// Project
The NANOCEL project
Period of deployment
2016 – 2017
Project no.
PIII-C4-PCFI-2016/2017-03
Coordinator
"Victor Babeș" University of Medicine and Pharmacy in Timișoara
Project manager
Prof. Dr. Virgil Păunescu
Funding
135,000 RON
General objectives:
(1) identification of cell death mechanisms in the presence of Fe3O4 nanoparticles;
(2) evaluating the influence of these nanoparticles in the formation and development of tumors.
Specific objectives:
(1) Morphological, phenotypic, genotypic and functional characterization in vitro of solid tumors in the presence of magnetite nanoparticles (Fe3O4);
(2) Evaluation of the mechanisms of cell death induced by nanoparticles;
(3) Identification in vitro of the characteristic markers resulting from the interaction of tumor cells exposed to nanoparticles with the tumor microenvironment;
(4) The in vivo evaluation of the ability of tumor cells exposed to nanoparticles to form and develop tumors
Project summary
During the process of tumorigenesis, tumor cells proliferate uncontrollably, evading the regulatory mechanisms of cell death. Several types of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II), necrosis (type III), mitotic catastrophe, anchorage-dependent mechanisms – anoikis, excitotoxicity, Wallerian degeneration, skin cornification. This project proposes to investigate a possible new mechanism for inducing tumor cell death in vitro and in vivo – enucleation. The influence of Fe3O4 nanoparticles in colloidal suspensions on human tumor cell lines will be tested. Magnetic particles (MNPs) will be synthesized by combustion and coated with a double layer of oleic acid, interacting with tumor cells. In vitro studies will be focused on morphological and ultrastructural changes, functional studies, immunophenotypic markers and gene expression induced in tumor cells and resulting nuclei, using a modern methodology. Through in vivo models, the ability of tumor cells treated with magnetic nanoparticles to form tumors in immunodeficient mice will be detected, followed by the characterization of the generated tumors and associated metastases. This project will provide evidence regarding enucleation as a potential mechanism of tumor cell death, generating new directions in tumor biology research and the development of therapeutic agents capable of exploiting this behavior.
Results
- The method of highlighting the effect of MNP on normal and tumor cells by SEM (Fig. 1)
- The method of highlighting the effect of MNP on normal and tumor cells by TEM (Fig. 2 and Fig. 3)
- The flow cytometric method for the analysis of changes induced by MNP at the cellular level (Fig. 4)
- The immunocytochemical method for morphological and immunophenotypic evidence of MNP-induced changes at the cellular level (Fig. 5)
- PBMC isolation protocols;
- Protocols for tumor cell-tumor microenvironment co-culture systems;
- Panel of characteristic markers of tumor cells treated with MNPs after interaction with immune cells;
- Protocols for GFP labeling of tumor cells;
- Protocols for tumor formation in immunosuppressed mice (Fig. 6);
Full articles
- Oprean C, Ivan A, Bojin F, Cristea M, Soica C, Drăghia L, Caunii A, Paunescu V & Tatu C. Selective in vitro anti-melanoma activity of ursolic and oleanolic acids. Toxicol Mech Methods. 2018 Feb;28(2):148-156. doi: 10.1080/15376516.2017.1373881 (IF = 1.994, Nanocell)
- Danciu C, Bojin F, Pinzaru I, Dehelean C, Ambrus R, Popescu A, Păunescu V, Hancianu M, Minda D and Şoica C. Rutin and Its Cyclodextrin Inclusion Complexes: Physico-chemical Evaluation and in vitro Activity on B164A5 Murine Melanoma Cell Line. Current Pharmaceutical Biotechnology, 2017, 18; (IF = 1.819, Nanocel)
- Oprean C, Mioc M, Csányi E, Ambrus R, Bojin F, Tatu C, Cristea M, Ivan A, Danciu C, Dehelean C, Paunescu V, Soica C. Improvement of ursolic and oleanolic acids' antitumor activity by complexation with hydrophilic cyclodextrins . Biomed Pharmacother. 2016;83:1095-1104. (IF = 2.023; Nanocel)
- Oprean C, Zambori C, Borcan F, Soica C, Zupko I, Minorics R, Bojin F, Ambrus R, Muntean D, Danciu C, Pinzaru IA, Dehelean C, Paunescu V, Tanasie G. Anti-proliferative and antibacterial in vitro evaluation of the polyurethane nanostructures incorporating pentacyclic triterpenes. Pharm Biol. 2016;54(11):2714-2722. (IF = 1.241; Nanocel)
- Tatu C, Panaitescu C, Marusciac L, Sisu AM, Cristea M, Puscasiu DA, Tanasie G. Adhesion and Secretory Profile of Mesenchymal Stem Cells Upon Contact with Some Biomaterials. REV.CHEM.(Bucharest), 2017; 68 (9): 2079-20182. (IF = 1.412; Nanocel)
Fig. 1. SEM images corresponding to mesenchymal cells (MSC) and tumor cells (SK-BR-3), before and after treatment with magnetic colloidal suspensions: A – mesenchymal cells (MSC) not treated with magnetic colloidal suspensions (control); B – mesenchymal cells treated with colloidal suspension 1F; C – mesenchymal cells treated with colloidal suspension 2F; D – SK-BR-3 tumor cells not treated with magnetic colloidal suspensions (control); E – tumor cells treated with colloidal suspension 1F; F – tumor cells treated with colloidal suspension 2F.
Fig. 3. TEM images corresponding to SK-BR-3 tumor cells, following treatment with magnetic colloidal suspensions; A and B – tumor cells treated with colloidal suspension 2F (Fe3O4 → precipitation); C and D – tumor cells treated with colloidal suspension 1F (Fe3O4 → combustion).
Fig. 5. Immunocytochemical analysis of mesenchymal cells (MSC), respectively tumor cells (SK-BR-3) labeled with cytoskeletal protein Vimentin, respectively with oncoprotein Her2; A – untreated mesenchymal cells (control); B – mesenchymal cells treated with colloidal suspension 1F; C – mesenchymal cells treated with colloidal suspension 2F; D – untreated tumor cells (control); E – tumor cells treated with colloidal suspension 1F; F – tumor cells treated with colloidal suspension 2F.
Fig. 2. TEM images corresponding to cells not treated with magnetic colloidal suspensions: A – adherent mesenchymal cells, grown on cell inserts; B – mesenchymal cells (MSC) in suspension; C – adherent tumor cells (SK-BR-3), cultivated on cell inserts; D – tumor cells in suspension.
Fig. 4. Flow cytometric analysis of mesenchymal cells (MSC) before and after treatment with magnetic colloidal suspensions: A, B and C – untreated mesenchymal cells (control); D, E and F – mesenchymal cells treated with colloidal suspension 2F; G, H and I – mesenchymal cells treated with colloidal suspension 1F.
Fig. 6. Tumor development in an immunosuppressed CD1 Nu/Nu mouse animal model