Advances in the Study of Cerium Oxide Nanoparticles: New Insights

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Advances in the Study of Cerium Oxide Nanoparticle: New Insights into Anti-amyloidogenic Activity Katarina Siposova, Veronika Huntosova, Yulia Shlapa, Lenka Lenkavska, Mariana Macajova, Anatolii Grigorievich Belous, and Andrey Musatov ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00816 • Publication Date (Web): 30 Mar 2019 Downloaded from http://pubs.acs.org on March 31, 2019

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Mixing & Heating

+

Ce(NO3)2

CeO2 - NPs

(aq)

(ThT FLUORESCENCE)

N NH H44O OH H

MASS OF AMYLOID FIBRILS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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protofibrils mature fibrils

Insulin monomer

cross β monomers

oligomers nuclei

CeO2-NPs

lag-phase + nucleation

elongation

TIME

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steady-state

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Advances in the Study of Cerium Oxide Nanoparticle: New Insights into Anti-amyloidogenic Activity Katarina Siposova1*, Veronika Huntosova2*, Yulia Shlapa3, Lenka Lenkavska4, Mariana Macajova5, Anatolii Belous3, Andrey Musatov1

1Institute 2Center

of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosice, Slovakia for Interdisciplinary Biosciences, Technology and innovation park, P.J. Safarik University in Kosice, Jesenna 5, 041 54 Kosice, Slovakia

3Department

of Solid State Chemistry, Institute of General Inorganic Chemistry, Ukrainian Academy of Sciences, 32/34 Prospect Palladina, Kyiv, 03680, Ukraine

4Department

of Biophysics, Faculty of Science, P.J. Safarik University in Kosice, Jesenna 5, 041 54 Kosice, Slovakia

5Institute

of Animal Biochemistry and Genetics, Centre of Biosciences, Slovak Academy of Sciences, Dubravska cesta 9, 840 05 Bratislava, Slovakia

*Corresponding

author e-mails: [email protected]; [email protected]

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Abstract There seems to be general agreement that oxidative stress is involved in many pathological conditions including Parkinson’s, Alzheimer’s and other neurodegenerative diseases, and overall aging. Cerium oxide nanoparticles, also known as nanoceria (CeO2-NPs), have shown promise as catalytic antioxidants, based on their ability to switch between Ce3+ and Ce4+ valence states. In the present work we have synthesized and characterized CeO2-NPs, examined the effect of CeO2-NPs on amyloidogenesis of insulin, and analyzed the impact of CeO2-NPs on oxidative stress and biocompatibility in vitro in three types of invasive cancer cells, and in vivo in the pre-clinical model of chorioallantoic membrane of quail embryos (CAM). The different experimental techniques revealed a high stability and homogeneity of the synthesized by precipitation from reversal microemulsion “naked” CeO2-NPs. The CeO2-NPs were 5-6 nm in diameter (TEM), monodispersed and have a ζ +46.9 mV zeta potential in Milli-Q water. We demonstrated for the first time that CeO2-NPs affect insulin fibrillation in a dose-dependent manner. The inhibiting, IC50, and disassembling, DC50, concentrations were calculated to be ~100 ± 3.5 µg/mL, and ~200 ± 5.5 µg/mL, respectively. Furthermore, CeO2-NPs demonstrated reliable biocompatibility and sufficient uptake by glioma and breast cancer cells. The presence of high concentration of CeO2NPs within the cells resulted only in local changes in metabolic activity and generation of oxidative stress at a low level. Moreover, high biocompatibility with CeO2-NPs was shown in vivo in the CAM.

Keywords: Cerium oxide nanoparticles, synthesis, insulin amyloidogenesis, biocompatibility, oxidative stress

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Introduction Cerium oxide nanoparticles (CeO2-NPs) are used increasingly in nanotechnology and particularly in bioresearch. Cerium possesses shielded 4f-electrons (electronic configuration [Xe]4f26s2), which are responsible for the attractive optical, magnetic, and chemical properties of the rare earth elements 1. Due to low redox potential of the Ce4+/Ce3+ redox couple (~1.52 V), cerium possesses unique ability to switch oxidation states between III and IV, based on experimental condition. Moreover, Ce3+ is associated with oxygen vacancies

2-3.

