Objectives:

i) the development, characterization and optimization of the EC/ncFET detection device;
ii) development, characterization and optimization of the secondary proton sources obtained after the interaction of the high-power laser beam with solid targets;
iii) assembling the experimental set-up by integrating the EC/ncFET sensor with immobilized cellular cultures within the irradiation chamber;
iv) characterization of radiation effect on cellular cultures.

Project Plan: Stages and activities

I           System Design

I.1        Design of EC / ncFET sensors

I.2        Design of seals / cells

I.3        Protocol design for colorimetric measurements

I.4        Protocol design for ex-situ quantification of ROS / RNS

I.5        Preparation of Specific Complex Sensors (1)

I.6        Colorimetric determinations of extracellular ROS (1)

I.7        In vitro test of biocompatibility of the sensor (1)

I.8        Sensor testing in Proton Radiation Environment (1)

II         Development, characterization and optimization of the detection system

II.1       Preparation of specific complex sensors

II.2      In vitro test of biocompatibility of the sensor

II.3       Colorimetric determinations of extracellular ROS

II.4       Testing the sensor in the environment with proton radiation

II.5       Assessment of cellular irradiation (1)

III        Radiation impact on the cells

III.1     Proton irradiation and ROS / RNS detection

III.2     Molecular response of cell irradiation

III.3     Assessment of cellular irradiation (2)

INCDFM (CO)– Enache Teodor Adrian (Project leader), Diculescu Victor, Ignat-Barsan Madalina, Enculescu Monica, Preda Nicoleta, Matei Elena, Florica Camelia, Costas Andreea, Evanghelidis Alexandru, Beregoi Mihaela;

INLFPR (P1) – Sima Felix, Axente Emanuel, Jipa Florin, Iosub Stefana, Diplasu Constantin, Marcu Aurelian, Serbanescu Mihai, Ungureanu Razvan, Cojocaru Gabriel, Achim Alexandru, Giubega Georgiana, Stancu Elena, Badita Eugenia, Mirela Trupina, Tudor Nicolae, Stoicu Marian, Craciun Florenta;

IFIN-HH (P2) – Bacalum Mihaela, Radu Mihai, Acasandrei Maria-Adriana, Dorobantu Ioan, Neagu Livia, Savu Iulia Diana, Mustaciosu Cosmin-Catalin, Temelie Mihaela, Zorila Bogdan, Popescu Roxana-Cristina, Polifron Victoria, Andrei Papusa;

IBCPNS (P3) – Butoi Elena, Manduteanu Ileana, Calin Manuela, Tucureanu Monica Madalina, Macarie Razvan Daniel, Ciortan Letitia, Vadana Mihaela, Constantinescu Cristina Ana, Mesca Gabriela, Balaceanu Elena.

Results obtained during the period: 18.10.2017 – 31.12.2019.

