Milestone 1 (1-6 months) [Leaded by IGIC, Ukraine Partner]

Research planned in the frames of the Milestone 1:

I. Synthesis of CeO² nanoparticles by the precipitation in the reverse microemulsions and mixed solutions. Investigation of the effect of the conditions of synthesis on the properties of CeO² nanoparticles (size, size distribution, crystallochemical properties, chemical state of cerium, surface charge) and their antioxidant behavior.

Data, which will be included in the report:

1) Results of the synthesis of CeO² nanoparticles, investigations of their physical-chemical properties [IGIC, Ukraine is responsible in collaboration with UALI, Span].

2) Results of the cytomorphological investigations of CeO² and measurements of the antioxidant properties on the cells is if it is possible [IEPOR, Ukraine is responsible].

3) Results of the measurement and analysis of antioxidant (peroxidase- and superoxide dismutase - like) activities of newly synthesized “naked” CeO² nanoparticles in vitro [IEP, Slovakia is responsible].

Milestone 2 (7-18 months) [Leaded by IEPOR, Ukraine Partner]

Research planned in the frames of the Milestone 2:

II. Study of the impact of the parenteral injection of CeO² nanoparticles on the level of the oxidative stress and other manifestations of the acute radiation syndrome.

It is planned to inject CeO² nanoparticles parenteral (intravenously and intraperitoneally) into two groups of the animals (exanimated group and intact group). After this, animal are killed and two types of investigations are performed: 1) blood plasma will be examined for the antioxidant activity indicators (it is expected that cerium dioxide will decrease the fraction of the free radicals); 2) blood cells will be tested to clarify the impact of CeO² nanoparticles on the on the blood formula of irradiated animals [IEPOR, Ukraine is responsible].

In this project stage it is also planned to investigate the effect of the nanocrystalline cerium dioxide on the formation of amyloid fibrils caused by the acute radiation injuries [IEP, Slovakia is responsible]; and the kinetics of the amyloid fibrils formation due to the influence of the nanocrystalline cerium dioxide. Experimental and modelling parts of work. Evaluation of the mechanism of the cerium dioxide inhibition of the oligomers formation: secondary and primary nucleation pathways experimental and modelling data analyses [NOVA id FCT, Portugal is responsible].

III. Development the biomarker scales for assessing the absorption properties of carbon enterosorbents, samples of highly activated carbon adsorbents obtained from different types of natural and synthetic raw materials, evaluating their physicochemical and structural characteristics and optimizing the technology for obtaining oral dispersible forms (formulations) of enterosorbents.

This stage means the test of the sorbents using different dyes to determine of the optimal parameters (molar weight, charge etc.) of the sorbent and to clarify the optimal technological conditions for preparation and standardization of the sorbent. The background for the technology of preparation: granulated sorbent (primary granules) are activated, grinded in special mill; secondary granules of the sorbent are formed using the obtained powder and binder compound (for example, starch). It is important to work out the amount of the binder compound since the sorbent should well decay when it is injected oral into the animals [IEPOR, Ukraine is responsible].

Data, which will be included in the report:

1) Results of the biological investigations of CeO2 nanoparticles, data of their antioxidant behavior in the blood plasma of the animals and impact on the blood formula in the irradiated animals.

2) Results of the physical-chemical properties of cerium dioxide nanoparticles, which will not be included into the report for the previous Milestone.

3) Results of antiamyloidogenic (inhibition and disaggregation) activity of “naked” newly synthesized CeO2 nanoparticles and analysis of the cytotoxicity, biocompatibility and biodistribution of CeO2-NPs using cell lines and in vivo experiments [IEP, Slovakia responsible].

4) Results on design of successful synthesis routes to prepare activated carbon materials with a large surface area and well-developed porous structure starting from different biomass residues. Textural and chemical characterization of the synthesized materials. Evaluation of potential binders to conform the synthesized carbons into tablets or monoliths [UALI, Spain is responsible].

5) Results of the kinetics parameters of the amyloid fibrils formation in the presence of the nanocrystalline cerium dioxide. Mechanism of the cerium dioxide inhibition of the oligomers formation distinguishing between secondary and primary nucleation pathways [NOVA id FCT, Portugal is responsible].

Milestone 3 9-27 months) [Leaded by IEPOR, Ukraine Partner]

Research planned in the frames of the Milestone 3:

IV. Investigation of the correlation between physical-chemical characteristics of the synthesized enterosorbents and their efficiency in treatment of the acute radiation syndrome.

At this stage, it is planned the combined injection of the sorbent (oral) and cerium dioxide (parenteral) into the two groups of the animals and study of the effect of such combination on the antioxidant behavior (tests of the blood plasma) as well as on the blood formula (tests of the blood cells). Cerium dioxide is planned to be injected via two ways: intravenously and intraperitoneally to compare, which way of injection is more effective. Additionally, it is supposed to investigate the morphology of the internal organs to determine the impact of CeO2 nanoparticles on their activity in the irradiated animals [IEPOR, Ukraine is responsible].

