zebrafish, the barb hatches within 24 hpf.
The question is still open as to whether the effects observed were caused by ceria nanoparticle penetration into the chorion. It has been established previously that silica nanoparticles do not penetrate zebrafish chorion [44]. Similarly, upon keeping zebrafish in the nanoceria-containing media, approximately 37 % of ceria nanoparticles bind with the chorion and only 0.07 % get through [45].
Citrate-stabilized cerium dioxide sols mixed with doxorubicin appeared to have the most toxic effect. A minimal effect was observed for suspended ceria nanopowders. An intermediate effect was registered for non-stabilized cerium dioxide sols.
Results obtained can be explained taking into account the peculiarities of doxorubicin adsorption on ceria nanoparticles. The interaction in the adsorption system depends on the charge of both adsorbent and adsorbate; the electrostatic difference potential of doxorubicin is unevenly distributed among functional groups, thus the position of functional groups will determine the orientation of the molecule. Calculations made indicate two possible orientations of a dox-orubicin molecule on the ceria surface - vertical and planar (see Appendix, Fig. A1). In the first case, interaction with the surface proceeds via a nitrogen group, which has a positive charge when protonated. Obviously, this orientation will be favorable on the negatively charged surface. In the second case, interaction of the molecule with the surface proceeds via the п-electron system of fused aromatic rings and acidic -OH phenol groups, having a negative charge when doxorubicin is ionized. This "flat" orientation of doxorubicin is favorable on the positively charged surface, wherein the molecule occupies the area corresponding to about eight cerium atoms. According to calculations of the electronic structure [46], the sum of the atomic partial charges of the isolated molecule of doxorubicin is zero; in the aquatic environment, the total charge of the molecule is slightly positive, and in complex with DNA is slightly negative, but the overall pattern of the charge distribution is the same.
Unlike weak electrostatic adsorption on gold [47], citric acid is strongly chemisorbed on the ceria surface and cannot be substituted by doxorubicin; the interaction of doxorubicin with citrate-stabilized ceria occurs via the layer of citric acid molecules. The citrate adsorbs on the surface in an ordered manner (see Appendix, Fig. A2(A)), and the grafting density is one citrate molecule per 3 - 4 Ce atoms. Our data indicate that "citrate-stabilized" ceria nanoparticles have negative Z-potential in the whole range of biologically relevant pH values. In turn, for "naked" ceria nanoparticles, the Z-potential typically has a positive charge in the range of pH < 8 (see Appendix, Fig. A3). Several authors have shown that the interaction of various citrate-coated nanoparticles with organic molecules including doxorubicin can proceed by formation of hydrogen bonds [48] or by more complicated surface interactions [49]. According to the calculation referred to above, the doxorubicin molecule would preferably have a vertical orientation on the surface of a nanoparticle coated with dissociated carboxylic groups. In this case, the nitrogen atom of the antibiotic interacted with the carboxyl group of the citrate. Fig. A2(B) shows that the grafting density of doxorubicin on a citrate-coated ceria surface is one molecule per 4 - 5 Ce atoms. Some of the citric acid molecules did not participate in the interaction with the antibiotic; these molecules assure the colloidal stability of ceria nanoparticles.
The fraction of atoms located on the surface of a nanoparticle strongly depends on its size. The corresponding dependence for nanoceria is shown in Figs. A4 and A5. For 6 nm ceria nanoparticles, the proportion of surface-located atoms is about 40 %; for 25 nm nanoceria, about 12 %. The amount of adsorbed doxorubicin molecules for these types of nanoparticles varies correspondingly. For example, 10 mg of citrate-coated ceria nanoparticles can adsorb 2.8 mg of doxorubicin; 10 mg of the same "naked" ceria nanoparticles can adsorb 1.58 mg of doxorubicin; 10 mg of ceria nanopowder can adsorb 0.47 mg of doxorubicin (see Appendix). As the present work has demonstrated, the toxicity of doxorubicin-ceria conjugated changes in the same manner: citrate-coated nanoceria > "naked" nanoceria > ceria nanopowder.
Results obtained demonstrated that there exists a synergetic effect of ceria nanoparticles and doxorubicin action; the effect was expressed in a high incidence of embryonic malformations in fish. This effect was more pronounced in
tiger barbs than in zebrafish, which, in the authors' opinion, was due to some peculiarities of their embryogenesis. It was also found that differently prepared CeO2 nanoparticles demonstrated different efficiency. Thus, CeO2 stable sols application had a stronger effect than a suspended nanopowder. The method of nanoceria stabilization also played a considerable role in the synergetic action of nanoparticles and doxorubicin. Citrate-stabilized cerium dioxide nanoparticles had significantly higher effects than non-stabilized nanoparticles. A certain minimum ratio of nanoparticles and doxorubicin had to be reached to provide a synergetic effect, which substantially depended on the cerium surface atoms available, i.e. on the nanoparticles' size and on the presence of the stabilizer.
Ceria nanoparticles (both non-stabilized and citrate-stabilized aqueous colloid solutions and water redispersible ceria nanopowder) have no embryotoxic effect on Danio rerio and Puntius tetrazona. Nevertheless, nanoceria has been shown to increase greatly the toxic effect of doxorubicin on fish embryogenesis. This synergetic effect of ceria nanoparticles and doxorubicin depends strongly on the concentration of components, as well as on particle size and the presence of a stabilizer.
Acknowledgements
This work was partially supported by research grants of Russian foundation for Basic Research No. 18-2905023 mk.
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Synergetic action of ceria nanoparticles and doxorubicin on the early development of two fish species.. Appendix
Fig . A1. The calculated adsorption of a doxorubicin molecule on the surface of a ceria cluster: A -planar orientation; B - vertical orientation
Fig. A2. The calculated adsorption of citrate molecules on the surface of a ceria cluster (A) and doxorubicin molecules on a citrate-coated surface of a ceria cluster (B)
Fig . A3. The dependence of the Z-potential of the "naked" and citrate-stabilized ceria nanoparticles on pH
Fig. A4. The calculated ratio of surface atoms to the total number of atoms as a function of ceria particle size
Fig. A5. The calculated number of surface and bulk cerium atoms in octahedral-shaped CeO2 nanoparticles as a function of particle size
Calculation of doxorubicin amount which could be adsorbed on 10 mg of cerium dioxide particles.
M = 10/MWCeO2 • R/S • MWDox = 31.6 • R/S,
where
M - the amount of doxorubicin, mg;
MWCeO2 - the molecular weight of ceria, 172 g/mol;
R - the fraction of the cerium atoms at the surface of the particle;
S - the "landing area" (the number of cerium atoms occupied by one molecule or grafting density) of dox-orubicin;
MWDox - the molecular weight of doxorubicin, 543.5 g/mol. "ceria (citrate)" (size 6 nm), R = 0.4, S = 4 - 5, M « 2.8 mg of doxorubicin; "ceria (naked)" (size 6 nm), R = 0.4, S = 8, M = 1.58 mg of doxorubicin; "ceria (nanopowder)" (size 24 nm), R = 0.12, S = 8, M = 0.47 mg of doxorubicin.