The main goal of this project was the development of a sensor with dual, parallel detection, by integrated in one sensor platform (EC/ncFET detection device) a classical electrochemical (EC) three-electrodes system and a nanowire-channel (nc) field effect transistor (FET) for analysing and quantifying the effects of laser-induced proton beam on cell cultures.
The first stage of this project was dedicated to the design of: (i) masks for photolithography; (ii) cell chamber and (iii) biological protoclos to be applied on irradiated cell cultures. Also, the performance of different metal nanostructured electrodes for cell culture biocompatibility and electrochemical detection of H₂O₂ was tested. Nevertheless, the effect of proton beam radiation on the electrodes substrate materials was investigated. For this, different nanostructured materials such as Au, Ag and Pt were irradiated at CETAL-PW using aluminum targets with 10 mm thickness. The proton beam energy was estimated at about 5 MeV. After irradiation the electrodes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry and compared with unirradiated electrode.
Electrochemical impedance spectroscopy of Au, Ag and Pt electrodes, deposited as thin films on Si/SiO₂ wafers, was performed in 0.1 M NaPB pH 7.0 before and after irradiation with protons. Au and Ag electrodes exhibited a similar EIS profile, whit a linear Warburg region at high frequencies followed by an unfinished semicircle for medium to low frequency range. Pt electrodes had the diffusion Warburg region shorter and the semicircle was better defined and almost complete. The spectra were fitted using an equivalent circuit comprising a cell resistance R_Ω in series with a Warburg element (Z_w) in series with a parallel combination of a charge transfer resistance (R_ct) and a double layer constant phase element (CPE_dl).
Au and Pt electrode behaviour did not change significantly after irradiation, while the Ag electrode exhibited an EIS spectra with higher impedance value after irradiation. The cyclic voltammetric profile of Ag confirmed these differences in electrochemical properties, with much smaller faradaic currents after irradiation of the sample. Au and Pt electrode had similar CV profiles before and after irradiation.
The effects of different metal electrode materials, Au and Ag, on the endothelial EAhy926 cell culture viability was investigated and compared with the cell culture on the TCP wells (as positive control). After 72 h incubation the cell viability and metal induced cytotoxicity were determined using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Lactate dehydrogenase (LDH), respectively. All samples were run in triplicate in the same assay.
For the fabrication of sealing chamber, Polydimethylsiloxane (PDMS), known for both mechanical and biocompatibility properties was used. The PDMS was prepared by combining in a 1:10 ratio a specific curing agent and an elastomer base. The mixture was next placed on a flat plate and heated at 100 °C for 3 minutes where the cross-linking process is occurring. After cooling at room temperature, the PDMS material was cut with precision to silicon sensor dimensions and around cells location. An oxygen plasma treatment was used to bond the PDMS material on the sensor surface. The cells chamber was further isolated from surrounding environment by a thin Kapton foil, with a thickness of 60 µm, through a self-adhesive process on the PDMS surface. The final structure was fixed in a Teflon mount and kept for 4 hours in a vacuum chamber at 10^-3 mbar pressure. No deformation was observed during or after the vacuum test.
The experiments for proton irradiation on B16 cells (melanoma cells) and EA.hy926 cells (endothelial cells) were performed at a Cyclotron TR19, IFIN-HH. After optimization of the set-up and dosimetry, the radiobiological experiments were performed at doses ranging between 0 and 2 Gy and the effect against cell cycle and detection of O2•-  after proton irradiation were investigated.
The OCP (open circuit potential) of components electrodes of electrochemical sensor developed at INCDFM was measured in culture media and for B16 cells before and after proton irradiation and in the presence of H₂O₂. It was observed that the OCP increase for cell culture exposed to stress conditions. While OCP recorded only for culture media was stabilized at a potential around -150 mV, for B16 cell culture the OCP value was 50 mV but increase after cell exposure at H₂O₂ and proton irradiation.
Also, following irradiation at the specified time points, the medium from the cells was collected, and the cells were incubated with 5 μM MitoSOX™ Red for 10 min. Afterwards the cells were washed and lysed with trypsin. The medium was collected and transferred to a 96 black well plate and the fluorescence was recorded at Mithras plate reader (Berthold) with the excitation set at 485 nm and the emission at 590 nm. Each experiment was done in duplicates and repeated at least three times. It was observed that the production of superoxide species is increasing with time and doe for both cell lines, with the highest concentration found at 24h after irradiation.
Other experiments, using the fluorescent dye H2DCFDA (Invitrogen), were performed in order to evaluate how proton radiation influences the formation of reactive oxygen species (ROS). After irradiation with similar doses as reported above, the cells were left for 2h, 4h and 24h and at the specified time points, the cells were loaded in PBS with the fluorescent dye for 30 min at 37^oC. After the time passed, the cells were washed and the fluorescence was monitored at a plate reader with and excitation set at 485 nm and the emission recorded at 520 nm. If in the cells are present oxygen species, from the non-fluorescent form of the dye the acetate groups are cleaved leaving only the fluorescent group DCF. Increase fluorescence in correlated with increase ROS. In figure 2 are presented the normalized values of ROS recorded after 2h, 4h and 24h prior to proton irradiation.
It was found that the amount of reactive oxygen species increases with time and the highest level is found at 24h after irradiation. In addition, the gene and protein expression for different molecules associated with pro and anti-oxidative status were investigated using RT-PCR and Western Blot. The peroxidase activity measured was increased in cells exposed to proton irradiation for both cell lines. Since the production of reactive oxygen species (ROS) was also increased by proton irradiation, the increased of peroxidase activity along with ROS underlie a defence response of cells to irradiation. In addition, the NADPH oxidase, an important source for ROS production in phagocytes and in vascular cells, was two time increased in irradiated cells with 2Gy suggesting an increased oxidative stress induced by proton irradiation.
The effect of irradiation on catalase activity was investigated and it was observed that enzyme activity is significant decreased in endothelial cells whereas the melanoma B16 cells exhibited lower catalase activity compared with endothelial cells and was not affected by proton irradiation. As an effect of increased reactive oxygen species produced by pro-oxidative enzymes and a reduced defence mechanism confirmed by decrease of catalase activity, the NO production and its bioavailability was also reduced. However, the levels of NO were significantly decreased by proton irradiation in both type of cells, while the AGE products remain unmodified.
In order to investigate the changes produced as gene expression of molecules involved in oxidative/anti-oxidative response, we performed PCR using ARN isolated from control or irradiated cells. The results indicate that gene expression of investigated pro-oxidant NADPH oxidase subunits are significantly increased while the anti-oxidative SOD is reduced by 2Gy exposure. The gene expression of NO was not affected by cell irradiation. In the case of melanoma cells, there is a small increase of the NDPH oxidase subunits NOX1 and NOX4, but not at significant level. The NADPH subunit p22, was significantly increased in B16 cells irradiated wit 1Gy while SOD was not significantly affected by irradiation.
The protein expression of anti-/pro-oxidative molecules were investigated by WB assay. After irradiation, cells were homogenised in RIPA lysis buffer and used for electrophoresis separation. The protein expression results confirmed the gene expression showing that while the protein expression of pro-oxidative molecules (p47phox, NOX1 and NOX4) is increased by proton irradiation, the protein expression of eNOS (NO synthase) is significantly reduced by 2Gy irradiation in both type of cells. Endothelial NOS, also known as nitric oxide synthase 3, generates NO in blood vessels. NO produced by eNOS in the vascular endothelium plays crucial roles in regulating vascular tone, cellular proliferation, leukocyte adhesion and platelet aggregation.