The relative

amount of cerium ions Ce3+ and Ce4+ is a function of particle size 4. In general, the fraction of Ce3+ ions in the particles increases with decreasing particle size 5. Pure CeO2 has a fluorite-type structure, with a special Fm3m group

2, 5-6.

Therefore, CeO2-NPs have been utilized in various

fields of applications such as areas of catalysis, electrochemistry, photochemistry, and materials science

7-10.

properties

In bioscience the great attention is paid to CeO2-NPs because of their antioxidant

3, 5.

In fact, it was demonstrated that due to regenerative properties, low reduction

potential and the coexistence of both Ce3+/Ce4+ on their surfaces, CeO2-NPs possess ability for multi-enzymatic scavenging of reactive oxygen species, exhibit superoxide dismutase and catalase enzymes mimetic activities in a redox-state dependent manner, and can be used in regenerative medicine, bioanalysis, drug delivery, and bioscaffolding 3, 5, 11-14. There are also some studies using CeO2-NPs to investigate the effect of NPs on cancer (for review see Brenneisen & Reichert 15). It is generally accepted that oxidative stress plays a role in aging and in a variety of human diseases, such as cardiovascular and neurodegenerative disorders, inflammatory, autoimunne diseases, and arthritis 5, 16-17. In turn, a number of human diseases, including the amyloidosis and other neurodegenerative diseases, arise from the deposition of highly-ordered and stable, fibrillar protein aggregates, known as amyloid fibrils. These disorders are caused by a combination of genetic and environmental factors, including oxidative or metabolic stress and/or changes in the 3 ACS Paragon Plus Environment

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intracellular conditions (ageing), suggesting that spontaneous events can destabilize a misfoldingprone protein or impair the clearance mechanisms, leading to the accumulation of misfolded aggregates 18-21. Although the proteins causing amyloid diseases show sequence, size and function diversity, they all form similar amyloid fibrils consist of the same cross-β structure i.e., β-strands arranged perpendicular to the long fibril axis 21-23. It was demonstrated that even ordinary globular proteins that are not related to disease (e.g., myoglobin or cytochrome c) can form amyloid fibrils under certain conditions 24-26, suggesting that fibril formation is a general property of polypeptide chain

22-23, 27.

Knowledge of the mechanism of amyloid formation, the factors affecting either

stimulation or inhibition is crucial for understanding of both, aging and age-related neurodegenerative diseases. Strategies for therapeutic intervention are based on: (i) reducing the population of oligomeric species (pre-fibrillar species) by disrupting the processes of their formation, (ii) suppressing the early stages of protein aggregation, for example by binding to specific amyloidogenic species and by reducing the risk of nucleation and proliferation of pathogenic agents, (iii) or by promoting the pathways of their removal (for review see Knowles 23).

Therefore, the use of active agents that interfere with amyloid fibril formation may have

therapeutic potential for the treatment of amyloidosis. Moreover, such approach also can help to elucidate the molecular mechanism of amyloidogenesis. It was demonstrated that NPs can significantly affect the protein amyloid fibrillation. For example, it was shown that NPs can enhance the rate of amyloid fibrillation 28-31, or significantly reduce the formation of amyloid fibrils 29, 32-35.

Moreover, previous studies demonstrated that nanosized materials can be used in detection

and diagnosis of amyloid fibril formation

36-38.

However, evidence of CeO2-NPs ability to affect

protein amyloidogenesis is very limited 29, 31. In the present work we have synthesized and characterized CeO2-NPs, examined the effect of CeO2-NPs on amyloidogenesis of insulin, and analyzed the impact of cerium oxide nanoparticles 4 ACS Paragon Plus Environment


on oxidative stress and biocompatibility in vitro in three types of invasive cancer cells: one glioma (U87 MG) and two breast cancer cells lines (BT 474 and SK BR 3); and in vivo in the pre-clinical model of chorioallantoic membrane (CAM) of quail embryos.


