The purpose of this multidisciplinary project is to evaluate the efficacy of the studied theranostic agents biodistributed in biological media using Transmission Electron Microscopy techniques.

The studied theranostic agents containing nanoparticles of iron oxide (maghemite - γ-Fe2O3) and cerium oxide will agglomerate, after their injection in mice, in macrophages inside phagolysosomes. The evolution of these nanoparticles will be followed  on a long period of time up to 120 days after administration. In time, because of the acidic medium from phagolysosomes, the nanoparticles will be degraded, most of them will change their crystalline structure. This biotransformation will be evidenced by electron diffraction patterns and EELS spectra.
These results will be compared with the ones obtained in an acidic environment mimicking the biologic one, as well as in 2D and 3D cancerous cell cultures.

Dr. Valentin-Adrian Maraloiu - Principal Investigator

Dr. Raluca Florentina Negrea - Postdoc

Drd. Catalina Gabriela Mihalcea - PhD Student

Drd. Cristian Radu - PhD Student

Phase I: Characterization of  CO-ION platform with controlled size and geometry and its biodegradation in an environment mimicking the biological one 

Starting from Fe₃O₄ and CeO₂ nanoparticles with determined morpho-structural characteristics, several CO-ION platforms were synthetized in which the thickness of the CeO₂ shell around Fe₃O₄ nanoparticles varied. Some of these platforms were thermally treated. Using Transmission Electron Microscopy techniques (CTEM, SAED, HRTEM EDS si EELS), morphology, crystalline structure and chemical composition of nanocomposites before and after thermal treatment were determined. It was proved that thermally untreated nanocomposites kept the initial characteristics of  Fe₃O₄ and CeO₂ nanoparticles. In the case of thermally treated nanocomposites, some changes of morphological characteristics of initial Fe₃O₄ and CeO₂ nanoparticles were observed: Fe₃O₄ nanowires disappeared and the size of  Fe₃O₄ and CeO₂ nanoparticles slightly increased.

The degradation of the chosen CO-ION platform was studied using an acidic medium similar with the one found in lysosomes. The biodegradation experiments were conducted in citric acid at various values of pH and certain time periods after the insertion of nanocomposites in acidic medium. The investigations carried out using CTEM, SAED, HRTEM EDS and EELS techniques demonstrated the acidic medium didn't provoked changes of morphology, crystalline structure or chemical composition  of nanocomposites in the period of 3 weeks.

Phase II. Biodegradation of CO-ION platform in vitro si in vivo

The second phase of the project took place between January 1 – December 31 2023, in the frame of 6 activities. This phase was dedicated to the determination of the way in which biodegradation of CO-ION platform take place in vitro and in vivo at certain time intervals after internalization.

For the study of biodegradation of CO-ION platform in vitro 2D and 3D (spheroids) cell cultures were used starting from liver (Huh7) and kidney (Vero) cell lines. Cultures were incubated with initial nanoparticles of O₄ and CeO₂, as well as CO-ION platform, at two concentrations (25 µg/mL and 200 µg/mL) for 1, 2, 3 and 5 days. Both cytotoxicity analyses and the results obtain from Transmission Electron Microscopy techniques demonstrated the fact that at low concentrations studied nanoparticles are not toxic, while at high concentrations there is a small toxicity. TEM images showed that the distribution of nanoparticles in cells is heterogenous, that they are located only in cytoplasm once they are internalized by cells. TEM techniques also demonstrated that there are no crystallinity changes in 5 days (for 2D cultures) or in 15 day (for spheroids).

For the study of biodegradation of CO-ION platform in vivo C57BL/6 mice were used intravenously injected with CO-ION platform with a dose of 25 ug/mL. Ex vivo samples from different organs (liver, kidney, spleen, brain) at different time intervals (1, 7, 15, 30, 60, 90 and 120 days) after injection were prepared by fixation, postfixation, dehydration and resin imbedding, sections of 100 nm thickness from resin blocks being cut using Leica UC7 ultramicrotome.

TEM images showed that the distribution of nanoparticles in cells and organs is heterogenous, that they are located in cytoplasm. On the other hand it can be observed that in time the size of CO-ION platform agglomerations decreases and that the agglomeration become more dispersed. Also, ferritin agglomerations can be observed which it can be intrinsic (as a result of normal metabolic processes) or extrinsic (as a result of the biotransformation of Fe₃O₄ nanoparticles in ferritin). Also, in CTEM images no sign of toxicity induced by the presence of nanoparticles in organs can be observed.

Phase III. Dissemination of the results 

Beside the article published in 2023, there are three other manuscripts there are being prepared:

1. “Fe₃O₄/CeO₂ Nanoplatform with “Core/Shell”-Like Structure: biodegradation and biotransformation in mimicking intracellular environment”; authors: Valentin-Adrian Maraloiu, Catalina Mihalcea, Cristian Radu, Yuliia Shlapa, Anatolii Belous;
2. “Cytotoxicity and biodistribution of Fe₃O₄/CeO₂ Nanoplatform with “Core/Shell”-Like Structure in 2D / 3D healthy cell cultures”; authors: Valentin-Adrian Maraloiu, Catalina Mihalcea, Cristian Radu, Aida Selaru, Sorina Dinescu, Yuliia Shlapa, Anatolii Belous;
3. “Biodegradation and biotransformation of Fe₃O₄/CeO₂ Nanoplatform with “Core/Shell”-Like Structure in murine model” authors: Valentin-Adrian Maraloiu, Catalina Mihalcea, Cristian Radu, Cosmin Mustaciosu, Yuliia Shlapa, Anatolii Belous.

The results of phase II will be presented in a poster presentation at the 9th International Workshop of Materials Physics which will take place between May 14 - May 16 2024 in Magurele, Romania. The presentation will be entitled „Cytotoxicity and biotransformation of Cerium Oxide-Iron Oxide platform in cells cultures and murine model”.

Published articles:

1.  Shlapa, Y; Siposova, K; Veltruska, K; Maraloiu, VA; Garcarova, I; Rajnak, M; Musatov, A; Belous, A - Design of Magnetic Fe₃O₄/CeO₂ "Core/Shell"-Like Nanocomposites with Pronounced Antiamyloidogenic and Antioxidant Bioactivity; ACS APPLIED MATERIALS & INTERFACES; 15 (42); 49346-49361; DOI: 10.1021/acsami.3c10845 

Conferences:

1.   Oral presentation: "Biodistribution, biodegradation and cytotoxicity of Cerium Oxide-Iron Oxide platform in healthy cells",

Authors: Valentin-Adrian MARALOIU, Yuliia Shlapa, Anatolii Belous, Aida Selaru, Sorina Dinescu

5-th Conference of Romanian Electron Microscopy Society, 18-27 Octomber 2023, Cluj-Napoca, Romania

2. Poster presentation:  "Cytotoxicity and biotransformation of Cerium Oxide-Iron Oxide platform in cells cultures and murine model"

Authors: Valentin-Adrian Maraloiu, Catalina Mihalcea, Cristian Radu, Yuliia Shlapa, Anatolii Belous, Aida Selaru, Sorina Dinescu, Cosmin Mustaciosu

9-th International Workshop of Materials Physics, 14-16 May 2024, Magurele, Romania