Data, which will be included in the report:

1) Results of the biological investigations of the combined injection of the sorbent and CeO2 NPs and data of their antioxidant behavior in the blood plasma of the animals and the impact on the blood formula of the irradiated animals; data of the effect of this combination on the morphology of the internal organs.

2) Results of the physical-chemical properties of cerium dioxide nanoparticles, which will be used for these biological investigations [IGIC, Ukraine is responsible].

3) Enhancement of anti-amyloidogenic activity of NPs. Coating of the CeO2-NPs surface with strong anti-amyloidogenic biopolymers such as detergents or phospholipids [IEP, Slovakia is responsible].

Milestone 4 (28-36 months) [Leaded by IGIC, Ukraine Partner]

Research planned in the frames of the Milestone 4:

V. Study of the effectiveness of the oral and parenteral injection of the optimized composites based on the nanocrystalline cerium dioxide and high-active microparticles of carbon for treatment of the ARS in rats.

At this stage, it is planned to synthesize the composites based on the previously standardized sorbents and cerium dioxide nanoparticles with different ratios of the components and investigation of their physical-chemical parameters. Study of the effectiveness of the injection of the optimized drugs based on the cerium dioxide and high-active carbon microparticles in treatment the experimental acute radiation sickness in rats and compare of the results to the data obtained at the previous stages will be also done [IGIC, Ukraine is responsible].

Data, which will be included in the report:

This Milestone will finish by the Final Report. It will include the main results obtained at the previous stages. Additionally, this report will include the report for the Milestone 4, divided into 2 parts:

1) Data of preparation of the nanocomposites and study of their physical-chemical properties [IGIC, Ukraine is responsible]. UALI, Spain will also evaluate different protocols to incorporate CeO2 nanoparticles into the carbon framework

2) Biological studies of the synthesized nanocomposites (performed on the animals) and data about their effectiveness in treatment of the experimental acute radiative sickness in rats [IEPOR, Ukraine is responsible].

3) Results of the measurement and analysis of antioxidant (peroxidase- and superoxide dismutase - like) activities of coated CeO2 nanoparticles in vitro. Analysis of the cytotoxicity, biocompatibility and biodistribution of coated CeO2-NPs using several cell lines and in vivo experiments. Screening of the effect of synthesized composites (carbon enterosorbents) on amyloid fibril formation (last task can be done only if the carbon enterosorbents will be stable in solution.) [IEP, Slovakia is responsible].

4) Nanoformulation studies: Investigation of the possible interaction of the nanocrystalline cerium dioxide with the nanoscale carbon in a form of the C60 Fullerene and Fullerenol in the solutions [NOVA id FCT, Portugal is responsible].

Equipments: 

Equipment purchased by IGIC team during Milestone 1: 

1. Litesizer 500 Anton Paar GmbH. Particles characterization in solutions: Particles size measurement via dynamic light scattering (DLS); Determination of zeta potential values for nanoparticles in solutions; Measurements of refractive index and molecular weight. 

Equipment purchased by IGIC team during Milestone 2: 

1. RETSCH PM 100 Planetary Ball Mill Grinding Jar 125 ml. Is used for grinding powders in the RETSCH PM 100 Planetary Ball Mill;

2. pH meter MP 511, ULAB;

3. Vacuum drier camera for nanomaterials processing and drying samples in vacuum;

4. High-pressure reactor “Ukrorgsyntez” for hydrothermal synthesis of nanomaterials and nanocomposites.

Equipment purchased by IEPOR team during Milestone 3:

1. Spectrofluorimeter RF-6000, Schimadzu. The equipment is used for: measuring the characteristic fluorescence spectra of solutions of macromolecules and ligands, fluorescence response of probes to processes occurring in biological systems at the molecular level; studying the native structure of proteins and nucleic acids and to determine the dynamics of the relationship between protein structure and function.

Equipment purchased by IGIC team during Milestone 4: 

1. Laboratory centrifuge MPW-380 with rotor Falcon for nanoparticles’ separation and manipulations;

2. Spectrophotometer ULAB 102UV for quantitative analysis during the synthesis;

3. Drying oven SP-100K for drying, vaporing the samples, temperature treatment of synthesized materials.

Equipment purchased by IEPOR team during Milestone 4: 

1. Multimode microplate reader Varioscan LUX, Thermo Fisher. The equipment is used for analytical analysis of the cells, allowing kinetic reactions to be monitored directly from the start of the reaction, which is relevant for "fast" luminescence reactions, Ca2 studies and other fast kinetic reactions. The ability to add reagents in any order or at any step of the kinetic assay allows sequential multi-step assays such as proteins and DNA binding, ATP or reporter gene assays.