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2.

MATERIALS AND METHODS

2.1.

CeO2 nanoparticles synthesis and characterization techniques

CeO2 nanoparticles (CeO2-NPs) were synthesized by precipitation in reversal microemulsions according to the scheme shown on Fig. 1. An aqueous solution of 1.9 M cerium (III) nitrate was used as the starting reagent. A concentrated aqueous solution of ammonia was used as a precipitator. Triton X-100 and n-butyl alcohol were used to form the microemulsions as a surfactant and co-surfactant, respectively. Cyclohexane was used as an organic medium. Initially, two microemulsions (designated as M1 and M2) were prepared. Each of them contained aqueous solution of salt (М1) or precipitator (М2), surfactant Triton X-100, butyl alcohol and cyclohexane in the ratio of 1.1:1:1.3:3.2 (weight to weight ratios). The M1 solution was added dropwise for 1 hour with constant stirring to M2 solution at room temperature. Reaction mixture, obtained after precipitation, was heated to 70 °C and incubated at this temperature for 1 hour under constant stirring. Obtained CeO2-NPs were separated by centrifugation (5000 rpm), washed by ethyl alcohol and bi-distilled water for several times and dispersed in 100 mL of bi-distilled water. M2

M1

Mixing and heating of the mixture

CeO2 nanoparticles formation

+ Ce(NO3)2

(NH3∙H2O)

aqueous solutions

aqueous solution

Figure 1. Scheme of synthesis of CeO2-NPs by precipitation in reversal microemulsions.

Synthesized NPs were studied by the X-ray analysis via powder method using diffractometer DRON-4 (CuKα radiation). The parameters of the unit cell were estimated by



Rietveld method using software package FULL PROF 39. Degree of crystallinity of CeO2-NPs was calculated from the parameters of baseline of X-ray pattern by the method of mathematical modeling using software package of Origin Pro 9.0. Particles size and morphology were studied by transmission electron microscope (TEM) JEOL JEM-1230. Samples were prepared by deposition of CeO2-NPs on the copper grid coated by formvar. Particles size was measured from the obtained TEM-images using the software package Image Tool 3. The size distribution of NPs was calculated and corresponding histograms were plotted using OriginPro 8.5 SR1. Hydrodynamic diameter of CeO2-NPs in water suspension and their zeta-potential were analyzed by dynamic light scattering (DLS) method at temperature of 22 °C using ZetaSizer 3 (Malvern Instruments) equipped by He-Ne laser (25 mW, λ = 633 nm) with the angle of detection 90 °C. 2.2.

Insulin amyloid fibrillation

Insulin (human recombinant, expressed in yeast, I2643; Sigma-Aldrich, Inc., St. Louis, MO; US) was dissolved in 70 mM glycine buffer pH 2.7, containing 80 mM NaCl (hereinafter referred to as Gly-NaCl buffer, pH 2.7) to a final concentration of 10 μM. The solution was incubated in an Eppendorf comfort thermomixer at 65 °C for 2 h under constant agitation (1200 rpm) with or without NPs. 2.3.

Thioflavin T fluorescence assay

For quantification of insulin amyloid formation alone and in the presence of CeO2-NPs, Thioflavin T (ThT, T3516; Sigma-Aldrich, Inc., St. Louis, MO; US) fluorescence assays were carried out using a 96-well plate by a Synergy MX (BioTek) spectrofluorometer. After process of


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fibrillation was completed, ThT was added to each sample to reach a final ThT to insulin molar ratio of 2:1. Samples were processed and the fluorescence intensities were measured as described previously 40, 41. The data presented are the results of three independent experiments. The error bars are the average deviation for repeated measurements of three separate samples. 2.4.