Articles:

V.C. Diculescu, T.A. Enache, Voltammetric and mass spectrometry investigation of methionine oxidation; J Electroanal Chem 834 (2019) 124. IF: 3.218; AIS: 0.73

Conferences:

• Mihaela Bacalum , Simona Dîrleci , Mihai Straticiuc , Ion Burducea , Radu Andrei , Decebal Iancu , Radu Vasilache , Diana Savu and Mihai Radu; Set-up optimization of IFIN-HH 3 MV Tandetron TM and proton beam dosimetry for radiobiology experiments;  44th European Radiation Research Congress, Pecs, Hungary, August 21-25, 2018;
• Mihai Radu, Liviu Craciun, Mihaela Temelie, Mihaela Bacalum, Mihai Straticiuc, Ana; New radiobiology setup for proton irradiation adapted at the TR19 cyclotron of IFIN-HH; Chiriacescu, Tiberiu Esanu, Radu Vasilache, and Diana Savu; 44th European Radiation Research Congress, Pecs, Hungary, August 21-25, 2018;
• Mihaela Bacalum, Simona Dîrleci, Mihai Straticiuc, Ion Burducea, Radu Andrei, Decebal Iancu, Radu Vasilache, Diana Savu and Mihai Radu; Optimization and dosimetry of IFIN-HH 3 MV TandetronTM proton beam for radiobiology experiments, , International Satellite Meeting of Radiobiology of the 15th National Conference of Biophysics, September 10, 2018;
• Florin Jipa, Stefana Iosub, Cristina Nita, Emanuel Axente, Felix Sima "Biosystems manufacturing in photosensitive glasses by high repetition rate picosecond laser", Oral presentation, RSBMB Conference, Bucharest, Romania, September 5-7, 2018.
• Florin Jipa, Stefana Iosub, Bogdan Calin, Emanuel Axente, Felix Sima, Koji Sugioka "3D embedded structures fabrication in photosensitive glasses by high-repetition-rate picosecond laser", Oral presentation, Symposium X : Photon-assisted synthesis and processing of materials in nano-microscale of the E-MRS 2018 Spring Meeting, Strasbourg, France, June 18-22, 2018.
• Mihaila A, Vadana M, Bacalum M, Raileanu M, Ciortan L, Caciun L, Esanu TR, Manduteanu I, Simionescu M, Butoi E. Effects of low-dose proton radiation on oxidative stress produced in endothelial and epithelial cells. The 37^th Annual Scientific Session of the Romanian Society for Cell Biology and the 11^th National Congress with International Participation, Constanta, Romania, 20-23 Iunie, 2019.
• M. Radu, M. Temelie, M. Bacalum, R. Popescu, D. Savu, L. Craciun, D. Niculae, M. Straticiuc, I. Burducea, P. Vasos, D. Stutman; Advances in Bio-medical Application of Ionizing Radiation at IFIN-HH & ELI-NP; 19th International Balkan Workshop on Applied Physics and Material Science, Constanta, Romania, July 16-19, 2019;
• T.A Enache, M. Enculescu and VC. Diculescu, Redox Mechanism of Azathioprine and its Interaction with DNA, 70th Annual Meeting of the International Society of Electrochemistry (ISE); 4-9 August 2019; Durban, Africa de Sud
• M Răileanu, M Straticiuc, R Andrei, D Iancu, M Radu and M Bacalum, PIM; Generation of Reactive oxygen species (ROS) in hepatocarcinoma cells after proton irradiation; , Cluj-Napoca, 25 - 27 September 2019;
• D. Savu, M. Temelie, R. Popescu, M. Bacalum, M. Straticiuc, L. Craciun, M. Radu; New developments in radiobiology with biomedical applications for cancer therapy, , 35th Congress of Turkish Physics Society, Bodrum, Turcia, September 4-8, 2019;