NANOCEL Project

NANOCEL Project

In vitro and in vivo evaluation of cellular death mechanisms induced by magnetic nanoparticles interacting with tumor cells 

Duration: 2016 – 2017

Project no.: PIII-C4-PCFI-2016/2017-03

Acronym: NANOCEL

Budget: 135.000 RON

Coordinator: ”Victor Babeș” University of Medicine and Pharmacy Timișoara

Project director: Prof. Dr. Virgil Păunescu 

General objectives: (1) identification of cell death mechanisms in the presence of Fe3O4 nanoparticles (MNPs); (2) evaluation of MNPs influence in tumor formation and development.

Specific objectives: (1) Morphologic, phenotypic, genotypic, and functional characterization of solid tumors in vitro in presence of magnetite nanoparticles (Fe3O4); (2) Evaluation of mechanisms involved in nanoparticles-induced cellular death;  (3) In vitro identification of characteristic markers that appear as a result of nanoparticles interactions with tumor cells and tumor microenvironment cells; (4) In vivo evaluation of tumor cells ability to form and develop solid tumors in presence of nanoparticles.

Abstract:

During the tumorigenesis process, tumor cells have an uncontrolled proliferation, thus eluding the regulatory mechanisms of cellular death. There are few major morphologies of cell death that have been described so far: apoptosis (type I), cell death associated with autophagy (type II), necrosis (type III), mitotic catastrophe, and anchorage-dependent mechanisms – anoikis, excitotoxicity, Wallerian degenerescence, skin cornification. Here, we show for the first time a possibly novel mechanism inducing tumor cell death under in vitro conditions-enucleation. We pursued the influence of colloidal suspensions of Fe3 O4 nanoparticles on tumor cell lines.  Magnetite nanoparticles were prepared by combustion synthesis and double layer coated with oleic acid, thus interacting with tumor cells. The in vitro studies were focused on morphological and ultrastructural changes, functional studies, immunophenotypical markers and gene expression – induced on tumor cells and the resulted nuclei, using modern technologies. The in vivo models will be used for confirming tumor cells ability to form solid tumors in immunodeficient mice, while revealing the potential of magnetite nanoparticle to inhibit tumor development. The primary tumors and metastases generated in vivo will be fully characterized. This project will bring new evidence regarding enucleation, as a potential mechanism of tumor cell death, which might open new horizons in cancer biology research and development of therapeutic agents capable of exploiting this behavior. 

Results:

  • Methods for demonstrating the MNPs effect on normal and tumor cells, using scanning electron microscopy (SEM) (Fig. 1)
  • Methods for demonstrating the MNPs effect on normal and tumor cells, using scanning electron microscopy (SEM) (Fig. 2 and 3)
  • Flowcytometric method to evaluate the changes induced by MNPs at cellular level (Fig. 4)
  • Immunocytochemical method for evaluation of morphological and immunophenotypical changes induced by MNPs at cellular level (Fig. 5)
  • PBMCs isolation protocols;
  • Protocols for co-culturing systems required for tumor cells – tumor microenvironment cells interactions;
  • Panel of characteristic markers of MNPs-treated tumor cells after interaction with immune cells;
  • Protocols for GFP staining of tumor cells;
  • Protocols for tumor formation in immunosuppressed mice (Fig. 6);

Articles in extenso:

  1. 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, Nanocel)
  2. 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)
  3. 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)
  4. 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)
  5. 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. CHIM. (Bucharest), 2017; 68 (9): 2079-20182. (IF = 1.412; Nanocel)
Fig. 1. SEM images of mesenchymal stem cells (MSCs) and tumor cells (SK-BR3 cell line) before and after treatment with colloidal magnetic suspension: A – untreated mesenchymal stem cells (MSCs) (control); B – colloidal suspension 1F-treated mesenchymal stem cells (MSCs); C – colloidal suspension 2F-treated mesenchymal stem cells; D – untreated tumor cells SK-BR-3 (control); E – colloidal suspension 1F-treated tumor cells; F – colloidal suspension 2F-treated tumor cells.
Fig.2. TEM images corresponding to untreated cells: A – adherent mesenchymal stem cells (MSCs), grown on cell culture inserts; B – mesenchymal stem cells (MSC) in cellular suspension; C – adherent tumor cells (SK-BR-3), grown on cell culture inserts; D – cellular suspension of SK-BR3 tumor cells.
Fig.3. TEM images of SK-BR3 tumor cells treated with colloidal magnetic 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. 4. Flowcytometric analysis of mesenchymal stem cells (MSCs) before and after treatment with magnetic colloidal suspensions: A, B and C – untreated MSCs (control); D, E and F – colloidal suspension 1F-treated MSCs; G, H and I – colloidal suspension 2F-treated MSCs.
Fig.5. Immunocytochemical analysis of mesenchymal stem cells (MSCs) and tumor cells (SK-BR3), stained for cytoskeleton protein Vimentin and Her2 oncoprotein; A – untreated MSCs (control); B – 1F colloidal suspension-treated MSCs; C – 2F colloidal suspension-treated MSCs; D – untreated SK-BR3 tumor cells (control); E – 1F colloidal suspension-treated tumor cells; F – 2F colloidal suspension-treated tumor cells.
Fig.6. Tumor development in murine model – CD1 Nu/Nu immunosuppressed mice.

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