Determination of CeO2 anti-amyloidogenic activity

The ability of CeO2-NPs to affect insulin amyloid aggregation, and to disassemble preformed amyloid fibrils, was initially examined using ThT assay. A stock solution of CeO2-NPs was prepared by dissolving of synthesized NPs in ultrapure water to final concentration of 20 mg/mL. The final insulin concentrations were always 10 µM, which correspond to 58 μg/mL. For the inhibition assay, different concentrations of CeO2-NPs were added to protein in Gly-NaCl buffer, pH 2.7, followed by incubation and fluorescence measurements under conditions described above. The concentration-dependent effect of CeO2-NPs has been tested in the range of 0.01:1 to 1:20 protein to CeO2-NPs ratio (mg/mg). Anti-amyloid disassembling activity of CeO2-NPs was investigated after incubation of preformed amyloid aggregates of insulin (always 10 µM of protein, equaled to 58 μg/mL; fixed concentration) with CeO2-NPs at the protein to nanoparticles ratio in the range of 0.01:1 to 1:20 protein to CeO2-NPs (mg/mg) at 37 °C for 24 hours. The extent of disassembling activity was detected by the ThT fluorescence assay. The IC50 (the concentration of NPs required to inhibit fibril formation by 50%) and DC50 (the concentration of NPs required to disassembly pre-formed fibrils by 50%) values were determined from fitting the dose-dependent experimental data using non-linear least-squares analysis with the sigmoidal logistic 4 parameters equation as described recently 40-42.



2.5.

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Atomic force microscopy

Atomic Force Microscopy (AFM) images were obtained using a Scanning Probe Microscope (Veeco di Innova, Bruker AXS Inc., Madison, USA). The AFM images were recorded in a tapping mode using an NCHV cantilever (Bruker AXS Inc., Madison, USA) at scan rate 0.250.5 kHz. The resolution of the image was 512 pixels per line (512 x 512 pixels/image). No smoothing or noise reduction was applied. Samples for AFM visualization were prepared as described recently 40, 41. 2.6.

Cell cultures

The U87 MG (human glioma, Cells Lines Services, Germany), BT474 (human breast ductal carcinoma, a gift from prof. Pluckthun laboratory, University of Zurich, Switzerland) and SK BR 3 (human breast adenocarcinoma, a gift from prof. Pluckthun laboratory, University of Zurich, Switzerland) cells were grown according propagation protocol presented in Lenkavska et al. 2.7.

43.

Confocal fluorescence microscopy

The intensity of Rhodamine 123 (Rh123, 5 µM, 30 min, ThermoFisher Scientific, USA) and MitoTracker® Orange CMTM/Ros (MTO, 0.4 µM, 30 min, ThermoFisher Scientific, USA) was detected in living cells. Confocal fluorescence microscopy system was used as described in Tomkova et al. 44. While Rh123 was excited at 488 nm and detected at 520 ± 30 nm 555 nm, MTO was excited at 555 nm and detected above 580 nm. The fluorescence images were collected before and after 24 h of administration of 5 and 50 µg/mL CeO2-NPs. Transmission (white light) images were detected with the same microscope and with a 20X Fluar (NA=0.75, , Zeiss, Germany). For these experiments, U87 MG, SK BR 3 and BT 474 cells were 72 h incubated with 0-200 µg/mL CeO2-NPs in complete cell culture medium. 9 ACS Paragon Plus Environment

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2.8.

Time-resolved fluorescence microscopy

MTO fluorescence lifetimes were detected in cells, according to protocol described in our previous work 44, before and 24 h after administration of 5 and 50 µg/mL CeO2-NPs in complete cell culture medium. With our setup and filters adjustments, we have avoided autofluorescence produced by cells without MTO labeling. 2.9.

MTT-assay

Standard cell viability test was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, Sigma-Aldrich, Germany) assay as described in Lenkavska et al. 43. MTT-assay in control cells was compared with MTT-assay in cells 24 h or 48 h subjected to 0 200 µg/mL CeO2-NPs in complete cell culture medium. The errors represent SD or SEM from the mean values of experimental data (performed in triplicates). The level of significance was estimated with T-test: *p

Advances in the Study of Cerium Oxide Nanoparticles: New Insights

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